Purification and characterization of a highly unusual tetrameric d -lactate dehydrogenase from the muscle of the giant barnacle, Balanus nubilus Darwin

ARCHIVES OF BIOCHEMISTRY AND BIOPIWXC~ Vol. 166, No. 2, March, pp. 266-274, 1978

Purification and Characterization of a Highly Unusual Tetrameric DLactate Dehydrogenase from the Muscle of the Giant Barnacle, Balanus nubilus Darwin W. ROSS ELLINGTON

AND GEORGE

Department of Chemistry, Pomona College, Clnremord,

L. LONG California

91711

Received June 27, 1977; revised September 6, 1977 n-Lactate dehydrogenase from the depressor muscle of the giant barnacle, Balunus nubilus Darwin, was purified to homogeneity. The molecular weight of this enzyme, as judged by meniscus depletion sedimentation equilibrium and gel filtration, corresponds to a tetrameric subunit organization unlike the n-lactate dehydrogenases from the horeseshoe crab, Limulus polyphemus. and the polychaete, Nerei.s kens, which are dimeric. It is concluded thet substrate stereospecificity and the degree of subunit organization are two independent parameters in the evolution of lactate dehydrogenases. The amino acid composition of B. nubilus n-lactate dehydrogenase shows general similarities to both the Limulus enzyme and the n-lactate dehydrogenase from the lobster, Homarus americanus, except for an unusually high cysteine content (10 residues per subunit). The isoelectric point of the barnacle enzyme is 5.0. B. nub&s Dlactate dehydrogenase is clearly a muscle-type enzyme, as it displays very little substrate inhibition at high pyruvate concentrations. The catalytic properties of this enzyme, including high reactivity with a-ketobutyrate and a-hydroxybutyrate, lowered pH optimum (7.5) for lactate oxidation, and relative insensitivity to oxamate, also set it apart from other animal n-lactate dehydrogenases.

In 1968, Long and Kaplan (1) reported the existence of n-specific diphosphopyridine nucleotide-linked lactate dehydrogenases (EC 1.1.1.28) in the animal kingdom. Independent of Long and Kaplan, Michejda et al. (2) discovered the existence of a n-lactate dehydrogenase in tissue extracts of the land snail, Helix pomatiu. These n-lactate dehydrogenases were found to be distributed in well-defined taxonomic units within the arthropods, annelids, and mollusks (3). Detailed catalytic and physicochemical studies of the n-lactate dehydrogenases from the horseshoe crab, Limulus polyphemus, and the polychaete, Nereis k-ens, revealed that these enzymes are dimeric, with molecular weights approximately one-half (70,000) those of the tetrameric, vertebrate L-lactate dehydrogenases (4). Invertebrate L-lactate dehydrogenases have been demonstrated to be tetrameric, including those of the brine shrimp, Artemia salina (51, the crayfish, Or-cone&es Zimnosus (61, and the fruit fly,

Drosophila (7). The L-lactate dehydrogenase from the tail muscle of the lobster, Homarus americanus, can exist in either dimeric or tetrameric forms depending on ionic conditions (8). Several studies suggest that not all invertebrate n-lactate dehydrogenases are dimeric. The molecular weights, as judged by analytical gel filtration, for crude preparations of lactate dehydrogenases from the barnacle, BaL anus nubilus, and the land snail, Helix uspersa, are around 140,000 (9, 10). We have purified to homogeneity and partially characterized the n-lactate dehydrogenase (NAD linked) from the depressor muscle of the giant barnacle, Balanus nubilus Darwin. Molecular weight determinations of this enzyme indicate that it is tetrameric at a wide range of ionic strengths. Several catalytic properties of B. nubilus n-lactate dehydrogenase indicate that this enzyme may be more closely aligned with invertebrate L-lactate dehydrogenases than with other n-lactate de265

0003-9861/78/1862-0265$02.00/O Copyright 8 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

