Guanylate Cyclase and the cNO-cGMP Signaling Pathway

7/27/2019 Guanylate Cyclase and the cNO-cGMP Signaling Pathway

1/17

Review

Guanylate cyclase and the cNO/cGMP signaling pathway

John W. Denninger a, Michael A. Marletta aYbYcY*

a 5315A Medical Sciences I, Department of Biological Chemistry, University of Michigan Medical School,

1150 West Medical Center Drive Ann Arbor, MI 48109-0606, USAb Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109-0606, USA

c Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065, USA

Received 13 August 1998; received in revised form 30 November 1998; accepted 16 December 1998

Abstract

Signal transduction with the diatomic radical nitric oxide (NO) is involved in a number of important physiological

processes, including smooth muscle relaxation and neurotransmission. Soluble guanylate cyclase (sGC), a heterodimeric

enzyme that converts guanosine triphosphate to cyclic guanosine monophosphate, is a critical component of this signaling

pathway. sGC is a hemoprotein; it is through the specific interaction of NO with the sGC heme that sGC is activated. Over

the last decade, much has been learned about the unique heme environment of sGC and its interaction with ligands like NO

and carbon monoxide. This review will focus on the role of sGC in signaling, its relationship to the other nucleotide cyclases,

and on what is known about sGC genetics, heme environment and catalysis. The latest understanding in regard to sGC will

be incorporated to build a model of sGC structure, activation, catalytic mechanism and deactivation. 1999 Elsevier

Science B.V. All rights reserved.

Keywords: Guanylate cyclase; Cyclic guanosine monophosphate; Nitric oxide; Carbon monoxide; Heme

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

2. Historical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

3. Signaling with the cNO/cGMP pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

4. The nucleotide cyclase family: sGC, pGC and AC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

0005-2728 / 99 / $ ^ see front matter 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 0 0 5 - 2 7 2 8 ( 9 9 ) 0 0 0 2 4 - 9

Abbreviations: AC, adenylate cyclase; ATP, adenosine 5P-triphosphate; L11385, NH2-terminal heme binding domain of the L1 subunit

of soluble guanylate cyclase; cAMP, cyclic adenosine 3P,5P-monophosphate; cGMP, cyclic guanosine 3P,5P-monophosphate; CO, carbon

monoxide; CO-sGC, soluble guanylate cyclase with nitric oxide bound in the distal heme coordination site; EDRF, endothelium derived

relaxing factor; EPR, electron paramagnetic resonance spectroscopy; FTIR, Fourier transform infrared spectroscopy; GTP, guanosine

5P-triphosphate; cNO, nitric oxide; NO32 , nitrite; NO3

3 , nitrate; NOS, nitric oxide synthase; NO-sGC, soluble guanylate cyclase with

nitric oxide bound in the distal heme coordination site; PDE, phosphodiesterase; pGC, particulate guanylate cyclase; sGC, soluble

guanylate cyclase; RR, resonance Raman spectroscopy

* Corresponding author, at address a. Fax: +1 (734) 6475687; E-mail: [email protected]

Biochimica et Biophysica Acta 1411 (1999) 334^350


2/17

5. Soluble guanylate cyclase genetics and isoforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339

6. Purication from mammalian tissue and expression systems . . . . . . . . . . . . . . . . . . . . . . . 341

7. Heme environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

8. Catalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

9. Model of sGC structure and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346

1. Introduction

Soluble guanylate1 cyclase (sGC) is the only con-

clusively proven receptor for nitric oxide (cNO), a

signaling agent produced by the enzyme nitric oxide

synthase (NOS) from the amino acid L-arginine. As

shown in Fig. 1, sGC catalyzes the conversion of

guanosine 5P-triphosphate (GTP) to cyclic guanosine

3P,5P-monophosphate (cGMP). Because it is one of

two enzymes (the other is the membrane-bound pep-

tide-receptor guanylate cyclase) that produce cGMP,

and because it is the only denitive receptor for cNO,

sGC is intimately involved in many signal transduc-tion pathways, most notably in the cardiovascular

system (e.g., in the regulation of vascular tone and

platelet function) and in the nervous system (e.g. in

neurotransmission and, possibly, long-term potentia-

tion and depression). Soluble guanylate cyclase is a

heterodimeric protein composed of K and L subunits;

in addition, it is a hemoprotein (see Fig. 2). cNO

binds to the sGC heme; this binding event activates

the enzyme. The activation of sGC and the subse-

quent rise in cGMP concentration are what allow

sGC to transmit an cNO signal to the downstream

elements of the signaling cascade-cGMP-dependent

protein kinase, cGMP-gated cation channels and

cGMP-regulated phosphodiesterase. As will be de-

scribed below, nearly 30 years of study of sGC

have given us an outline of sGC structure and func-

tion; however, many facets of sGC structure and

function, especially at the molecular level, remain

to be discovered.

2. Historical perspective

Although both sGC and its product, cGMP, were

identied in the 1960s, it has been only within thelast 10 years that the complete outline of the cNO/

cGMP signaling pathway has been understood. After

the discovery of cAMP in 1958 [1] and the initial

elucidation of its role in physiology (for early reviews

see [2,3]), the search for other physiologically rele-

vant cyclic nucleotides began. In 1963, cGMP was

detected in the urine of rats [4]; several years later,

guanylate cyclase was found in mammalian tissues

[5^9]. In 1977, cNO 9 both as cNO gas and as cNO

released from vasodilators like nitroglycerin and

nitroprusside 9 was shown to activate guanylate

cyclase [10^13]. At that point, though, cNO was

unknown in biological systems, except when exoge-

nously supplied by drugs like nitroglycerin, so it was

assumed that cNO activation of sGC was a non-

physiological, if useful, phenomenon.

The story soon became clear, however. In 1980,

Furchgott et al. were able to demonstrate the exis-

tence of a substance produced by the endothelium

that was required to mediate relaxation of blood

1 Although we prefer `guanylate' cyclase (as does the Index

Medicus and the Oxford Dictionary of Biochemistry and Molec-

ular Biology), `guanylyl' cyclase is also used. Neither of the com-

mon names for this enzyme is technically correct: `guanylate'

refers to the anion of guanylic acid (guanosine monophosphate)

and `guanylyl' refers to the acyl group derived from guanylic

acid.

J.W. Denninger, M.A. Marletta / Biochimica et Biophysica Acta 1411 (1999) 334^350 335


3/17

vessels [14]. They designated this substance endothe-

lium-derived relaxing factor (EDRF). In 1986, Ignar-

ro and Furchgott independently suggested that

EDRF was cNO (reviewed in [15]); the next year, it

was demonstrated that this was the case [16,17]. Atthe same time, our laboratory and others were un-

raveling the story of nitrite (NO32 ) and nitrate (NO3

3 )

production in mammals. After initial observations by

Tannenbaum and coworkers that mammals produce

NO33 and that NO3

3 synthesis is increased following

immune stimulation [18^20], it was shown that NO32and NO33 were produced by immunostimulated mac-

rophages from the amino acid arginine (reviewed in

[21,22]). Shortly thereafter, our laboratory and

others demonstrated that a soluble enzyme in murine

macrophages, later puried and named nitric oxide

synthase, was producing cNO from the amino acid

arginine [23^25]. At this point, the major compo-

nents of the cNO/cGMP signaling pathway had

been identied: nitric oxide synthase produces cNO,

which activates soluble guanylate cyclase, leading to

a subsequent increase in the concentration of cGMP.

3. Signaling with the cNO/cGMP pathway

Since the initial elucidation of the cNO/cGMP sys-

tem, many good examples of signal transduction bythis system have come to light, some better charac-

terized than others. They include: smooth muscle

relaxation and blood pressure regulation [26]; plate-

let aggregation and disaggregation [27]; and neuro-

transmission both peripherally, in non-adrenergic,

non-cholinergic (NANC) nerves [26], and centrally,

in the processes of long term potentiation and de-

pression [28]. Understanding the cNO/cGMP path-

way, however, requires an understanding of cNO's

role as a signaling agent, the alternative targets forcNO, the alternative activators of sGC and, natu-

rally, the targets for cGMP.

It was initially a surprise that cNO was produced

physiologically: prior to the demonstration of cNO

synthesis by nitric oxide synthase, the biological rele-

vance of cNO was as a pollutant; for example, as a

component of automobile exhaust. Given the phys-

ical properties of cNO, however, it is now clear thatcNO is uniquely well suited to its role as a signaling

agent. Because it is small and carries no charge, cNO

diuses freely across cell membranes, obviating the

need for a transport system. cNO is relatively unreac-

tive for a radical species, so that it can diuse from a

cell in which it is produced to neighboring cells with-

out being destroyed before it reaches its target. Be-cause cNO is oxidized to NO32 and NO

3

3 under phys-

iological conditions, no separate mechanism for its

destruction is required [29^31]; however, at low con-

centrations of cNO and in the absence of proteins

and metals which contribute to its destruction, the

decomposition of cNO will be slow [29]. Most impor-

tantly, the electronic structure of cNO makes it an

excellent ligand for heme, allowing it to bind to the

sGC heme at a low, non-toxic concentration.

The one well-dened target for cNO, of course, is

sGC; however, several paths for cNO signal trans-

duction which do not rely on the production of

cGMP by sGC have been proposed, though they

are less clearly dened than the cNO/cGMP pathway.

