Adrenal cortex adenylate cyclase

Naunyn-Schmiedeberg's Arch. Pharmacol. 291, 89--100 (1975) �9 by Springer-Verlag 1975

Adrenal Cortex Adenylate Cyclase Solubilization of Adenylate Cyclase

and Guanyl Nucleotide Binding Sites

If. Glossmann with the technical assistance of K. Klapettek

Pharmakologisches Institut der Justus-Liebig-Universit~t Giessen

Received May 7 / Accepted August 1, 1975

Summary. Adenylate cyelase activity and binding sites for 5'-guanylyl-imidodiphosphate [Gpp(Ntt)p] have been solubilized from bovine adrenal cortex membranes with Triton N-101. Solubilized adenylate cyclase is stimulated by Gpp(NH)p and fluoride ion but not by ACTH. Pretreatment of particulate membrane fractions with Gpp(Ntt)p, hormone and Gpp(NtI)p or fluoride prior to detergent extraction leads to an activated solubilized enzyme which is nearly independent from added stimulants. Adenylate cyclase solubilized from membranes pretreated with Gpp(NH)p can be separated from guanyl nucleotide binding sites without loss of activity.

Key words: Detergent, 5'-Guanylyl-Imidodiphosphate -- Adenylate Cyclase.

5'-guanylyl-imidodiphosphate is a GTP analogue which stimulates adenylate cyclase (Harwood et al., 1973; Londos et al., 1974; Lefkowitz, 1974; Spiegel and Aurbach, 1974; Rodbcll et al., 1974; Eber t and Schwabe, 1974). Gpp(NH)p and other slowly metabolized GTP analogues appear to act via nueleotide binding sites present in membrane fractions containing adenylate cyclase and hormone receptors. The evidence for the existence of these guanyl nueleotide specific binding sites was indirect and rested on effects observed on either adenylate cyclase or hormone binding (Rodbell et al., 1971 ; Glossmann et al., 1974). Recently at tempts have been made to identify and characterize these sites with labeled GTP or GTP analogues. (Rodbell et al., 1974; Spiegel and Aurbach, 1974; Pfeuffer and ttelmreich, 1975; Lefkowitz, 1975; Glossmann, 1975.) A remarkable feature of the guanyl nueleotide binding sites is their apparent abundance by one or two orders of magnitude compared to hormone receptors. I t is not known ff all of these sites are connected to the effects of Gpp(NIt)p seen with adenylate cyclase and hormone receptors and what function they may have in addition to

Send o//print requests to: It. Glossmann, Pharmakologisches Institut der Justus- Liebig-Universit~t, D-6300 Giessen, Frankfurter Stral~e 197, Federal Republic of Germany.

90 H. Glossmann

these effects. W e have therefore a t t e m p t e d to solubil ize G p p ( N H ) p b ind ing sites toge the r wi th G p p ( N K ) p responsive adeny la t e eyelase to fac i l i ta te the i r charac te r iza t ion .

Materials1 and Methods [(x-a2p]-ATP, [3~I] cyclic AMP were from New England Nuclear (Dreieichen-

hain, Germany). [a/i] Gpp(NH)p (8.7 C/retool) was from International Chemical and Nuclear Corporation, Irvine, California, U.S.A. Triton | ~N-101 (Nonyl-phenoxy- polyethox-ethanol) was from Sigma Chemical Company, Miinchen, Germany. Polycthyleneglycol 6000 and porcine ?-globulin were from Serva, /ieidelbcrg, Germany. 1-methyl-3-isobutylxanthine was from EGA Chemic, /ieidenheim, Germany. Unlabeled nucleotides, creatine phosphate and creatine phosphokinase were from Boehringer, Mannheim, Germany.