266

ELLINGTON

hydrogenases. The results presented herein tend to support the idea that the degree of subunit organization and the substrate stereospecificity are two independent parameters in the evolution of lactate dehydrogenases. EXPERIMENTAL PROCEDURES Determination of lactate dehydrogenase activity. Lactate dehydrogenase activity was routinely assayed in an assay system consisting of 50.0 rnM potassium phosphate buffer (pH 7.51, 0.14 mM NADH, and 6.7 mM sodium pyruvate. Activity in the direction of lactate oxidation was measured in an assay system consisting of 50.0 mM potassium phosphate buffer (pH 7.51, 1.4 mM NAD+, and 67 mM lithium n-lactate. Kinetic experiments were performed in a Gilford 2400 recording spectrophotometer. Thermoequilibration was rigorously maintained at 25°C. One unit of enzyme activity is defined as that amount of enzyme which elicits a change in absorbance at 340 nm of 1.0 unitlmin (l.O-cm light path) in the above assay system. (This represents the oxidation of 0.40 pmol of NADH/GO s in the assay mixture.) Protein content of partially purified extracts was determined by the method of Warburg and Christian (11). Protein determinations on purified enzyme were performed by the method of Lowry et al. (12). Electrophoresis. Starch gel electrophoresis was performed on enzyme extracts by the method of Fine and Costello (13) at pH 7.0. Gel slices were stained for lactate dehydrogenase activity and for general protein. Polyacrylamide disc electrophoresis was performed on purified enzyme preparations according to the method outlined by Gabriel (14) using a Tris/barbital buffer system. Under these conditions, the pH of the separating gel was 8.0. Analytical gel filtration. Molecular weight determinations on Sephadex G-100 were performed by the method of Andrews (151. All operations were carried out at 3°C using an elution buffer of 0.05 M potassium phosphate (pH 7.0) containing 14.0 mM 2-mercaptoethanol. Markers used were as follows: bovine liver glutamate dehydrogenase (250,000 M,), beef heart lactate dehydrogenase (140,000), chicken muscle triose phosphate dehydrogenase (140,000), yeast hexokinase (102,000), pig heart malate dehydrogenase (67,000), and ovalbumin (45,000). Ultracentrifugal analyses. Molecular weights of native and acid-dissociated proteins were determined with a Beckman Model E analytical ultracentrifuge using a double-sectored cell according to the method of Yphantis (16). Initial enzyme concentrations were 100-200 pg/ml in 0.05 M potassium phosphate buffer (pH 7.0) containing 1.4 mxe 2-mercaptoethanol. Runs with native enzyme were conducted at 20°C and lasted for 18-24 h. Acid dissociation of

AND LONG the enzyme was produced by dialysis against 0.01 N HCl containing 1.4 mM 2-mercaptoethanol. Runs were performed at 6°C for 18 h. Data from ultracentrifugation runs were treated as described by Long and Kaplan (41, assuming a partial specific volume of 0.74. Isoelectric focusing. Isoelectric focusing of purified B. nubilus n-lactate dehydrogenase were performed in a 110-ml LKB column according to the method of Haglund (17). Columns of 1% LKB Ampholine solutions (pH 3.5-10 or 4-6) were stabilized by a lo-layered sucrose gradient containing 14.0 mM 2-mercaptoethanol. Focusing runs were performed at 4°C with an initial voltage of 500 V (~3 W). Runs usually lasted 48 h. After attainment of a constant current, 1.5-ml fractions were removed and assayed for enzyme activity and pH. Amino acid analyses. Amino acid analyses of hydrolysates of B. nubilus n-lactate dehydrogenase were done by a single-column method on a Beckman amino acid analyzer (Model 119B) as described by Taylor and Oxley (18). Hydrolyses were carried out in vocuo for 24, 48, and 72 h in constantly boiling HCl at 105°C. Cysteine was determined aRer performate oxidation by the method of Hirs (19). All values were normalized to the number of residues per subunit of 36,000 molecular weight. Materials. Specimens of B . nubilus were collected at the municipal pier in Monterey, California. Depressor muscle was removed by dissection, washed in seawater, and frozen at -20°C. Tissues maintained at -20°C retained high levels of lactate dehydrogenase activity for extended time periods. n-Lactic acid was purchased from Calbiochem, La Jolla, California. DEAE-cellulose, Sephadex, 2mercaptoethanol, enzyme-grade ammonium sulfate, hydrolyzed starch, pyruvate, NAD+, NADH o-hydroxybutyrate, a-ketobutyrate, oxamate, and oxalate were purchased from Sigma Chemical Co., St. Louis, Missouri. Beef liver glutamate dehydrogenase, beef heart lactate dehydrogenase, yeast hexokinase, pig heart malate dehydrogenase, and ovalbumin were also from Sigma. We thank C.-Y. Lee for the gift of [8(6-aminohexyl)-5’-AMP]-Sepharose. PURIFICATION OF B. nubilus MUSCLE n-LACTATE DEHYDROGENASE All preparative procedures were carried out in the cold (3°C) with buffers containing 14.0 mM 2mercaptoethanol. Step 1. Cruo!e extract. Three hundred grams of freshly thawed B. nubilus depressor muscle was homogenized in 3 vol of 0.05 M potassium phosphate buffer (pH 7.0) using two 15-s bursts of a Waring Blendor. The homogenate was centrifuged for 2i min at 10,000 rpm in a GSA head of an RC-2B refrigerated centrifuge. The sediment was discarded. The supematant was assayed for pyruvate

