Polynucleotide kinase from rat-liver nuclei. Purification and properties.

A polynucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of deoxyribonucleic acid in the presence of adenosine triphosphate, has been purified 260-fold with a yield of 14% from 0.15 M NaCl extracts of rat liver nuclei. The purified enzyme has a pH optimum of 5.5. The enzyme is reversible inhibited by p-chloromercuribenzoate. The S0.5 value (ligand concentration required for a half-maximal activity) for ATP is 2.5 muM. A bivalent cation is essential for the reaction and S0.5 values for Mg2+, Ca2+ and Mn2+ are 3.3 mM, 4 mM and 0.05 mM respectively. Pyrophosphate remarkable inhibits the activity with I0.5 value (ligand concentration required for a half-maximal inhibition) of 0.2 mM, and sulfate, with I0.5 of 0.5 mM, whereas phosphate weakly inhibits the activity with I0.5 of about 20 mM. An apparent molecular weight of the purified enzyme is estimated to be 8 X 10(4) by gel filtration on a column of Sephadex G-150, and the Stokes radius of the enzyme molecule is shown to be about 0.36 nm. Sucrose density gradient centrifugation reveals that the enzyme has a sedimentation coefficient of about 4.4 S.     

Purification and physical-chemical properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver

Acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver was purified by protamine sulfate precipitation, ion exchange chromatography on DEAE-A-25 Sephadex, gel filtration on Sephadex G-75, amethopterin-AH-Sepharose 4B affinity chromatography, and finally, gel filtration on Sephadex G-100. The enzyme preparation was homogeneous as judged by ultracentrifugation studies, SDS-polyacrylamide gel electrophoresis and gel filtration. The N-terminal amino acid was detected to be histidine and the complete amino acid composition is reported. The enzyme contains one disulfide bridge and two cysteine residues/mol monomer. The isoelectric point was estimated to be 4.8. The molecular weight was determined to be 32900 by high-speed sedimentation equilibrium analysis, 33000 by Sephadex G-100 gel filtration and 31600 by SDS-disc gel electrophoresis. The sedimentation coefficient from conventional sedimentation velocity runs was 3.1 S observed by ultraviolet optics. 'Active enzyme centrifugation' showed a sedimentation constant of 5.0 and 4.8 S for the purified enzyme and crude extract from pigeon liver, respectively, indicating that the enzyme forms a dimer under conditions of catalysis. It could be demonstrated that the inhibitor amethopterin was noncompetitive with respect to the acetyl donor and the acetyl acceptor. Acetyl-CoA:arylamine N-acetyltransferase was examined in different organs of pigeon. The enzyme was not inducible by 1,3-phenylenediamine and hexobarbital in vivo.     

Proline iminopeptidase from Bacillus coagulans: purification and enzymatic properties

Proline iminopeptidase [EC 3.4.11.5] was purified about 2,700-fold from cell-free extract of Bacillus coagulans by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose, and hydroxyapatite, and gel filtration on Sephadex G-150. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.3 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid including proline, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminus. Pro-D-amino acid bonds were also susceptible to the enzyme. The enzyme was completely inhibited by p-chloromercuribenzoate (PCMB) and partially by proline but not by metal chelators, diisopropylphosphorofluoridate (DFP), or phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by incubation with 2-mercaptoethanol. These results and the chromatographic profile on PCMB-T-Sepharose suggest that the enzyme is a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0, and the molecular weight of the enzyme was estimated to be 40,000 by gel filtration on Sephadex G-100 and 35,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, indicating that the enzyme exists as a monomer.     

Pipecolic acid biosynthesis in Rhizoctonia leguminicola. II. Saccharopine oxidase: a unique flavin enzyme involved in pipecolic acid biosynthesis