Adenylate cyelasc (EC 4.6.1.1.) assays were performed at 30~ in 50 mM Tris- HC1 buffer p t I 7.6, 1 mM cyclic AMP, 2 mM 1-methyl-3-isobutylxanthine, 0.6 mg per ml of creatine phosphokinase, 2 mM crcatine phosphate, 5 mM MgCI~, [a.02p]. ATP (0.1 raM, 100--200 cpm/pmol)and the listed additions (final assay volume: 150 tA). [32p] cyclic AMP was isolated as described by Solomon et al. (1974).

Solubilization of adenylate cyclase and Gpp(NH)p binding sites: Particulate bovine adrenal cortex plasma membranes (Glossmann and Gips,

1974) were incubated at 1 ~ C with Triton | ~-101 (6 mg detergent per 1 mg of membrane protein). The solubilization mixture was 10 mM in Tris-FfC1 (p/-I 7.4) and contained 1 mM MgCI 2 and 1.5 mM mercaptoethanol. After 40 rain incubation and periodic shaking the mixture was centrifuged at 4~ (150000 x g; 60 rain). The supernatant was carefully decanted and kept on ice.

Binding of [3H] Gpp(NH)p to detergent solubilized plasma membrane fractions was determined with the polyethyleneglycol precipitation technique following the procedures outlined by Cuatrecasas (1972): Solubilized membrane preparations were incubated with [~ Gpp(Nl~I)p, 10 mM Tris-RC1 pH 7.4, 1.5 mM EGTA for 20--30 rain at 30~ (final volume: 100 ~1). 20 ~1 of 2.5% (w/v)porcine ?-globulin in 0.1 M phosphate buffer (pH 7.4) was added, followed by 2 ml of ice-cold 12.5~ (w/v) polyethylcneglycol 6000. The sample were agitated, kept for 10 rain in ice water and were filtered over iViillipore | HAWP filters. The filter was rinsed once with 2 ml of 6.25 (w/v) polyethyleneglycol 6000. Trapped radioactivity on filters was determined by liquid scintillation counting. Protein-bound [oH] Gpp(TN/i)p was also determined by a centrifugation technique. Samples containing 5 or 10 tzl of detergent-solubilized [a/I] Gpp(N/I)p labeled binding sites were mixed~with 10 mM Tris-/IC1 buffer (pH 7.4) and various additions (final volume: 100 ~1). The same sequence of operations was used as above except that samples were centrifuged (8000 • g, 40 rain).

The pellet was rinsed once with polyethyleneglycol 6000 (6.25~ w/v), dissolved in l~ (w/v) sodiumdodecylsulfate and radioactivity determined as above.

Pretreatment of membranes with stimulants: Bovine adrenal cortex membranes were preincubated for 15 rain at 30~ in a medium containing 20 mM NaHCO S, 1 mM dithiothreitol, 0.25 mM EDTA, 4 mM MgC12 and the listed additions (final volume: 0.5 ml). The membranes were diluted 80-fold with ice-cold 20 mM

1 The abbreviations used are: ACTHI_2~, corticotropin (1--24) tetracosa peptide; Gpp(NH)p, 5'-guanylyl-imidodiphosphate; cyclic AMP, cyclic adenosine 3':5'-monophosphate; EDTA, ethylenediaminotetraacetic acid; EGTA, ethylen- glycol-bis(fl-aminoethylether)~,~'-tetraacetic acid.

Solubilized Adenylate Cyclase 91

NaHC03 containing 1 mM dithiothreitol and centrifuged at 2~ for 35 min at 40000• g. The supernatant was decanted and the pellet rinsed twice with 5 ml of distilled water. Subsequently the pellet was solubilized as described above.

Column chromatography of detergent solubilized plasma membranes was performed at 4 ~ C on a Sephadex | G-200 column (0.9 • 54 era) equilibrated with 0.25% (w/v) Triton | N-101 in 10 mM Tris-tIC1 pH 7.4, 1 mM MgCl~ and 1.5 mM mercaptoethanol.

Protein was determined according to Lowry et al. (1951). 0.1 ml of 10~ (w/v) sodiumdodeeylsulfate was added to all samples (1.2 ml) prior to measurement of optical density.