BARNACLE

MUSCLE

n-LACTATE

reducing ability and protein content as described under Experimental Procedures. Step 2. Ammonium sulfate fractionation. Powdered ammonium sulfate was added to the supematant with stirring over a period of 30 min until the solution was 45% saturated. Percentage saturation was determined by Table I in Ref. (20) and no correction for temperature was made. The solution was stirred for an additional 30 min and then centrifuged as above. The supernatant was brought to 65% saturation, stirred for 30 min, and then centrifuged. The pellet was resuspended in a minimal volume of extraction buffer and dialyzed against 2 liters of extraction buffer. At the end of 2 h, the buffer was changed and dialysis was continued for an additional 4 h. Step 3. Sephadex G-200 chromatography. The above sample was clarified by centrifugation and then was applied by layering to a Sephadex G-200 column (4.4 x 108 cm, 1640 ml) which had been preequilibrated with 0.05 M potassium phosphate buffer (pH 7.0). Fractions were monitored for enzyme activity and protein. The ratio of the column void volume over the elution volume of B. nubilus n-lactate dehydrogenase was 0.68. Fractions containing enzyme activity were pooled (total volume = 275 ml) and dialyzed against 2 liters of 0.01 M potassium phosphate buffer (pH 7.0) for 2 h, followed by a buffer change and dialysis for an additional 4 h. Step 4. DEAE-cellulose-22 chromatography. The above sample was applied to a DEAE-cellulose-22 column (4.4 x 32 cm, 500 ml) which had been preequilibrated with 0.01 M potassium phosphate buffer (pH 7.0). A linear gradient in buffer from 0 to 0.3 M KC1 (total volume = 2 liters) was started. The enzyme was eluted as a single peak of activity at 0.14 M KCl. Fractions containing enzyme activity were pooled and dialyzed against 2 liters of 0.01 M potassium phosphate buffer (pH 7.0) for 2 and then 4 h. Step 5. Afinity chromatography. The above sample (total volume = 196 ml) was applied slowly (21 ml/h) to a [8(6-aminohexyll-5’-AMP]-Sepharose column (1.6 x 12.4 cm, 25 ml) which had been preequilibrated with 0.01 M potassium phosphate buffer (pH 7.0). The column was then flushed with eluting buffer until the absorbance of the effluent at 280 nm approached zero. Enzyme was eluted by passing one column volume of 0.2 mM reduced NAD-pyruvate adduct in running buffer through the column. The reduced NAD-pyruvate adduct was synthesized and purified according to the procedure of Everse et al. (21). In some cases, a linear gradient of adduct (0 to 0.2 mM) was used to elute the enzyme. Figure 1 is a typical elution profile when a gradient was used. The large increase in absorbance at 280 nm is due to the adduct. Fractions containing enzyme

DEHYDROGENASE 30

-

20 EU/ml *--a

-

267 1.5

:

1.0 A

280 .-. ,0.5

IO -

40

00 Fraction

FIG. 1. Elution profile for the affinity chromatography step in the purification of B. nubilus Dlactate dehydrogenase. Enzyme was eluted with a linear gradient of reduced NAD-pyruvate adduct (0 to 0.2 mM). Enzyme activity (enzyme units per milliliter) and 280-nm absorption are plotted against fraction number. The high absorbance (280 nm) accompanying and following the enzyme elution is due to the reduced NAD-pyruvate addict. activity were pooled, brought to 75% saturated ammonium sulfate, stirred for 45 min, and then centrifuged at 15,000 rpm in an SS-34 head of an RC-2B centrifuge. The resulting pellet was resuspended with a minimal volume of extraction buffer and dialyzed for 4 h against 250 ml of the same. Purified preparations were stored at 3°C and showed a 510% loss in activity per month of storage. RESULTS