The fungal parasite Rhizoctonia leguminicola produces two indolizidine alkaloids, slaframine and swainsonine, of physiological interest. These alkaloids are biosynthesized from pipecolic acid which in turn is derived from L-lysine in this fungus as shown in the accompanying paper (Wickwire, B.M., Harris, C.M., Harris, T.M., and Broquist, H.P. (1989) J. Biol. Chem. 265, 14742-14747): L-lysine----saccharopine----delta 1----piperideine-6- carboxylate----pipecolate. This paper concerns the discovery, purification, and properties of a flavoenzyme, termed saccharopine oxidase, which carries out the oxidative cleavage of saccharopine as follows: Saccharopine + O2----delta 1-piperidine-6-carboxylate + glutamate + H2O2 The enzyme was purified 2,000-fold to homogeneity (polyacrylamide gel electrophoresis) in 14% yield from R. leguminicola mycelia, and had a native molecular mass of about 45,000 daltons by gel filtration (fast protein liquid chromatography Superose). Evidence for the presence of a flavin in the enzyme was drawn from these considerations: (a) the enzyme, while oxidatively cleaving saccharopine, concomitantly reduces 2,6-dichlorophenolindophenol; (b) the purified enzyme has a fluorescence spectrum typical of flavins; and (c) the enzyme requires oxygen and produces hydrogen peroxide. Good correlation was shown with purified saccharopine oxidase between disappearance of saccharopine with the concomitant appearance of delta 1-piperideine-6-carboxylate plus glutamate. The enzyme has a pH optimum about 6 and a Km for saccharopine of 0.128 mM. The enzyme apparently exists in R. leguminicola to shunt saccharopine, a major lysine metabolite, into a secondary pathway of lysine metabolism leading to pipecolate and subsequently to slaframine and swainsonine.     

Rat kidney L-alpha-hydroxy acid oxidase: isolation of enzyme with one flavine coenzyme per two subunits.

L-alpha-Hydroxy acid oxidase (listed as EC 1.4.3.2, L-amino acid: O2 oxidoreductase) has been purified 100-fold from rat kidney to apparent homogeneity by gel electrophoresis. A subunit molecular weight of 47,500 was found by sodium dodecyl sulfate gel electrophoresis, but in contrast to previous reports, the enzyme has been found to have a molecular weight of ca. 200,000 by Sephadex gel filtration and by dodecyl sulfate gel electrophoresis of the enzyme cross-linked with dimethyl suberimidate. A somewhat higher value was found by sedimentation equilibrium, but a tetrameric structure for the active enzyme is definitely established. The enzyme was found to contain the FMN coenzyme at a concentration of one FMN/102,000 daltons or one flavine/two subunits, a highly unusual finding. This ratio was determined from spectroscopic analysis of the FMN in lyophilized samples of the enzyme and by titration of the coenzyme with the flavine specific enzyme inactivator 2-hydroxy-3-butynoate. The enzyme has the same specific activity as a crystalline sample of the enzyme reported to have twice as much flavine/milligram.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.     

Beta-glucuronidase of rat preputial gland. Crystallization, properties, carbohydrate composition, and subunits.

Rat preputial gland beta-glucuronidase [ED 3.2.1.31] was purified by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G-200 and crystallization. The purified enzyme appeared homogeneous on electrophoresis in polyacrylamide gel, and on analytical ultracentrifugation and had a molecular weight of approximately 320,000, and a sedimentation coefficient of 12S. SDS polyacrylamide gel electrophoresis indicated that the enzyme consisted of subunits with molecular weight of 79,000, so the native enzyme appeared to be a tetramer. The Km with p-nitrophenyl beta-D-glucosiduronic acid as substrate was about 0.53 mM. The enzyme had a single pH optimum at 4.5. The enzyme had a very low content of sulphur-containing amino acid and contained 5.7 per cent carbohydrate, consisting of mannose, glucose, fucose, galactose, and glucosamine in a ratio of 44;9;6;2;41. Sialic acid was not detected in the crystallized enzyme.     

Purification and Characterization of the Bifunctional Enzyme Lysine-Ketoglutarate Reductase-Saccharopine Dehydrogenase from Maize

The first enzyme of the lysine degradation pathway in maize (Zea mays L.), lysine-ketoglutarate reductase, condenses lysine and [alpha]-ketoglutarate into saccharopine using NADPH as a cofactor, whereas the second, saccharopine dehydrogenase, converts saccharopine to [alpha]-aminoadipic-[delta]-semialdehyde and glutamic acid using NAD+ or NADP+ as a cofactor. The reductase and dehydrogenase activities are optimal at pH 7.0 and 9.0, respectively. Both enzyme activities, co-purified on diethylaminoethyl-cellulose and gel filtration columns, were detected on nondenaturing polyacrylamide gels as single bands with identical electrophoretic mobilities and share tissue specificity for the endosperm. The highly purified preparation containing the reductase and dehydrogenase activities showed a single polypeptide band of 125 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native form of the enzyme is a dimer of 260 kD. Limited proteolysis with elastase indicated that lysine-ketoglutarate reductase and saccharopine dehydrogenase from maize endosperm are located in two functionally independent domains of a bifunctional polypeptide.     

