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d -Lactate oxidation in a marine gastropod and a sea urchin
Comp. Biochem. Physiol., 1972, Vol. 42B,pp. 123 to 129. Pergamon Press. Printed in Great Britain
D-LACTATE OXIDATION IN A MARINE GASTROPOD AND A SEA URCHIN* C. S. H A M M E N and SUSAN C. L U M Department of Zoology, University of Rhode Island, Kingston, Rhode Island (Received 26 October 1971)
Abstract--1. Lactate dehydrogenase activities in extracts of a gastropod, Monodonta sagittifera, and a sea urchin, Arbacia lixula, were determined by change in light absorbance of a dye mixture on addition of salts of lactic acid in each isomeric form. 2. The Monodonta extract catalyzed the oxidation of D-lactate only, with an apparent Michaelis constant of 180 raM. 3. Weight-specific enzyme activity of Monodonta was proportional to the - 0"18 power of total body weight. 4. The sea urchin appeared to contain two enzymes of equal activity, one specific for L-lactate and one specific for D-lactate. INTRODUCTION AS COMPARATIVEbiochemistry has become more comparative, some interesting exceptions to the principle of biochemical unity have come to light. One generalization, that lactic acid is the exclusive end-product of anaerobic glycolysis in animal tissues, has been abandoned as various species of invertebrates have been shown to form other products. Numerous parasitic flatworms and nematodes, and at least a few species of mollusks, produce succinic acid rather than lactic acid. Succinate formation occurs by means of carbon dioxide fixation with pyruvate or phosphoenolpyruvate, followed by reversal of several steps of the citric acid cycle (Bueding, 1962; Hammen, 1966; Awapara & Simpson, 1967; Gilles, 1970; Saz, 1971). The relative reversability of lactate and succinate oxidoreductases (Hammen, 1969) suggests that representatives of some other invertebrate groups may also produce succinate in preference to lactate during anaerobiosis, especially certain sponges, brachiopods and barnacles. Another recently discarded generalization is that only one stereoisomer of lactic acid, L-lactic, is involved in animal metabolism. Long & Kaplan (1968) discovered that the horseshoe crab Limulus, five species of arachnids, a marine gastropod and a polychaete have lactate dehydrogenases specific for D-lactate. Soon after this discovery, the oyster Crassostrea virginica was also found to oxidize only D-lactate (Hammen, 1969). Such specificity has since been found in another pelecypod, another gastropod, an amphineuran and a cephalopod (Hammen, 1971). In their survey of eighteen species of invertebrates, Long & Kaplan (1968) * Work carried out at Laboratorio Oceanografico de Canarias, Santa Cruz de Tenerife, Islas Canarias. 123
124
C. S. HAMMENAND SUSAN C. LUM
f o u n d that "in each animal, the lactate dehydrogenases are specific for only one isomer of lactate". Apparently even this modest generalization has exceptions. I n this report are presented results of experiments showing the presence of a lactate dehydrogenase specific for D-lactate in another marine gastropod, ~Vlonodonta sagittifera (Lamarck), and the variation of its activity with substrate concentration. I n addition, a sea urchin, Arbacia lixula (Busch) is shown to have enzymes for the oxidation of both o-lactate and L-lactate, the first instance of double specificity known in animals. MATERIALS AND M E T H O D S The animals used in this study were collected from 2 January to 9 February 1970 at Las Caletillas, Tenerife, Islas Canarias. Monodonta sagittifera was very abundant, and its size and position on the rocky shore were reminiscent of the familiar Littorlna littorea of North Atlantic coasts. At Lanzarote, some 270 km to the east, both Littorina striata and Monodonta turbinata are found (Duffus & Duffus, 1968). Arbacia lixula specimens occurred in the shallow subtidal zone, and weighed 33'1-50"0 g. A single experiment was done with a specimen of Diadema antillarum (Philippi) which was captured at a depth of 5 m, and weighed 127"9 g. The experiments were done the same day in the biochemistry laboratory of the Universidad de La Laguna. Lactate dehydrogenase activity was measured by decolorization of dichlorophenolindophenol (DCIP) with phenazine methosulfate (PMS) as intermediate electron carrier, the method of Arrigoni & Singer (1962). The enzyme source was the supernatant of a tissue