First, cNO was shown to inhibit mitochondrial cyto-

chrome oxidase at concentrations of cNO found

physiologically [32,33]. Second, cNO was reported

to activate calcium-dependent potassium channels

in vascular smooth muscle directly, though it is pos-

sible that this only happens with levels of cNO higher

than typically seen in vivo [34]. Third, cNO was

shown to inhibit activation of NF-UB [35,36].

Fourth,c

NO was found to activate G-proteins(such as protooncogene p21ras) by enhancing the

rate of nucleotide exchange, leading to an increase

in the GTP-bound form. In fact, a p21ras cysteine

residue (C118) was shown to be S-nitrosylated in the

presence of cNO [37^39]. Fifth, caspase, a cysteine

protease involved in apoptosis, was shown to be in-

hibited by S-nitrosylation of its active-site cysteine

[40]. Finally, both hemoglobin and the cardiac calci-

um release channel (i.e., the ryanodine receptor) were

shown to be S-nitrosylated in vivo; moreover, this

change resulted in a modication of their function

[41^43]. As these alternative cNO targets become bet-

ter dened, it will be important to ensure that they

are physiologically relevant. cNO, especially at high

concentration, can react with O2 through a series of

reactions to form N2O3, which is a potent nitrosating

agent reacting capable of reacting with nucleophiles

such as the sulfur of cysteine residues. As a result, in

vitro experiments with high cNO concentrations may

lead to higher than normal levels of S-nitrosocysteine

J.W. Denninger, M.A. Marletta / Biochimica et Biophysica Acta 1411 (1999) 334^350336


4/17

formation. Therefore, while there is no doubt that S-

nitrosylation occurs in vivo, we believe its physiolog-

ical signicance will not be established until two ob-

servations are made: rst, that S-nitrosylation 9 at

in vivo levels ofc

NO9

modies the function oftarget proteins and, second, that in vivo concentra-

tions of S-nitrosylated proteins can elicit a physio-

logical response.

In addition to other targets for cNO, there are

other potential physiological activators of sGC.

These fall into two categories 9 non-cNO nitroge-

nous compounds and carbon monoxide (CO).

Although most investigators are now convinced

that cNO itself is the signaling agent which activates

sGC, there are still some who believe that there are

other nitrogenous products (not cNO) which acti-

vates sGC. Many alternatives have been proposed,

such as nitrosothiols. It is not clear, however, that

these agents are present in high enough concentra-

tion in vivo to perform any signicant signaling func-

tion. Likewise, it has been hypothesized that CO,

which binds to the sGC heme and weakly activates

the enzyme, may act as a signaling agent [44^46]. In

contrast to cNO, which activates sGC approx. 400-

fold, CO only activates known isoforms of sGC ap-

prox. 5-fold [47]. Additionally, CO activation re-

quires saturating concentrations of CO, a clearly

non-physiological alternative. Although CO is pro-duced by the enzyme heme oxygenase during the

breakdown of heme, it is dicult to imagine that

cells can provide an adequate supply of free heme

(which is toxic to cells) for signaling with CO. In

the absence of a more CO-sensitive sGC isoform or

a mechanism to increase the response of sGC to CO,

we will have to reserve judgment on this potential

pathway. Such a mechanism may have been found,

however. It has been shown recently that CO and the

synthetic compound YC-1 ([48]; see Fig. 4 for the

structure) synergistically activate sGC to levels sim-

ilar that of the cNO-stimulated enzyme [49,50]. If a

physiological counterpart for YC-1 is identied, this

could be a mechanism by which CO could act as a

signaling agent.

Regardless of the species that activates sGC, all

signal transduction through sGC (and pGC as well)

takes place through an increased concentration of

cGMP. Cyclic GMP, like cAMP, is a second messen-

ger; as such, it requires target proteins to have its

eect. Moreover, the diversity of targets allows

cGMP to have wide-ranging eects that can dier

by cell and tissue type. There are three known targets

for cGMP which mediate the transmission of thec

NO/cGMP pathway signal downstream from guany-late cyclase: cGMP-dependent protein kinase [51],

cGMP-regulated phosphodiesterase [52,53] and

cGMP-gated ion channels [54]. The rst of these,

cGMP-dependent protein kinase, phosphorylates tar-

get proteins in response to an increase in cGMP con-

centration. For example, in smooth muscle cells,

cGMP-dependent protein kinase phosphorylates the

inositol 1,4,5-triphosphate receptor, leading to a de-

crease in Ca2 concentration and, ultimately, smooth

muscle relaxation. The second, cyclic nucleotide

phosphodiesterase, catalyzes the hydrolysis of the

3P-phosphodiester bond of cAMP and cGMP to yield

AMP and GMP, respectively. Of the seven known

families, three (PDE2, PDE5 and PDE6) are allos-

terically regulated by cGMP and one (PDE3) is in-

hibited by the binding of cGMP in its substrate site.

Phosphodiesterases allow crosstalk between cGMP

and cAMP signaling pathways because they cause

the concentration of one cyclic nucleotide to inu-

ence the degradation of the other. The third, cyclic

nucleotide-gated ion channels, are non-specic cation

channels found in a variety of tissues. Most notably,

these channels are found in the retina and in olfac-tory epithelium where they are involved, respectively,

in visual phototransduction and in olfaction.

4. The nucleotide cyclase family: sGC, pGC and AC

Just as sGC is a component of the larger cNO/

cGMP signaling pathway, so is it a member of a

larger group of related enzymes: the nucleotide cy-

clases. This family of enzymes converts nucleotide

triphosphates (GTP and ATP) to cyclic nucleotide

monophosphates (cGMP and cAMP) and includes

adenylate cyclase (AC), particulate guanylate cyclase

(pGC) and soluble guanylate cyclase. As a family,

these enzymes are involved in a broad array of signal

transduction pathways mediated by the cyclic nucleo-

tides cAMP and cGMP-vision, blood pressure regu-

lation, response to hormones and many others. An

evolutionarily conserved catalytic domain denes

these three groups of enzymes as a single family; in



5/17

other words, the genes that code for the catalytic

portions of these enzymes are derived from a com-

mon ancestor. This is unsurprising, given that they

catalyze virtually identical reactions. Moreover, be-

cause of their relatedness and similar function, in-

sights into the structure, catalytic mechanism andpossibly even regulation of one member of this fam-

ily may be generalizable to other members.

Adenylate cyclase, for example, has been studied

for nearly four decades. As a result, there is a rich

literature of ndings with AC that may, because of

the similarity of AC to sGC, prove useful in under-

standing sGC. Unlike the guanylate cyclases, which

contain both receptor and catalytic functions within

the same protein, ACs are stimulated by hormone

receptors acting through heterotrimeric G-proteins

[55]. AC is an integral membrane protein and is as-

sumed to be monomeric. All of the known mamma-

lian isoforms are activated by GsK; other activators

are isoform specic [56]. AC is also activated by the

small molecule forskolin, a diterpene derivative iso-

lated from the Indian plant Coleus forskohli. Forsko-

lin was originally shown to have a variety of cardio-

vascular eects [57] and was later shown to activate

AC [58]; it has served as a useful tool in the study of

AC, but no mammalian counterpart has been iden-

tied. As shown in Fig. 2, AC contains two groups

of six transmembrane helices and two intracellular

loops. The intracellular loops each contain a region

9 C1 and C2, respectively 9 now known to come

together in a head-to-tail fashion to form a function-

al catalytic domain. (Because the domain is made upof two regions from a single polypeptide chain, it is

sometimes referred to as `pseudo-heterodimeric'.)

Two crystal structures of catalytic domains have, in

fact, been solved recently. The rst was a structure of

a C2C2 homodimer [59], the second of a C1C2 heter-

odimer [60]; both were engineered constructs which

lacked the transmembrane domains and were, thus,

soluble. The crystal structure of C1C2 supports the

conclusion that this pseudo-heterodimeric catalytic

domain has two pseudo-symmetric binding sites:

one binds substrate, the other forskolin. In addition,

GsK binds to a site in the plane of the catalytic do-

main opposite the substrate binding site.

Particulate guanylate cyclase has also provided in-

sight into the structure and function of sGC. After

the purication of cGMP and guanylate cyclase, it

was observed that guanylate cyclase activity could be

separated into a membrane-bound (particulate) and

cytosolic (soluble) fractions. As its name implies,

particulate guanylate cyclase is the membrane-bound

Fig. 1. The guanylate cyclase reaction and cNO signal transduction.



6/17

guanylate cyclase. In a sense, pGC is a prototypic

cell-surface receptor enzyme: it contains an extracel-

lular peptide receptor domain and an intracellularcatalytic domain separated by a single transmem-

brane domain (Fig. 2). pGC is now generally thought

to be a homodimer of subunits of approx. 120 kDa

[61]. Seven subclasses of mammalian pGC (GC-A

through GC-G) have been identied. Three of these

have known peptide ligands (GC-A, atrial natriuretic

peptide and brain natriuretic peptide; GC-B, C-type

natriuretic peptide; GC-C, the heat stable enterotox-

ins ofEscherichia coli and guanylin); the rest (GC-D,

olfactory epithelium; GC-E and GC-F, retina;

GC-G lung, intestine, skeletal muscle) are so-called

`orphan' receptors because their activating ligands

have not been determined [55,61,62]. For the pur-

poses of understanding sGC, pGC is useful because

it helps to dene the characteristics that are specic

to guanylate cyclases, for example, the identity of

the amino acids which allow the dierent members

of the nucleotide cyclase family to distinguish GTP

from ATP [63]. Also, given the similarity of their

catalytic domains, it is possible that the general

mechanism of activation of pGC will be similar to

that of sGC.