Results

Solubilization of Adenylate Cyclase P r e l i m i n a r y exper imen t s wi th nonionic detergents , showed t h a t

Tri ton | N-101 could solubflize adeny la t e cyclase a c t i v i t y f rom bovine ad rena l cor tex membranes and t h a t the de tergent -so lubi l ized a c t i v i t y was s t imu la t ed b y Gpp(NH)p . A n example of a solubfl izat ion exper imen t , where the concen t ra t ion of de te rgen t was var ied , is shown in Table 1. U p to 20 or 30 ~ of the t o t a l adeny l a t e cyelase a c t i v i t y p resen t in par- t i cu la te p r epa ra t i ons could be e lu ted a t op t ima l de t e rgen t /p ro t e in ra t ios . The solubflized enzyme could no t be sed imen ted b y centr i - fuga t ion (150000 • g, 60 or 90 rain) and fi l tered th rough H A W P ni t ro- cellulose filters w i thou t loss of ac t iv i ty . The solubil ized enzyme was

Table 1. Solubilization of adenylate cyclase by Triton N-101. Bovine adrenal cortex membranes were incubated with varying concentrations of Triton+ N-101 but otherwise treated as described unter "Materials and Methods". 100 [zl aliquots of the solubilized material were tested for adenylate cyclase activity in the presence or absence (basal activity) of the listed stimulants. NaF was 12 mM and Gpp(NH)p 5 IzM. 0.2 mM MnC12 was present in the adenylate cyclase assay medium. The apparent yield of adenylate cyclase in the solubilized material is expressed as the percentage of the total activity found in membranes kept in the solubilization

medium (detergent omitted)

Triton Detergent/ ~ Adenylate cyelase activity (pmoles/mg • 15 min) N-lOl Protein Protein (w/v) ratio solubili- no ad- ~ NaF ~ Gpp(NH)p ~

(mg/mg) zed dition yield a yield yield (basal)

none -- -- 343 100 2413 100 820 100 (control) 2 54 70 66 13 275 8.0 186 16 1 27 56 140 23 561 13 341 24 0.25 6.7 56 132 22 710 17 470 33 0.125 3.3 48 149 20 845 17 570 33 0.062 1.7 31 109 10 377 5 260 10

a Control = lO0~

92 H. Glossmann

150-

E

x

E \ 100- {D

O E

I:L

50- <

40. ,tD 30- ,,J 0 20.

>- 10. O C.

i . . . . . . - . - G - 7 - . . . .

250-

200.

150.

100-

50-

~1 ~ 0 0.0t

. . . . . . . . . Basal activity . . . . . . . . . . . .

. I l l I I I ! I I I

o.o~ o.I i ~o ~oo o.I i 1o 1oo Nuc leo t ide Concentrat ion GTP [)JM]

[ u M ]

Fig. 1. (a left) Stimulation of detergent-solubilized adenylate cyclase by Gpp(NH)p. Adenylate cyclase activity was tested in the absence (basal activity) or presence of Gpp(NH)p or GTP. 30 Fg of solubilized protein was used per assay. Incubation time was 15 rain. Means from duplicate determinations. (b right) Inhibition of the Gpp(NH)p stimulation by GTP. Adenylate cyclase activity of solubilized membranes (32.5 Bg of solubilized protein per assay) was measured with 5 BM Gpp(NH)p in the presence of increasing concentrations of GTP. Incubation time was 15 rain.