homogenate, prepared by grinding about 1 g tissue with nine times its weight of Tris buffer, 0"25 M, pH 7"38, and centrifuging for 10 rain at threequarters maximum speed in a Viher clinical centrifuge. The absorbance at 600 nm was measured with a Beckman D U spectrophotometer with the assay mixture at room temperature, 21°C. Sodium lactate was from E. Merck, Darmstadt; the lithium salts of D-lactate and L-lactate, DCIP and PMS were from Calbiochem, Los Angeles; and Tris from Sigma, St. Louis. Activities were calculated from changes in absorbance in the first minute of the reaction. RESULTS Rates of the lactate dehydrogenase activity in M. sagittifera extract were: blank, 0.006; L-lactate, 0.045; o-lactate, 0.121; and oL-lactate, 0"184 # m o l e / m i n per g tissue. T h u s the activity with D-lactate was 66 per cent of the activity with the racemic mixture at equivalent p-lactate concentration, and the activity with L-lactate was 24 per cent of the activity with oL-lactate. T h e low activity with L-lactate was p r e s u m e d to be an artefact of the crude enzyme preparation. T h e reciprocals of reaction velocity and substrate concentration are plotted against each other ( L i n e w e a v e r - B u r k method) in Fig. 1. I n addition to the Monodonta results, this graph shows results of experiments with Mytilus edulis which were done on 28 N o v e m b e r 1969 at Banyuls-sur-Mer, France, and experim e n t s with muscle of Crassostrea virginica, which were done on 30 August 1968 at Noank, Connecticut. T h e Michaelis constants for o-lactate indicated by Fig. 1 are Mytilus, 105 m M ; Monodonta, 180 m M ; and Crassostrea, 260 m M . T h e body proportions of M. sagittifera of various sizes, from a total weight of 1.0 to 3.8 g, are shown in T a b l e 1. As total weight increased, the fractional shell weight increased f r o m 58.1 to 69.4 per cent, and the fractional tissue weight
125
D - L A C T A T E O X I D A T I O N I N A M A R I N E G A S T R O P O D A N D A SEA U R C H I N
decreased from 30.3 to 23.3 per cent. Weight-specific lactate dehydrogenase activity, also shown in Table 1, decreased with size. With activity (y) in nmoles min per g, and weight (W) in rag, the relation was y = 2.64W -°as.
20
I V
l0
- 8
0
I0
IZS
FIC. 1. Lactate dehydrogenase reaction catalyzed by extracts of three species of mollusks. Reciprocal of velocity plotted against reciprocal of substrate concentration. O, M. edulis; A, M. sagittifera; 0 , C. virginica. TABLE
1--BODY
P R O P O R T I O N S A N D LACTATE D E H Y D R O G E N A S E A C T I V I T Y O F VARIOUS SIZES.
ACTIVITIES IN
~mole/minP E R
M. sagittifera
OF
g TISSUE
Total weight (g)
Shell (g)
Tissue (g)
Fluid (g)
Enzyme activity
1.000 1.033 1.773 2.413 3.785
-0.600 1.133 1.547 2.625
0.283 0.313 0.460 0.650 0.885
-0"120 0"180 0.210 0"275
0.134 0.121 O.108 0"086
T h e double specificity of lactate dehydrogenase from Arbacia lixula was suggested by three experiments which were done between 26 August and 5 September 1969 at Banyuls-sur-Mer. It was confirmed with two further experiments on the same species at Tenerife; results of these five experiments are summarized in Table 2. It is clear that each stereoisomer supported a rate just one-half
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C . S . HAMMEN AND SUSAN C. LUM
TABLE 2--LACTATE DEHYDROGENASEACTIVITY OF i . lixula EXTRACTSWITH ISOMERS OF LACTIC ACID SALTS (/zmole/min p e r g)
Place and date of experiment Banyuls-sur-Mer 26 August 1969 Net Percentage 28 August 1969 Net Percentage 5 September 1969 Net Percentage
Blank
~)
L
DL
0"193 --
0"274 0-081 44 0'510 0'203 60 0"332 0"163 37
0"266 0"073 39 0-470 0-163 48 0-338 0'169 39
0-378 0'185
0-069 0"095 48 0"103 0"125 53
0'086 0"112 56 0"099 0'121 52
0"173 0-199
0"307 -0"169 --
Tenerife 23 January 1970 Net Percentage 9 February 1970 Net Percentage
-0"026 --0.022 --
Sum of o and L
83 0'644 0'337 108 0-605 0.436 76
104 0.213 0"235 105
t h a t of t h e m i x t u r e c o n t a i n i n g b o t h in e q u a l c o n c e n t r a t i o n . T h u s t h e u r c h i n e i t h e r has t w o e n z y m e s in e q u a l a m o u n t o r a single e n z y m e l a c k i n g stereospecificity. A single e x p e r i m e n t w i t h a l a r g e r sea u r c h i n , D. antillarum, y i e l d e d s i m i l a r results, as s h o w n in F i g . 2. T h e rates ( ~ m o l e / m i n p e r g) w e r e : b l a n k , 0.056; D-lactate,
o.o5o
"~.
~ .