Purification and properties of L-lysine-alpha-ketoglutarate reductase from human placenta.

l-Lysine-α-ketoglutarate reductase has been extensively purified from human placenta. The enzyme is active in the formation of saccharopine from l-lysine and α-ketoglutarate and possesses a stringent substrate specificity. Steady-state product inhibition studies indicate the possibility of either of two basic reaction mechanisms. The first is an ordered reaction mechanism in which α-ketoglutarate, l-lysine, and NADPH bind to the enzyme followed by the release of NADP and saccharopine. The second mechanism involves an initial binding of NADPH. This is followed by either the ordered addition of α-ketoglutarate and l-lysine with the occurrence of an E-NADPH-saccharopine dead-end complex or by the random addition of α-ketoglutarate and l-lysine with the formation of an E-NADPH-sac-charopine-l-lysine dead-end complex. No inhibition of the forward reaction or stimulation of the reverse reaction by the addition of ammonium sulfate was found; other investigators, working with other mammalian tissue have reported such effects. A molecular weight estimate of 480,000 for both l-lysine-α-ketoglutarate reductase and saccharopine dehydrogenase was obtained on gel filtration. No indication of separation of the two activites was obtained throughout the purification procedure, and the presence of detergents had no effect on the sedimentation rate in the ultracentrifuge or on the migration rate in gel filtration.     

Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate-Nitrate Reductase of Aspergillus nidulans

The reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans was purified over 200-fold by use of salt fractionation, gel filtration, and ion-exchange chromatography. The purified enzyme was specific for NADPH and catalyzed reduction of nitrate, cytochrome c from isolated mitochondria of Aspergillus, and mammalian cytochrome c. An S(0.725) (20, w) of 7.8 was derived with sucrose density gradient centrifugation, and a Stokes radius of 6.4 nm was derived by gel filtration on Sephadex G-200. From these values, a molecular weight of 197,000 was computed, assuming v = 0.725 cm(3)/g. The spectral properties of the purified enzyme suggested a flavine component was present but revealed no pattern indicative of a hemoprotein. A cytochrome c, similar to the cytochrome c from isolated mitochondria, was found unassociated with the nitrate reductase after ion-exchange chromatography. No NADPH-nitrate reductase activity was detected in isolated mitochondria. Spectrally discernable reduction of the flavine component of the enzyme at 450 nm was noted after reaction with NADPH. This reduction was inhibited by p-chloromercuribenzoate but not by KCN. The addition of nitrate to NADPH reduced enzyme caused a reoxidation of the flavine component via a reaction which was inhibited by KCN but not by p-chloromercuribenzoate. The half-life of the purified enzyme at 37 C was 20 min for NADPH-nitrate reductase and 35 min for NADPH-cytochrome c reductase.     

Purification of a derepressible arylsulfatase from Chlamydomonas reinhardti. Properties of the enzyme in intact cells and in purified state.

Arylsulfatase (aryl-sulfate sulfohdydrolase, EC 3.1.6.1) has been purified from SO4-2-minus-starved cells of Chlamydomonas reinhardti. The enzyme was isolated from acetone-powder extract by (NH4)2SO4 precipitation, Sephadex G-200 filtration and ion-exchange chromatography. Only one fraction of aryl-sulfatase was found. The final preparation was homogenous by the criteria of sedimentation, diffusion and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of about 150 000, estimated by ultracentrifugation and gel filtration, and an isoelectric point of 9.0. The properties of the enzyme as investigated in intact cells and in the purified state were found to be very similar except for the temperature optimum. Imidazole strongly increased the enzyme by increasing the V, but reduced the affinity for the substrate. The enzyme activity was competitively inhibited by borate with a greater affinity for borate than for the substrate. The Chlamydomonas enzyme is a Type I arylsulfatase since it was inhibited by CN-minus, but not SO4-2-minus and phosphate.     

Purification and characterization of a strictly specific beta-D-fucosidase from Aspergillus phoenicis.

