Mechanism of lactic acid oxidation catalyzed by lactate dehydrogenase from the tail muscle of Homarus americanus

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 191, No. 2, December, pp. 666672, 1978

Mechanism of Lactic Acid Oxidation Catalyzed by Lactate Dehydrogenase from the Tail Muscle of Homarus americanus’ RONALD D. EICHNER

NATHAN

AND

0. KAPLAN’

Department of Chemistry, University of California, San Diego, La Jolla, California 92093 Received April 12, 1978; revised July 18, 1978 The mechanism of lactic acid oxidation in the tail muscles of Homarus americanus was studied. In solutions of intermediate ionic strength (0.55) time-course progress curves for lactic acid oxidation as catalyzed by lactate dehydrogenase exhibited a lag period. Evidence is presented which indicates that the lactate dehydrogenase found in the tail muscles of the lobster exists in two distinct physical and kinetic forms. The equilibrium of these forms is dependent upon the ionic strength of the reaction mixture. In low ionic strength solutions, the enzyme exists as a tetrameric species with an apparent K,,, for lactic acid of 1.1 M; in high ionic strength solutions, the enzyme exists as a dimer and the corresponding K,,, is 0.028 M. At intermediate ionic strengths, an equilibrium between the two physical and kinetic species exists which is modulated by the NADH mole-fraction ([NADH]/[NADH + NAD+]) and, in turn, this modulation results in sigmoidal time-course progress curves. The role of this enzyme is discussed as affected by in vivo ionic strength, temperature and levels of oxidized and reduced nicotine adenine dinucleotides.

Kaloustian and Kaplan (1,2) reported that the oxidation of lactic acid by lactate dehydrogenase from the tail muscles of the East Coast lobster exhibited anomalous kinetic patterns; time-course progress curves as well as substrate saturation curves were sigmoidal In addition NADH was shown to modulate the lactate oxidation reaction; if NADH was included in the initial reaction mixture, hyperbolic substrate saturation curves were observed. It should be emphasized that these studies were conducted in solutions of intermediate ionic strength (approximately 0.55). Two recent communications from this laboratory (3,4) have characterized the physical, chemical, and kinetic properties of two LDHs? purified from the East Coast lobster (isolated from the tail muscles and ’ This work was supported in part by grants from the American Cancer Society (BC-60-R) and the National Institutes of Health (USPHS CA 11683). * To whom correspondence should be addressed. 3 Abbreviations used: NADH mole-fraction, [NADH]/([NADH + NAD+]); c, ionic strength defined by p = EErn;c: where m = molarity and c = ionic charge on a given ion, summed for all ions in solution; LDH, lactate dehydrogenase.

walking leg muscles of this organism). The aggregation state of both of these proteins was dependent upon ionic strength, in 0.1 M Tris, the molecular weight was found to be 145,000,corresponding to that of a tetramerit species; in 1.3 M ammonium sulfate, the corresponding value was 75,000, consistent with the formation of a dimer. It should be noted that no evidence has been obtained for the existence of an active monomer. These changes in molecular weight as a function of ionic strength were concomitant with changes in the sedimentation coefficients of these proteins. Time course progress curves and substrate saturation curves were exponential and hyperbolic, respectively, for lactic acid oxidation under conditions of either low or high ionic strength, apparent K,,, values for both lactic acid and NAD+ were reduced by more than 90% when determinations were made in high ionic strength solutions. Finally, the enzymes isolated from the tail muscles and walking leg muscles of the East Coast lobster were characterized as kinetic “hearttype” LDH and “muscle-type” LDH, respectively. The delineation of the physical and ki666

0003-9861/78/1912-0666$02.00/O Copyright 0 1978 by Academic Press, Inc. *11 .&At. nF m”rnA,.r+ir\n in en., Fn- r~~arrr.rl

LACTIC

ACID

BY Homarus americanus LACTATE

OXIDATION

AND

METHODS

Materials. NAD’ was obtained from Cyclo-Chemical Corp. in acid form; all other pyridine nucleotides were obtained from P-L Biochemicala, Inc., in the form of sodium salts; Tris was purchased from Sigma Chemical Company; L-lithium lactic acid and sodium pyruvate from Calbiochem; all reagents were of the highest grade available commercially. The LDHs from lobster tissues used in this study were purified and assayed as described previously (3). Initial velocities were extrapolated from the tangent at time zero of the time courses. Stopped-flow measurements. Stopped-flow determinations provide an accurate measurement of the amount of LDH activity in crude enzyme solution; by using a higher concentration of enzyme in the assay mixture than that used for measuring normal steady state velocities, dissociation of the inactive ternary complex (LDH-NAD+-pyruvate) to active LDH is minimized. Tails and legs were excised from live resting lobsters and the tissues were separated from exoskeleton and gastrointestinal tract. These muscles were then sepa-