Purification of B. nubilus muscle D-lactate dehydrogenase. B. nubilus n-lactate

dehydrogenase was purified 662-fold with a final specific activity of 516 e.u./mg of protein and a 32% recovery of enzyme activity. A summary of the purification statistics for a typical preparation of enzyme is listed in Table I. It is evident that the gel filtration and ion-exchange steps afforded the greatest purification. The final affinity chromatography step did not result in a substantial increase in specific activity, but this step removed several minor protein contaminants. The resulting enzyme preparation was judged homogeneous by electrophoretic criteria and by its behavior in the analytical ultracentrifuge. B. nubilus lactate dehydrogenase was electrophoretically homogeneous on both starch and polyacrylamide gel electrophoretic systems. Figure 2 is a photograph of disc gels stained for lactate

268

ELLINGTON

AND LONG

TABLE SUMMARY

OF

PIJRIFICATION

STATISTICS

I

FOR A TYPICAL PUIUFICATION DEHYDROGENME

SkP

Volume

Total e.u.

Initial extract 45% Ammonium sulfate supernatant 65% Ammonium sulfate pellet aRer dialysis Sephadex G-200 DEAE-cellulose-22 AMP-Sepharose

980 1,025

18,OOW 16,555

(ml)

OF

Total protein

Specific activity

Purification (n-fold)

Percentage recovery 100 91

20.8 c 12.9 g

0.8 1.3

1.0 1.7

9.9

12.6

a4

30.0 443.0 516.0

38.5 567.9 661.5

64 49 32

62

15,113

1.5 g

275 196 7.3

11,550 8,771 5,800

385.0 mg 19.8 mg 11.2 mg

a Protein and enzyme activities were determined

B. nubilus MUSCLE D-LACTATE

as described in Experimental

Procedures.

Physicochemical properties of B. nubilus D-lactate dehydrogenase. B. nubilus

FIG. 2. Photograph of the polyacrylamide disc gels of purified B. nubilus lactate dehydrogenaae. Gels A and B were stained for lactate dehydrogenase activity and general protein, respectively. Electrophoresie was performed in a TrYbarbital system in the cold at 2 mA per gel for 90 min. The arrow indicates the position of the tracking dye front.

dehydrogenase activity and general protein. The disc gels are clearly homogeneous with respect to the enzyme.

n-lactate dehydrogenase is found as a single isoenzymic form in muscle tissue, as judged by its migration in several electrophoretic systems. The electrophoretic mobility of this enzyme was relatively high on starch gel at pH 7.0 (Fig. 3). Its electrophoretic mobility was intermediate to that of the lactate dehydrogenases from the muscle of the horeseshoe crab, L. polyphemus, and the tail of the lobster, H. americanus. Isoelectric focusing of B. nubilus n-lactate dehydrogenase yielded an isoelectric point of 5.0. For comparative purposes L. polyphemus n-lactate dehydrogenase was subjected to isoelectric focusing over a number of pH ranges. These determinations gave an isoelectric point of 4.7. The isoelectric point of B. nubilus n-lactate dehydrogenase is consistent with its electrophoretic mobility with respect to H. americanus L-lactate dehydrogenase, which has an isoelectric point of 5.8 (8). The barnacle enzyme was quite unstable at its isoelectric point, as evidenced by only a lo-1696 recovery of the total amount of enzyme activity subjected to focusing. Molecular weight determinations for B. nubilus n-lactate dehydrogenase by ultracentrifugation and gel filtration are 142,000 and 160,000, respectively. These values are consistent with a tetrameric structure for the enzyme. Meniscus depletion sedimentation equilibrium molecular weight determination of acid-dissociated enzyme yields a value of 36,000, which is

BARNACLE

/

MUSCLE

D-LACTATE

+

0 6 ---

9

ABC

D

J FIG. 3 Starch gel electrophorogram of purified B. nubilus n-lactate dehydrogenase (A), H. americanus L-lactate dehydrogenase (B), L. polyphemus n-lactate dehydrogenase (Cl, and chicken heart Llactate dehydrogenase (D). Electrophoresis was conducted for 15.5 h at a voltage gradient of 10 V/cm. Gel slices were stained for lactate dehydrogenase activity.