5. Soluble guanylate cyclase genetics and isoforms

Since the original identication of sGC, a great

deal of information about sGC genetics has become

available. Thus far, a total of 12 sGC cDNAs have

been cloned from ve dierent species (for identity

and similarity comparisons see Fig. 3). The rst were

cloned on the basis of protein sequence from puried

bovine and rat K1L1 heterodimer. The cDNA se-

quences for bovine and ratL

1 subunits, respectively,

were reported in 1988 [64,65], followed by the bovine

and rat K1 subunits in 1990 [66,67]. Subsequently,

K1L1 pairs were cloned from Homo sapiens (origi-

nally called K3 and L3) ([68,69], localized to chromo-

some 4q32, [70]), Drosophila melanogaster [71^73]

and the sh Oryzias latipes [74]. Putative sGC se-

quences exist in the Caenorhabditis elegans genome,

but these have yet to be conrmed with any empiri-

cal studies beyond the sequencing itself.

Fig. 2. Schematic representation of sGC, pGC and AC. Transmembrane helices are shown in purple, the conserved sGC, pGC and

AC catalytic regions are shown in orange and the conserved NH2-terminal domains of the sGC K1 and L1 subunits are shown in

green. The sGC L1 subunit also binds prosthetic heme, which is ligated by histidine 105.



7/17

In addition to the cloning of K1L1 pairs, the

cDNAs for two additional sGC subunits have been

cloned: L2 from rat kidney [75] and K2 from human

fetal brain ([76], localized to chromosome 11q21-q22

[77]. In transient transfection experiments, the K2

subunit has been shown to form an active hetero-

dimer with the L1 subunit [76]. Although the L2 sub-

unit has been recently reported to form an active

heterodimer with the K1 subunit in transient expres-

sion experiments [78], similar experiments in our lab-

oratory (unpublished results) and others (unpub-

lished results, see [79,80]) were unable to generate

an active heterodimer containing the L2 subunit.

Thus, the issue of the L2 subunit partner remains

to be conclusively proven. A unique feature of the

L2 subunit is that its predicted amino acid sequence

contains a COOH-terminal consensus sequence

(-CVVL) for isoprenylation/carboxyl-O-methylation

[75], but it is not known whether such a modication

exists on the mature polypeptide. Because full-length

sequences of the K2 and L2 subunits from species

other than the ones from which they were originally

isolated have yet to be identied, one might question

their signicance. However, a partial sequence for

the bovineK

2 has been determined [76] and a puta-

tive L2 subunit partial sequence exists in a mouse

placenta EST database (GenBank accession No.

AA023957, from the Soares mouse placenta library).

These subunits, then, may well be common to all

mammals and perhaps to other sGC-containing spe-

cies.

The potential existence of novel sGC isoforms (i.e.,

in addition to the K1L1) raises an important issue

about the distinction between sGC subunits and

Fig. 3. Sequence identity and similarity among the predicted amino acid sequences of known sGC subunits. Translated cDNA se-

quences for the indicated sGC subunits were compared using the program Gap with gap weight = 12 and length weight= 4 (Wisconsin

Package Version 9.1, Genetics Computer Group (GCG), Madison, WI, USA). The denitions of the three letter species codes above

are: bov, bovine, Bos taurus ; hum, human, H. sapiens ; rat, Rattus rattus ; fsh, Medaka sh O. latipes ; dro, D. melanogaster. GenBank

accession numbers for the cDNA sequences are as follows: bovine K1, X54014; human K1, X66534; rat K1, M57405; O. latipes K1,AB000849; Drosophila K1, U27117; human K2, X63282; bovine L1, Y00770; human L1, X66533; rat L1, M22562; O. latipes L1,

AB000850; Drosophila L1, U27123; rat L2, M57507.



8/17

sGC isoforms. Our denition of an sGC isoform is a

catalytically competent heterodimer (in principle,however, it could be a homodimer). For AC (which

exists as a monomer) and pGC (which exists as a

homodimer), the identication of dierent isoforms

9 with dierent tissue or developmental distribution,

dierent regulation and/or dierent kinetic parame-

ters 9 has been relatively simple: each distinct gene

product is a distinct isoform. For sGC, the picture is

somewhat more complicated. Given that four distinct

sGC subunits have been cloned, there are 16 possible

homodimeric and heterodimeric combinations of

sGC subunits. Each of these combinations, if shown

to exist in vivo, could be rightly called an isoform.

The only isoform, therefore, that has been shown to

exist at the protein level in animal tissues is the K1L1.

Again, as mentioned above, putative K2L1 and K1L2

isoforms have been demonstrated in transient mam-

malian expression experiments [76,78], but until these

subunits are shown to exist in vivo and, ideally, pu-

ried from tissue sources, they cannot be considered

bona de sGC isoforms. Because the role (even the

existence) of sGC isoforms other than the K1L1 is

poorly understood, our discussion of sGC character-

istics will, by necessity, focus on the K1L1 isoform.

An additional curiosity about the heterodimeric

nature of sGC is the apparent requirement for co-expression of sGC subunits. For example, sGC K1

and L1 subunits, when expressed separately in tran-

sient mammalian expression systems, have no guany-

late cyclase activity [67,73,81]. In fact, there is still no

detectable guanylate cyclase activity when the soluble

fractions from cells expressing the K1 and L1 subu-

nits individually are mixed [73,81]. Only when the K1

and L1 subunits are co-expressed (i.e., produced in

the same cell) does basal and cNO-stimulated activity

result [67,73,81]. The sGC requirement for co-expres-

sion is similar to that of bacterial luciferase, which

has been investigated extensively in the Baldwin lab-

oratory ([82], for a review of co-translational protein

folding, see [83]). It is possible that sGC, like bacte-

rial luciferase, has one subunit that can, in addition

to normal heterodimerization, form a kinetically

trapped homodimer or fold to a dimerization-incom-

petent monomer.

6. Purication from mammalian tissue and expression

systems

Although the existence of other isoforms of sGC

besides the K1L1 has been suggested by cloning and

expression experiments, only the K1L1 has been pu-

ried from mammalian tissue. In the past, a number

of dierent sources for sGC, including lung, liver,

and brain, were used for purications. Early puri-

cations (see, for example, [84,85]) produced enzyme

with little or no bound heme [86]. Because of the

high sGC content and ready availability, bovine

lung is now used as a source for purication from

mammalian tissue. The groups currently purifying

sGC from bovine lung use a variety of purication

procedures [47,87^92]. Most of the more recent pu-

rications have been modied to increase the yield of

heme-containing sGC. In addition, yields have been

increased: purication from tissue can yield up to 3^

5 mg of pure protein (S. Kim, M.A. Marletta, un-

published data; [92]).

Because isolation of sGC from bovine lung is typ-

ically arduous, there has been strong motivation for

Fig. 4. Activators and inhibitors of sGC. Shown in panel A are

compounds known to interact directly with sGC. As described

in the text, YC-1 is an activator of sGC that acts synergistically

with CO to yield activity comparable to that observed withcNO. ODQ is a relatively potent inhibitor of the enzyme,

although the mechanism of action is not clearly established. It

has been reported to oxidize the ferrous heme moiety [124].

Shown in panel B are compounds that have been reported to

aect catalytic activity. Methylene blue and LY83583 are re-

ported to inhibit the enzyme and isoliquiritigenin has been re-

ported to activate the enzyme. Studies with these compounds

have typically been carried out with crude preparations of the

enzyme and so the exact mechanism of action is not clear.



9/17

the development of alternative sources of sGC,

namely, the development of expression systems.

Both transient and stable expression of sGC in mam-

malian expression systems using COS cells (an Afri-

can green monkey kidney cell line) and HEK293 cells(a human embryonic kidney cell line) has successfully

produced active cyclase in several laboratories

[67,73,76,78,81]. Unfortunately, while these systems

have been useful for exploring the catalytic activity

of sGC isoforms and mutants, the low level of pro-

tein produced has made them unsuitable as sources

of puried protein. In order to take advantage of an

expression system that can generate amounts of sGC

suitable for purication, several labs (including ours)

have used expression and purication of sGC in the

baculovirus/Sf9 system [93^96]. In general, purica-

tion of sGC from baculovirus/Sf9 expression systems

is more straightforward because it requires a lower

purication factor. Obviously, the ability to express

heterodimeric sGC at high levels in E. coli would be

a tremendous boon to the eld. Many laboratories

have doubtless tried to do just that and have been, to

this point, unsuccessful 9 and here we can judge

only by our own experience, given the lack of pub-

lished reports 9 because sGC tends to form insolu-

ble inclusion bodies when expressed in E. coli. To

our knowledge, the only sGC construct that has

been successfully overexpressed in E. coli is the L1subunit heme binding region that has been expressed

and puried in our laboratory [97,98].