0

4 TIME,mm

8

12

0

667

rately added to 2 volumes (w/v) of cold 0.2 M sodium phosphate, pH 7.5, homogenized and then filtered. The solutions were placed in syringe 1 and then connected to an Aminco-Morrow stopped-flow apparatus. Syringe 2 contained 2.7 mre pyruvate, 0.4 mru NADH in 0.2 M sodium phosphate, pH 7.5. Activities were then measured at various time intervals. Zero time indicates the beginning of tissue homogenization; after 1 h, enzyme samples were dialyzed against 2 liters of buffer overnight (v/v = l/500) and then assayed for activity the following morning. NAD’ and NADH levels in lobster tails. NAD’ and NADH levels were determined as described by Ciotti and Kaplan (5). For NAD+ levels, 10 g of fresh lobster tail muscle were quickly homogenized in 50 ml of 5% trichloroacetic acid and then centrifuged. The sample was subsequently neutralized with sodium hydroxide and sodium bicarbonate and then subjected to the methyl ethyl ketone procedure for NAD+ analysis. The fluorescence of the resulting adduct was monitored at 450 nm; excitation occurred at 340 nm. The NADH level was determined by homogenizing 10 g of fresh lobster tail in 50 ml of boiling sodium carbonate, pH 10.5. The sample was centrifuged and neutralized. The fluorescence of methyl ethyl ketone adducta was measured on a sample treated with and without alcohol dehydrogenase and acetaldehyde. All other methods have been described previously (3,4). Sedimentation coefficients. .SZO.~ values were determined according to the method of Kemper and Everse

netic properties of the lactate dehydrogenase from the tail muscles of the lobster in solutions of both low and high ionic strengths can be utilized to understand the complex intermediate case where anomalous kinetic profiles are observed. It is the purpose of this paper to define the mechanism of lactic acid oxidation in the tail muscles of the East Coast lobster especially as this mechanism is affected by the ionic strength and temperature of the sea water in which the organism resides. Measurements of in vivo levels of oxidized and reduced nicotine adenine dinucleotides were correlated with enzymatic regulation. MATERIALS

DEHYDROGENASE

(6). RESULTS

Figure 1 illustrates typical time-course progress curves for lactic acid oxidation catalyzed by LDH from the tail muscles of the East Coast lobster. Reaction conditions were identical for all three cases except for the variables, ionic strength of the reaction mixture, and enzyme concentration. In so-

I

2

TIME .mln

3

0

I

2

3

TIME.m,”

FIG. 1. Effect of ionic strength on shape of time-course progress curves for lactic acid oxidation by lobster tail lactate dehydrogenase. Ammonium sulfate was used to adjust the ionic strengths of the reaction solutions. Assays were carried out at 20 mu Lactate and 0.23 mru NAD+; the amount of enzyme used in the assays at ionic strengths of 0.1, 0.55, and 3.0 were 54 cg, 40 pg, and 8 pg, respectively.

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KAPLAN

quivalent binding sites for the coensyme or substrate. (An alternative explanation is that the coenqme binds to the protein in a positively cooperative manner.) Thus the kinetics have the general characterization of a second order autocatalytic reaction.

5.0 -

2.0-

3 ;:

IO-

3 2

:9.5 -

0.2 -

WI,.,.,. 1.00

0.50

0.20

0.10 0.05

[NAD’], mM FIG. 2. Hill plot for the forward reaction catalyzed by lobster tail lactate dehydrogenase. ~-Lactate present at 6.7 x lo-’ M for all determinations; solutions buffered with 0.2 M sodium phosphate, pH 7.5 (JI = 0.6).