D
<3
o.oio
o . 15c
I
I
I
I
2
3
rain
FIO. 2. Lactate d e h y d r o g e n a s e reaction catalyzed b y extract of D. antillarum. A b s o r b a n c e change at 600 n m on s u p p l y i n g various substrates : buffer only (none),
L-lactate (L), D-lactate (D), racemic mixture (DL).
D-LACTATEOXIDATIONIN A MARINEGASTROPODANDA SEAURCHIN
127
0"167; L-lactate, 0"181; DL-lactate, 0"310; after subtracting the blank, the separate isomers made up 44 and 49 per cent of the total activity. DISCUSSION The demonstration of enzyme activity for oxidation of D-lactate only in M. sagittifera confirms and extends the discovery of specificity for D-lactate in a marine gastropod, Polinices heros (Long & Kaplan, 1968). Two species of pelecypods, an amphineuran, a cephalopod and another gastropod share this trait (Hammen, 1971). Although no scaphopod or monoplacophoran has been examined, it seems likely that all mollusks have lactate dehydrogenases active only with the D-isomer. If the two kinds of enzymes are expressions of alleles, the Mollusca are genetically uniform with respect to them, while the Arthropoda and Annelida are not, since both types of specificity were found in various classes of the latter phyla (Long & Kaplan, 1968). This suggests that all classes of mollusks originated from a single ancestral stock, and that this stock was more closely related to polychaetes than to other annelids, and more closely related to arachnids than to other arthropods. A. lixula contained enzymes which catalyzed the oxidation of both D-lactate and L-lactate at equal rates, and in sum equal to the rate for DL-lactate. The same result was obtained with D. antillarum, a larger urchin not in the same family as Arbacia. Since every lactate dehydrogenase examined so far, regardless of its source, is specific for only one isomer, it is probable that both types of lactate dehydrogenase occur together in sea urchins. This is interesting from the evolutionary point of view because echinoderms have been considered for a long time closely related to the presumed invertebrate ancestor of the chordates. Biochemical evidence in support of this hypothesis includes the presence of both typically invertebrate and typically vertebrate phosphagens (N-phosphoryl arginine and N-phosphoryl creatine) in the muscles of certain urchins (Baldwin, 1964). The presence of an extra metabolite, such as creatine phosphate, or an extra enzyme, such as D-lactate dehydrogenase, implies extra genes directing their formation. The minimum DNA content per haploid nucleus of the sea urchin, Paracentrotus lividus approaches 20 per cent of the content of a mammalian cell, indicating a greater total genome than those of other invertebrates, which range from about 3 to 7 per cent (Britten & Davidson, 1969). Among echinoderms other than urchins, a holothurian and a crinoid appear to have only one lactate dehydrogenase, specific for L-lactate (Hammen, 1971). The value of having both types is unknown. In the case of a bacterium, Lactobacillusplantarum, the ability to oxidize both isomers may be useful as a means of converting one carbon skeleton into another (Dennis & Kaplan, 1960). Since the majority of animals have only one type, possession of both must confer little or no selective advantage on them. In order to form hypotheses about the physiological role of invertebrate lactate dehydrogenases, more of their basic properties must be known. Most of the information to date is concerned with pyruvate reduction only, and at a single
128
C. S. HAMMEN AND SUSAN C . LUM
concentration of pyruvate, often related in no way to the endogenous concentration of pyruvate in the tissue from which the enzyme was obtained. A first step is to determine the effect of various lactate concentrations on velocity of the reaction, summing up the results in a Michaelis constant (K,n). Values of K,,~(lac) and K m (pyr) for a number of vertebrate enzymes were given by Fondy & Kaplan (1965). For L-lactate enzymes of the muscle type, Km (lac) ranged from 25 to 110 raM, and K m (pyr) from 0.14 to 1.0 raM. Enzymes from marine invertebrates, listed in Table 3, have a Km (pyr) in the same range, TABLE 3--MICHAELIS CONSTANTS OF LACTATE DEHYDROGENASES FROM MARINE INVERTEBRATES
Species
Reference
Mollusca Crassostrea virginica Mytilus edulis Monodonta sagittifera Loligo pealei (axoplasm)
Hammen (1969) This paper This paper Roberts et al. (1958)
Arthropoda Homarus americanus (muscle) Limulus polyphemus Chthamalus depressus
Kaloustian & Kaplan (1969) Long & Kaplan (1968) Hammen (1972)
Lactate Pyruvate (raM) D(mM) L(mM) 0'20 N N 0'64
260 105 180 N
A A A N
0"4 0"1 N
A N A
2"5 A 345
N = not determined; A = absent. 0.10-0.64mM. The Km (lac) values, however, extend over a greater range, from 2.5 mM in lobster tail muscle to 345 mM in a barnacle extract. The mollusks generally had a K m (lac) greater than those of vertebrates. This suggests that lactate oxidation would be very slow in mollusk tissues until D-lactate concentrations had increased to high levels. Lactate concentrations of 4.6 + 1-9 mg/g net weight were reported in oyster mantle (Hammen & Wilbur, 1959), and up to 0.044 mg/g in Mytilus californianus tissues (Moon & Pritchard, 1970). Assuming 80 per cent water in these tissues, the endogenous lactate concentrations would be about 64 and 6 mM, respectively. Thus, the oyster had an endogenous lactate concentration about ten times that of M. californianus, and a Km (lae) about 2.5 times that of M. edulis, indicating a correlation between normal levels of substrate and properties of enzyme. The weight-specific activity of Monodonta extracts decreased with body weight according to the empirical relation y = 2-64W -°'18. The weight-specific oxygen consumption of the limpet Patella vulgata decreased in a similar manner, with b = - 0.27 in the equation y = K W b (Davies, 1966). Thus there are two factors tending to reduce the metabolism of larger gastropods, a greater fraction of metabolically inert material, and a reduced activity per unit weight of active material.
