Stereospecificity of some invertebrate lactic dehydrogenases

RICE UNIVERSITY

Stereospecificity of Some Invertebrate Lactic Dehydrogenases

Mary Jacobs Scheia

A TEESIS SUBMITTED IN PARTIAL FULFILLMENT1 OF TEE REQUIREMENTS FOR TEE DEGREE OF

Master of Arts

Thesis Director's signature:

May, 1970

ABSTRACT

Stereospecificity of Some Invertebrate Lactic Dehydrogenases

by

Mary Jacobs Scheid

1. It has been assumed that only among unicellular organisms did nicotin¬

amide adenine dinucleotide (NAD)-linked lactic dehydrogenases occur.

All higher animals were thought to possess L- specific lactic dehydro¬

genases. It was discovered recently, however, that many species of

invertebrates possess lactic dehydrogenases specific for the D(-)

isomer of lactic acid. A study of lactic dehydrogenase stereospecifi¬

city in organisms from ten invertebrate phyla has been carried out in

order to supplement the available data on the phylogenetic distribution

of D-specific lactic dehydrogenases.

2. D-specific lactic dehydrogenases were found in annelids, acanthocephala,

molluscs, and arthropods. All of the platyhelminths and echinoderms

tested had enzymes specific for L(+) lactic acid. In the annelids

the oligochaetes and the closely related hirudinea possess L-specific

lactic dehydrogenases while the polychaetes which utilized lactic

acid utilized the D(-) isomer. Among the four classes of molluscs

which have been studied all possessed D-specific lactic dehydrogenases.

In the arthropods the chelicerates tested were specific for L~lactic

acid while the mandibulates were specific for D-lactic acid. These

results indicate that the occurrence of D-lactic dehydrogenases

apparently falls along previously established taxonomic lines.

3. A brief review of the terminal pathways of anaerobic glycolysis in

the phyla studied has also been included. Many invertebrates do not

produce lactic acid as the primary end product of anaerobic carbohy¬

drate catabolism as in vertebrate tissues. Rather they produce low

levels of lactic acid and varying quantities of succinic acid and

fatty acids. The pathway usually proposed for succinic acid produc¬

tion in these organisms involves the degradation of glucose via the

Embden-Meyerhof-Parnas pathway to the level of phosphoenolpyruvate

or pyruvate and then partial reversal of the tricarboxylic acid cycle.

While succinic acid production is widespread among invertebrates under

conditions of anoxia or low oxygen tension, its occurrence cannot be

directly correlated with the distribution of D-specific lactic

dehydrogenases.

ACMCWIEDGEMENTS

I wish to express my appreciation to Dr. Jorge Awapara for his

valuable advice and encouragement throughout the course of this inves¬

tigation. I would also like to thank Dr. F.M. Fisher and Dr. J.B. Walker

for reading and criticizing this manuscript.

TABLE OF COHTEKTS

Introduction 1

Experimental Procedures 8

Materials 8

Methods 10

Results 13

Discussion . . . 21

Conclusions 35

References 37

Stereospecificity of Some Invertebrate

Lactic Dehydrogenases

INTRODUCTION

The conversion of carbohydrate to lactic acid by the Embden-Meyerhof

Parnas pathway is the major route of glucose catabolism in vertebrate

tissues. The intermediate enzymatic steps of this sequence can occur

either under aerobic or under anaerobic conditions. Usually, however,

the terminal steps of glycolysis occurring in the presence of oxygen

differ from those which occur in its absence; the pathways diverge at

the level of pyruvic acid. In the presence of oxygen pyruvic acid may

enter the tricarboxylic acid cycle where its complete oxidation occurs.

Under anaerobic conditions it is reduced to lactic acid by the enzyme

lactic dehydrogenase. Lactic dehydrogenase serves to reoxidize the

reduced form of nicotinamide adenine dinucleotide (WADIl) which is pro¬

duced by the oxidation of 3-phosphoglyceraldehyde to 3~phosphoglyceric

acid during glycolysis. Without this reoxidation of NADH by lactic

dehydrogenase, anaerobic carbohydrate degradation could not continue.

Lactic dehydrogenase from vertebrate sources, which is specific

for the L(+) isomer of lactic acid, has five different isozymic forms

(Markert and Moller, 1959)* Each isozyme is a tetramer consisting of

four dissociable polypeptide subunits. These subunits are of two dif¬

ferent kinds, both having a molecular weight of 35^000, "but with dif¬

ferent net changes (Appella and Markert, 1961) and different amino acid

sequences. The two types of subunits, designated the nM” (Cahn et al.,

1962) or nAn type (Markert, 1962) which is found predominantly in skele¬

tal muscle and the MHM or nBn type which occurs primarily in heart muscle

2

are each encoded by a separate gene (Markert and Ursprung, 1962). The

five isozymes of lactic dehydrogenase are formed by the random asso¬

ciation of these subunits into tetrameric molecules. Thus,, in skeletal

muscle the isozyme (lDH-5) predominates while in heart muscle

(iDH-l) is found. The distribution of the lactic dehydrogenase isozymes

varies among different species and different tissues although many of the

properties of the two types of subunits appear to be relatively similar

among the vertebrates. Differences in some properties of the subunits

have been related to evolutionary change in the vertebrates (Kaplan,

1965)* The occurrence of a particular isozyme of lactic dehydrogenase

in a certain tissue is apparently related to the energy requirements of

that tissue and to the availability of oxygen. Thjs indicates that the

isozymes of lactic dehydrogenase have different functional roles (Dawson

et al., 1964). The isozyme, which is readily inhibited by pyruvic

acid, is found primarily in tissues in which energy is continuously

required. Inhibition of the Hij. enzyme by pyruvic acid assures that this

substance is channeled toward the tricarboxylic acid cycle where it is

oxidized and energy produced. In tissues in which the metabolism is

primarily anaerobic, as in skeletal muscle, energy is supplied by gly¬

colysis. Thus a form of lactic dehydrogenase which is not inhibited by

pyruvic acid is necessary to prevent the glycolytic pathway from coming

to a standstill. The form of lactic dehydrogenase serves this need.

As mentioned above, all the isozymes from vertebrate tissues which

have been studied thus far are apparently specific for the L(+) isomer

of lactic acid. In microorganisms, hen-/ever, a wide variety of lactic

dehydrogenases exist, some of which are specific for the D(-) isomer of

- 3 -

lactic acid and some of which are not linked to HAD. Isozymes similar

to those mentioned above, however, have not been discovered in bacteria

(Tarmy and Kaplan, 1968a). In the bacterium Lactobacillus plantarum

(=arabinosus) two NAD-linked dehydrogenases have been purified and char¬

acterized: one is specific for L(+) lactic acid and the other for D(-)

lactic acid (Dennis and Kaplan, 1959> i960). The former has a molecular

weight of 155,000 which is approximately the same size as the vertebrate

dehydrogenases while the D-specific enzyme has a molecular weight of

60,000 to 70,000 (Tarmy and Kaplan, 1968a). In E. coll an NAD-linked

D-specific lactic dehydrogenase, but not an L-specific one, has been

isolated and shown to have a molecular weight of 115,000. This enzyme

(Tarmy and Kaplan, 1968a,b) and a similar enzyme from Butyribacterlum

rettgerl (Wittenberger, 1966; Wittenberger and Fulco, 1967), are rela¬

tively incapable of catalyzing the oxidation of lactic acid to pyruvic

acid in the presence of NAD although they do carry out this conversion

in the presence of coenzyme analogues. NAD-linked D-specific lactic

dehydrogenases have also been isolated from the cellular slime mold

Polyspondylium pallidum (Garland and Kaplan, 1967) and in several

species of fungi in the order Leptomitales (Gleason et al., 1966).