7. Heme environment

Although there has been some debate in the past,

it is clear now that sGC binds one protoporphyrin

IX type heme per heterodimer. The early history of

the determination that sGC is a hemoprotein is re-

viewed in [99]. After initial observations that heme

was needed to allow stimulation of sGC by cNO in

crude preparations [100,101], sGC was shown to con-

tain a prosthetic heme moiety that was important in

activation by cNO and cNO donors [85,102^104]. Be-

cause the early purications yielded sGC with heme

stoichiometries less than one (often far less than one)

[86], it was assumed that the heme stoichiometry was,

in fact, one. After the cloning of both K and L sGC

subunits and the cloning of some of the pGC iso-

forms, it became clear that the COOH-terminal por-

tion of both sGC subunits was most likely catalytic

in nature. In addition, as our laboratory noted

[47,87], the NH2-terminal portions 9 upstream

from the conserved catalytic regions9

of both theK and L subunits were homologous to each other

(i.e., 30% identical and 39% similar), though less ho-

mologous at the extreme NH2 terminus. This obser-

vation led us to hypothesize that both subunits might

bind heme. Initially, our heme stoichiometry deter-

mination for sGC supported that hypothesis [87];

however, recent work in our laboratory has demon-

strated that there had been an error in the quanti-

tative amino acid analysis used to determine sGC

concentration. As a result, we have denitively deter-

mined the heme stoichiometry to be one heme per

heterodimer ([93]; S. Kim, P.E. Brandish, M.A. Mar-

letta, unpublished data).

The hypothesis that the sGC heme is ligated by a

histidine residue, much like the well-studied heme

proteins hemoglobin and myoglobin, is not new;

however, it has been only within the last several

years that that suspicion has been conrmed and

the ligand identied as histidine 105 of the L1 sub-

unit (the numbering applies to bovine, human and

rat L1). Early observations of the sGC heme Soret

(i.e., the maximal heme absorbance peak) as isolated

[102] hinted that sGC was likely to bind heme in away similar to deoxyhemoglobin and deoxymyoglo-

bin, namely, as a ve-coordinate histidyl complex. It

was not until two relatively recent observations were

made that this picture began to become clear. First, a

mutant of sGC in which histidine 105 of the bovine

L1 subunit had been mutated to phenylalanine

(H105F) and transiently expressed in COS cells was

found to have lost stimulation by cNO and, in addi-

tion, to lack any heme absorbance [94]. Second, elec-

tronic absorption spectroscopy on pure sGC revealed

a spectrum that was entirely consistent with a histi-

dine-ligated heme, namely, a Soret peak at 431 nm

and a broad K/L band at 562 nm [47,105]. Additional

observations with electronic absorption spectroscopy

[106], magnetic circular dichroism spectroscopy [106],

resonance Raman spectroscopy (RR) [107,108] and

Fourier transform infrared spectroscopy (FTIR)

[109] conrmed that the heme ligand was histidine.

Subsequent observations in our laboratory deni-

tively identied L1-His105 as the heme ligand: the



10/17

NH2-terminal portion of the L1 subunit (L11385) ex-

pressed and puried from E. coli was capable of

binding heme with spectral characteristics virtually

identical to the heterodimeric enzyme [97]; mutants

of His105 did not bind heme [98]; and heme bindingto the H105G mutant could be rescued by including

imidazole during growth and purication [98].

The majority of groups studying sGC purify the

enzyme in a ve-coordinate, high-spin histidyl com-

plex that resembles deoxyhemoglobin and deoxy-

myoglobin in terms of ligation, oxidation state and

spectral characteristics. Unlike hemoglobin and my-

oglobin, however, sGC has an extremely low anity

for oxygen, which fails to bind to the sGC heme even

under 100% O2 [47]. The molecular basis for this

property is still under investigation. The failure of

sGC to bind oxygen enables sGC to act as a receptor

for cNO by allowing cNO to bind directly to the

ferrous-deoxy heme; in contrast, cNO will react

with ferrous-oxy complexes (like oxyhemoglobin) to

give nitrate and ferric heme [110]. The Soret position

for sGC heme (431 nm) coupled with a broad K/L

peak at 562 nm [47,105] is consistent with ve-coor-

dinate high-spin histidyl-ligated proteins and model

compounds [111]. Despite this seeming consensus,

however, there have been reports that sGC, as iso-

lated, is a mixture of ve-coordinate high-spin histid-

yl heme and six-coordinate low-spin bis-histidylheme [95,106,108,112]. It is not clear whether heme

reconstitution, or some other dierence in procedure,

is the cause of these multiple populations of enzyme.

Extensive study of the sGC heme in its cNO-bound

(NO-sGC) and CO-bound (CO-sGC) forms has giv-

en us a clear picture of the characteristics of these

species. NO-sGC is a ve-coordinate nitrosyl heme,

in which the Fe-His bond has been broken

[47,92,107,108,112,113]. A combination of electronic

absorption spectroscopy, electron paramagnetic res-

onance spectroscopy (EPR) and RR has been used to

show this. When CO is bound to the sGC heme, the

Fe-His bond apparently remains intact, forming a

six-coordinate heme [47,92,106,108,112]. Observa-

tions by electronic absorption spectroscopy and RR

have demonstrated this to be the case. Table 1 shows

a summary of electronic absorption spectroscopy

maxima for sGC in its as isolated, CO-bound andcNO-bound forms; Table 2 shows RR and FTIR

vibrations for the heme ligands of these forms.

8. Catalysis

Several lines of evidence have converged to unam-

biguously dene the catalytic domain of sGC : se-

quence comparisons to AC and pGC to dene anevolutionarily conserved region, site-directed muta-

genesis to produce inactive enzyme, the construction

of deletion mutants that retain catalytic function,

and the creation of mutants with altered substrate

specicity. The rst method that was used to dene

the catalytic regions of AC, pGC and sGC was align-

ment of their predicted amino acid sequences (once

the cDNA sequences became available). These align-

ments revealed a highly homologous region approx.

200 amino acids long that was common to all three

enzymes. Two regions were found in the AC se-

quence (C1 and C2), one region in pGC monomers

and one region in each half of the sGC heterodimer

(K1 and L1). Several additional pieces of evidence

showed that these regions in the K1 and L1 subunits

of sGC came together to form the catalytic domain.

First, the mutation of two aspartic acid residues in

the sGC K1 subunit each abolished catalytic activity

in co-expressed heterodimers [114]. Second, Wedel

and coworkers demonstrated that co-expressed

NH2-terminal deletion mutants of K1 (deletion of

the rst 366 amino acids) and L1 (deletion of the rst

305 amino acids) retained catalytic activity [115]. Fi-nally, any remaining doubt that these regions formed

the catalytic domain was removed by the recent nd-

ing that sGC can be made into an cNO-responsive

adenylate cyclase with changes to these regions

alone: by mutating one residue in the K1 subunit

and two in the L1 subunit in the putative catalytic

regions that were predicted 9 on the basis of the AC

Table 1

Peak positions and extinction coecients from electronic ab-

sorption spectroscopy of sGC

sGC form Soret L K Por-Fea

Ferrous 431 (111) 562 (12) 770 (0.2)

CO complex 423 (145) 541 (12) 567 (12)

NO complex 399 (85) 537 (11) 572 (11)

Ferric 391 (110) 511 (14) 642 (4)

Values for this table were drawn from [47,105]. Extinction coef-

cients (mM31 cm31) are given in parentheses.aPor-Fe refers to the putative porphyrin to iron charge transfer

band.



11/17

catalytic domain crystal structures 9 to be important

in nucleotide base binding [59,60], Sunahara and

coworkers were able to change the nucleotide specif-

icity of sGC [116].

Many aspects of the catalytic mechanism of sGCare poorly understood. The type of reaction cata-

lyzed by sGC 9 the attack of an activated hydroxyl

on the K-phosphate of a nucleotide triphosphate 9 is

a common one. This is, for example, the same reac-

tion catalyzed in the polymerization of deoxynucleo-

tide triphosphates in the synthesis of DNA; however,

in the case of DNA it is an intermolecular attack of

the 3P-hydroxyl on an K-phosphate. In fact, after

publication of the AC C2C2 structure, it was sug-

gested that AC catalytic domain contains a fold sim-

ilar to the `palm' domain in prokaryotic DNA poly-

merase I [117]. It is possible that some of the features

of the DNA polymerase mechanism will hold true

for the nucleotide cyclases. Thus, the outline of the

reaction is clear: the 3P-hydroxyl is activated in some

way to become more nucleophilic and attacks the K-

phosphate, causing the release of the L- and Q-phos-

phates as inorganic pyrophosphate. What is not clear

is how the 3P-hydroxyl is activated, whether pyro-

phosphate release is concerted or proceeds through

a pentavalent phosphorous transition state and what

role divalent metal cations play in the mechanism.

Despite these questions, however, some possibilitiesfor the sGC mechanism have been eliminated. For

example, the displacement of PPi by the 3P-hydroxyl

of GTP has been shown to occur with inversion of

stereochemistry, which indicates that the reaction

proceeds by a single displacement reaction [118].

9. Model of sGC structure and function

In order to synthesize some of the latest sGC de-

velopments into a model 9 albeit a working model

9 we have divided sGC enzymology into four sub-

areas that represent what are, in our view, the most

important open questions: structure, activation, cat-

alytic mechanism and deactivation.

No X-ray crystal structure of sGC or of any sGC

domain has been solved; as a result, any model of

sGC structure must draw heavily on related proteins:

The high degree of homology (approx. 30% identity

and 40% similarity) between the catalytic domain of

AC and the catalytic domain of sGC, in addition to

their almost identical catalytic function, suggests that

these catalytic domains are descended from a com-

mon ancestor and, as such, are likely to share a

common three-dimensional fold. In fact, the Hurley

group has used the AC catalytic domain structure

determined in his laboratory to create a homology

model of the catalytic domain of sGC [63]. Such

models have been used to correctly predict the amino

acid residues that control the substrate specicity of

both ACs and GCs. For example, the substrate spe-cicity of retinal guanylate cyclase (a pGC) was

changed from GTP to ATP [119], sGC was con-

verted into an cNO-activated AC and AC was con-

verted into a non-specic (but still GsK-activated)

purine nucleotide cyclase [116]. As a result, the

X-ray crystal structures of the AC C1C2 heterodimer

[60] and C2C2 homodimer [59] appear to be excellent

models for the sGC catalytic domain. We would con-

cur, then, with the prediction made by others [63],

that the COOH-terminal portions of the K and L

sGC subunits form a head-to-tail catalytic domain.