D-LACTATE OXIDATION I N A MARINE GASTROPOD AND A SEA U R C H I N
129
Acknowledgements--We sincerely thank Sr. Carmelo Garcia Cabrera and Sr. Antonio Gonzalez Gonzalez for providing laboratory space and equipment, and Sr. Angelo Rodriguez de Leon for many services which facilitated our research at Tenerife. REFERENCES ARRIGONI O. & SINGER T. P. (1962) Limitations of the phenazine methosulphate assay for succinic and related dehydrogenases. Nature, Lond. 193, 1256-1258. AWAPARA J. & SIMPSON J. W. (1967) Comparative physiology: metabolism. Ann. Rev. Physiol. 29, 87-112. BALDWINE. (1964) Ain Introduction to Comparative Biochemistry, 4th edn. University Press, Cambridge. BRITTEN R. J. & DAVXDSONE. H. (1969) Gene regulation for higher cells: a theory. Science 165, 349-357. BUEDINO E. (1962) Comparative aspects of carbohydrate metabolism. Fedn Proc. Fedn Aim. Socs exp. Biol. 21, 1039-1046. DAVIESP. S. (1966) Physiological ecology of Patella--1. The effect of body size and temperature on metabolic rate. jY. mar. Biol. Ass. U.K. 46, 647-658. DENNIS D. & KaPLaN N. O. (1960) D- and L-lactic acid dehydrogenase in Lactobacillus plantarum. ~. biol. Chem. 235, 810-818. DuPFuS J. H. & DtrFFUS C. M. (1968) Some enzymes present in marine mollusca of the Canary Island of Lanzarote. Experientia 24, 1114-1115. FONDY T. P. & KaPLAN N. O. (1965) Structural and functional properties of the H and M subunits of lactic dehydrogenases. Ann. N. Y. Aicad. Sci. 119, 888-903. GILLES R. (1970) Intermediary metabolism and energy production in some invertebrates. Airchs int. Physiol. Biochim. 78, 313-326. HAMMEN C. S. (1966) Carbon dioxide fixation in marine invertebrates--V. Rate and pathway in the oyster. Comp. Biochem. Physiol. 17, 289-296. HAMrVmNC. S. (1969) Lactate and succinate oxidoreductases in marine invertebrates. Mar. Biol. 4, 233-238. HAMMEN C. S. (1971) Metabolism of brachiopods and bivalve mollusks. In Aictas del I Simposio Internacional de Zoofilogenia (Edited by ALVARADOR., GADEAE. & DE HAROA.), pp. 471-478. Universidad de Salamanca, Salamanca. HAMMEN C. S. (1972) Lactate oxidation in the upper-shore barnacle, Chthamalus depressus. Comp. Biochem. Physiol. (In press.) LONG G. L. & KAPLANN. O. (1968) D-Lactate specific pyridine nucleotide lactate dehydrogenase in animals. Science 162, 685-686. MOON T. W. & PRITC~RD A. W. (1970) Metabolic adaptations in vertically separated populations of Mytilus californianus Conrad. jY. Exp. Mar. Biol. Ecol. 5, 35-46. REGNOUF F. & VAN TaOAI N. (1970) Octopine and lactate dehydrogenase in marine mollusc muscles. Comp. Biochem. Physiol. 32, 411-416. ROBERTSN. R., COELHOR. R., LOWRYO. H. & CRAWFORDE. J. (1958) Enzyme activities of giant squid axoplasm and axon sheath, ft. Neurochem. 3, 109-115. Saz H. J. (1971) Facultative anaerobiosis in the invertebrates: pathways and control systems. Aim. Zool. 11,125-135. Key Word Index--Lactate dehydrogenase in invertebrates; sea urchin L D H ; gastropod L D H ; Arbacia li~da L D H ; Monodonta sagittifera L D H ; and L- and D-Lactate dehydrogenase.