Until recently it has been assumed that only among unicellular

organisms were NAD-linked D-specific lactic dehydrogenases found. All

higher animals were thought to possess L-specific lactic dehydrogenases.

It has been discovered, however, that many species of invertebrates

possess lactic dehydrogenases which are specific for the D(-) isomer

of lactic acid (Long and Kaplan, 1968). Long and Kaplan found these

enzymes in polychaetes, molluscs, and arthropods and have purified a

- k -

D-specific enzyme from the horseshoe crah Limulus polyphemus. This

enzyme has a molecular weight of 65,000 as determined by gel filtration

and ultracentrifugation and exists in electrophoretically distinguish¬

able isozymic forms. These authors conclude that there are two major

catalytically and physically distinguishable forms of this D-specific

enzyme, one associated with the muscle and one with the heart. This

latter isozyme is apparently more susceptible to pyruvic acid inhibi¬

tion than is the former. In a more recent study on the lactic dehy¬

drogenase from this arthropod, Massaro (1970) was able to confirm the

stereospecificity of the D-lactic dehydrogenase, but in contrast to

Long and Kaplan he found that the molecular weight as determined by

gel filtration and column chromatography and compared to other enzymes

of known molecular weight is 140,000, the same as vertebrate L-lactic

dehydrogenases. (He obtained similar results for the D-specific enzyme

from the pelecypod Msrcenaria mercenaria♦) In addition, Massaro found

that the Limulus lactic dehydrogenase exists in five isozymic forms

whose distributions vary from tissue to tissue. On the basis of his

results Massaro postulates that the Limulus enzyme is a tetramer con¬

sisting of two types of subunits and that the five isozymes are formed

by the various associations of these subunits, a situation analogous to

that occurring in vertebrates.

In another arthropod, the lobster, Homarus americanus, lactic dehy¬

drogenase from the tail muscle has been characterized chemically and

catalytically (Kaloustian et ad.., 19&9; Kaloustian and Kaplan, 1969)•

In contrast to that of Limulus the enzyme from Homarus tail muscle is

specific for L-lactic acid. This enzyme has at least three isozymes,

- 5 -

two of which were separated and shown to have molecular weights similar

to vertebrate lactic dehydrogenases. These authors have also shown that

NADH has a regulatory effect on the enzyme, activating the reduction of

NAD at low NADH concentrations and inhibiting this reduction at high

NADH concentrations. No proposals are made about the role that this

control mechanism might play in the carbohydrate metabolism of crusta¬

cean muscle (Kaloustian and Kaplan, 1969). Homarus americanus and Limulus

polyphemus are members of two separate subphyla of the arthropods, the

mandibulates and the chelicerates respectively. In early studies (Kaplan

and Ciotti, 1961) on the lactic dehydrogenases from several species of

arthropods using coenzyrne analogues, marked differences in the enzymes

from the two groups were noted. Long and Kaplan (1968) tested the stereo¬

specificity of the lactic dehydrogenases from several arthropods and found

that all the mandibulates tested have enzymes specific only for L~lactic

acid while the chelicerates have D-specific enzymes. This suggests that

there may be phylogenetic significance in the distribution of D-lactic

dehydrogenases.

The discovery of the existence among invertebrates of two perhaps

very similar lactic dehydrogenases which differ in the stereospecificity

of their substrates but which exist in isozymic forms is of interest both

with regard to phylogenetic relationships and with regard to carbohydrate

metabolism. For many years it has been known that in some invertebrates

which live in anaerobic or partially anaerobic environments lactic acid

is not the sole or even the primary endproduct of carbohydrate catabolism

(von Brand, 19^6) even though most of the individual reactions of the

Embden-Meyerhof-Parnas pathway have been detected in invertebrates,

- 6 -

including the lactic dehydrogenase reaction. This has been demonstrated

in many species of parasitic helminths and in many species of molluscs.

These invertebrates do not produce lactic acid in quantities correspond¬

ing to the theoretical quantities which would obtain under anaerobic

conditions if glucose were degraded in exactly the same manner as in

vertebrate tissues. Rather they produce low levels of lactic acid and

varying quantities of succinic acid and volatile fatty acids in the

absence of oxygen (Humphrey, 1944; von Brand, 1946; Fairbain, 1954;

Bueding and Farrow, 1956; Agosin, 19573 Hammen and Wilbur, 19593 Mansour,

19593 Fairbain et al., I96I; Simpson and Awapara, 1964, 1966). In many

of these invertebrates it is thought that the production of succinic

acid may to varying extents replace lactic acid production under anaero¬

bic conditions. Succinic acid is one of the intermediates of the tri¬

carboxylic acid cycle; however, under anaerobic conditions it cannot be

produced by what are usually considered the forward reactions of this

pathway. Thus the pathway generally proposed for the formation of suc¬

cinate under anaerobic conditions in invertebrates involves a partial

reversal of the tricarboxylic acid cycle. Glycolysis proceeds via the

Embden-Meyerhof-Parnas pathway to the level of phosphcenolpyruvic acid

(PEP) or pyruvic acid which is then carboxylated and enters the tri¬

carboxylic acid cycle. The formation of succinic acid by the reversal

of this cycle provides a means for reoxidizing the NADH produced in the

glyceraldehyde phosphate dehydrogenase reaction, effectively bypassing

the lactic dehydrogenase reaction necessary for this oxidation in

anaerobic vertebrate glycolysis.

- 7 -

Because of the occurrence among some invertebrates of such a modi¬

fied pathway for anaerobic glucose catabolism which can bypass lactic

dehydrogenase and the occurrence of a pathway similar to the Embden-

Meyerhof-Parnas pathway in others., it was felt that a systematic study

of lactic dehydrogenases in representative invertebrate phyla was a

necessary adjunct to studies on carbohydrate metabolism. This survey

of the stereospecificity of invertebrate lactic dehydrogenases was under¬

taken to determine if there is any phylogenetic pattern in the distri¬

bution of the D-specific and L-specific enzymes and if there is any

relationship between lactic dehydrogenase specificity and pathways of

anaerobic carbohydrate catabolism in invertebrates.