Although beta-D-fucosidase (beta-D-fucohydrolase, EC 3.2.1.38) has been isolated from various sources, all those enzymes were associated with a high activity of beta-D-galactosidase and/or beta-D-glucosidase. We have purified a specific beta-D-fucosidase in electrophoretically homogeneous form from crude extracts of Aspergillus phoenicis by polyethyleneglycol 8000-phosphate buffer aqueous two-phase separation, and successive chromatography on DEAE-Sephadex A-50, hydroxyapatite, and Sephadex G-100 columns. The molecular weight of the enzyme was estimated to be 57,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 50,000 to 60,000 by gel filtration on Sephadex G-100. The enzyme showed optimum activity at pH 6.0 and 40 degrees C; it was stable in the pH range 5.5-6.5 and below 35 degrees C. The Km and the Vmax values for pNP-beta -D-fucoside were 2.4 mM, and 12.8 mumol.min-1.mg-1, respectively. The enzyme was strongly inhibited by sulfhydryl group reagents, p-chloromercuribenzoate, n-ethylmaleimide, and iodoacetate. It was also inhibited by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, diethyl pyrocarbonate, and N-bromosuccinimide. Thus, -SH and -COOH groups and histidyl and tryptophyl residues were essential for enzyme activity. The purified beta-D-fucosidase showed high specificity toward p-nitrophenyl-beta-D-fucoside. The enzyme was inhibited by D-fucose and D-fucono-gamma-lactone, but not by D-galactose, D-galactono-gamma-lactone, D-glucose, or D-glucono-gamma-lactone; the latter compounds are specific inhibitors of beta-D-galactosidase and beta-D-glucosidase, respectively. Thus, this enzyme is the most strictly specific beta-D-fucosidase when compared with those previously reported.     

Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain

l-Tryptophan-activating enzyme [l-tryptophan-tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different K(m) and V(max.) values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg(2+), ATP, in any combination.     

Studies on the Pectolytic Enzyme

Pectinesterase was extracted from the pulp of tomato fruit (Lycopersicum esculentum var. Hikari) pericarp with 250 mM potassium phosphate buffer, pH 8.0, and purified about 60 folds by means of ammonium sulfate fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100 column. The enzyme preparation thus obtained was confirmed to be homogeneous state both ultracentrifugationally and disk electrophoretically. The sedimentation coefficient of this enzyme was calculated to be 3.17 S.     

Purification and properties of one component of acid phosphatase produced by Aspergillus niger.

One component, the i form, of acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) produced by Aspergillus niger was purified from the mycelial extract. The purified enzyme was homogenous on Sephadex G-200 gel filtration, disc electrophoresis and heat inactivation. The purified enzyme was studied and the following results were obtained: 1. The enzyme catalyzed the hydrolysis of a wide variety of phosphomonoesters, but not that of bis(p-nitrophenyl)phosphate, adenosine 3',5'-cyclic monophosphate, fructose 1,6-diphosphate, adenosine 5'-diphosphate or adenosine 5'-triphosphate. 2. Fluoride, orthophosphate, arsenate, borate, molybdate and (+)-tartrate acted as inhibitors. This enzyme was inactivated by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, and was not affected by p-chloromercuribenzoate, N-acetylimidazole, p-diazobenzenesulfonic acid and tetranitromethane. From these results, tryptophan was estimated to play an important role in the enzyme activity. 3. The apparent molecular weight was 310000 by Sephadex G-200 gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggested that the molecular weight of the subunit was approximately 89000. 4. The purified enzyme contained 29% carbohydrate consisting of glucosamine, mannose and galactose. The amino acid composition of this enzyme was not specific compared with other known acid phosphatases.     

Purification of Cytosine Deaminase from Pseudomonas aureofaciens

Cytosine deaminase from Pseudomonas aureofaciens was purified about 480-fold by means of ammonium sulfate fractionation, ethyl alcohol fractionation, chromatography on columns of DEAE-Sephadex A–50 and hydroxylapatite and by gel filtration on a Sephadex G–200 column. The enzyme was homogeneous by the criteria of sedimentation and electrophoretic analysis. The molecular weight of the purified enzyme was estimated to be 630,000 by gel filtration and consisted of twelve to sixteen identical subunits having a molecular weight of about 45,000. The enzyme catalyzed the deamination of cytosine and 5-methylcytosine.     

Properties of partially purified saccharopine dehydrogenase from human placenta.

Saccharopine dehydrogenase was previously purified 380-fold from human placenta. The enzyme was shown to catalyze the formation of α-aminoadipic-δ-semialdehyde and glutamate from saccharopine, to have a molecular weight of 480,000 on gel filtration, and not to be separable from l-lysine-α-ketoglutarate reductase. Additional properties of the saccharopine dehydrogenase are now described. The pH optimum for the conversion of saccharopine to glutamate and α-aminoadipic-δ-semialdehyde is 8.5 in Tris-HCl buffer and 8.9 in 2-amino-2-methyl-1,3-propanediol buffer. The specificity of the enzyme for Saccharopine and NAD and the inhibition by glutamate and product analogs were tested. It was found the NADP was the only cofactor that could replace NAD in the enzyme reaction and that several NAD analogs were reaction inhibitors. Glutamate was found to be only moderately effective as an inhibitor. Initial velocity studies revealed that the enzyme has an ordered reaction mechanism. The true K_m values for saccharopine and NAD are 1.15 mm and 0.0645 mm, respectively.     

