Characterization of a flavoprotein iodotyrosine deiodinase from bovine thyroid. Flavin nucleotide binding and oxidation-reduction properties.

A stable apoprotein has been prepared from a soluble purified bovine thyroid iodotyrosine deiodinase, previously shown to be an FMN-containing flavoprotein requiring dithionite for enzymatic activities. The apoprotein binds FMN (Ka = 1.47 x 10(8) M-1) with an almost complete restoration of enzymatic activity. It can also bind FAD (Ka = 0.58 x 10(8) M-1) with partial restoration of activity, but does not bind riboflavin. Photoreduction of the holoenzyme in presence of excess of its free cofactor, FMN, supported enzyme activity at a level of 50% of that obtained with dithionite; substituting FAD or riboflavin for FMN produced, respectively, 20 and 11% of the dithionite-supported activity. The oxidation-reduction potential (E1) of the couple semiquinone/fully reduced enzyme is -0.412 V at pH 7 and 25 degrees C. The value (E2) for the oxidized/semiquinone couple is -0.190 V at pH 7 and 25 degrees C. Potentiometric titrations with sodium hydrosulfite suggests that the enzyme is reduced in two successive 1-electron oxidation-reduction steps. Effects of pH on E1 suggest ionization of the protonated flavin with an ionization constant of 5.7 x 10(-7). The highly negative oxidation-reduction potential for the fully reduced enzyme species and the apparent requirement for full reduction for enzymatic activity suggests that in NADPH-mediated microsomal deiodination an NADPH-linked electron carrier of suitably negative midpoint potential is a probable intermediate.     

Purification and characterization of thiamine triphosphatase from bovine brain.

Soluble thiamine triphosphatase (EC 3.6.1.28) of bovine brain has been purified 68,000-fold to an electrophoretically homogeneous state with an overall recovery of 5.5% by hydrophobic chromatography on Toyopearl HW-60, Sephadex G-75 gel filtration, DEAE-Toyopearl 650M chromatography and Blue Sepharose CL-4B chromatography. The enzyme has an absolute specificity among thiamine and nucleoside phosphate esters for thiamine triphosphate and shows no nonspecific phosphatase activities. Thiamine triphosphatase is composed of a single polypeptide chain with molecular mass of 33,900 kDa as estimated by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 8.7 and is dependent on divalent metal ions. Mg2+ has been found to be the most effective among cations tested. A study of the reaction kinetics over a wide range of thiamine triphosphate concentrations has revealed a biphasic saturation curve being described by higher-degree rational polynomials.     

Purification and characterization of a protein-tyrosine kinase from bovine thymus

A protein-tyrosine kinase has been isolated from a soluble extract of bovine thymus based on its ability to phosphorylate the tyrosine-containing peptide angiotensin I. The purification procedure employs sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-agarose, butyl-agarose, and Sephadex G-75. The purified enzyme (p40) is a monomer of Mr = 40,000. The p40 kinase contains an ATP-binding site as determined by photoaffinity labeling experiments and catalyzes an intramolecular autophosphorylation reaction that leads to its modification on tyrosine. Of several proteins tested only the cytoplasmic domain of the erythrocyte band 3 protein serves as a good substrate for p40 (Km = 12 microM). Increasing concentrations of NaCl stimulate the phosphorylation of angiotensin I, inhibit the phosphorylation of band 3, and have no effect on the autophosphorylation of p40. At low concentrations of NaCl, Mn2+ is the preferred divalent cation. Peptide mapping experiments indicate that p40 is distinct from pp60src and from the major phosphotyrosine containing proteins of T and B lymphocyte membranes.     

Purification and characterization of a 2'-phosphodiesterase from bovine spleen

Interferon-induced 2',5'-oligoadenylates are transiently produced during viral infection and are believed to play a role in the interferon-mediated inhibition of replication of at least some viruses. 2',5'-Oligoadenylates must be catabolized but are resistant to degradation by most known ribonucleases. A 2'-phosphodiesterase that degrades 2',5'-oligoadenylates was purified 1500-fold from a low speed homogenate of bovine spleen by precipitation at pH 5.2, ammonium sulfate fractionation, differential ultrafiltration, and successive chromatography on DEAE-Sephacel, hydroxylapatite, and a fast protein liquid chromatography Mono P column. No other 2-5A-degrading activity was observed during the purification procedure. The molecular mass of the enzyme estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 65,000. The enzyme is distinct from bovine spleen phosphodiesterase II. The 2'-phosphodiesterase cleaves 2',5'- and 3',5'-linked oligonucleotides, as well as branched oligoadenylate, A(2'pA)(3'pA), but appears to be most active on 3',5'-oligoribonucleotides. The enzyme cleaves 5'-AMP from the 2' terminus of 2',5'-oligoadenylates and appears to require a free 2' terminus and a 3'-oxygen on the penultimate nucleotide. Substrate length, 5'-phosphorylation, and base composition do not appear to be critical factors in determining enzyme activity. The effects of pH, Mg2+, Mn2+, EDTA, phosphate, 2-mercaptoethanol, and N-ethylmaleimide are also described. This enzyme may be involved in the catabolism of the interferon-induced 2',5'-oligoadenylates and other 2',5'-linked RNAs in the cell.     

Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction-active disulfide

The flavoprotein mercuric reductase catalyzes the two-electron reduction of mercuric ions to elemental mercury using NADPH as an electron donor. It has now been purified from Pseudomonas aeruginosa PAO9501 carrying the plasmid pVS1. In this plasmid system, where the mer operon is on the transposon Tn501, mercuric reductase comprises up to 6% of the soluble cellular protein upon induction with mercurials. The purification is a rapid (two-step), high yield (80%) procedure. Anaerobic titrations of mercuric reductase with dithionite revealed the formation of a charge transfer complex with an absorbance maximum around 540 nm. Striking spectroscopic similarities to lipoamide dehydrogenase and glutathione reductase were observed. These two enzymes, which catalyze the transfer of electrons between pyridine nucleotides and disulfides, are flavoproteins which contain an oxidation-reduction-active cysteine residue at the active site. The expectation that mercuric reductase contains a similar electron acceptor was confirmed when it was shown that mercuric reductase has the capacity to accept four electrons per FAD-containing subunit, and that two thiols become kinetically titrable by 5,5'-dithiobis-(2-nitrobenzoate) upon reduction with NADPH. These are characteristic features of the disulfide reductase class of flavoproteins. Further similarities with at least one of these enzymes, lipoamide dehydrogenase, include the E/EH2 midpoint potential (-269 mV), fluorescence properties, and extinction coefficients of E and EH2. Preliminary observations relevant to an understanding of the mechanism of mercuric reductase are discussed.     

Purification and characterization of a cathepsin D protease from bovine chromaffin granules.

Purification and potential tachykinin and enkephalin precursor cleaving enzymes from bovine chromaffin granules was undertaken using as substrates the model precursors 35S-(Met)-beta-preprotachykinin [35S-(Met)-beta-PPT] and 35S-(Met)-preproenkephalin [35S-(Met)-PPE]. Purification by concanavalin A-Sepharose, Sephacryl S200, and chromatofocusing resulted in a chromaffin granule aspartyl protease (CGAP) that preferred the tachykinin over the enkephalin precursor. CGAP was composed of 47-, 30-, and 16.5-kDa polypeptides migrating as a single band in a nondenaturing electrophoretic gel system, and coeluting with an apparent molecular mass of 45-55 kDa by size-exclusion chromatography. These results suggest that two forms exist: a single 47-kDa polypeptide and a complex of 30 + 16.5-kDa-associated subunits. CGAP was optimally active at pH 5.0-5.5, indicating that it would be active within the acidic intragranular environment. Cleavage at basic residues was suggested by HPLC and HVE identification of 35S-(Met)-NKA-Gly-Lys as the major acid-soluble product generated from 35S-(Met)-beta-PPT. Neuropeptide K was cleaved at a Lys-Arg basic residue site, as determined by identification of proteolytic products by microsequencing and amino acid composition analyses. Structural studies showed that the three CGAP polypeptides were similar to bovine cathepsin D in NH2-terminal sequences and amino acid compositions, indicating that CGAP appears to be a cathepsin D-related protease or cathepsin D itself. The 47- and 16.5-kDa polypeptides of CGAP possessed identical NH2-terminal sequences, suggesting that the 16.5-kDa polypeptide may be derived from the 47-kDa form by proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a novel proline-directed protein kinase from bovine brain.

A novel protein kinase which phosphorylates a synthetic peptide substrate (RRPDAHRTPNRAF) has been purified approximately 200,000-fold from bovine brain. This peptide contains the consensus sequence for phosphorylation by the p34cdc2 kinase. The purification procedure took advantage of the phenomenon that this novel brain kinase, in partially purified extracts, chromatographed on a gel filtration column as a high molecular weight complex which dissociated in buffer containing 1 M NaCl. The purified native enzyme was estimated to be approximately 63,000, and displayed two bands of M(r) = 33,000 and 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On Western immunoblot, the M(r) = 33,000 peptide reacted strongly with antibodies specific for a conserved amino-terminal sequence, weakly with antibodies to the conserved PSTAIRE sequence, and not at all with antibodies to the carboxyl terminus, of HeLa cell p34cdc2. The brain kinase and p34cdc2 were similar in displaying good activity toward the parent peptide substrate, but no activity toward peptide analogues in which the -T-P- motif was substituted with either -T-G- or -T-A-. Both kinases showed marked preference in phosphorylating a peptide derived from H1 histone (KTPKKAKKPKTPKKAKKL), and both kinases could be phosphorylated by the src-family tyrosine kinase, p56lyn, purified from bovine spleen. However, the brain kinase did not co-purify with a subunit having a molecular weight corresponding to known cyclins, nor did it undergo specific interaction with p13suc1 beads, suggesting that this enzyme is distinct from p34cdc2.     