EXPERIMENTAL PROCEDURES

Materials

With some exceptions,, most of the invertebrates used in this study

were obtained from commercial suppliers. Whenever possible they were

used immediately upon arrival or collection. Abnormal and unhealthy

animals were discarded as soon as they were detected and only apparently

healthy organisms were used in the experiments. Marine organisms were

maintained in aquaria containing synthetic salt water (instant Ocean.

Aquarium Systems, Inc.) or natural sea water from Galveston, Texas. The

aquaria were equipped with pumps to recirculate and aerate the water.

The temperature in these aquaria ranged from 15°C to 20°C. The free-

living terrestrial helminths used in these experiments were kept in

containers lined with moistened filter paper for at least 12 hours prior

to the assay in order that the contents of the gut might be voided. All

parasitic species, with the exception of Fasciola hepatica, were used as

soon as they were removed from the host. Fasciola hepatica was kept in

a saline solution at 38°C for two hours after its removal until complete

egestion had occurred. Below is a list of the animals used and their

sources.

Phylum Platyhelminthes Bipalium kewense Fasciola hepatica

Hymenolepis diminuta

Phylum Nernertea Cerebratulus sp Pacific Bio-Marine

Collected from a local nursery Host livers obtained from a local

abbatoir Gift of Professor C.P. Read

- 9 -

Phylum Acanthocephala Moniliformis dublus

Phylum Annelida Arenicola marina

Chaetopterus variopedatus Cirratulus grandls

Enchytraeus sp Lumbrlcus terrestris Nereis limhata

Phylum Sipunculida Dendrost omum pyroldes

Phylum Echiurida Urechis eaupo

Phylum Brachiopoda Laqueus californieus

Phylum Mollusca Anadara oyalis

Aquipecten irradians

Busycon sp

Chaetopleura apiculata Ensls directus

Helix aspersa Mercenaria mercenaria Mopa.lia muscosa Mytilus edulis

Otala lactea Polinlces sp

Rang!a cuneata

Spisula solidlssima

Strophocheilus oblongatus

Phylum Arthropoda Callinectes sapidus

Cancer irroratus

Gift of Mr, Robert McAlister

Marine Biological Laboratory, Woods Hole

Pacific Bio-Marine Marine Biological Laboratory,

Woods Hole Carolina Biological Supply Carolina Biological Supply Gulf Specimen Co., Inc.

Gulf Specimen Co., Inc.

Pacific Bio-Marine

Pacific Bio-Marine




Gulf Specimen Co., Inc. Marine Biological Laboratory,

Woods Hole Collected from a local nursery Pacific Bio-Marine Pacific Bio-Marine Marine Biological Laboratory,

Woods Hole Gift of Professor J.W. Campbell Marine Biological Laboratory,

Woods Hole Collected from Trinity Bay,

Point Barrow, Texas Marine Biological Laboratory,

Woods Hole Gift of Professor J.W. Campbell



10 -

Carcinus maenas

Libinia emarginata

Llmulus polyphemus

Phylum Echinodermata Arbacia punctlata

Astropecten brazlliensis Cucumarla rubra Lytechinus plctus Patirla minlata Strongylocentrotus purpuratus



Gulf Specimen Co., Inc.


Pacific Bio-Marine Gulf Specimen Co., Inc. Pacific Bio-Marine Pacific Bio-Marine Marine Biological Laboratory,

Woods Hole

All chemicals were obtained from the Sigma Chemical Company, St. Louis,

Missouri.

Methods

Tissue Preparation:

Organs or whole animals were first washed in cold 0.1 M potassium

phosphate buffer, pH J.0, and blotted on filter paper. After weighing,

the tissues were minced and homogenized with a hand homogenizer in two

to three volumes (g/ml) of 0.1 M potassium phosphate buffer, pH J.0,

The homogenate was then centrifuged at 12,000 x g for 20 minutes in a

refrigerated Sorval centrifuge, model RC-2. Any material which remained

on the surface of the centrifuged preparation was removed by filtration

through glass wool or with a Pasteur pipette. The pellet was discarded

and the supernatant used for the assay. Early attempts to dialyze the

supernatant fraction led to a decrease in activity in some cases so dialysis

was not used in any of the experiments reported here.

Enzyme Assay:

Lactic dehydrogenase activity was assayed spectrophotometrically

using a Cary model it- recording spectrophotometer. All assays were

11 -

carried out at room temperature. Both the oxidation of lactic acid to

pyruvic acid and the reduction of pyruvic acid to lactic acid were

measured.

The oxidation of lactic acid was assayed by measuring the rate of

reduction of NAD at 3A0 rap,. The reaction was carried out in a quartz

cuvette with a 1 cm light path. The reaction mixture used to assay an

enzyme specific for D(~) lactic acid contained 100 rnM potassium phosphate

buffer, pH 7 < 0; 100 mM D(-) lithium lactate; 8 mM NAD; and an amount of

enzyme which gave a linear change in optical density between 0.010 and

0.090 per minute for at least 75 seconds. The final volume of the reac¬

tion mixture was 1.0 ml. The reaction mixture used for an L(+) specific

enzyme contained 100 mM potassium phosphate buffer, pH 7•0; A0 mM L(+)

lithium lactate; A rnM NAD; and an amount of enzyme which gave a linear

change in optical density between 0.010 and 0.090 per minute for at

least 75 seconds. The coenzyme was added to initiate the reaction.

Blanks were run using only enzyme and coenzyme and only enzyme and sub¬

strate. Any activity obtained in the blanks was subtracted from that

obtained with the complete reaction mixture to give the final activity.

The D(-) lithium lactate obtained from Sigma Chemical Company

contained approximately 5% L~lactate. This meant that an enzyme

specific for only L-lactic acid might show activity when tested for

activity with D-lactate. For this reason assays were carried out using

final concentrations of L(+) lactate equivalent to the amounts of L-

lactate contaminating the D-isomer in assays for the D-specific enzyme.

In all cases the activity of an L-specific enzyme using these concen¬

trations of L-lactate was equal to or greater than any noted using the

12 -

D-form of the isomer. None of the D-specific enzymes showed activity

with these concentrations or higher concentrations of Is-lactate.

The reduction of pyruvate was assayed by measuring the oxidation

of NADH at 3^0 m^. The reaction mixture contained 100 mM potassium

phosphate buffer, pH 7•0; 3*0 mM sodium pyruvate; 0.15 mM NADH; and an

amount of enzyme which gave a linear change in optical density between

0.010 and 0.090 per minute for at least 75 seconds. Coenzyme was added

to initiate the reaction and blanks were run using only enzyme and

coenzyme and only enzyme and substrate.

Protein Determination:

For all enzyme assays, protein was determined by the method of

Lowry et al. (l95l) using the Cary 14 spectrophotometer.

Enzyme Units:

One unit of enzyme activity is defined as the reduction of one jjmole

of NAD or the oxidation of one ^moie of NADH by one gram of protein in

one minute under the conditions of the assay employed.