MOLECULAR

WEIGHTS

OF B. nubilus

Method

roughly 25% of the weight of the native enzyme. The molecular tieight of B. nubiZus n-lactate dehydrogenase at high ionic conditions (CL = 1.6) is also consistent with a tetrameric subunit organization (M, = 154,000). The values obtained at high ionic strength, and particularly at low pH, must be taken with caution since no consideration of possible primary and secondary charge effects has been made. In all sedimentation equilibrium experiments, the protein appeared to be homogeneous as judged by the existence of a single, straight line when the log of protein concentration is plotted vs the radius of rotation squared. The system appeared to be at equilibrium since no differences were observed between the 18- and 24-h measurements. On Sephadex G-100 the barnacle enzyme coeluted with the tetrameric beef heart L-lactate dehydrogenase, but eluted much earlier than the n-lactate dehydrogenase from L. polyphemus, which has been demonstrated to be dimerit (4). The results of the molecular weight determinations are summarized in Table II. The amino acid composition of B. nubiZus n-lactate dehydrogenase is listed in Table III. The amino acid composition of the barnacle enzyme shows no pronounced differences from the compositions of L. polyphemus (4) and H. americanus (18) lactate dehydrogenases except for its

TABLE II D-LACTATE DEHYDROGENASE AND GEL FILTRATION and buffer

AS DETERMINED

system

(1) Meniscus depletion sedimentation equilibrium phate buffer with 1.4 mM 2-mercaptoethanol, 17,250 rpm (2) Meniscus depletion sedimentation equilibrium phate buffer with 1.4 mM 2-mercaptoethanol sulfate, pH = 7.0) at a speed of 19,160 rpm (3) Meniscus depletion sedimentation equilibrium 2.3) at a speed of 33,450 rpm Gil00 gel filtration (50 mM potassium (4) Sephadex 14 mM 2-mercantoethanol. DH = 7.01

269

DEHYDROGENASE

(50 mM potassium phospH = 7.0) at a speed of (50 mM potassium phosand 0.5 M ammonium (acid

dissociated,

phosphate

buffer

pH with

BY ULTBACENTRIFIJGATION Molecular

weight

142,000

-t 3,000’

154,OOOb =

36,000 160,000

2 16,000

0 Fringe displacements were measured using a comparator (Nikon Model 6C Shadow graph). The log of protein concentration (y-coordinate displacement) was routinely plotted vs the comparator x coordinate, rather than against the radius of rotation squared (91, as described by Yphantis (16). The difference between the two methods was found to be 1%. b Corrected for density effects due to the high salt concentration.

270

ELLINGTON AND LONG TABLE HI

AMINOACIDCOMPOSITION OFB. nubilus ~-LACTATE DEHYDZWGENABP

Residue CYS

Asx Met Th+ Se+ Glx pro

GUY Ala Val Ile LeU Tyr Phe Try His Arg Lvs

“gIesJr 10.0 25.7 2.0 21.5 13.6 31.7 17.3 26.6 26.1 20.3 22.1 32.6 7.1 15.3 14.0 16.3 21.6

0 Values are calculated as the number of residues per subunit (M, = 36,000). b Values extrapolated from 24, 48, and 72 h to zero time.

higher cysteine content (10 residues per 36,000 M,). Histidine content for B. nubiZus n-lactate dehydrogenase is also much higher than for the other two invertebrate lactate dehydrogenases. In addition, the serine content of the barnacle enzyme is less than that of threonine, which is not the case for the enzymes from L. polyphemus and H. americanus. All three lactate dehydrogenases have remarkably similar amounts of threonine, glycine, alanine, isoleucine, and leucine. Catalytic properties of B. nubilus D-kctate dehydrogenase. Catalytic constants of B. nubilus n-lactate dehydrogenase for

substrates and substrate analogs are listed in Table IV. It is evident that this enzyme has a high capacity for the utilization of a-ketobutyrate and a-hydroxybutyrate. The apparent K,s (3.6 and 67 mM, respectively) are similar to those of the normal physiological substrates, but Vs are somewhat lower. The apparent K, for pyruvate reduction under the stated pH conditions was the high value of 1.5 mM (Table Iv>. B. nubilus n-lactate dehydrogenase behaves as a typical muscle-type enzyme in showing very little substrate inhibition by