D-LACTATE OXIDATION IN A MARINE GASTROPOD AND A SEA URCHIN* C. S. H A M M E N and SUSAN C. L U M Department of Zoology, University of Rhode Island, Kingston, Rhode Island (Received 26 October 1971)
Abstract--1. Lactate dehydrogenase activities in extracts of a gastropod, Monodonta sagittifera, and a sea urchin, Arbacia lixula, were determined by change in light absorbance of a dye mixture on addition of salts of lactic acid in each isomeric form. 2. The Monodonta extract catalyzed the oxidation of D-lactate only, with an apparent Michaelis constant of 180 raM. 3. Weight-specific enzyme activity of Monodonta was proportional to the - 0"18 power of total body weight. 4. The sea urchin appeared to contain two enzymes of equal activity, one specific for L-lactate and one specific for D-lactate. INTRODUCTION AS COMPARATIVEbiochemistry has become more comparative, some interesting exceptions to the principle of biochemical unity have come to light. One generalization, that lactic acid is the exclusive end-product of anaerobic glycolysis in animal tissues, has been abandoned as various species of invertebrates have been shown to form other products. Numerous parasitic flatworms and nematodes, and at least a few species of mollusks, produce succinic acid rather than lactic acid. Succinate formation occurs by means of carbon dioxide fixation with pyruvate or phosphoenolpyruvate, followed by reversal of several steps of the citric acid cycle (Bueding, 1962; Hammen, 1966; Awapara & Simpson, 1967; Gilles, 1970; Saz, 1971). The relative reversability of lactate and succinate oxidoreductases (Hammen, 1969) suggests that representatives of some other invertebrate groups may also produce succinate in preference to lactate during anaerobiosis, especially certain sponges, brachiopods and barnacles. Another recently discarded generalization is that only one stereoisomer of lactic acid, L-lactic, is involved in animal metabolism. Long & Kaplan (1968) discovered that the horseshoe crab Limulus, five species of arachnids, a marine gastropod and a polychaete have lactate dehydrogenases specific for D-lactate. Soon after this discovery, the oyster Crassostrea virginica was also found to oxidize only D-lactate (Hammen, 1969). Such specificity has since been found in another pelecypod, another gastropod, an amphineuran and a cephalopod (Hammen, 1971). In their survey of eighteen species of invertebrates, Long & Kaplan (1968) * Work carried out at Laboratorio Oceanografico de Canarias, Santa Cruz de Tenerife, Islas Canarias. 123
124
C. S. HAMMENAND SUSAN C. LUM
f o u n d that "in each animal, the lactate dehydrogenases are specific for only one isomer of lactate". Apparently even this modest generalization has exceptions. I n this report are presented results of experiments showing the presence of a lactate dehydrogenase specific for D-lactate in another marine gastropod, ~Vlonodonta sagittifera (Lamarck), and the variation of its activity with substrate concentration. I n addition, a sea urchin, Arbacia lixula (Busch) is shown to have enzymes for the oxidation of both o-lactate and L-lactate, the first instance of double specificity known in animals. MATERIALS AND M E T H O D S The animals used in this study were collected from 2 January to 9 February 1970 at Las Caletillas, Tenerife, Islas Canarias. Monodonta sagittifera was very abundant, and its size and position on the rocky shore were reminiscent of the familiar Littorlna littorea of North Atlantic coasts. At Lanzarote, some 270 km to the east, both Littorina striata and Monodonta turbinata are found (Duffus & Duffus, 1968). Arbacia lixula specimens occurred in the shallow subtidal zone, and weighed 33'1-50"0 g. A single experiment was done with a specimen of Diadema antillarum (Philippi) which was captured at a depth of 5 m, and weighed 127"9 g. The experiments were done the same day in the biochemistry laboratory of the Universidad de La Laguna. Lactate dehydrogenase activity was measured by decolorization of dichlorophenolindophenol (DCIP) with phenazine methosulfate (PMS) as intermediate electron carrier, the method of Arrigoni & Singer (1962). The enzyme source was the supernatant of a tissue homogenate, prepared by grinding about 1 g tissue with nine times its weight of Tris