If the pseudo-heterodimeric AC catalytic domain is

any guide, sGC may well have only one substrate-

binding site.

As for the rest of the protein, the lack of direct

evidence (even by analogy) makes it more dicult to

choose among the possibilities ; still, there are nd-

ings upon which we can continue to build the model.

The homology between the K and L subunits of sGC

(33% identity and 42% similarity overall, although

Table 2

Resonance Raman and FTIR vibrations for sGC heme ligands

sGC form cm31 Description

Ferrous

X(Fe-His) 204 Fe-His stretch

CO complexX(Fe-CO) 472 Fe-CO stretch

N(Fe-C-O) 562 Fe-C-O bending

X(C-O) 1987 C-O stretch

NO complex

X(Fe-NO) 525 Fe-NO stretch

X(N-O) 1677 N-O stretch

The values in this table were drawn from [107,109]. Similar val-

ues have been reported by other laboratories [91,92,108,112];

some dierences have been reported for reconstituted sGC

[91,108,112].



12/17

higher in the COOH-terminal catalytic region than in

the NH2-terminal regulatory region) suggests that

the fold of the two subunits may be pseudo-symmet-

rical. The K and L subunits of sGC have a predicted

amphipathic helical region just NH2-terminal to thepredicted catalytic domain. These helices may be

evolutionarily related to the predicted amphipathic

helices NH2-terminal to the catalytic region of

pGC, which have been demonstrated to mediate di-

merization [120]. This conclusion is supported by the

homodimerization of the L11385 [97] and by the di-

merization of K1 and L1 subunit NH2-terminal dele-

tion mutants [115], which retain the putative dimeri-

zation helices. The conformational change which

shifts the AC catalytic domain to its active state

upon binding of GsK may be similar to the confor-

mational change which shifts the sGC catalytic do-

main to its active state when cNO binds to the sGC

heme. It is possible, then, that the sGC heme domain

(the NH2-terminal portion of the L subunit) abuts

the catalytic domain in the same way that GsK abuts

the AC catalytic domain [60]. This would put both

the K and L subunit NH2-terminal domains in the

plane of the catalytic domain (which is roughly

disk-shaped) 9 the heme domain opposite the sub-

strate site (in the same way GsK is opposite the AC

substrate site) and the K subunit NH2-terminal por-

tion next to the substrate site (opposite the site anal-ogous to where forskolin binds in the AC structure).

Our model of sGC activation by cNO does not

dier substantially from the following simplied pic-

ture. Binding of cNO to the sGC heme severs the

bond between the heme Fe and the ligating histidine,

presumably puckers the heme toward the distal nitro-

syl ligand and, through some combination of

changes in heme geometry, causes a conformational

change that activates the enzyme. Recent work, how-

ever, has rened our view of the heme environment

of sGC in a way that allows us to rene our working

model of activation. As mentioned above, YC-1 is an

activator of sGC that has the interesting property of

synergistically activating sGC in the presence of CO

(see Fig. 4 for the structure of YC-1 and other sGC

activators and inhibitors). Electronic absorption

spectroscopy studies have shown that sGC activated

by YC-1 and CO has a six-coordinate heme, even

though its level of activity is similar to the cNO-acti-

vated enzyme [49,121]. Therefore, the breaking of the

Fe-His bond which occurs with cNO binding may be

correlated with the molecular event that brings about

activation, but may not directly cause it. In other

words, it is possible that the breaking of the Fe-His

bond can be eliminated as a step in the activation ofsGC. There may be, then, some other eect of cNO

binding to the heme that leads to activation. We are

currently pursuing this possibility in our laboratory.

Our model of the sGC catalytic mechanism is

based largely on proteins that catalyze similar reac-

tions. In addition to this, we have incorporated what

is known about the nucleotide cyclase catalytic mech-

anisms through crystal structures and mutational

studies. The active site functions/characteristics that

need to be elucidated are as follows: the number of

substrate binding sites; the number of Mg2 ions

participating in substrate binding and/or catalysis;

the amino acid residues important for substrate bind-

ing; the amino acid residues involved in Mg2 bind-

ing; and the amino acid residues involved in the

catalytic mechanism (e.g., activation of the 3P-OH

and transition-state stabilization). There are almost

certainly no more than two substrate-binding sites in

sGC; given that the catalytic domain of sGC, like

that of AC, is made up of homologous but non-iden-

tical components, we hypothesize that there is only

one substrate-binding site, as in AC. This hypothesis

also ts in well with the obvious asymmetry of thesGC regulatory region: the L1 subunit binds heme,

the K1 does not. As for important residues, the mu-

tation of rat K1D513 and K1D529 each abolished

catalytic activity when transiently expressed with

wild-type L1 subunit [114], but it is dicult to pin-

point the role that these residues may be playing

without further study.

The nal component of our model of sGC struc-

ture and function is the question of deactivation,

which remains an open question because of the fol-

lowing observation: the deactivation of sGC in or-

gan bath studies has suggested that the half-life of

sGC activation is on the order of minutes [17],

whereas the half-lives of most nitrosyl-heme com-

plexes are longer. Two hypotheses have been ad-

vanced to explain this discrepancy. First, the sGC

heme may simply have dierent characteristics that

allow cNO to dissociate more rapidly. Second, there

may be some extrinsic factor 9 a small molecule or

protein 9 which assists in the deactivation either by



13/17

directly interacting with the ferrous-nitrosyl heme or

via an allosteric eect that increases the rate of cNO

dissociation from the heme. Studies of the sGC

heme, for example, showed that its properties were

dierent from most histidine-ligated heme proteinsand model compounds: the half-life (extrapolated

to 37C) for cNO dissociation was on the order of

2 min [122]. Recent work in our laboratory has

shown that thiol compounds like glutathione, at con-

centrations similar to that found in vivo, increase the

rate of cNO release from the heme [93]. Compounds

that trap cNO, like oxyhemoglobin, also increase the

rate of cNO release from the sGC heme [93,122].

There has been a report that the presence of Mg2-

GTP increases the rate of deactivation [123]; how-

ever, we have found that a precipitate which forms

under these conditions complicates the determination

of such rates [93]. We believe, therefore, that the

relatively fast cNO o-rate for the sGC heme [122]

and contribution of thiol compounds like glutathione

account for most, if not all, of the in vivo rate of

deactivation.

10. Conclusion

We have attempted in this review to provide some

insight into the structure and function of solubleguanylate cyclase. In doing so, we have placed sGC

in its larger context by discussing the history of the

sGC eld, the physiological importance of the cNO/

cGMP pathway and its components, and the nucleo-

tide cyclase family to which sGC belongs. We have

delved into the current understanding of sGC by

discussing what is known about sGC isoforms, ex-

pression and purication, heme environment and cat-

alysis. And nally, we have presented a working

model of sGC structure, activation, catalytic mecha-

nism and deactivation. Naturally, a great deal of

work remains to be done to complete our under-

standing of sGC. Two important milestones in

achieving that progress will be the identication

and characterization of sGC subunits and isoforms

from several species and the development of an sGC

expression system capable of producing large quanti-

ties of the heterodimeric enzyme. With or without

these breakthroughs, the most pressing problems to

address for sGC are : (1) determining the structure,

(2) understanding the catalytic mechanism and (3)

piecing together the molecular details of activation

and deactivation. As these questions are answered,

a complete view of sGC structure and function will

emerge.

Acknowledgements

We would like to thank all of the Marletta lab for

many thought-provoking discussions and for their

insightful comments on this manuscript.

References

[1] T.W. Rall, E.W. Sutherland, Formation of a cyclic adenine

ribonucleotide by tissue particles, J. Biol. Chem. 232 (1958)

1065^1076.

[2] J.G. Hardman, G.A. Robison, E.W. Sutherland, Cyclic nu-

cleotides, Annu. Rev. Physiol. 33 (1971) 311^336.

[3] G.A. Robison, R.W. Butcher, E.W. Sutherland, Cyclic

AMP, Academic Press, New York, 1971.

[4] D.F. Ashman, R. Lipton, M.M. Melicow, T.D. Price, Iso-

lation of adenosine 3P,5P-monophosphate and guanosine

3P,5P-monophosphate from rat urine, Biochem. Biophys.

Res. Commun. 11 (1963) 330^334.

[5] A.A. White, G.D. Aurbach, Detection of guanyl cyclase in

mammalian tissues, Biochim. Biophys. Acta 191 (1969) 686^697.

[6] G. Schultz, E. Bohme, K. Munske, Guanyl cyclase. Deter-

mination of enzyme activity, Life Sci. 8 (1969) 1323^1332.

[7] N.D. Goldberg, S.B. Dietz, A.G. O'Toole, Cyclic guanosine

3P,5P-monophosphate in mammalian tissues and urine,

J. Biol. Chem. 244 (1969) 4458^4466.

[8] E. Ishikawa, S. Ishikawa, J.W. Davis, E.W. Sutherland, De-

termination of guanosine 3P,5P-monophosphate in tissues

and of guanyl cyclase in rat intestine, J. Biol. Chem. 244

(1969) 6371^6376.