pyruvate. The percentages of maximal velocity at 20 and 40 mM pyruvate were 95 and 89%, respectively. There was.8 nearly 20-fold difference (18.2) in maximal rates of pyruvate reduction over lactate oxidation (Table IV). The enzyme has been shown to lack the ability to oxidize or lactate (3). The pH optimum for B. nubilus n-lactate dehydrogenase in the direction of pyruvate reduction is 6.5, which is typical of other lactate dehydrogenases (Fig. 4). The pH optimum for lactate oxidation was 7.5. At pHs greater than 8.0, there was a large decrease in enzyme activity. Since most lactate dehydrogenases show pH optima for lactate oxidation in the alkaline range, the possibility that alkaline conditions. of the assay system resulted in the inactivation of B. nubilus n-lactate dehydrogenase was investigated. Incubation of this enzyme at pH 3.4 and 9.4 results in rapid inactivation of the enzyme, which progresses with further incubation (Table V). The net effect of this inactivation of the enzyme at alkaline pHs is to shifi the pH optimum for lactate oxidation to a more neutral value. Inhibition of B. nubilus n-lactate dehydrogenase by oxamate is competitive with respect to pyruvate (Figs. 5a and b). The sensitivity of this lactate dehydrogenase TABLE IV CATALYTIC CONSTANTS FOR B.

nubilus

D-LACTATE

DEHYDBOOENABP

Substrate

K,

VW

Pyruvateb a-Ketobutyrateb

1.5 x 10-a 3.6 x 1O-3

1.6

liLactat& nL-cr-Hydroxybutyratee

4.7 x 10-Z 6.7 x 10-Z

NADHd NAD+e

1.0 x 10-5 1.9 x lo-’

Appa;;t

1.3 18.2

a Apparent K,s and Vs were determined graphically by double-reciprocal and/or Eadie-Hofstee plots. Ratios of V/V refer to the apparent maximum velocities for the two substrates in each group. b 0.14 mM NADH. c 1.4 rnro NAD+. d 6.7 mr.rpyruvate. e 67.0 rnM n-lactate.

BARNACLE

MUSCLE

n-LACTATE

271

DEHYDROGENASE

Ki as extrapolated from the Dixon yields a value of 27.5 mM.

100

plot

DISCUSSION

%

The discovery of n-specific NAD-linked lactate dehydrogenases in animals (1, 2)

max. act.

50

0 7 8 9 PH FIG. 4. The effect of pH on the activity of B. nubilus n-lactate dehydrogenase in the direction of pyruvate reduction (triangles) and lactate oxidation (circles). Enzyme activities are calculated as percentages of maximal activity. Buffer in all cases consisted of 0.05 M potassium phosphate. 5

6

TABLE

V STABILITY OF PURIFIED B. nubilus D-LACTATE DEHYDROGENABE AT ALKALINE ~HSQ Buffer pH Relative enzyme activity (I) 6.5 a.4 9.4

Initial

90 min

100 63 40

106 40 7

b

D A small amount of purified enzyme (-16 pg) was diluted in 1.0 ml of 0.05 M Trie/HCl buffer of various pHs. Enzyme activity was determined initially within a few minutes after dilution and at the end of 90 min. Assay conditions were at pH 7.6. Enzyme was incubated on ice.

to oxamate, however, is not particularly high, as the apparent Ki (130 mM) is nearly 100 times higher than the apparent K, for pyruvate. Oxalate is an effective inhibitor of B. nubilus n-lactate dehydrogenase (Figs. 6a and b). The oxalate inhibition is clearly competitive with respect to lactate, as indicated by the double-reciprocal plot (Fig. 6a). The Dixon plot (Fig. 6b) is distinctly nonlinear at the higher oxalate concentrations. The value of the apparent

-IjO

- Ii0

40

io

100 ’

Oxornate (mM1 FIG. 5. Oxamate inhihitbn of B. nub&s n-lactate dehydrogenaee. (a) Double-reciprocal plot of l/ velocity ver5us l/pyruvate concentration (millimolar) in the presence of no oxamate (A), 30 mre oxamate (B), 60 mM oxamate (0, and SO rniu oxamate (D). (b) Dixon plot of l/velocity versus oxamate concentration at 2.0 mre pyruvate (A), 1.5 mru pyruvate (B), 1.0 rnrd pyruvate (Cl, and 0.5 mM pyruvate (D).