buffer, 0"25 M, pH 7"38, and centrifuging for 10 rain at threequarters maximum speed in a Viher clinical centrifuge. The absorbance at 600 nm was measured with a Beckman D U spectrophotometer with the assay mixture at room temperature, 21°C. Sodium lactate was from E. Merck, Darmstadt; the lithium salts of D-lactate and L-lactate, DCIP and PMS were from Calbiochem, Los Angeles; and Tris from Sigma, St. Louis. Activities were calculated from changes in absorbance in the first minute of the reaction. RESULTS Rates of the lactate dehydrogenase activity in M. sagittifera extract were: blank, 0.006; L-lactate, 0.045; o-lactate, 0.121; and oL-lactate, 0"184 # m o l e / m i n per g tissue. T h u s the activity with D-lactate was 66 per cent of the activity with the racemic mixture at equivalent p-lactate concentration, and the activity with L-lactate was 24 per cent of the activity with oL-lactate. T h e low activity with L-lactate was p r e s u m e d to be an artefact of the crude enzyme preparation. T h e reciprocals of reaction velocity and substrate concentration are plotted against each other ( L i n e w e a v e r - B u r k method) in Fig. 1. I n addition to the Monodonta results, this graph shows results of experiments with Mytilus edulis which were done on 28 N o v e m b e r 1969 at Banyuls-sur-Mer, France, and experim e n t s with muscle of Crassostrea virginica, which were done on 30 August 1968 at Noank, Connecticut. T h e Michaelis constants for o-lactate indicated by Fig. 1 are Mytilus, 105 m M ; Monodonta, 180 m M ; and Crassostrea, 260 m M . T h e body proportions of M. sagittifera of various sizes, from a total weight of 1.0 to 3.8 g, are shown in T a b l e 1. As total weight increased, the fractional shell weight increased f r o m 58.1 to 69.4 per cent, and the fractional tissue weight
125
D - L A C T A T E O X I D A T I O N I N A M A R I N E G A S T R O P O D A N D A SEA U R C H I N
decreased from 30.3 to 23.3 per cent. Weight-specific lactate dehydrogenase activity, also shown in Table 1, decreased with size. With activity (y) in nmoles min per g, and weight (W) in rag, the relation was y = 2.64W -°as.
20
I V
l0
- 8
0
I0
IZS
FIC. 1. Lactate dehydrogenase reaction catalyzed by extracts of three species of mollusks. Reciprocal of velocity plotted against reciprocal of substrate concentration. O, M. edulis; A, M. sagittifera; 0 , C. virginica. TABLE
1--BODY
P R O P O R T I O N S A N D LACTATE D E H Y D R O G E N A S E A C T I V I T Y O F VARIOUS SIZES.
ACTIVITIES IN
~mole/minP E R
M. sagittifera
OF
g TISSUE
Total weight (g)
Shell (g)
Tissue (g)
Fluid (g)
Enzyme activity
1.000 1.033 1.773 2.413 3.785
-0.600 1.133 1.547 2.625
0.283 0.313 0.460 0.650 0.885
-0"120 0"180 0.210 0"275
0.134 0.121 O.108 0"086
T h e double specificity of lactate dehydrogenase from Arbacia lixula was suggested by three experiments which were done between 26 August and 5 September 1969 at Banyuls-sur-Mer. It was confirmed with two further experiments on the same species at Tenerife; results of these five experiments are summarized in Table 2. It is clear that each stereoisomer supported a rate just one-half
126
C . S . HAMMEN AND SUSAN C. LUM
TABLE 2--LACTATE DEHYDROGENASEACTIVITY OF i . lixula EXTRACTSWITH ISOMERS OF LACTIC ACID SALTS (/zmole/min p e r g)
Place and date of experiment Banyuls-sur-Mer 26 August 1969 Net Percentage 28 August 1969 Net Percentage 5 September 1969 Net Percentage
Blank
~)
L
DL
0"193 --
0"274 0-081 44 0'510 0'203 60 0"332 0"163 37
0"266 0"073 39 0-470 0-163 48 0-338 0'169 39
0-378 0'185
0-069 0"095 48 0"103 0"125 53
0'086 0"112 56 0"099 0'121 52
0"173 0-199
0"307 -0"169 --
Tenerife 23 January 1970 Net Percentage 9 February 1970 Net Percentage
-0"026 --0.022 --
Sum of o and L
83 0'644 0'337 108 0-605 0.436 76
104 0.213 0"235 105
t h a t of t h e m i x t u r e c o n t a i n i n g b o t h in e q u a l c o n c e n t r a t i o n . T h u s t h e u r c h i n e i t h e r has t w o e n z y m e s in e q u a l a m o u n t o r a single e n z y m e l a c k i n g stereospecificity. A single e x p e r i m e n t w i t h a l a r g e r sea u r c h i n , D. antillarum, y i e l d e d s i m i l a r results, as s h o w n in F i g . 2. T h e rates ( ~ m o l e / m i n p e r g) w e r e : b l a n k , 0.056; D-lactate,
o.o5o
"~.
~ .