RESULTS

With one exception, all of the invertebrates studied, both in this

survey and elsewhere (Long and Kaplan, 1968; Hammen, 1969; Massaro, 1970),

which possess lactic dehydrogenase in detectable levels, possess an enzyme

which is specific for only one of the isomers of lactic acid. Hamrnen

(1969) reported that in the echinoderm Arbacia lixula lactic dehydrogenase

capable of oxidizing both isomers of lactic acid at the same rate was

detected. This does not correspond with any of the results obtained in

other echinoderms although this particular species was not used in the

present survey. As mentioned previously, Lactobacillus plantarum has

two NAD-linked lactic dehydrogenases, each specific for one of the iso¬

mers of lactic acid; these enzymes, however, have very different cata-

lytical properties (Dennis and Kaplan, i960). The fact that with this

one exception none of the invertebrates studied possess a lactic dehy¬

drogenase which can catalyze the oxidation of both isomers of lactic

acid, in conjunction with the demonstration by Massaro (1970) of the

D-specific enzyme from Limulus polyphemus which is apparently physically

similar to the L-enzyme from Homarus americanus and to vertebrate lactic

dehydrogenases, suggests that a mutation of one gene locus may be respon¬

sible for the differentiation of the L- and D-specific lactic dehydrogenases

(Long and Kaplan, 1968).

If indeed the differences in the D- and L-specific lactic dehydro¬

genases lie in the mutation of a single gene locus, the results of this

survey and the others included seem to indicate that it did not occur

- 14 -

randomly among various invertebrate species. It is apparent that the

distribution of lactic dehydrogenases falls along previously determined

taxonomic divisions. Without more detailed studies of these enzymes,

however, their distribution with regard to morphological evolution can

be treated only very superficially.

D-specific lactic dehydrogenases were found in annelids, acantho-

cephala, molluscs, and arthropods. All of the platyhelminths and echino-

derms tested had enzymes specific for L(+) lactic acid. Among the anne¬

lids, the oligochaetes and the hirudinea are accepted as closely related;

the oligochaetes are thought to be ancestral to the hirundinea (Dales,

1963)* The organisms tested in both of these groups have L-specific

lactic dehydrogenases, while among the more distantly related pclychaetes,

those which utilize lactic acid have D-specific enzymes. Before the

discovery of WAD-linked D-specific enzymes among the invertebrates,

Kaplan and Ciotti (1961) compared the lactic dehydrogenases from five

annelids, including the polychaete, Nereis vlrens, two oligochaetes and

two leeches, with regard to the activities with three coenzyme analogues.

The results indicated that the enzymes from the two earthworms were

closely related as were those from the two leeches, while the lactic

dehydrogenase from Nereis virens was significantly different from both

the leech lactic dehydrogenases and the earthworm lactic dehydrogenases.

These results agree with those obtained on the stereospecificity of the

enzymes from members of different annelid classes. In the echiurids,

which are thought to be closely related to the annelids, Urechis caupo

possesses an L-specific lactic dehydrogenase.

- 15 -

The distribution of B-lactic dehydrogenases among the molluscs is

apparently widespread. Of the molluscs which have "been studied, which

include members of four of the five major classes, those which can oxi¬

dize lactic acid possess a dehydrogenase specific for the D-isomer of

lactic acid. Many molluscs, hcwever, possess octopine dehydrogenase

rather than lactic dehydrogenase (Regnouf and van Thoai, 1970). Among

the arthropods, on the other hand, the lactic dehydrogenases occur in

two groups which are in line with the main divisions of this phylum.

The mandibulates, which include the insects and the crustaceans, have

enzymes specific for L-lactic acid. The chelicerates, including the

horseshoe crab Limulus polyphemus and the arachnids possess D-specific

enzymes. In studies utilizing coenzyme analogues, Kaplan et al. (i960)

demonstrated that the mandibulate lactic dehydrogenases were all closely

related; these enzymes were more active with the acetylpyridine analogue

of NAD than with NAD itself. Moreover, they exhibited activity with the

thionicotinamide analogue of NAD. The chelicerate dehydrogenases, how¬

ever, were more active with NAD and were unable to utilize the thionico¬

tinamide analogue. Kaloustian and Kaplan (1969a) found that dialysis

of the L-lactic dehydrogenase from the lobster Homarus americanus led

to a decrease in the activity of the enzyme when NAD is used but not

when the acetylpyridine analog is used. The authors suggest that this

is due to the removal of NADH on dialysis. The differences in the ac¬

tivities of the two groups of enzymes in members of the arthropods is

in agreement with the main divisions of the phylum and with the distri¬

bution of D- and L-specific lactic dehydrogenases.

Table I

Lactic Dehydrogenases in Invertebrate Tissues

nmoles coenzyme utilized/g protein/min D(-)lactate! L(+)lactate Pyruvate

Phylum Coelenterata

Class Hydrazoa Hydra8,

+2

Phylum Platyhelminthes

Class Turbellaria Bipalium kewense

(whole organism) —3 113 552

Class Cestoda Hymenolepis diminuta

(whole organism) 434 1662

Class Trematoda Fasciola hepatica

(whole organism) 22 301

Phylum Nemertea

Class Anopla Cerebratulus

(whole organism) trace

Phylum Acanthocephala

Order Archiacanthocephala Moniliformis dubius 325 — 3677

Phylum Annelida

Class Polychaeta Arenicola marina

(whole organism) (body wall)

13

- 17 -

umoles coenzyme utilized/g protein/min D(-)lactate!.(+) lactate Pyruvate

Chaetopterus variopedatus (whole organism, posterior) 39 — 371

Cirratulus grandis (whole organism) (hody wall) I

I I

I l

I

I 1

1 t

1 1

LO

H

00

Nereis limbata (whole organism)

Nereis virensa ii

+

Class Oligochaeta Enchytraeus

(whole organism) 151 1058 Lumbricus terrestris

(body wall) Lumbricusa

44l 2756 +

Class Hirudinea Haemopsis grandis8. +

Phylum Sipunculida

Dendrostomum pyroides (intestine) (retractor muscle)

3 51

Phylum Echiurida

Order Echiurinea Urechis caupo

(body wall) 12 81

Phylum Brachiopoda

Class Articulata Laqueus californicus

(whole organism)

Class Inarticulata Glottidia pyrimidata^1 +

Phylum Mollusca

Class Amphineura Chaetopleura apiculata

~(whole organism) 23 102

- 18 -

jjmoles coenzyine utilized/g proteln/mln D(-)lactate £(+)lactate Pyruvate

Middendorffia capreariW3

Mopalia muscosa (digestive gland)

+

7 53

Class Gastropoda Acmaea unicolor13

Busycon (kidney)

+

trace 3 Helix aspersa

(hepatopancreas) 26 279 Otala lactea

(hepat opancreas) to 376 Poliniees

(hepatopancreas 151 822 Poliniees herosa

Strophocheilus ohlongatus (hepatopancreas)