lutions of both low (0.1) and high (3.0) ionic strengths, the time-course progress curves were exponential. This observation in addition to the fact that double reciprocal plots of l/v vs. l/s are linear (data not shown), suggest that the protein exists as a discrete catalytic species in these solutions. In contrast, in intermediate ionic strengths (0.55) a lag period is evident in the timecourse progress curves and sigmoidal double reciprocal plots are obtained. However, linear double reciprocal plots result under these conditions if sufficient levels of NADH are included in the reaction mixtures (2). A Hill plot representation of lactic acid oxidation is depicted in Fig. 2. The logarithm of V/V--V is plotted against the logarithm of NAD+ concentration. A slope is calculated between 1.7 and 2.0 from this plot. It has been demonstrated with the tail enzyme that double reciprocal plots were nonlinear with coenxyme or substrate (2) however, when l/Z? is plotted against the reciprocal of the velocity, linear graphs are obtained. These results are consistent with the hypothesis that in solutions of intermediate ionic strength there are two none-

Effect of NADH Mole-Fraction on Lactic Acid Apparent Michaelis Constant for Lobster Tail LDH The effect of reduced coenxyme on the Michaelis constant of lactate for lobster tail LDH at intermediate ionic strengths is shown in Table I. The initial NADH molefractions and corresponding apparent If,,, of lactic acid are presented. If the initial reaction conditions contain no NADH, a K,,, for lactate of 0.32 M is calculated. As the mole-fraction of NADH was increased, the K,,, decreased, until at an NADH mole-fraction of 0.10, a K, of 0.095 M was reached. Further additions of NADH resulted in no further decrease in the K,,, of lactate. At an NADH mole-fraction of 0.20, the K, of lactate had increased to 0.18 M. Effect of NADH Mole-Fraction on the Sedimentation Coefficient of Lobster Tail LDH Sign&ant changes in the sedimentation coefficient (820,~)occurred when the lactic acid oxidation as catalyzed by lobster tail LDH was observed (by monitoring the increase in optical density at 340 nm which is due to the formation of NADH) at an intermediate ionic strength and varying conTABLE

I

EFFECT OF NADH MOLE-FRACTION ON APPARENT MICHAELIS CONSTANT OF LACTIC ACID FOR LOBSTER TAIL LACTA~ DEHYDROGENASE’ NADH

mole-fraction 0 0.05 0.10 0.14 0.20

Km of lactate, M 0.32 0.13 0.065 0.102 0.18

f f f f f

0.050 0.014 0.011 0.011 0.020

a All assays were conducted in 0.1 M sodium pyrephosphate, pH 6.9. This corresponds to an ionic strength of 0.55. Initial NAD+ concentration was 9.3 x lo-’ M in all cases. Double reciprocal plots of l/u versus l/g were linear. All determinations were at room temperature, 23°C. Values are means f standard deviation where n = 4.

LACTIC

ACID

OXIDATION

BY Homarus americanus LACTATE

DEHYDROGENASE

669

centrations of NADH were included in the reaction solutions. Table II sumarizes the results of such studies. With no NADH present initially, the sedimentation coefficient was 6.2. As the NADH mole-fraction was increased, there was a decrease in ~20,~ until at an NADH mole-fraction of 0.10, a value of 5.0 was reached. Further increases in the NADH mole-fraction resulted in an increase in the ~20,~until the original value of 6.2 was achieved. Similar changes were noted when the sedimentation of the enzyme was monitored at 280 nm in the presence of various NADH mole-fractions without added substrates. In Vivo Levels of NAD’ and NADH in Tail Muscles of Homarus americanus NAD’, 420 + 20 nmol, was found per gram of wet weight of lobster tail muscles; for NADH, 35 & 5 nmol were found. Using these values, the NADH mole-fraction in this muscle is 0.07 & 0.01. This value is intermediate between those found in skeletal muscle, typically 0.02-0.04, and those found in heart or aerobic tissue, approximately 0.2-0.3 (7-11). Ternary Complex in Homarus americanus During the course of purifying the enzyme from the tail muscles, procedures, which in the purification of LDH from other sources yielded substantially less than 100%of the enzyme activity, produced TABLE

II

EFFECT OF NADH MOLE-FRACTION ON SEDIMENTATION COEFFICIENT OF THE FORWARD REACTION CATALYZED BY LOBSTER TAIL LACTATE DEHYDROGENASE~ NADH

mole-fraction 0 0.05 0.10 0.20

sm. w 6.2 5.6 5.0 6.0

+ f f f.

0.1 0.1 0.1 0.1

a Active enzyme centrifugation was employed in these determinations. The ionic strength was maintained at 0.55 using 0.1 M sodium pyrophosphate. NADH mole-fraction is defined as the molar ratio of NADH to total nicotine adenine dinucleotides. Sedimentation coefficients, sob, were corrected for temperature, density and viscosity, yielding the appropriate sm.W.