D
<3
o.oio
o . 15c
I
I
I
I
2
3
rain
FIO. 2. Lactate d e h y d r o g e n a s e reaction catalyzed b y extract of D. antillarum. A b s o r b a n c e change at 600 n m on s u p p l y i n g various substrates : buffer only (none),
L-lactate (L), D-lactate (D), racemic mixture (DL).
D-LACTATEOXIDATIONIN A MARINEGASTROPODANDA SEAURCHIN
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0"167; L-lactate, 0"181; DL-lactate, 0"310; after subtracting the blank, the separate isomers made up 44 and 49 per cent of the total activity. DISCUSSION The demonstration of enzyme activity for oxidation of D-lactate only in M. sagittifera confirms and extends the discovery of specificity for D-lactate in a marine gastropod, Polinices heros (Long & Kaplan, 1968). Two species of pelecypods, an amphineuran, a cephalopod and another gastropod share this trait (Hammen, 1971). Although no scaphopod or monoplacophoran has been examined, it seems likely that all mollusks have lactate dehydrogenases active only with the D-isomer. If the two kinds of enzymes are expressions of alleles, the Mollusca are genetically uniform with respect to them, while the Arthropoda and Annelida are not, since both types of specificity were found in various classes of the latter phyla (Long & Kaplan, 1968). This suggests that all classes of mollusks originated from a single ancestral stock, and that this stock was more closely related to polychaetes than to other annelids, and more closely related to arachnids than to other arthropods. A. lixula contained enzymes which catalyzed the oxidation of both D-lactate and L-lactate at equal rates, and in sum equal to the rate for DL-lactate. The same result was obtained with D. antillarum, a larger urchin not in the same family as Arbacia. Since every lactate dehydrogenase examined so far, regardless of its source, is specific for only one isomer, it is probable that both types of lactate dehydrogenase occur together in sea urchins. This is interesting from the evolutionary point of view because echinoderms have been considered for a long time closely related to the presumed invertebrate ancestor of the chordates. Biochemical evidence in support of this hypothesis includes the presence of both typically invertebrate and typically vertebrate phosphagens (N-phosphoryl arginine and N-phosphoryl creatine) in the muscles of certain urchins (Baldwin, 1964). The presence of an extra metabolite, such as creatine phosphate, or an extra enzyme, such as D-lactate dehydrogenase, implies extra genes directing their formation. The minimum DNA content per haploid nucleus of the sea urchin, Paracentrotus lividus approaches 20 per cent of the content of a mammalian cell, indicating a greater total genome than those of other invertebrates, which range from about 3 to 7 per cent (Britten & Davidson, 1969). Among echinoderms other than urchins, a holothurian and a crinoid appear to have only one lactate dehydrogenase, specific for L-lactate (Hammen, 1971). The value of having both types is unknown. In the case of a bacterium, Lactobacillusplantarum, the ability to oxidize both isomers may be useful as a means of converting one carbon skeleton into another (Dennis & Kaplan, 1960). Since the majority of animals have only one type, possession of both must confer little or no selective advantage on them. In order to form hypotheses about the physiological role of invertebrate lactate dehydrogenases, more of their basic properties must be known. Most of the information to date is concerned with pyruvate reduction only, and at a single
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C. S. HAMMEN AND SUSAN C . LUM
concentration of pyruvate, often related in no way to the endogenous concentration of pyruvate in the tissue from which the enzyme was obtained. A first step is to determine the effect of various lactate concentrations on velocity of the reaction, summing up the results in a Michaelis constant (K,n). Values of K,,~(lac) and K m (pyr) for a number of vertebrate enzymes were given by Fondy & Kaplan (1965). For L-lactate enzymes of the muscle type, Km (lac) ranged from 25 to 110 raM, and K m (pyr) from 0.14 to 1.0 raM. Enzymes from marine invertebrates, listed in Table 3, have a Km (pyr) in the same range, TABLE 3--MICHAELIS CONSTANTS OF LACTATE DEHYDROGENASES FROM MARINE INVERTEBRATES
Species
Reference
Mollusca Crassostrea virginica Mytilus edulis Monodonta sagittifera Loligo pealei (axoplasm)
Hammen (1969) This paper This paper Roberts et al. (1958)
Arthropoda Homarus americanus (muscle) Limulus polyphemus Chthamalus depressus
Kaloustian & Kaplan (1969) Long & Kaplan (1968) Hammen (1972)
Lactate Pyruvate (raM) D(mM) L(mM) 0'20 N N 0'64
260 105 180 N
A A A N
0"4 0"1 N
A N A
2"5 A 345
N = not determined; A = absent. 0.10-0.64mM. The Km (lac) values, however, extend over a greater range, from 2.5 mM in lobster tail muscle to 345 mM in a barnacle extract. The mollusks generally had a K m (lac) greater than those of vertebrates. This suggests that lactate oxidation would be very slow in mollusk tissues until D-lactate concentrations had increased to high levels. Lactate concentrations of 4.6 + 1-9 mg/g net weight were reported in oyster mantle (Hammen & Wilbur, 1959), and up to 0.044 mg/g in Mytilus californianus tissues (Moon & Pritchard, 1970). Assuming 80 per cent water in these tissues, the endogenous lactate concentrations would be about 64 and 6 mM, respectively. Thus, the oyster had an endogenous lactate concentration about ten times that of M. californianus, and a Km (lae) about 2.5 times that of M. edulis, indicating a correlation between normal levels of substrate and properties of enzyme. The weight-specific activity of Monodonta extracts decreased with body weight according to the empirical relation y = 2-64W -°'18. The weight-specific oxygen consumption of the limpet Patella vulgata decreased in a similar manner, with b = - 0.27 in the equation y = K W b (Davies, 1966). Thus there are two factors tending to reduce the metabolism of larger gastropods, a greater fraction of metabolically inert material, and a reduced activity per unit weight of active material.