X*

13 — 73

Class Pelecypoda Anadara ovalis

(mantlej 26 Aquipecten irradians

(mantle) 3 34 . Ensis directus

(mantle) 6 • 70 Mercenaria mercenaria

(mantle) 2 53 Mercenaria mercenaria0 + Mytilus edulis

(mantle) „ 31 Mytilus edulis^3

Kangia cuneata (mantle)

+

6 46 Spisula solidissima

Xmantle) 3 58

Class Cephalopoda Eledone cirrosa0 +

Phylum Arthropoda Suhphylum Chelicerata

Class Meristomata Limulus polyphemus '(muscle) 355 — 2838

- 19 -

jjmoles coenzyme utilized/g protein/min

D(- ") lactate li(+)lactate Pyruvate

Limulus polyphemusa

Limulus polyphemusc

Class Arachnida

Phlocus phalangioide sa

Centuroides sculpturatusa

Dugesiella hentzia

Geolycosaa

Subphylum Mandibulata

Class Crustacea

Carcinus maenas

("muscle)

Calllnectes sapidus

(muscle)

Cancer Irroratus

(muscle)

Homarus americanusa

Liblnia emarginata

(muscle

Oniscusa

Penaes seriferusa

Class Clrripeda

Chthamalus depressusa

Class Insecta

Melanopus bruneria

Porthetrla dispar**

Class Chilipoda Scolopendra polymorphaa

Class Diplopoda

Spirobolusa

Phylum Echinodermata

Class Asteroidea

Astropecten braziliensis

(digestive tract) Patiria mlniata

(digestive tract)

+

+

+ +

+ +

394 1773

176 570

469 +

2157

4oo +

1333

+

+

4*

4*

4-

+

91 509

trace 18

- 20 -

uTnoles coenzyme utilized/g protein/mln

D(-)lactateL(+)lactate Pyruvate

Class Holothuroidea Cucumaria rubra

~ (digestive tract)

Class Echinoidea

Lytechinus pictus

(digestive tract) —

(gonads) —

.Arbacia punctulata

(digestive tract) —

Arbacia lixula"*3 +

Eat Kidney 286 577

-*■ Substrate utilized in the assay. 2 + indicates that activity vTas reported by another investigator. 3 indicates that no activity was detected. a from Long and Kaplan (1968). h from Hammen (1969)• c from Massaro (1970)*

DISCUSSION

In order to evaluate the role which D-specific lactic dehydrogenases

play in the carbohydrate metabolism of invertebrates, a brief review of

terminal glycolysis in members of the phyla studied in this survey is

included which incorporates the results presented above.

Phylum Platyhe.bminthes

Most of the data available on the terminal glycolytic processes of

the flatworms is limited to the parasitic cestodes and trematodes. Very

little is known of the end products of carbohydrate catabolism in the

turbellarians (head, 1968). No research on the glycolytic enzymes of

turbellarians has been done, although Bryant and Janssens (1969) found

that succinic acid accumulated in the terrestrial planarian Geoplana

caerulea when a variety of radioactive compounds, including glucose, were

administered. In homogenates of Polycelis nigra, radioactivity from la¬

beled glucose was incorporated primarily into succinic acid and alanine

(Bryant and Smith, 1963)- In the present survey an L(+)-specific lactic

dehydrogenase was found in the planarian, Bipalium kewense.

Among the cestodes which have been studied a variety of glycolytic

end products are produced. In the larval scolices of the costode

Echinococcus granulosus there are differences in the products produced

aerobically and anaerobically, although under both conditions lactic

acid accounts for at least 50$ of the carbohydrate utilized. Aerobically

pyruvic, acetic and succinic acids as well as small amounts of ethanol

22 -

are produced. Anaerobically, no pyruvate is formed, but succinic acid

(which is produced only in small quantities aerobically) accounts for

3O/0 of the end products, (Agosin, 1957)* Glycolytic enzymes (Agosin and

Aravena, 1959) as well as several tricarboxylic acid cycle enzymes

(Agosin and Repetto, 1963) have been found in extracts of E. granulosus

scolices. To account for the production of succinic acid Agosin and

Repetto (1965)^ on the basis of NaH^'COg incorporation studies, conclude

that CO2 fixation into pyruvic acid occurs via malic enzyme, followed by

a partial reversal of the tricarboxylic acid cycle. In adult and larval

Taenia taenlaeformis a similar situation exists with regard to succinic

and pyruvic acid production (von Brand and Bowman, 1961). In studies

utilizing glucose and glycerol as substrates, von Brand et al. (1968)

found that the amount of succinic acid produced depended on the amount

of substrate consumed as' well as on the amount of oxygen available, while

lactic acid synthesis was independent of these factors.

The cestode Hymenolepis diminuta, an intestinal parasite, is capable

of utilizing oxygen when it is present (Bueding, 19^9; Read, 1956), al¬

though its environment is relatively anaerobic. Lactic acid has been

reported as an end product in this helminth in amounts varying from l4$>

to 98$ (Laurie, 1957; Read, 1956; Fairbain et al., 1961). The results

of the present survey indicate that the H. diminuta lactic dehydrogenase

is specific for the L(+) isomer of lactic acid. In studies on irradiated

strains of H. diminuta, Fairbain et al. (196.1) reported that succinic acid

is the primary end product of carbohydrate fermentation. From studies

utilizing radioactive glucose and radioactive COg, Scheibel and Saz (1966)

postulate that H. diminuta catabolizes carbohydrate by the glycolytic

- 23 -

pathway to pyruvic acid, which is followed by CO2 fixation and reduction

to succinic acid. They further indicated that the production of succinic

acid decreases in the presence of oxygen while lactic acid accumulation

is not affected.

As in the cestodes, trematodes produce a wide variety of metabolic

end products. In the blood fluke Schistosoma mansoni the major product

of carbohydrate catabolism is lactic acid, which accounts for over 80$

of the metabolized glucose (Bueding, 1950)* Neither glucose metabolism

nor lactic acid production is affected by cyanine dyes or the presence

or absence of oxygen indicating that the metabolism of Schistosoma is

primarily anaerobic. Read (1968) has pointed out that the schistosomes

have evolved from intestinal parasites, and this may account for the

occurrence of this type of metabolism. In electrophoretic studies on

dehydrogenases from S. mansoni, Conde-del Pino _et al. (1966) found only

a single band of lactic dehydrogenase. Bueding and Saz (1968) have

assayed pyruvate kinase and PEP carboxykinase in three parasitic hel¬

minths including S. mansoni, Hymenolepis diminuta, and the nematode

Ascaris lumbricoides. These authors propose that glycolysis leading

to the production of either succinic acid or lactic acid is controlled

by the competition between pyruvate kinase and PEP carboxykinase.