Means from duplicate experiments

Table 2. Influence of Triton N-101 on particulate adenylate cyclase in bovine adrenal cortex plasma membranes. Bovine adrenal cortex plasma membranes (0.73 mg of protein per ml) were incubated in the adenylate cyclase assay medium in the presence and absence of Triton | N-101. Fluoride (as NaF) was 12 raM, ACTHI_2~ was 0.6 ~g/ml and Gpp(NH)p was 5 ~M. 0.2 mM MnCl 2 was present in the adenylate cyelase assay medium. Triplicate samples were incubated for 15 rain at 30~ and the results are expressed in pmolcs cyclic AMP produced per mg of

protein • 15 min

Incubation Final concentration of Tr i~n N-101 (w/v) conditions 0 0.20/0 0.40/0 0.8~

Basal activity 219 107 97.6 76 Fluoride 2416 2021 1376 1195 ACTH 497 104 92.7 67 Gpp(NH)p 595 841 666 585

s t imula ted by Gpp(NH)p and fluoride ion (as NaF) as shown in Table i . GTP, in contras t to Gpp(NH)p had li t t le effect on soluble adenyla te cyelase (Fig. 1 a). GTP could, however, inh ib i t the effects of Gpp(NH)p


0 1.0. E 0..

"0 c "

0

0., ' ~ 0.5.

0.4- ~--~ 0.3- 0 0.2- -r" 0.1'

o 0

66.2 nM

s , I I

10 20 30 40

Protein [pg/asaay]

100-

:< 50-

=__., 10

4 0 10 -s 10 .7 10 -s 10 -5 10 -4

Gpp [NH]p (Molar)

Fig.2 (a left) Binding of [3tt] Gpp(I~H)p to detergent solubilized membranes. The binding assay mixture ("Materials and Methods") contained 66.2 nM [3H] Gpp(NH)p and increasing amounts of solubilized membrane protein. Triplicate experiments (Millipore filtration). (b right) Saturation of Gpp(I~ItI)p binding sites. Aliquots of solubilized plasma membranes (32 ~zg of protein) were incubated in the binding assay mixture (see "Materials and Methods") with 66.2 nM [SH] Gpp(NH)p and unlabeled Gpp(NH)p as indicated on the figure. B 0 is the fraction of total tracer Gpp(NI-I)p bound in the absence and B in the presence of unlabeled nucleotide

B 0 was 0.2. Means from duplicate experiments (Millipore filtration)

on solubilized adenylate eyclase (Fig. l b). The same observation has been made with particulate adenylate cyclase from bovine adrenal cortex (Glossmann and Gips, 1974).

There was little or no effect of ACTH on solubilized adenylate eyelase whether Gpp(NH)p was present or not. Addition of detergent to particulate adenylate cyclase preparations (Table 2) leads to a marked inhibition of basal activity, a loss of the hormone response and an increase (at low detergent concentrations) of the response to Gpp(NtI)p. The solubilized adenylate cyclase, when kept a t 1 ~ C in concentrated solution, retained the general properties mentioned above for 48 hrs, though a detailed investigation of the stability has not been performed.

Gpp(NH)T Binding Sites in the Solubilized Material

Bovine adrenal cortex membranes contain binding sites for Gpp(NH)p (Glossmann, 1975), and similar binding sites for Gpp(NH)p were found in the solubflized membrane fraction. The amount of bound tracer Gpp(NIt)p increased with increasing concentrations of solubflized membrane protein (Fig.2a) and binding of Gpp(NH)p was a saturable process as shown by the displacement curve in Fig.2b. The value for Ka (association constant) of the Gpp(NH)p binding sites was 10 ~M -1

94 It. Glossmann

~ & z 8

o. ID

:I: ~ 30 Z ~

.0.6

'~ 3" I Adenylate cyclase ~ - ~ ' ~ .e.

/J :::k 0 o ~ "~ 2- I / ~, ~ , ", -0.5 20

8 ~ / z . / - / ',, " / ; '~' ", ~o . . . , , -o.4

"~ \ - .~IGpp [NH]p binding 1

" i ,,', i ,,, , ...,. -o.3 10

< ~ 1- ,, 'q i . --0.2 / / ' , , i ", "... - ; ' ,'~ V ' "*- . . . . . . -,. "*',. !