FIG. 3. Rates of pyruvate reduction by lobster tail and leg lactate dehydrogenase following disruption of the tissues. Tails (B) and legs (A) were removed from live resting lobsters and the tissues treated as described in text.

remarkable yields (3). Kaplan et al. (12-14) have shown that LDH activity is substantially reduced upon initial isolation from chicken heart as compared to later points in time; the corresponding enzyme from chicken muscle did not exhibit this phenomenon. Furthermore, if the heart enzyme were isolated in the presence of pyruvate, little or no increase in activity was observed with time. These authors suggested that a large proportion of the chicken heart LDH may exist in vivo as the ternary complex (LDH-NAD+-pyruvate), thus accounting for an initial inhibition of pyruvate conversion to lactate. A subsequent slow dissociation of this complex upon dilution would then account for the apparent increase in enzymatic activity as a function of time after isolation. Figure 3 illustrates an analogous experiment for both lobster tail and leg LDH. The lobster leg protein behaves predominantly like a vertebrate muscle-type LDH. There is only a slight increase in enzymatic activity with time, even after dialysis. It should also be remembered that crude extracts of lobster leg tissues contain approximately 15% lobster tail LDH subunits in its LDH composition (3). The capacity of lobster tail LDH to reduce pyruvate is substantially increased with time; after 1 h, the activity was 200%of its original value. After

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dialysis for 15 h against 0.005 M sodium phosphate, pH 7.5 (v/v : l/200), the activity reached 315%of the initial measurement. It should be noted that lobster tail LDH has been shown to form the abortive ternary complex (4). Thus, lobster tail LDH, like the chicken heart enzyme, may exist in viuo in a large part as the ternary complex. DISCUSSION

The delineation of the physical and kinetic properties of LDH from the tail muscles of the East Coast lobster in both low and high ionic strength solutions is necessary for the understanding of the complex intermediate case where both sigmoidsl double reciprocal plots and time-course progress curves possessing a lag period are observed. The physical and kinetic properties of the tetramer and dimer are summarized in Fig. 4. Note that the apparent K,,, for lactic acid has changed from a value of 1.1 M for the tetramer to 28 mM for the dimer; this represents approximately a 40-fold increase in the affinity for lactate over that of the tetramer. Furthermore, the apparent K,,,for NAD+ has decreased by a factor of 7 in comparing the tetrameric state to the dimerit state. No ~significant changes were observed for the apparent Km's of pyruvate and NADH (4). The velocity vs. substrate plots of the lactate oxidation are hyperbolic for both protein states. These protein states correspond to a molecular weight of 145,000 with a sedimentation coefficient of 7.3 for

K m, Lactate

LOW

HIGH

IONIC STRENGTH

IONIC STRENGTH

83G

8

-I

I hi

28 x IO-’ M

1.0 x lo-3.f

1.4 ): lO+M

Klneflc Profile

Hyperbolic

Hyperballc

Molecular

145,000

79,000

73

39

K, , NAD’

sI, 20

Weight

FIG. 4. Physical and kinetic parameters of lobster tail lactate dehydrogenase in low and high ionic strength solutions. Low ionic strength corresponds to 0.1 M Tris, high ionic strength, to 0.1 M Tris, 1.1 M ammonium sulfate. The kinetic profile refers to the substrate-saturation curve.

KAPLAN

the protein in low ionic strength solutions, and a molecular weight of 75,090 with a sedimentation coefficient of 3.9 for the protein in the high ionic strength solutions. We conclude that there exists two protein states, a tetramer with an impaired ability of catalyzing lactic acid oxidation due to its relatively low affinities for lactate and NAD+, and a dimer, which is capable of oxidizing lactic acid due to its relatively high affinities for the substrate and cofactor. The equilibrium between these two protein states in vitro can be mediated by ionic strength. Figure 5 summarizes the kinetic and

0

0.05 0.10 0.15 0.20 0.25 NADH/(NADH+NAD+)