[9] J.G. Hardman, E.W. Sutherland, Guanyl cyclase, an enzyme

catalyzing the formation of guanosine 3P,5P-monophosphate

from guanosine triphosphate, J. Biol. Chem. 244 (1969)

6363^6370.[10] S. Katsuki, W. Arnold, C. Mittal, F. Murad, Stimulation of

guanylate cyclase by sodium nitroprusside, nitroglycerin and

nitric oxide in various tissue preparations and comparison to

the eects of sodium azide and hydroxylamine, J. Cyclic

Nucleotide Res. 3 (1977) 23^35.

[11] N. Miki, Y. Kawabe, K. Kuriyama, Activation of cerebral

guanylate cyclase by nitric oxide, Biochem. Biophys. Res.

Commun. 75 (1977) 851^856.

[12] W.P. Arnold, C.K. Mittal, S. Katsuki, F. Murad, Nitric

oxide activates guanylate cyclase and increases guanosine



14/17

3P:5P-cyclic monophosphate levels in various tissue prepara-

tions, Proc. Natl. Acad. Sci. USA 74 (1977) 3203^3207.

[13] K. Schultz, G. Schultz, Sodium nitroprusside and other

smooth muscle-relaxants increase cyclic GMP levels in rat

ductus deferens, Nature 265 (1977) 750^751.

[14] R.F. Furchgott, J.V. Zawadzki, The obligatory role of en-dothelial cells in the relaxation of arterial smooth muscle by

acetylcholine, Nature 288 (1980) 373^376.

[15] S. Moncada, R.M. Palmer, E.A. Higgs, The discovery of

nitric oxide as the endogenous nitrovasodilator, Hyperten-

sion 12 (1988) 365^372.

[16] L.J. Ignarro, G.M. Buga, K.S. Wood, R.E. Byrns, G.

Chaudhuri, Endothelium-derived relaxing factor produced

and released from artery and vein is nitric oxide, Proc.

Natl. Acad. Sci. USA 84 (1987) 9265^9269.

[17] R.M. Palmer, A.G. Ferrige, S. Moncada, Nitric oxide release

accounts for the biological activity of endothelium-derived

relaxing factor, Nature 327 (1987) 524^526.

[18] D.A. Wagner, V.R. Young, S.R. Tannenbaum, Mammalian

nitrate biosynthesis: incorporation of 15NH3 into nitrate is

enhanced by endotoxin treatment, Proc. Natl. Acad. Sci.

USA 80 (1983) 4518^4521.

[19] L.C. Green, K. Ruiz de Luzuriaga, D.A. Wagner, W. Rand,

N. Istfan, V.R. Young, S.R. Tannenbaum, Nitrate biosyn-

thesis in man, Proc. Natl. Acad. Sci. USA 78 (1981) 7764^

7768.

[20] L.C. Green, S.R. Tannenbaum, P. Goldman, Nitrate syn-

thesis in the germfree and conventional rat, Science 212

(1981) 56^58.

[21] M.A. Marletta, M.A. Tayeh, J.M. Hevel, Unraveling the

biological signicance of nitric oxide, Biofactors 2 (1990)

219^225.

[22] M.A. Marletta, Nitric oxide: biosynthesis and biological sig-nicance, Trends Biochem. Sci. 14 (1989) 488^492.

[23] J.B. Hibbs Jr., R.R. Taintor, Z. Vavrin, E.M. Rachlin, Ni-

tric oxide: a cytotoxic activated macrophage eector mole-

cule, Biochem. Biophys. Res. Commun. 157 (1988) 87^94.

[24] M.A. Marletta, Mammalian synthesis of nitrite, nitrate, ni-

tric oxide, and N-nitrosating agents, Chem. Res. Toxicol. 1

(1988) 249^257.

[25] J.M. Hevel, K.A. White, M.A. Marletta, Purication of the

inducible murine macrophage nitric oxide synthase. Identi-

cation as a avoprotein, J. Biol. Chem. 266 (1991) 22789^

22791.

[26] T.D. Warner, J.A. Mitchell, H. Sheng, F. Murad, Eects of

cyclic GMP on smooth muscle relaxation, Adv. Pharmacol.

26 (1994) 171^194.

[27] W.A. Buechler, K. Ivanova, G. Wolfram, C. Drummer, J.M.

Heim, R. Gerzer, Soluble guanylyl cyclase and platelet func-

tion, Ann. NY Acad. Sci. 714 (1994) 151^157.

[28] S.R. Jarey, S.H. Snyder, Nitric oxide: a neural messenger,

Annu. Rev. Cell Dev. Biol. 11 (1995) 417^440.

[29] R.S. Lewis, W.M. Deen, Kinetics of the reaction of nitric

oxide with oxygen in aqueous solutions, Chem. Res. Toxicol.

7 (1994) 568^574.

[30] J.M. Fukuto, Chemistry of nitric oxide: biologically relevant

aspects, Adv. Pharmacol. 34 (1995) 1^15.

[31] J.F. Kerwin Jr., J.R. Lancaster Jr., P.L. Feldman, Nitric

oxide: a new paradigm for second messengers, J. Med.

Chem. 38 (1995) 4343^4362.

[32] C. Giulivi, Functional implications of nitric oxide producedby mitochondria in mitochondrial metabolism, Biochem. J.

332 (1998) 673^679.

[33] G.C. Brown, Nitric oxide regulates mitochondrial respira-

tion and cell functions by inhibiting cytochrome oxidase,

FEBS Lett. 369 (1995) 136^139.

[34] V.M. Bolotina, S. Najibi, J.J. Palacino, P.J. Pagano, R.A.

Cohen, Nitric oxide directly activates calcium-dependent po-

tassium channels in vascular smooth muscle, Nature 368

(1994) 850^853.

[35] D. Sekkai, F. Aillet, N. Israel, M. Lepoivre, Inhibition of

NF-kappaB and HIV-1 long terminal repeat transcriptional

activation by inducible nitric oxide synthase 2 activity,

J. Biol. Chem. 273 (1998) 3895^3900.

[36] M. Spiecker, H.B. Peng, J.K. Liao, Inhibition of endothelial

vascular cell adhesion molecule-1 expression by nitric oxide

involves the induction and nuclear translocation of Ikappa-

Balpha, J. Biol. Chem. 272 (1997) 30969^30974.

[37] H.M. Lander, P.K. Sehajpal, A. Novogrodsky, Nitric oxide

signaling: a possible role for G proteins, J. Immunol. 151

(1993) 7182^7187.

[38] H.M. Lander, J.S. Ogiste, S.F. Pearce, R. Levi, A. Novog-

rodsky, Nitric oxide-stimulated guanine nucleotide exchange

on p21ras, J. Biol. Chem. 270 (1995) 7017^7020.

[39] H.M. Lander, D.P. Hajjar, B.L. Hempstead, U.A. Mirza,

B.T. Chait, S. Campbell, L.A. Quilliam, A molecular redox

switch on p21(ras). Structural basis for the nitric oxide-

p21(ras) interaction, J. Biol. Chem. 272 (1997) 4323^4326.[40] J. Li, T.R. Billiar, R.V. Talanian, Y.M. Kim, Nitric oxide

reversibly inhibits seven members of the caspase family via

S-nitrosylation, Biochem. Biophys. Res. Commun. 240

(1997) 419^424.

[41] J.S. Stamler, L. Jia, J.P. Eu, T.J. McMahon, I.T. Demchen-

ko, J. Bonaventura, K. Gernert, C.A. Piantadosi, Blood ow

regulation by S-nitrosohemoglobin in the physiological oxy-

gen gradient, Science 276 (1997) 2034^2037.

[42] L. Xu, J.P. Eu, G. Meissner, J.S. Stamler, Activation of the

cardiac calcium release channel (ryanodine receptor) by

poly-S-nitrosylation, Science 279 (1998) 234^237.

[43] A.J. Gow, J.S. Stamler, Reactions between nitric oxide and

haemoglobin under physiological conditions, Nature 391

(1998) 169^173.

[44] T. Ingi, J. Cheng, G.V. Ronnett, Carbon monoxide: an en-

dogenous modulator of the nitric oxide-cyclic GMP signal-

ing system, Neuron 16 (1996) 835^842.

[45] A. Verma, D.J. Hirsch, C.E. Glatt, G.V. Ronnett, S.H.

Snyder, Carbon monoxide: a putative neural messenger, Sci-

ence 259 (1993) 381^384.

[46] R. Zakhary, K.D. Poss, S.R. Jarey, C.D. Ferris, S. Tone-

gawa, S.H. Snyder, Targeted gene deletion of heme oxygen-



15/17

ase 2 reveals neural role for carbon monoxide, Proc. Natl.

Acad. Sci. USA 94 (1997) 14848^14853.

[47] J.R. Stone, M.A. Marletta, Soluble guanylate cyclase from

bovine lung : activation with nitric oxide and carbon mon-

oxide and spectral characterization of the ferrous and ferric

states, Biochemistry 33 (1994) 5636^5640.[48] S.-C. Kuo, F.Y. Lee, C.-M. Teng, United States patent No.

5,574,168, 1996.

[49] J.R. Stone, M.A. Marletta, Synergistic activation of soluble

guanylate cyclase by YC-1 and carbon monoxide: implica-

tions for the role of cleavage of the iron-histidine bond dur-

ing activation by nitric oxide, Chem. Biol. 5 (1998) 255^

261.

[50] A. Friebe, G. Schultz, D. Koesling, Sensitizing soluble gua-

nylyl cyclase to become a highly CO-sensitive enzyme,

EMBO J. 15 (1996) 6863^6868.