272

ELLINGTON

AND LONG

this observation was that n-lactate dehydrogenases are a distinct group of dimeric enzymes and that there is a correlation between substrate stereospecificity and the degree of subunit organization. Our results clearly indicate that the molecular weight ofB. nubilus n-lactate dehydrogenase, as judged by sedimentation equilibrium and gel filtration, corresponds to that of a tetramer. The enzyme elutes distinctly earlier on Sephadex G-100 than L. polyphemus n-lactate dehydrogenase and also another dimeric enzyme, pig heart malate dehydrognease. The molecular weight of B. nubilus n-lactate dehydrogenase is similar to the values of 140,000 to 145,000 for vertebrate L-lactate dehydrogenases (22). It is of interest that the molecular weight .i .i of the barnacle enzyme does not appear to be influenced by the ionic strength of the medium. Unlike the L-lactate dehydrogenb ase from another crustacean, H. americanus (8), there was no decrease in the I apparent molecular weight of B. nubilus v n-lactate dehydrogenase when the determination was made at a high ionic strength (p ~1.6). Ki = 28mM n-Lactate dehydrogenases from microorganisms show some diversity in molecular weights, with values ranging from 65,000 and 68,000 for Leuconostoc mesenteroides (23) and Lactobacillus plantarum (241, respectively, to 133,000 for Escherichiu coli (25). The slime mold, Polysphondylium palidum, appears to have a A& tetrameric n-lactate dehydrogenase (23). It appears that substrate stereospecificity -20 46 ;0 and enzyme quaternary structure are two independent variables in the evolution of Oxalate (mM) FIG. 6. Oxalateinhibition ofB. nubilus D-lactate lactate dehydrogenase. The similarities in amino acid contents dehydrogenase. (a)Double-reciprocal plot of l/velocof the lactate dehydrogenases from B. nuity versusl/D-lactateconcentration(millimolar) in bilus, L. polyphemus, and H. americanus the presence of no oxalate (A), 10 mM oxalate (B), 20 mMoxalate (C), and40 mMoxalate(D). (b) Dixon occur primarily in the aliphatic amino plot of l/velocity versus oxalate concentrationat acids: These amino acids serve primarily 5 mM D-lactate(A), 10 mM o-lactate (B), 20 mM a nonspecific structural role and would n-lactate CC!), and 30mMD-lactate(D). not be expected to vary significantly from one protein to another. Differences are was later accompanied by the observation evident, however, in the catalytically that n-lactate dehydrogenases from two functional residues such as histidine, serdistantly related, invertebrate species ine, methionine, and cysteine. L. polyphewere dimeric in structure (4). The temptmus n-lactate dehydrogenase appears to ing conclusion that could be drawn from have a high amount of acidic residues,

BARNACLE

MUSCLE

D-LACTATE

correlating well with its low isoelectric point of 4.7 and its highly anodal electrophoretic mobility on starch gel at pH 7.0. The high cysteine content of B. nubilus Dlactate dehydrogenase is of interest. It is not known whether there are any disulfide bridges in this enzyme, although they ap pear to be absent in vertebrate L-lactate dehydrogenases (22). Investigation of the catalytic properties of B . nubilus n-lactate dehydrogenase revealed some striking features. The enzyme has a relatively high capacity for the utilization of cY-ketobutyrate and cy-hydroxybutyrate. The maximal velocities with these compounds approach the values for the normal physiological substrates. Gleason et al. (9) have also observed that crude, cell-free preparations of B. nubilus muscle have a high capacity for a-ketobutyrate reduction. In addition, these workers observed that the n-lactate dehydrogenases from L. polyphemus and the tarantula reduced cw-ketobutyrate at a much lower rate (9). GLactate dehydrogenase from the brine shrimp, Artemia salina, utilizes (Yketobutyrate at a rate similar to that observed for B. nubilus n-lactate dehydrogenase (5). There is considerable diversity in the relative abilities of the heart and muscle isoenzymes of vertebrate Glactate dehydrogenases to utilize a-ketobutyrate and cY-hydroxybutyrate (26, 27). The depressor muscle of B. nubilus is a typical fast, striated muscle (28) with no apparent myoglobin content. It is therefore not surprising that the n-lactate dehydrogenase from this muscle displays very little substrate inhibition at high pyruvate concentrations. The high ratio of maximal velocities for pyruvate reduction/ lactate oxidation is similar to values for a number of M, vertebrate L-lactate dehydrogenases and to the value of 38 for leg muscle L-lactate dehydrogenase of the lobster, H. americanus (8). Although muscle enzymes, the n-lactate dehydrogenases from the horseshoe crab and tarantula have ratios which are considerably lower (9). The pH optimum for lactate oxidation of B. nubilus n-lactate dehydrogenase approaches neutrality, as is the case for L-