FIG. 5. Dependence of rate of lactic acid oxidation, apparent K,,, of lactic acid, and protein aggregation state of lobster tail lactate dehydrogenase on NADH mole-fraction at an intermediate ionic strength. The ionic strength for these determinations was 0.55 by adjusting a 0.1 M Tris solution with ammonium sulfate. The velocity measurements plotted as a function of NADH mole-fraction were obtained from individual rate profile using the same protein concentration in each determination; the initial concentrations of lactate and NAD+ were 40 mu and 0.93 mu, respectively. The apparent K,,, values were derived from Table I. The apparent V,, extrapolated to saturating lactic acid concentration and at 0.93 rn~ NAD+ was 10 times greater when determined in low ionic strength solutions (0.05 M Tris, pH 8.9) as compared to high ionic strength solution (0.05 M Tris, 1.1 M ammonium sulfate, pH 8.9). Therefore as in the case of Fig. 1, one would expect a 35-fold increase in velocity due exclusively to enhanced lactate binding; however, a somewhat different value is observed due to the combined effects of enhanced lactate and NAD’ binding at high ionic and a depressed apparent V,,,, at high ionic strength.

LACTIC

ACID

OXIDATION

BY Homarus

physical properties of lobster tail LDH in solutions of intermediate ionic strength. At low levels of NADH, the enzymatic reaction proceeds with a relatively slow finite rate. As the NADH mole-fraction is increased, so is this velocity until a maximum is reached. The velocity then decreases with further addition of NADH due to significant back-reaction from product build-up. The apparent K,,, for lactic acid decreases from a value of 0.32 M to a value of 0.095 M as the NADH mole-fraction is increased. Further production of the NADH results in an increase in the apparent K,. This change is paralleled by variations in the aggregation state of the protein. The ratios of tetramers to dimers can be determined from sedimentation coefficients at various NADH mole-fractions (Table II). Evidence has been presented which indicates that s~,~ values and molecular weights have similar dependencies on ionic strength (3); therefore, weighted-average molecular weights for these protein mixtures can be extrapolated from the sm,w measurements. In addition, active monomers have not been observed. From this, ratios of tetramers to dimers can be obtained if only tetramers and dimers are present under these reaction conditions. With no NADH present, the ratio of tetramers to dimers is 2.5. As NADH increases, this ratio is reduced until a minimum value of 0.5 is reached. Further increases in the NADH mole-fraction results in the establishment of the initial aggregation state of the protein. There are three alternative mechanisms which might apply to this LDH catalyzed oxidation of lactate. First, that purely allosteric mechanism proposed by Kaloustian et al. (2) is applicable by itself but only if one ignores the ultracentrifugal data. Second, the existence of a stable trimer at intermediate ionic strengths with the kinetic properties previously reported (2) and either the ability to undergo reversible changes in conformation in response to NADH mole-fraction or ability to undergo reversible changes in aggregation state (dissociation to a more active dimer) in response to NADH mole-fraction (remember ~20,~at intermediate ionic strength is dependent upon the NADH mole-fraction).

americanus

LACTATE

DEHYDROGENASE

671

Third, given an equilibrium between a dimer and tetramer at intermediate ionic strength, a conformational change, in response to NADH mole-fraction, in the tetramer (without a change in the equilibrium between tetramer and dimer) could also lead to time-course progress curves possessing a lag period and changes in SZO,~. It is our opinion that the simplest mechanism that explains ah the data is the one proposed involving a dissociation of the inactive tetramer to active dimers in response to NADH mole-fraction. Since an in vitro mechanism has been delineated for lactic acid oxidation by LDH from the tail muscles of the East Coast lobster, one might ask if this mechanism is of physiological importance to the organism. Homarus americanus is at osmotic equilibrium with the surrounding sea water. Thus, one finds an in uiuo ionic strength approximating that of the organism’s surroundings, about 0.55 eq/liter (15). This concentration corresponds to that at which most of the kinetic and physical properties of the protein have been determined and that concentration where the protein exhibits marked sigmoidal time courses. Thus, the ionic strength in the lobster is capable of establishing an equilibrium between the LDH tetramers and dimers that results in an effective increase in the afhnity for lactate over that of the tetramer. This affinity can be further enhanced through fluctuations in the NADH molefraction (Table I). Data on in uiuo levels of NADH and NAD+ indicate an NADH mole-fraction in lobster tail muscles of 0.07. This molar ratio of NAD+ to NADH could further enhance the affinity of lobster tail LDH for the substrate, lactate. Thus, the ionic strength in the lobster coupled with modulations in the NADH mole-fraction is capable of mediating lactic acid oxidation in the tail muscles of the East Coast lobster. Homarus americanus is a salt water arthropod, living in the Atlantic Ocean, off the coast of Maine. It is a benthic organism, feeding off the bottom of the ocean, and is predominantly quiescent in nature. Sudden bursts of activity are exhibited only for defensive purposes as in escaping natural