Purification and properties of a fructose-1,6-diphosphate activated L-lactate dehydrogenase from Staphylococcus epidermidis.

L-(+)-lactate dehydrogenase (LDH) from Staphylococcus epidermidis ATCC 14990 was purified by affinity chromatography. The purified enzyme was specifically activated by fructose-1,6-diphosphate (FDP). The concentration of FDP required for 50% maximal activity was about 0.15 mM. The enzyme activity was inhibited by adenosine diphosphate (ADP) and oxamate. The inhibition by ADP appeared to be competitive with respect to reduced nicotinamide adenine dinucleotide (NADH). The catalytic activity of the LDH for pyruvate reduction exhibited an optimum at pH 5.6. The enzyme is composed of four, probably identical, subunits. Sephadex gel filtration and sedimentation velocity at pH 5.6 Yielded molecular weights of about 130 000 and 126 000, respectively. The molecular weight at pH 6.5 and 7.0 was found to be only about 68 000. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and sedimentation velocity at pH 2.0 or 8.5 revealed monomeric subunits with an approximate molecular weight of 36000. The thermostability of the heat labile enzyme was increased in the presence of FDP, NADH and pyruvate. The purified LDH exhibited an anomalous type of kinetic behavior. Plots of initial velocity vs. different concentrations of pyruvate, NADH or FDP led to saturation curves with intermediary plateau regions. As a consequence of these plateau regions the Hill coefficient alternated between lower and higher n-values. Some distinguishing properties of the S. epidermidis LDH and other LDHs activated by FDP are discussed.     

The Structures of Myocotrienins I. and II,a Novel Class of Ansamycin Antibiotic

Endopolygalacturonase I [EC 3.2.1.15], the major component of endopolygalacturonases causing silver-leaf symptoms, was purified from culture liquids of Stereum purpureum by column chromatographies on CM-52 and Sephadex G-100. The purified enzyme was homogeneous on Polyacrylamide gel electrophoresis and ultracentrifugation. The sedimentation coefficient (S_20,W) was determined to be 3.21 S, and the molecular weight was estimated to be 40,000 by gel filtration, 41,000 by SDS-polyacrylamide gel electrophoresis and 44,000 by sedimentation equilibrium. The enzyme had an isoelectric point of pH 8.5. The optimal pH of the enzyme was 3.5 for trigalacturonic acid, 4.0 for tetragalacturonic acid, and 4.5 for pectic acid. The enzyme was stable in the range of pH 4.0 to 9.0 and up to 70%C for 30 min. The amount of the enzyme which was required to induce silver-leaf symptoms on apple trees was 20 μg/tree.     

Diphosphopyridine nucleotide-linked D-lactate dehydrogenases from the horseshoe crab, Limulus polyphemus and the seaworm, Nereis virens. I. Physical and chemical properties

d-lactate dehydrogenase has been purified from horseshoe crab (Limulus polyphemus) skeletal muscle and the seaworm (Nereis virens). The purified Limulus dehydrogenase was shown to be a dimer, with a molecular weight of approximately 70 000. Sephadex gel filtration and equilibrium sedimentation yield molecular weights of about 80 000 and 70 000 respectively. Acid dissociation yields a molecular weight species of about 35 000. The native enzyme has an s^o₂₀^w of 3.95. Extrapolation of para-hydroxymercuribenzoate inhibition curves to 100% inhibition corresponds to two molecules of para-hydroxymercuribenzoate bound per molecule of enzyme. Studies on the stoichiometric binding of reduced coenzyme show two molecules bound per molecule of enzyme. The number of tryptic peptides has been found to be one-half that expected from the amino acid composition. The electrophoretic pattern of isoenzymic forms can be best interpreted as suggesting that the enzyme is dimeric. In vitro high salt, freeze-thaw hybridizations of the isolated Limulus muscle isoenzymes yield the electrophoretic pattern predicted by a dimeric structure.The physical properties ot Nereis lactate dehydrogenase have been found to be similar to those for the Limulus muscle lactate dehydrogenase.