30 35 40 45 50 55 FRACTION NUMBER

Fig. 3. Sephadex G-200 column chromatography of solublized membrane material. 2.2 mg of solubilized membrane protein was applied on the column (sample volume: 1 ml). The flow rate was 1.5 ml per hour and fractions of 0.45 ml were collected. After completion of the run adenylate cyclase activity (in the presence of 12 mM NaF and 0.2 mM MnC12) was tested with 100 F1 aliquots. Binding of [att] Gpp(NH)p (70 nM) was tested with 50 F1 ~liquots with Millipore filtration as described under "Materials and Methods". The void volume of the column, determined with Dextran blue was 15.5 ml (= Fraction 35). The recovery of adenylate cyclase activity was

22 ~ and of protein 80 ~

and is similar to the Ka of particulate binding si~es (12 ~M-1; Gloss- mann, 1975). About 70 pmoles of binding sites, characterized by a Ka of 10 FM -1, were found per one mg of solubilized membrane protein. As sometimes observed in binding studies with particulate preparations (Spiegel and Aurbach, 1974) deviations of the Scatchard plot occur a t higher concentrations of bound Gpp(NH)p. These observations could indicate the presence of low affinity, high capacity Gpp(NH)p binding sites in particulate and solubilized preparations. Alternatively, a systematic error in the calculation of bound and free ligand could be responsible for the deviation (Rodbard, 1973).

Chromatography of the solubillzed membrane material on Sephadex G-200 was performed and binding sites for Gpp(NtI)p were detected in the column effluent with polyethyleneglycol precipitation. Par t of the Gpp(NIt)p binding sites cluted at the void volume but most of the material capable of binding [all] Gpp(Ntt)p eluted at a position well separated from the void volume (Fig. 3). Adenylate eyclase act ivi ty was detectable in the column effluent in three out of four experiments. The


recovery of adenylate cyclase activity was poor (0, 5, 13 and 220/o respectively of the total activity apphed on the column). In the three experiments where adenylate cyclase activity was recovered it was found close to the void volume of the column but always well separated from most of the Gpp(NH)p binding sites. The same elution pat tern was found when solubilized membrane material was preincubated with [SH] Gpp(NH)p and chromatographed on Sephadex G-200 (not shown).

Solubilization o] Stimulant-Pretreated Membranes Particulate membrane fractions from bovine adrenal cortex which

were pretreated with Gpp(NH)p and subsequently washed show per- sistant activation of adenylate cyclase (Glossmann and Gips, 1975). Gpp(NH)p and to somewhat lesser extent fluoride ion "protected" adenylate cyclase against the inhibitory action of detergent added to particulate fractions (Table 2).

We have now tried to solubilize adenylate cyclase ifrom stimulant- pretreated membranes. An examples is given in Table 3. Pretreatment with hormone alone had very little effect on the activity of solubilized adenylate cyclase but preincubation with either fluoride ion or Gpp(NH)p lead to a more active solubilized adenylate cyclase. The highest activity of the solubflized enzyme was obtained by pretreatment with ACTtt and Gpp(NH)p, very similar to results obtained with particulate preparations. I t will be also noted from the data in Table 3 tha t subsequent addition of Gpp(NIt)p and fluoride ion only slightly enhances the activity of solubflized adenylate cyelase eluted from membranes pretreated with

Table 3. Effect of pretreatment of particulate membranes on solubilized adenylate cyclase. Bovine adrenal cortex membranes (0.6 mg of protein) were preincubated with or without the following additions: Gpp(NH)p: 8 ~M, ACTH,_2~ 8 ~g/ml and NaF 16 mM. Subsequently membranes were washed and solubilized as described under "Materials and Methods" Aliquots of the solubilized material were tested for adenylate cyclase activity; Gpp(Ntt)p was 5 {zM, ACTHl_24 0.6 ~g/ml and

NaF 12 mM, when present

Pretreatment conditions (Particulate membranes)