In the liver fluke, Fasciola hepatica, glucose metabolism is

anaerobic (Bueding, 19^9)• In this organism lactic acid accounts for

only 4-9$ of the glucose utilized under anaerobic conditions (Mansour,

1959)* Propionic acid is the major end product, with acetic acid also

produced. Prichard and Schofield (1968a) demonstrated the presence of

all of the glycolytic enzymes except pyruvic kinase; lactic dehydrogenase

- 24 -

levels were low in comparison to those from rat liver. Subsequent cletec

tion of an active PEP carboxykinase (Prichard and Schofield, 1968b), an

active malic dehydrogenase favoring the reduction of oxaloacetate (QAA),

and low levels of aconitase and isocitric dehydrogenase (Prichard and

Schofield, 1968c) has led these authors to propose that carbohydrate is

degraded to PEP via the Embden-Meyerhof-Parnas pathway. PEP is then car

boxylated to OAA by PEP carboxykinase and OAA in turn is converted to

succinic acid and fatty acid end products. The small amounts of lactic

acid produced arise from the decarboxylation of malic acid to pyruvic

acid which is then reduced by lactic dehydrogenase. This dehydrogenase

is specific for L(+) lactic acid.

Phylum Acanthocephala

Information on the carbohydrate metabolism of acanthocephalans is

limited; most research has been done on the intestinal parasites Monili¬

formis dubius and Macrocanthorhyneus hirudinaceus. Similar to other

parasitic helminths adapted to relatively ’anaerobic environments, these

organisms appear to have modified pathways of glucose catabolism.

Laurie (1959) demonstrated that under aerobic conditions 30°[o of

the acids excreted by Moniliformis dubius consisted of equal amounts

of acetic and formic acids, while lactic acid made up only 3$ of the

total acids. In more recent studies, succinic acid has been indicated

as an end product of carbohydrate metabolism (Graff, 1965; Bryant and

Nicolas, 1965)^ while ethanol has also been reported (Crompton and

Ward, 1967b). Eead (1961) reported the occurrence of lactic dehydro¬

genase in M. dubius which in this survey was found to be specific for

the D(-) isomer of lactic acid. On the basis of isotope incorporation

- 25 -

studies, Bryant and Nicolas (1965, 1966) have concluded that M. duhius

possesses a modified pathway of glucose catabolism leading to the forma¬

tion of pyruvic acid by the glycolytic pathway and followed by the car-

boxy lation of pyruvic acid to malic acid which in turn gives rise to

fumaric and succinic acids as well as OAA and aspartate.

Lactic acid is also an excretory product of other acanthocephalans

including Neoechinorhyncus emydis and N. pseudemydis (Dunagan, 1963)

and Polymorphus minutus (Crompton and Ward, 1967a) which secretes equal

amounts of succinic and lactic acid on incubation with glucose. Electro¬

phoretic studies on the lactic dehydrogenase from Macrocanthorhyncus

hirudinaceus (Dunagan and de Luque, 1966) indicated the presence of only

a single band of lactic dehydrogenase which the authors described as a

species variant of LDH^ of the mouse. DL-lactate was used in this study

so it is impossible to determine if this acanthocephalan, like M. dubius,

possesses a D-specific lactic dehydrogenase.

Phylum Annelida

The annelids, with the exception of the hirudinea, are primarily

free-living burrowing or tube-dwelling organisms. Of the two major

classes, the oligochaetes are primarily terrestrial and freshwater

species while the polychaetes are primarily marine. Although evidence

on the nature of carbohydrate metabolism of the annelids may, at best,

be called piecemeal, it appears that biochemical differences do exist

between these two groups.

In no polychaete species studied thus far has any evidence for

the presence of the enzymes of the glycolytic or tricarboxylic acid

pathways been presented. However, Clark (196^) reports that Nephtys

- 26 -

hombergi, an intertidal burrowing species subject to long periods of

low oxygen tension, accumulates lactic acid when subjected to low oxygen

tension. In a study on the anaerobic metabolism of Arenicola marina and

Owenia fusiformis, both intertidal tube dwellers, Dales (1958) found that

both worms utilized oxygen under aerobic conditions but they are capable

of withstanding lengthy periods without oxygen. Arenicola utilized more

glycogen, which is stored primarily in the body wall, during anaerobic

periods than aerobically while fasting. There is no indication that this

worm incurs an oxygen debt. Further, neither lactate nor pyruvate accumu¬

late in the body wall during aerobiosis or anaerobiosis suggesting that

glycogen is broken down to other end products. In support of this, nei¬

ther pyruvate nor lactate utilization was detected in body wall extracts

in this survey. In Owenia fusiformis which can survive up to 21 days

without oxygen, there is no decrease during anaerobiosis in the glycogen

content of the coelomic cells where carbohydrate is stored. Owenia

becomes completely quiescent when under anaerobic conditions while

Arenicola calms but still exhibits activity. Dales suggests that

survival of anaerobic periods may be due to an ability to reduce the

metabolic rate.

In Nereis virens, an intertidal polychaete, Long and Kaplan (1968)

have demonstrated the presence of a lactic dehydrogenase specific for

the D(-) isomer of lactic acid. In this survey the presence of a similar

enzyme was found in Chaetopterus variopedatus, a sublittoral tube dweller.

However, in three other polychaetes, including Arenicola marina, Nereis

limbata, and Cirratulus grandis, no lactic acid utilization could be

demonstrated.

- 27 -

In experiments on the metaholism of the oligochaete Lumbricus

terrestris Davis and Slater (3.928) demonstrated the accumulation of

lactic acid during anaerobic periods. Dastoli (196^) has presented

evidence for the occurrence of all the enzymes in the glycolytic path¬

way, including lactic dehydrogenase, in extracts from the body wall of

Lumbricus. Long and Kaplan (1968) demonstrated that the lactic dehy¬

drogenase from this organism is specific for the L isomer of lactic

and the results of the present survey confirm this. In another oligo¬

chaete, Enchytraeus, the lactic dehydrogenase also utilizes L(+) lactic

acid. O'Brien (1957); in studies on regeneration in the earthworm

Eisenia foetida found that lactic acid and pyruvic acid were normally

present in the tissues in a ratio of 6:1. However, if the worms were

placed under anaerobic conditions this ratio increased to approximately

63:1 leading the author 'to conclude that the anaerobic metabolism of

this earthworm is essentially similar to that found in vertebrates.

Coles (1967) has presented evidence for a modified pathway of carbo¬

hydrate catabolism in the oligochaete Alma emini, which inhabits swamps

in which the mud is anaerobic and which has a specially modified respira¬

tory organ. Although no determinations were made of metabolic end pro¬

ducts formed under aerobic conditions, under anaerobic conditions the

three major end products were acetic, propionic and a five-carbon fatty

acid, which together accounted for almost 100p of the glycogen utilized.

Tests for lactic acid accumulation during anaerobiosis were negative.

In Haemopsis grandis, a member of the Hirudinea, Long and Kaplan (1968)

found L-specific lactic dehydrogenase. As mentioned previously, the

distribution of D- and L-specific lactic dehydrogenases falls along

the established lines of taxonomic divisions among the annelids.