DEHYDROGENASE

273

lactate dehydrogenases from lobster tail and leg muscle (8) and the enzyme from the bacterium, Lactobadlus plantarum (29), but is not true for most other L-lactate dehydrogenases, which have alkaline pH optima. L-Lactate dehydrogenase from the fruit fly, Drosophila, also has a low pH optimum (7.51, and this has been reported to be related to the instability of this enzyme under alkaline conditions (7). The observation that B. nubilus &lactate dehydrogenase is rapidly inactivated at alkaline pHs indicates that a similar process is occurring for this enzyme. Preliminary electrophoretic evidence also suggests that alkaline pHs result in a dissociation of the protein into its constituent subunits and possibly smaller fragments. It is of interest that the animal species having lactate dehydrogenases which show this neutral pH optimum for lactate oxidation all belong to the same subphylum (Mandibulata) of the phylum Arthropoda. Oxalate and oxamate inhibit B . nubilus n-lactate dehydrogenase. In the case of oxamate, the inhibition is not pronounced, as the K, is two orders of magnitude greater than the apparent K, for pyruvate. Dennis and Kaplan (29) found that the n-lactate dehydrogenase from Lactobacillus plantarum was insensitive to oxamate inhibition. The n-specific enzymes from L. polyphemus and N. virens, on the other hand, were found to be very sensitive to oxamate inhibition (4). Preliminary experiments in our laboratory have also shown that the lactate dehydrogenases from the lobster, garden snail, and abalone are effectively inhibited by oxamate. The catalytic studies of B. nubilus n-lactate dehydrogenase with oxalate yield an apparent Ki which is an approximation, at best, due to the nonlinearity of the l/u vs inhibitor concentration plot. It is evident that oxalate is an effective inhibitor, as evidenced by its apparent K, being in the same range as that of the substrate apparent K,. The studies of Long and Kaplan (4) on L. polyphemus and N. virens n-lactate dehydrogenases revealed a number of features suggesting homology with animal Llactate dehydrogenases. Taylor and Oxley

274

ELLINGTON

(18) have recently reported the amino acid sequence of the substrate binding peptide and the loop region peptide of the coenzyme binding site for lobster and four vertebrate lactate dehydrogenases. These regions are all remarkably homologous. The results of the above studies and others clearly suggest that all animal lactate dehydrogenases arose from the same locus. This study reveals that the barnacle n-lactate dehydrogenase is homologous with other animal lactate dehydrogenases in several respects. However, the main point we wish to stress is how divergent the enzyme appears to be, by virtue of its tetrameric subunit structure, high sulfhydry1 content, high catalytic effectiveness with cY-ketobutyrate and cY-hydroxybutyrate, low pH optimum for lactate oxidation, and relative insensitivity to oxamate. This point is underscored by evidence showing the involvment of zinc in the enzyme (30): Atomic absorption analyses show the presence of 2 g-atoms of zinc per subunit when the enzyme is protected against sulfhydryl oxidation. Additionally, kinetic studies using zinc chelators show that zinc is required for maximal enzyme activity. ACKNOWLEDGMENTS We thank the staff of the Hopkins Marine Station of the Stanford University for providing bench space for dissection of barnacle specimens. We are also grateful to Dr. S. S. Taylor (University of California, San Diego) for performing amino acid analyses. This study was supported by NIH Grant GM-2286801 to GLL. REFERENCES 1. LONG, G. L., AND KAPLAN, N. 0. (1968) Science 162, 685-686. 2. MICHEJDA, J. W., WALA, R., ZERBE, T., AND TILGNPR, H. (1969) Bull. Sot. Amis Sci. L&t. Poznan 9, 181-191. 3. LONG, G. L. (1976) Comp. Biochem. Physid. 55B,

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