Adenylate cyclase activity (pmoles/mg • 15 rain) (solubilized preparations)

no addition Gpp(NH)p ACTH Gpp(NH)p NaF q- ACTH

No addition 44 Gpp(NH)p 404 ACTtt 57 Gpp(NH)p q- ACTH 823 NaF 218

135 50 142 190 521 396 501 561 189 70 187 246

1030 837 891 910 347 203 348 386

96 H. Glossmann

,o 1 ADENYLATE CYCLASE~ ~bound Gpp[NH]p

,~o t []-Gpp[..~p y, \_1 \ '~ / "1 "~ III+Gpp(NH]p / ~ 1 \

z "~ ~

o o. z ~

o 5o~ s~

o '20

0 10 '

30 FRACTION NUMBER

Fig.4. Gel filtration of adenylate cyclase solubilized from [3H] Gpp(NH)9 pretreated membranes. Bovine adrenal cortex plasma membranes (4.8 mg of protein) were pretreated with [8It] Gpp(NH)p (3 txM) and washed free from unbound nucleotide by eentrifugation ("Materials and Methods"). The rinsed membrane pellet contained 438 pmoles [311] Gpp(N11)p and was solubilized as described. The supernatant after centrifugation of the detergent suspended membrane pellet contained 3.6 mg of protein, 365 pmoles of [311] Gpp(N11]p and 90~ of the radioactivity was pre- cibitable by polyethyleneglycol. The residual pellet contained 1.9 mg of protein and 69 pmoles [311] Gpp(Ntt)p. An aliquot of the detergent solubilized material (1.5 ml containing 1.12 mg of protein) was applied on top of a Sephadex G-200 column. The flow rate was 3 ml per hour and 0.6 ml fractions were collected. 1.0 mg of protein was recovered (88~ 89 pmoles of [311] Gpp(NH)p were recovered in fractions 21--40 (780/0) and 12.3 pmoles [811] Gpp(N11)p (free nucleotide) was found in fractions 55--75. Adenylate cyelase activity was measured in the absence and presence of 5 ~M Gpp(NH)p. The specific activity of the starting material was 394 pmoles per nag of protein • 15 rain in the absence and 421 pmoles per mg of protein• 15 rain in the presence of 5 ~zM Gpp(N11)p. 97~ of the adenylate cyclase

activity was recovered in fractions 2 1 - 32

Gpp(Nt t )p (in the absence or presence of ACTtI) and fluoride ion. De- tergent solubilized material f rom s t imulant pretreated membranes was chromatographed over a Sephadex G-200 column as described above. I n Fig.4 the elution profile of a soluble adenylate eyclase eluted from membranes pretreated with [sH] Gpp(NtI )p is shown. I t was found tha t the detergent solubilized 800/0 of the [3H] Gpp(NH)p prelabeled binding sites and 75 ~ of the adenylate cyclase ac t iv i ty f rom part iculate membranes.


Table 4. Dissociation of [3H] Gpp(NH)p from detergent-solubilized prelabeled binding sites. Aliquots (10 ~l) from the detergent-solubilized [3H] Gpp(NH)p prelabeled membrane material used for the experiment shown in Fig.4 were incubated with the listed additions for 40 vain at 30 ~ in 10 ml~ Tris-HC1 pH 7.4 (final volume: 100 ~1). Bound [all] Gpp(NH)p was determined with polyethyleneglycol precipitation and eentrifugation. Unlabeled GTP and Gpp(NH)p were 0.1 raM, EDTA (Tris-salt) was 20 n ~ . 100~ of the radioactivity present in the solubilized material was found in the supernatant when acidified ethanol (1 part 1 n HC1 plus 8 parts ethanol) was used instead polyethyleneglycol for precipitation; 900/0 of the total radioactivity was precipitated by polyethyleneglycol in control samples

(quadruplicate determinations)

Additions [all] Gpp(NH)p bound (pmoles per mg of protein)

Buffer control 102 Gpp(NH)p 101 GTP 84 GDP 102 EDTA 22 EDTA q- Gpp(NH)p 22 EDTA + GTP 21 Buffer a 0

a Acidified ethanol was used for precipitation.