- 28 -

Phylum Mollusca

Evidence for the functioning of the glycolytic pathway in molluscan

tissues seems conclusive. Beames (1963) demonstrated the presence of

all the glycolytic enzymes from glucose to lactic acid (with the excep¬

tion of fructose diphosphatase which was not assayed) in the aquatic

snail Physa halei. He found that the lactic dehydrogenase activity in

this snail is low in comparison to that from vertebrate tissues. Bennett

and Nakada (1968) carried out a similar study on the bivalves kytilus

californianus and Haloitus refuscens and presented data supporting the

occurrence of the glycolytic pathway in these organisms; these authors

obtained low levels of lactic dehydrogenase as well. Bryant et al.

(1964), in studying four land snails, found that radioactivity from C-^

labeled glucose was incorporated into hexose phosphates, lactic acid,

and alanine.

While it appears that the glycolytic enzymes are found in molluscs,

these animals apparently possess a modified pathway with regard to lactic

acid production. Humphrey (19^4) noted that lactic acid and pyruvate

acid production could not account for the anaerobic utilization of glu¬

cose in oyster muscle. In studies on the metabolism of freshwater

snails, von Brand et al. (1950) found that all of the species examined

produced some lactic acid under anaerobiosis. Lactic acid in some cases

accounted for only 1$ of the total carbohydrate consumed, while in none

of the organisms did it account for more than 50$. Lactic acid was a

major end product anaerobically only in two species of Lymnaeidae,

neither of which was able to survive long periods of anaerobiosis. In

addition these authors found that the species least resistant to anaero¬

biosis accumulated the greatest amounts of lactic acid in their tissues,

- 29 -

while in the species able to withstand long periods without oxygen no

lactic acid accumulated but was excreted. In a study on the dehydro¬

genases from the digestive glands of several snails Coles (1969) found

that different species possess different numbers of lactic dehydrogenase

isozymes. Lactic dehydrogenase occurred as a single band in Biomphalaria

sudanica and B. naustis and as three bands in Pila ovata. No bands were

found in Lymnaea natalensis although von Brand et al. (1950) found lactic

acid to be a major end product in this species. Goldberg and Cather

(1963) described multiple bands of lactic dehydrogenase in the snail,

Argobuccinum oregonense. Long and Kaplan (1968) demonstrated that the

lactic dehydrogenase from the snail Pollnices heros was specific for the

D(-) isomer of lactic acid. In this survey the lactic dehydrogenase from

several gastropods, several pelecypods, and two amphineurans were found

to be D(-) specific. In'addition, lactic dehydrogenase from the cepha-

lopod Eledone cirrosa is also D-specific (Hammen, 1969)*

In addition to lactic acid, two other compounds have been demon¬

strated as end products of carbohydrate catabolism in molluscs: octopine

and succinic acid. Octopine is formed by the condensation of arginine

and pyruvic acid followed by a reduction in the presence of octopine

dehydrogenase, an NAD-linked enzyme (van Thoai and Robin, 1961). This

compound has been found primarily in the striated muscle of several

molluscan species including members of the Cephalopoda and Pelecypoda

(Roche et al-; 1952) and the Scaphopoda and Gastropoda (Regnouf and van

Thoai, 1970). Hie formation of octopine prevents the reduction of pyruvic

acid to lactic acid (Robin and van Thoai, 1961) and may account for the

low levels of lactic acid which occur in molluscan tissues. In a survey

- 30 -

of k-2 molluscs, Regnouf and van Thoai (1970) found only 13 species which

contained both octopine dehydrogenase and lactic dehydrogenase, while

four contained neither. In all other species the occurrence of the two

enzymes was mutually exclusive, leading the authors to conclude that the

two enzymes may be closely related. In the present survey all but two

of the molluscs studied utilized D-lactic acid. Anadara oyalis and

kfybilus edulis did not utilize either D- or L~lactic acid. These two,

as well as the other molluscs tested, utilized pyruvate in the absence

of arginine, the criterion used by Regnouf and van Thoai to indicate

the presence of lactic dehydrogenase. The low levels of lactic dehy¬

drogenase obtained in many of the molluscs tested in the present survey

may indicate the occurrence of octopine dehydrogenase in these species.

In the survey of Regnouf and van Thoai, Lfytilus edulis did not possess

lactic dehydrogenase but' had high levels of octopine dehydrogenase.

Hammen (1969), on the other hand, reports the presence of a D-specific

lactic dehydrogenase in this organism. Since octopine dehydrogenase

appears to occur primarily in striated muscle in molluscs the possibility

exists that there are differences in the tissue distribution of the two

dehydrogenases.

Succinic acid has been demonstrated as an end product of anaerobic

glycolysis in several species of molluscs (Simpson, 1965; Simpson and

Awapara, 1966). In the brackish water bivalve, Rangia cuneata, which

has very low levels of lactic dehydrogenase, 40$ of the glucose utilized

anaerobically is accounted for by the production of succinic acid.

Simpson and Awapara (1964, 1966) have demonstrated that succinic acid

is produced by the carboxylation of PEP by an active PEP carboxykinase

- 31 -

to yield OAA which then undergoes two reductions via a partial reversal

of the tricarboxylic acid cycle. The two reductive steps regenerate the

NAD produced during glycolysis. In Rangia, succinic acid and alanine

are formed in equimolar amounts anaerobically, alanine being formed by

the transamination of pyruvate (Stokes and Awapara, 1968). Aerobically

glucose is converted primarily to alanine, while very little succinic

acid is formed (Chen and Awapara, 1969b).

Phylum Slpunculida and Phylum Nemertea

Van Thoai and Robin (3.959) have discovered an active octopine dehy¬

drogenase in the sipunculid, Sipunculus nudus. In the present study,

no lactic acid utilization was demonstrated either in the intestine or

the retractor muscles of Dendrostomum pyroldes. In light of the fact

that octopine and lactic dehydrogenases are mutually exclusive in many

mol3-uscs (Regnouf and van Thoai, 1970), there is a possibility that octo¬

pine dehydrogenase occurs in D. pyroides. However, Robin (1964) did not

find octopine in the closely related Dendiostomum dyscrltum.

Octopine has also been found in two species of Nemertea, Cere-

bratulus occidentalis and Lineus pictifrons (Robin, 1964). No lactic

acid utilization could be demonstrated in Cerebratulus in the present

survey.

Phylum Arthropoda

Arthropoda, especially the insects and the crustaceans, have been

the subject of much biochemical investigation, and extensive reviews

which include carbohydrate metabolism have been prepared (Scheer and

Meenakshi, I96I; Chefurka, 1965a,b; Gilmour, 1965; Huggins and Munday,

- 32 -

1968). Among the insects, the enzymes of the classical Embden-Meyerhof-

Parnas pathway from glucose to lactic acid have been demonstrated (Chefurka,

1954; McGinnis et al., 1956; Ito and Horie, 1959)- Insect flight muscles,

which are usually well-tracheated and which have abundant mitochondria,

possess a modification of the classical pathway. In these tissues lactic

dehydrogenase activity is extremely low while the activity of crglycero-

phosphate dehydrogenase is very high (Zebe and McShan, 1957)* The crgly¬

cerophosphate dehydrogenase functions extramitochondrially to oxidize the

NADH produced glycolytically and thus can replace the function of lactic

dehydrogenase. Aerobically the extramitochondrial crglycerophosphate

dehydrogenase can function in conjunction with a flavoprotein-linked

intramitochondrial crglycerophosphate dehydrogenase to generate ATP via

the cytochrome sequence (Chefurka, 1965a).