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predators or in occasional attempts to catch prey. Energy for this activity exhibited by the tail muscles is primarily derived from substrate level phosphorylation. Thus, pyruvate must be converted to lactic acid so that NAD+ can be regenerated to allow glycolysis to continue. Initially, the LDH in the tail muscles of the lobster must function as a pyruvate reductase. However, these organisms are poikilotherms and thus are subject to environmental temperature modulations. These temperatures vary from 8 to 12°C. As a result of these low physiological temperatures, the affinity of the tail enzyme for pyruvate is markedly enhanced and, as a result, the protein is severely inhibited by high levels of pyruvate (4). The tail muscle of the organism, though, must be able to catalyze the reduction of pyruvate and it does so by utilizing the muscle-type LDH (lobster leg LDH) as a catalyst for this reaction. Approximately 25% of the lactate dehydrogenase in the tail muscles is composed of this type of subunit. The lactate dehydrogenase in the tail muscle is 75-80s inhibited, probably due to ternary complex, while those tissues composed predominantly of muscle-type enzyme (walking legs) are not dramatically inhibited (Fig. 3). Thus, one would conclude that the heart-type enzyme in the tail (lobster tail LDH) is inhibited for pyruvate reductase activity but the muscle-type LDH (lobster leg LDH) is not. This affords the lobster a mechanism in the tail muscles to produce lactic acid as an end product of glycolysis while the organism is in physically stressing situations. The question arises as to how this accumulated lactic acid is metabolized in the lobster. The metabolism of lactic acid in this organism must occur during the resting state. The lobster has no liver per se but instead a hepatopancreas, which possesses no LDH activity (4). Thus, lactate must be metabolized elsewhere. Ninety percent of

KAPLAN

all the LDH activity is localized in the tail muscles of the organism. It appears possible, then, that lactic acid oxidation occurs in the tail muscles of the lobster and that this unique enzymatic action has physiological importance to the East Coast lobster. Note added in proof. AU molecular weights were determined assuming a constant partial specific volume (V = 0.74 cm3/g). REFERENCES 1, KALOUSTIAN, H. D., AND KAPLAN, N. 0. (1969) J.

Biol. Chem. 244,2891-2901. 2. KALOUSTIAN, H. D., STOLZENBACH, F., EVERSE, J., AND KAPLAN, N. 0. (1969) J. Biol. Chem. 244.2902-2910. 3. EICHNER, R. D., AND KAPLAN, N: 0. (1977) Arch. Biochem. Biophys. 181,490-500. 4. EICHNER, R. D., AND KAPLAN, N. 0. (1977) Arch. B&hem. Biophys. 181,501-507. 5. CIO~~I, M. M., AND KAPLAN, N. 0. (1957) in Methods in Enzymology (Colowick, P., and Kaplan, N. O., eds.), Vol. 3, pp. 890-899, Academic Press, New York. 6. KEMPER, D. L., AND EVERSE, J. (1973) in Methods in Enzymology (Him, C. H. W., and Timasheff, S. N., eds.), Vol. 27D, pp. 67-82, Academic Press, New York. 7. JEDEIKEN, L. A., AND WEINHOUSE, S. (1955) J. Biol. Chem. 213,271-280. 8. JACOBSON, K. B., AND KAPLAN, N. 0. (1957) J.

Biol. Chem. 226,603-613. 9. GLOCK, G. E., AND MCLEAN, P. (1955) B&hem. J. 61,388-390. 10. PONDE, S. V., BHAN, A. K., AND VENKITASUBRAMANIAN, T. A. (1964) Anal. B&hem. 8, 446-462. 11. KANEKO, T., AND FIELD, J. B. (1969) Anal. Bio-

them. 29,193-202. 12. KAPLAN, N. 0. (1964) Brookhauen Symp. Biol. 17, 131-153. 13. EVERSE, J., BERGER, R. L., AND KAPLAN, N. 0. (1970) Science 169, 1236-1238. 14. KAPLAN, N. O., AND EVERSE, J. (1972) in Advances in Enzymatic Regulation (Weber, G., ed.), pp. 323-336, Pergamon Press, New York. 15. FLORKIN, M. (1960) in The Physiology of Crusteatea (Waterman, T. A., ed.), p. 141, Academic Press, New York.