The recovery of adenylate eyelase act ivi ty after gel filtration was 100~ and the specific act ivi ty of the enzyme increased about 3-fold. Most of the Gpp(NH)p binding sites were separable from adenylate cyelase and were enriched about 1.5-fold after gel filtration. The recovery of the Gpp(Ntt )p binding sites was 800/0 .

The bound labeled Gpp(NH)p could not be released from the solubflized binding sites by t rea tment with unlabeled Gpp(NH)p (Table 4). This is in contrast to findings with particulate binding sites where about 500/0 of the labeled bound GTP analogue exchanges with excess unlabeled nueleotide after 40 rain (Glossmann, 1975). The chelator EDTA released 78~ of the bound nucleotide, GTP 18~ and GDP none. 100~ of the bound nucleotide was released by t rea tment with acidified ethanol indicating tha t the binding is not covalent.

Discussion

The nonionic detergent Triton| N-101 was able to solubilize adenylate cyelase from bovine adrenal cortex membranes. The solubilized enzyme was stimulated by Gpp(NII)p and the Gpp(NH)p effects were inhibited by GTP. Fluoride ion also enhanced the solubifized adenylate cyclase

7 Naunyn-Scb_miedeberg's Arch. Pharmacol., Vol. 291

98 H. Glossmann

activity but ACTH had no effect. I t would require the use of labeled ACTIt to determine whether the detergent prevented the coupling process or the binding of the hormone to its receptors.

The loss of the hormone response is a common observation with detergent-sohibilized adenylate eyelase (Perkins, 1973) but Ryan and Storm (1974) recently succeeded to solubilize a hormone-sensitive adenylate cyelase from liver cell membranes with Triton X-305.

The stimulation of detergent-solubilized adenylate cyclase by Gpp(NH)p suggested that binding sites for guanyl nucleotides were present in the solubilized material. When [aH] Gpp(NH)p was used to pre-label particulate binding sites, it was possible to quantitative the extraction of binding sites by the detergent and follow their behavior on gel filtration. The association constant of the soinbilized material for Gpp(NtI)p could not be determined because the bound GTP analogue was only released by rather drastic procedures like treatment with EDTA or acidified ethanol. This prohibited the determination of the association constant of the Gpp(NYt)p-binding site complex. Deter- gent-sohibilized material extracted from membranes not pretreated with Gpp(NH)p contained binding sites for the GTP analogue with an association constant very similar to that determined on particulate membranes. Both findings strongly suggest that the majority of the solubilized Gpp(NH)p binding sites is identical to the particulate binding sites. Each method led to a solubilized adenylate cyclase which had different properties. Adenylate cyclase eluted from Gpp(NH)p pretreated membranes was nearly independent from added stimulants and was completely recovered after gel filtration. Adenylate cyclase eluted from membranes not exposed to the GTP analogue prior to detergent treatment was stimulated by Gpp(NH)p but could not be recovered to a large extent after gel chromatography. This loss could indicate a separation from necessary cofactors.

There are indications that activating factors can be isolated and separated from adenylate eyclase. Recently a calcium-binding protein has been identified as a regulator of detergent-dispersed adenylate cyelase from porcine cerebral cortex (Brostrom et al., 1975). Solubflized adenylate cyclase from bovine adrenal cortex was separable from Gpp(Ntt)p binding sites by gel filtration. Pfeuffer and Helmreich (1975) have reported similar findings with Lubrol PX solubflized adenylate cyelase from pigeon red cell membranes. Lubrol PX and Triton N-101 are mild detergents and they are often unable to dissociate protein- protein interactions in biological membranes (Helenius and Simons, 1975). I t is therefore possible that there are no strong protein-protein interactions between the catalytic subunit of adenylate cyelase and the majority of the guanyl nucleotide binding sites.