[51] S.M. Lohmann, A.B. Vaandrager, A. Smolenski, U. Walter,

H.R. De Jonge, Distinct and specic functions of cGMP-

dependent protein kinases, Trends Biochem. Sci. 22 (1997)

307^312.

[52] E. Degerman, P. Belfrage, V.C. Manganiello, Structure, lo-

calization, and regulation of cGMP-inhibited phosphodies-

terase (PDE3), J. Biol. Chem. 272 (1997) 6823^6826.

[53] M.D. Houslay, G. Milligan, Tailoring cAMP-signalling re-

sponses through isoform multiplicity, Trends Biochem. Sci.

22 (1997) 217^224.

[54] W.N. Zagotta, S.A. Siegelbaum, Structure and function of

cyclic nucleotide-gated channels, Annu. Rev. Neurosci. 19

(1996) 235^263.

[55] B.J. Wedel, D.L. Garbers, New insights on the functions of

the guanylyl cyclase receptors, FEBS Lett. 410 (1997) 29^33.

[56] J. Hanoune, Y. Pouille, E. Tzavara, T. Shen, L. Lipskaya,

N. Miyamoto, Y. Suzuki, N. Defer, Adenylyl cyclases: struc-ture, regulation and function in an enzyme superfamily, Mol.

Cell. Endocrinol. 128 (1997) 179^194.

[57] E. Lindner, A.N. Dohadwalla, B.K. Bhattacharya, Positive

inotropic and blood pressure lowering activity of a diterpene

derivative isolated from Coleus forskohli: forskolin, Arznei-

mittelforschung 28 (1978) 284^289.

[58] K.B. Seamon, W. Padgett, J.W. Daly, Forskolin: unique

diterpene activator of adenylate cyclase in membranes and

in intact cells, Proc. Natl. Acad. Sci. USA 78 (1981) 3363^

3367.

[59] G. Zhang, Y. Liu, A.E. Ruoho, J.H. Hurley, Structure of the

adenylyl cyclase catalytic core, Nature 386 (1997) 247^

253.

[60] J.J. Tesmer, R.K. Sunahara, A.G. Gilman, S.R. Sprang,

Crystal structure of the catalytic domains of adenylyl cyclase

in a complex with GsKWGTPQS, Science 278 (1997) 1907^

1916.

[61] H.J. Fulle, D.L. Garbers, Guanylyl cyclases: a family of

receptor-linked enzymes, Cell. Biochem. Funct. 12 (1994)

157^165.

[62] S. Schulz, B.J. Wedel, A. Matthews, D.L. Garbers, The clon-

ing and expression of a new guanylyl cyclase orphan recep-

tor, J. Biol. Chem. 273 (1998) 1032^1037.

[63] Y. Liu, A.E. Ruoho, V.D. Rao, J.H. Hurley, Catalytic

mechanism of the adenylyl and guanylyl cyclases: modeling

and mutational analysis, Proc. Natl. Acad. Sci. USA 94

(1997) 13414^13419.

[64] D. Koesling, J. Herz, H. Gausepohl, F. Niroomand, K.D.

Hinsch, A. Mu lsch, E. Bo hme, G. Schultz, R. Frank, Theprimary structure of the 70 kDa subunit of bovine soluble

guanylate cyclase, FEBS Lett. 239 (1988) 29^34.

[65] M. Nakane, S. Saheki, T. Kuno, K. Ishii, F. Murad, Mo-

lecular cloning of a cDNA coding for 70 kilodalton subunit

of soluble guanylate cyclase from rat lung, Biochem. Bio-

phys. Res. Commun. 157 (1988) 1139^1147.

[66] D. Koesling, C. Harteneck, P. Humbert, A. Bosserho, R.

Frank, G. Schultz, E. Bo hme, The primary structure of the

larger subunit of soluble guanylyl cyclase from bovine lung.

Homology between the two subunits of the enzyme, FEBS

Lett. 266 (1990) 128^132.

[67] M. Nakane, K. Arai, S. Saheki, T. Kuno, W. Buechler, F.

Murad, Molecular cloning and expression of cDNAs coding

for soluble guanylate cyclase from rat lung, J. Biol. Chem.

265 (1990) 16841^16845.

[68] G. Giuili, U. Scholl, F. Bulle, G. Guellaen, Molecular clon-

ing of the cDNAs coding for the two subunits of soluble

guanylyl cyclase from human brain, FEBS Lett. 304 (1992)

83^88.

[69] V. Chhajlani, P.A. Frandberg, J. Ahlner, K.L. Axelsson, J.E.

Wikberg, Heterogeneity in human soluble guanylate cyclase

due to alternative splicing, FEBS Lett. 290 (1991) 157^

158.

[70] G. Giuili, N. Roechel, U. Scholl, M.G. Mattei, G. Guellaen,

Colocalization of the genes coding for the K3 and L3 sub-

units of soluble guanylyl cyclase to human chromosome 4 at

q31.3-q33, Hum. Genet. 91 (1993) 257^260.[71] S. Yoshikawa, I. Miyamoto, J. Aruga, T. Furuichi, H. Oka-

no, K. Mikoshiba, Isolation of a Drosophila gene encoding a

head-specic guanylyl cyclase, J. Neurochem. 60 (1993)

1570^1573.

[72] W. Liu, J. Yoon, M. Burg, L. Chen, W.L. Pak, Molecular

characterization of two Drosophila guanylate cyclases ex-

pressed in the nervous system, J. Biol. Chem. 270 (1995)

12418^12427.

[73] S. Shah, D.R. Hyde, Two Drosophila genes that encode the

K and L subunits of the brain soluble guanylyl cyclase,

J. Biol. Chem. 270 (1995) 15368^15376.

[74] T. Mikami, T. Kusakabe, N. Suzuki, Molecular cloning of

cDNAs and expression of mRNAs encoding K and L sub-

units of soluble guanylyl cyclase from medaka sh Oryzias

latipes, Eur. J. Biochem. 253 (1998) 42^48.

[75] P.S. Yuen, L.R. Potter, D.L. Garbers, A new form of gua-

nylyl cyclase is preferentially expressed in rat kidney, Bio-

chemistry 29 (1990) 10872^10878.

[76] C. Harteneck, B. Wedel, D. Koesling, J. Malkewitz, E.

Bo hme, G. Schultz, Molecular cloning and expression of a

new K-subunit of soluble guanylyl cyclase. Interchangeability

of the K-subunits of the enzyme, FEBS Lett. 292 (1991) 217^

222.



16/17

[77] F. Yu, D. Warburton, S. Wellington, R.S. Danziger, Assign-

ment of GUCIA2, the gene coding for the K2 subunit of

soluble guanylyl cyclase, to position 11q21-q22 on human

chromosome 11, Genomics 33 (1996) 334^336.

[78] G. Gupta, M. Azam, L. Yang, R.S. Danziger, The L2 sub-

unit inhibits stimulation of the K1/L1 form of soluble gua-nylyl cyclase by nitric oxide. Potential relevance to regula-

tion of blood pressure, J. Clin. Invest. 100 (1997) 1488^1492.

[79] B. Mayer, D. Koesling, E. Bo hme, Characterization of nitric

oxide synthase, soluble guanylyl cyclase, and Ca2/calmodu-

lin-stimulated cGMP phosphodiesterase as components of

neuronal signal transduction, Adv. Second Messenger Phos-

phoprotein Res. 28 (1993) 111^119.

[80] D. Koesling, P. Humbert, G. Schultz, in: S.R. Vincent (Ed.),

Nitric Oxide in the Nervous System, Academic Press, San

Diego, CA, 1995, pp. 43^50.

[81] C. Harteneck, D. Koesling, A. So ling, G. Schultz, E. Bo hme,

Expression of soluble guanylyl cyclase. Catalytic activity re-

quires two enzyme subunits, FEBS Lett. 272 (1990) 221^223.

[82] A.C. Clark, S.W. Raso, J.F. Sinclair, M.M. Ziegler, A.F.

Chaotte, T.O. Baldwin, Kinetic mechanism of luciferase

subunit folding and assembly, Biochemistry 36 (1997)

1891^1899.

[83] A.N. Fedorov, T.O. Baldwin, Cotranslational protein fold-

ing, J. Biol. Chem. 272 (1997) 32715^32718.

[84] D.L. Garbers, Purication of soluble guanylate cyclase from

rat lung, J. Biol. Chem. 254 (1979) 240^243.

[85] M.S. Wolin, K.S. Wood, L.J. Ignarro, Guanylate cyclase

from bovine lung. A kinetic analysis of the regulation of

the puried soluble enzyme by protoporphyrin IX, heme,

and nitrosyl-heme, J. Biol. Chem. 257 (1982) 13312^13320.

[86] S.A. Waldman, F. Murad, Cyclic GMP synthesis and func-

tion, Pharmacol. Rev. 39 (1987) 163^196.[87] J.R. Stone, M.A. Marletta, Heme stoichiometry of heterodi-

meric soluble guanylate cyclase, Biochemistry 34 (1995)

14668^14674.

[88] A. Mu lsch, R. Gerzer, Purication of heme-containing solu-

ble guanylyl cyclase, Methods Enzymol. 195 (1991) 377^

383.

[89] P. Humbert, F. Niroomand, G. Fischer, B. Mayer, D. Koes-

ling, K.D. Hinsch, H. Gausepohl, R. Frank, G. Schultz, E.