Long and Kaplan (1968) demonstrated that the lactic dehydrogenases

from Melanopus bruneri and Porthetria dispar were specific for the L-

isomer of lactic acid. In addition, the enzymes from the chilopod

Scolopendra polymorpha and from the diplopod Spirobolus were also found

to be L-specific. Multiple bands of lactic dehydrogenase have been

found in the silk moths Sarnia cynthia and Hyalophora cecropia (Laufer,

1961) and in tissues of the grasshopper Melanoplus differentialis

(Aronsen et al», 1968).

Von Brand (1946) pointed out that many insects are able to survive

long periods of anaerobiosis even though their habitat may be an aerobic

one. Augenfeld (1966) in a study on eight genera of insects, found that

insects which obtain their oxygen by means of trachea had markedly lower

levels of lactic dehydrogenase than the insects which utilize gills or

- 33 -

body surface to obtain oxygen. He also found that there was no correla¬

tion between the length of time which an insect could survive in anaerobic

conditions and the lactic dehydrogenase activity in the tissues of that

insect. Chefurka (1958) noted that in the leg muscle of the cockroach

Periplaneta and pyruvic acid, lactic acid., and crglycerophosphate pro¬

duced did not account for the amount of fructose diphosphate utilized.

Price (1961) in studies on the housefly Musca domestica found that in

the early period of anaerobiosis pyruvate accumulates. Later the concen¬

tration of pyruvate decreased with a concurrent increase in alanine con¬

centration. He suggested that alanine is produced by the activity of

glutamic dehydrogenase and glutamate-alanine transaminase; coupling these

two enzymes would serve to reoxidize NADH.

In studies on crustacean muscle, Meyerhof and Lohmann (1928a,b)

found that carbohydrate was converted to lactic acid. Experiments

based on radioisotope incorporation indicate that glucose is oxidized

via the glycolytic pathway in the crabs Hemigraspus nudas, Cancer

magister and Carcinus maenas (Hu, 1958; Meenakshi and Scheer, I96I;

Huggins, 1966). As described previously, the lobster Homarus americanus

possesses an L-specific lactic dehydrogenase which is apparently physi¬

cally similar to vertebrate lactic dehydrogenases. In addition, lactic

dehydrogenases from several other crustaceans are L~specific as shown

in this survey.

Phylum Echinodermata

Very little data is available on the carbohydrate metabolism of

echinoderms although some studies have been carried out on sea urchin

eggs. Cleland and Rothchild (1952) in a study on the anaerobic

- 34 -

"breakdown of carbohydrate in unfertilized Echinus eggs found that whole

eggs produce lactic acid under anaerobic conditions. Yeas (1954) pre¬

sented further evidence supporting the occurrence of the glycolytic

pathway in the eggs of Lytechinus pictus and Strongylocentrotus purpura-

tus. This author detected lactic acid in unfertilized eggs as well as

lew levels of lactic dehydrogenase activity. In the present survey

lactic dehydrogenases from adults of three classes of echinoderms, the

Asteroidea, and Holothuroidea, and the Echinoidea, were all found to be

specific for the L(+) isomer of lactic acid. This is in contrast to

work done by Hammen (1969) in which the lactic dehydrogenase from Arbacia

llxula utilized both L- and D-lactic acid at the same rate.

Boyland (1928) found that the lactic acid content of fatigued muscles

of Holothuria nigra is greater than that of resting muscles. In a study

on the longitudinal muscles from the holothuroidean Stlchopus mollis,

Gay and Simon (1964) presented evidence that resting muscle incubated

in sea water had a very low oxygen uptake in comparison to resting ver¬

tebrate muscle and that lactic acid production was also very low. How¬

ever, under conditions of ionic stress such as increased potassium ion

concentration prolonged contraction occurs and oxygen uptake and lactic

acid production is stimulated. The authors conclude that the metabolism

in resting Stichopus muscle is not based on carbohydrate oxidation but

that the change in the ionic medium causes a switch to a carbohydrate

oriented metabolism.

- 35 -

CONCLUSIONS

From this investigation it is apparent that modifications of the

terminal portions of the Emhden-Meyerhof-Parnas pathway are widespread

among the invertebrates. The endproducts of glycolysis appear to he

characteristic for the individual organisms, although most which have

been studied seem to utilize carbohydrates via the glycolytic pathway

to the level of PEP or pyruvate. That the modifications of terminal

glycolysis are related to adaptations to a variety of anaerobic or

microaerophilic habitats is supported by the large number of cases

in which there is a shift in the end products produced aerobically

and anaerobically. No readily apparent correlation exists between

the occurrence of D(-) lactic dehydrogenases and the production of

succinic acid or the ability to withstand long periods of anoxia.

Members of the platyhelminths, for example, produce succinic acid during

anaerobic glycolysis and produce L-specific enzymes. Many organisms

which produce primarily lactic acid during anaerobiosis are also capable

of producing succinic acid by reversal of the tricarboxylic acid cycle.

Saz and Bueding (1966) have pointed out that in animals which live

in anaerobic environments succinic acid production may be more profitable

than lactic acid production. Both succinic acid production via a partial

reversal of the tricarboxylic acid cycle and lactic acid production ac¬

cording to the Embden-Meyerhof-Parnas pathway serve to reoxidize glyco-

lytically produced NADH. However, if the reduction of fumaric acid to

succinic acid is coupled with a cytochrome sequence energy may be produced.

- 36 -

This type of energy production has been indicated in the flatworm

Hymenolepis diminuta (Scheibel et al., 1966) and in the nematode Ascaris

lumbricoides (Seidman and Entner, 1961) which is also a succinate pro¬

ducer. The occurrence of such a system is suggested in the mollusc,

Rangia cuneata (Chen and Awapara, 1969) in which ilADH is thought to be

able to penetrate into the mitochondria.

According to the theory of Oparin (1962) the earth was originally

anaerobic and all life forms relied on anaerobic glycolysis as the main

source of energy. Only after photosynthetic forms evolved did mechanisms

of aerobic energy production develop. It seems probable, however, that

invertebrates now living in anaerobic environments developed from ances¬

tors which possessed primarily aerobic rather than anaerobic metabolisms

(von Brand, i960). This is evidenced by the fact that most organisms

which have a primarily anaerobic metabolism do utilize oxygen when it is

present, and further, very few can survive anaerobiosis indefinitely.

Because such mechanisms have arisen secondarily it is not unusual that

a variety of pathways of anaerobic glycolysis may have been developed

and that a variety of end products are produced.

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Documents

Stereospecificity of some invertebrate lactic dehydrogenases