Solubilized Adenylate Cyelase 99

The presence of guanyl nucleotide binding sites in solubflized membrane preparations (Lcfkowitz, 1975; Pfeuffer and Helmreich, 1975) can only suggest tha t these sites are responsible for the activation of adenylate cyelase. Separation and recombination experiments are needed to clarify the role of the binding sites. Solubilized membrane preparations can help to achieve this goal. From experiments with particulate membranes it is evident that the enzyme is activated ff the sites are occupied by GTP analogoues like Gpp(NH)p. This process is facilitated by hormone agonists like ACTH in adrenal cortex membranes or isoproterenol in pigeon erythroeyte ghosts and it can even occur in the absence of adenylate cyclase reagents.

The resulting activated adenylate cyclase is resistant to pertubation by nonionic detergents and can be extracted from stimulant-pretreated membranes. I t is remarkable that an important feature of hormonal activation of intact cells, reversibility, is lost by occupation of guanyl nueleotide binding sites with Gpp(Ntt)p in particulate membrane preparations. There are several questions which remain open.

The role of divalent cations (membrane-bound or free) in the activation process needs to be clarified. We have reported that uncomplexed EDTA in the millimolar range inhibited the binding of Gpp(Ng)p to adrenal cortex membranes. I f the chelator is added simultaneously or prior to Gpp(NH)p the activation is inhibited as well (Glossman~, 1975; unpublished results).

Evidence is also missing that guanyl nucleotide binding sites are plasma membrane specific 2. They have been found in pigeon red cell membranes (Pfeuffer and tIelmreich, 1975), ra t liver cell membranes (Rodbell et al., 1975), canine myocardial membranes (Lefkowitz, 1975), turkey red cell membranes (Spiegel and Aurbach, 1974) and as reported here and elsewhere (Glossmann, 1975) in bovine adrenal cortex plasma membranes. The nature of these guanyl nucleotide binding sites is not known. Their apparent abundance compared to hormone receptors should facilitabe their isolation and characterization.

Acknowledgements. We thank Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg for generous support.

References Brostrom, C. 0., Huang, u C., Breckenridge, B. ~cL., Wolff, D. J. : Identification

of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Proc. nat. Aead. Sci. (Wash.) 72, 64--68 (1975)

Cuatrecasas, P. : Isolation of the insulin receptor of liver and fat-cell membranes. Proc. nat. Aead. Sci. (Wash.) 69, 318--322 (1972)

2 Among other intracellular proteins like tubulin which bind guanyl nucleotides, elongation factor 2, which is subject to diphteria toxin catalyzed ADP-ribosylation, has high affinity for GTP, GDP and GTP analogues (tIenrikson et aI., 1975).

7*

100 H. Glossmann

Ebert, R., Sehwabe, U. : Biphasic effect of 5'-guanylyl-imidodiphosphate on fat cell adenylate cyclase. Naunyn-Schmiedeberg's Arch. Pharmacol. 286,297--313 (1974)

Glossmann, 1[.: Adrenal cortex adenylate cyclase: Specific binding sites for 5'- guanylyl-imidodiphosphate in partially purified plasma membranes from bovine adrenal cortex. Naunyn-Schmiedeberg's Arch. Pharmacol. 289, 99--109 (1975)

Glossmann, 1[, Baukal, A., Cart, K. J. : Angiotensin I I receptors in bovine adrenal cortex: Modification of angiotensin I I binding by guanyl nucleotides. J. biol. Chem. 249, 664--666 (1974)

Glossmann, H., Gips, H. : Adrenal cortex adenylate cyclase: Comparison between the action of GTP and 5'-guanylyl-imidodiphosphato on the particulate enzyme from bovine adrenal cortex and rat adrenals. Naunyn-Schmiedeberg's Arch. Pharmacol. 286, 239--249 (1974)

Glossmann, H., Gips, tI.: Bovine adrenal cortex adenylate cyclase: Properties of the particulate enzyme and effects of guanylnucleotides. Naunyn-Schmiede- berg's Arch. Pharmacol. 289, 77--97 (1975)

Harwood, J. P., LSw, H., Rodbell, M.: Stimulatory and inhibitory effects of guanyl nucleotides on fat cell adenylate cyclase. J. biol. Chem. 248, 6239--6245 (1973)

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