Bo hme, Purication of soluble guanylyl cyclase from bovine

lung by a new immunoanity chromatographic method,

Eur. J. Biochem. 190 (1990) 273^278.

[90] E.A. Dierks, J.N. Burstyn, The deactivation of soluble gua-

nylyl cyclase by redox-active agents, Arch. Biochem. Bio-

phys. 351 (1998) 1^7.

[91] E.A. Dierks, S.Z. Hu, K.M. Vogel, A.E. Yu, T.G. Spiro,

J.N. Burstyn, Demonstration of the role of scission of the

proximal histidine-iron bond in the activation of soluble

guanylyl cyclase through metalloporphyrin substitution stud-

ies, J. Am. Chem. Soc. 119 (1997) 7316^7323.

[92] T. Tomita, T. Ogura, S. Tsuyama, Y. Imai, T. Kitagawa,

Eects of GTP on bound nitric oxide of soluble guanylate

cyclase probed by resonance Raman spectroscopy, Biochem-

istry 36 (1997) 10155^10160.

[93] P.E. Brandish, W. Buechler, M.A. Marletta, Regeneration

of the ferrous heme of soluble guanylate cyclase from the

nitric oxide complex: acceleration by thiols and oxyhemo-

globin, Biochemistry (1998) in press.

[94] B. Wedel, P. Humbert, C. Harteneck, J. Foerster, J. Mal-

kewitz, E. Bo hme, G. Schultz, D. Koesling, Mutation ofHis-105 in the L1 subunit yields a nitric oxide-insensitive

form of soluble guanylyl cyclase, Proc. Natl. Acad. Sci.

USA 91 (1994) 2592^2596.

[95] G. Gupta, J. Kim, L. Yang, S.L. Sturley, R.S. Danziger,

Expression and purication of soluble, active heterodimeric

guanylyl cyclase from baculovirus, Protein Expr. Purif. 10

(1997) 325^330.

[96] W.A. Buechler, S. Singh, J. Aktas, S. Muller, F. Murad, R.

Gerzer, High-level expression of biologically active soluble

guanylate cyclase using the baculovirus system is strongly

heme-dependent, Adv. Pharmacol. 34 (1995) 293^303.

[97] Y. Zhao, M.A. Marletta, Localization of the heme binding

region in soluble guanylate cyclase, Biochemistry 36 (1997)

15959^15964.

[98] Y. Zhao, J.P. Schelvis, G.T. Babcock, M.A. Marletta, Iden-

tication of histidine 105 in the L1 subunit of soluble gua-

nylate cyclase as the heme proximal ligand, Biochemistry 37

(1998) 4502^4509.

[99] L.J. Ignarro, Haem-dependent activation of guanylate cy-

clase and cyclic GMP formation by endogenous nitric ox-

ide: a unique transduction mechanism for transcellular sig-

naling, Pharmacol. Toxicol. 67 (1990) 1^7.

[100] P.A. Craven, F.R. DeRubertis, D.W. Pratt, Electron spin

resonance study of the role of NOc catalase in the activa-

tion of guanylate cyclase by NaN3 and NH2OH. Modula-

tion of enzyme responses by heme proteins and their nitro-

syl derivatives, J. Biol. Chem. 254 (1979) 8213^8222.[101] P.A. Craven, F.R. DeRubertis, Restoration of the respon-

siveness of puried guanylate cyclase to nitrosoguanidine,

nitric oxide, and related activators by heme and hemepro-

teins. Evidence for involvement of the paramagnetic nitro-

syl-heme complex in enzyme activation, J. Biol. Chem. 253

(1978) 8433^8443.

[102] R. Gerzer, E. Bo hme, F. Hofmann, G. Schultz, Soluble

guanylate cyclase puried from bovine lung contains

heme and copper, FEBS Lett. 132 (1981) 71^74.

[103] L.J. Ignarro, J.N. Degnan, W.H. Baricos, P.J. Kadowitz,

M.S. Wolin, Activation of puried guanylate cyclase by

nitric oxide requires heme. Comparison of heme-decient,

heme-reconstituted and heme-containing forms of soluble

enzyme from bovine lung, Biochim. Biophys. Acta 718

(1982) 49^59.

[104] R. Gerzer, F. Hofmann, G. Schultz, Purication of a solu-

ble, sodium-nitroprusside-stimulated guanylate cyclase

from bovine lung, Eur. J. Biochem. 116 (1981) 479^486.

[105] J.R. Stone, M.A. Marletta, Spectral and kinetic studies on

the activation of soluble guanylate cyclase by nitric oxide,

Biochemistry 35 (1996) 1093^1099.

[106] J.N. Burstyn, A.E. Yu, E.A. Dierks, B.K. Hawkins, J.H.

Dawson, Studies of the heme coordination and ligand bind-



17/17

ing properties of soluble guanylyl cyclase (sGC): character-

ization of Fe(II)sGC and Fe(II)sGC(CO) by electronic ab-

sorption and magnetic circular dichroism spectroscopies

and failure of CO to activate the enzyme, Biochemistry

34 (1995) 5896^5903.

[107] G. Deinum, J.R. Stone, G.T. Babcock, M.A. Marletta,Binding of nitric oxide and carbon monoxide to soluble

guanylate cyclase as observed with resonance Raman spec-

troscopy, Biochemistry 35 (1996) 1540^1547.

[108] A.E. Yu, S.Z. Hu, T.G. Spiro, J.N. Burstyn, Resonance

Raman-spectroscopy of soluble guanylyl cyclase reveals dis-

placement of distal and proximal heme ligands by NO,

J. Am. Chem. Soc. 116 (1994) 4117^4118.

[109] S.Y. Kim, G. Deinum, M.T. Gardner, M.A. Marletta, G.T.

Babcock, Distal pocket polarity in the unusual ligand bind-

ing site of soluble guanylate cyclase : implications for the

control of cNO binding, J. Am. Chem. Soc. 118 (1996)

8769^8770.

[110] M.P. Doyle, J.W. Hoekstra, Oxidation of nitrogen oxides

by bound dioxygen in hemoproteins, J. Inorg. Biochem. 14

(1981) 351^358.

[111] E. Antonini, M. Brunori, Hemoglobin and Myoglobin in

their Reactions with Ligands, vol. 21, North-Holland Publ.

Co., Amsterdam, 1971.

[112] B. Fan, G. Gupta, R.S. Danziger, J.M. Friedman, D.L.

Rousseau, Resonance Raman characterization of soluble

guanylate cyclase expressed from baculovirus, Biochemistry

37 (1998) 1178^1184.

[113] J.R. Stone, R.H. Sands, W.R. Dunham, M.A. Marletta,

Electron paramagnetic resonance spectral evidence for the

formation of a pentacoordinate nitrosyl-heme complex on

soluble guanylate cyclase, Biochem. Biophys. Res. Com-

mun. 207 (1995) 572^577.[114] P.S. Yuen, L.K. Doolittle, D.L. Garbers, Dominant nega-

tive mutants of nitric oxide-sensitive guanylyl cyclase,

J. Biol. Chem. 269 (1994) 791^793.

[115] B. Wedel, C. Harteneck, J. Foerster, A. Friebe, G. Schultz,

D. Koesling, Functional domains of soluble guanylyl cy-

clase, J. Biol. Chem. 270 (1995) 24871^24875.

[116] R.K. Sunahara, A. Beuve, J.J.G. Tesmer, S.R. Sprang,

D.L. Garbers, A.G. Gilman, Exchange of substrate and

inhibitor specicities between adenylyl and guanylyl cy-

clases, J. Biol. Chem. 273 (1998) 16332^16338.[117] P.J. Artymiuk, A.R. Poirrette, D.W. Rice, P. Willett, A

polymerase I palm in adenylyl cyclase?, Nature 388

(1997) 33^34.

[118] P.D. Senter, F. Eckstein, A. Mu lsch, E. Bo hme, The stereo-

chemical course of the reaction catalyzed by soluble bovine

lung guanylate cyclase, J. Biol. Chem. 258 (1983) 6741^

6745.

[119] C.L. Tucker, J.H. Hurley, T.R. Miller, J.B. Hurley, Two

amino acid substitutions convert a guanylyl cyclase,

RetGC-1, into an adenylyl cyclase, Proc. Natl. Acad. Sci.

USA 95 (1998) 5993^5997.

[120] E.M. Wilson, M. Chinkers, Identication of sequences me-

diating guanylyl cyclase dimerization, Biochemistry 34

(1995) 4696^4701.

[121] A. Friebe, D. Koesling, Mechanism of YC-1-induced acti-

vation of soluble guanylyl cyclase, Mol. Pharmacol. 53

(1998) 123^127.

[122] V.G. Kharitonov, V.S. Sharma, D. Magde, D. Koesling,

Kinetics of nitric oxide dissociation from ve- and six-co-

ordinate nitrosyl hemes and heme proteins, including solu-

ble guanylate cyclase, Biochemistry 36 (1997) 6814^

6818.

[123] V.G. Kharitonov, M. Russwurm, D. Magde, V.S. Sharma,

D. Koesling, Dissociation of nitric oxide from soluble gua-

nylate cyclase, Biochem. Biophys. Res. Commun. 239

(1997) 284^286.

[124] J. Garthwaite, E. Southam, C.L. Boulton, E.B. Nielsen, K.Schmidt, B. Mayer, Potent and selective inhibition of nitric

oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazo-

lo[4,3-a]quinoxalin-1-one, Mol. Pharmacol. 48 (1995) 184^

188.


Documents

Guanylate Cyclase and the cNO-cGMP Signaling Pathway