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Acknowledgements--We sincerely thank Sr. Carmelo Garcia Cabrera and Sr. Antonio Gonzalez Gonzalez for providing laboratory space and equipment, and Sr. Angelo Rodriguez de Leon for many services which facilitated our research at Tenerife. REFERENCES ARRIGONI O. & SINGER T. P. (1962) Limitations of the phenazine methosulphate assay for succinic and related dehydrogenases. Nature, Lond. 193, 1256-1258. AWAPARA J. & SIMPSON J. W. (1967) Comparative physiology: metabolism. Ann. Rev. Physiol. 29, 87-112. BALDWINE. (1964) Ain Introduction to Comparative Biochemistry, 4th edn. University Press, Cambridge. BRITTEN R. J. & DAVXDSONE. H. (1969) Gene regulation for higher cells: a theory. Science 165, 349-357. BUEDINO E. (1962) Comparative aspects of carbohydrate metabolism. Fedn Proc. Fedn Aim. Socs exp. Biol. 21, 1039-1046. DAVIESP. S. (1966) Physiological ecology of Patella--1. The effect of body size and temperature on metabolic rate. jY. mar. Biol. Ass. U.K. 46, 647-658. DENNIS D. & KaPLaN N. O. (1960) D- and L-lactic acid dehydrogenase in Lactobacillus plantarum. ~. biol. Chem. 235, 810-818. DuPFuS J. H. & DtrFFUS C. M. (1968) Some enzymes present in marine mollusca of the Canary Island of Lanzarote. Experientia 24, 1114-1115. FONDY T. P. & KaPLAN N. O. (1965) Structural and functional properties of the H and M subunits of lactic dehydrogenases. Ann. N. Y. Aicad. Sci. 119, 888-903. GILLES R. (1970) Intermediary metabolism and energy production in some invertebrates. Airchs int. Physiol. Biochim. 78, 313-326. HAMMEN C. S. (1966) Carbon dioxide fixation in marine invertebrates--V. Rate and pathway in the oyster. Comp. Biochem. Physiol. 17, 289-296. HAMrVmNC. S. (1969) Lactate and succinate oxidoreductases in marine invertebrates. Mar. Biol. 4, 233-238. HAMMEN C. S. (1971) Metabolism of brachiopods and bivalve mollusks. In Aictas del I Simposio Internacional de Zoofilogenia (Edited by ALVARADOR., GADEAE. & DE HAROA.), pp. 471-478. Universidad de Salamanca, Salamanca. HAMMEN C. S. (1972) Lactate oxidation in the upper-shore barnacle, Chthamalus depressus. Comp. Biochem. Physiol. (In press.) LONG G. L. & KAPLANN. O. (1968) D-Lactate specific pyridine nucleotide lactate dehydrogenase in animals. Science 162, 685-686. MOON T. W. & PRITC~RD A. W. (1970) Metabolic adaptations in vertically separated populations of Mytilus californianus Conrad. jY. Exp. Mar. Biol. Ecol. 5, 35-46. REGNOUF F. & VAN TaOAI N. (1970) Octopine and lactate dehydrogenase in marine mollusc muscles. Comp. Biochem. Physiol. 32, 411-416. ROBERTSN. R., COELHOR. R., LOWRYO. H. & CRAWFORDE. J. (1958) Enzyme activities of giant squid axoplasm and axon sheath, ft. Neurochem. 3, 109-115. Saz H. J. (1971) Facultative anaerobiosis in the invertebrates: pathways and control systems. Aim. Zool. 11,125-135. Key Word Index--Lactate dehydrogenase in invertebrates; sea urchin L D H ; gastropod L D H ; Arbacia li~da L D H ; Monodonta sagittifera L D H ; and L- and D-Lactate dehydrogenase.