Purification and characterization of myosin from bovine retina

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca²⁺ and a low activity in the presence of Mg²⁺. The Mg²⁺-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

Purification and characterization of prostaglandin F synthase from bovine liver.

Prostaglandin D2 11-ketoreductase activity of bovine liver was purified 340-fold to apparent homogeneity. The purified enzyme was a monomeric protein with a molecular weight of about 36 kDa, and had a broad substrate specificity for porstaglandins D1, D2, D3, and H2, and various carbonyl compounds (e.g., phenanthrenequinone and nitrobenzaldehyde, etc.). Prostaglandin D2 was reduced to 9 alpha,11 beta-prostaglandin F2 and prostaglandin H2 to prostaglandin F2 alpha with NADPH as a cofactor. Phenanthrenequinone competitively inhibited the reduction of prostaglandin D2, while it did not inhibit that of prostaglandin H2. Moreover, chloride ion stimulated the reduction of prostaglandin D2 and carbonyl compounds, while it had no effect on that of prostaglandin H2. Besides, the enzyme was inhibited by flavonoids (e.g., quercetin) that inhibit carbonyl reductase, but was not inhibited by barbital and sorbinil, which are the inhibitors of aldehyde and aldose reductases, respectively. These results indicate that the bovine liver enzyme has two different active sites, i.e., one for prostaglandin D2 and carbonyl compounds and the other for prostaglandin H2, and appears to be a kind of carbonyl reductase like bovine lung prostaglandin F synthase (Watanabe, K., Yoshida, R., Shimizu, T., and Hayaishi, O., 1985, J. Biol. Chem. 260, 7035-7041). However, the bovine liver enzyme was different from prostaglandin F synthase of bovine lung with regard to the Km value for prostaglandin D2 (10 microM for the liver enzyme and 120 microM for the lung enzyme), the sensitivity to chloride ion (threefold greater activation for the liver enzyme) and the inhibition by CuSO4 and HgCl2 (two orders of magnitude more resistant in the case of the liver enzyme). These results suggest that the bovine liver enzyme is a subtype of bovine lung prostaglandin F synthase.     

Purification and characterization of dopamine beta-hydroxylase from bovine adrenal medulla.

A new purification procedure that permits large-scale purification of dopamine beta-hydroxylase from bovine adrenal medulla was developed. Whole adrenal medullas were extracted with 0.1% Triton X-100, and the enzyme was purified by precipitation with polyethylene glycol, chromatography on DEAE-cellulose, and adsorption to concanavalin A linked to agarose. The yield of protein and the specific activity were high compared with previously published methods. The enzyme appeared essentially homogenous by the criteria of polyacrylamide gel electrophoresis in the presence or absence of dodecylsulfate, and sedimentation velocity analysis. The purified protein was subjected to amino acid and carbohydrate analyses, and the results were compared with previously published data. We found about 3 mol of copper per mol of protein (tetramer of 290000 daltons). No free sulfhydryl groups could be found. Analysis for NH2-terminal amino acids with [14C]dansyl chloride revealed 2 residues of alanine and 2 residues of serine per tetramer. We found the NH2-terminal amino acid of chromogranin A to be leucine. The results of our analysis for amino acid composition and NH2-terminal amino acids do not support the suggestion that dopamine beta-hydroxylase and chromogranin A contain identical peptide chains.     

Purification and characterization of a phosphatidylinositol 4-kinase from bovine uteri

Phosphatidylinositol 4-kinase has been purified 10,148-fold to a specific activity of 2.7 mumol/mg/min from bovine uteri. This purification was accomplished by detergent extraction of an acetone powder, ammonium sulfate precipitation, and chromatography on MonoQ, S-Sepharose, MonoP, and hydroxylapatite columns. The purified enzyme has a molecular mass of 55 kDa and appears to be monomeric. Kinetic analyses of the enzymatic activity demonstrated apparent Km values of 18 microM and 22 micrograms/ml (approximately 26 microM) for ATP and phosphatidylinositol, respectively, optimal activity in the pH range of 6.0-7.0, and a sigmoidal dependence of enzymatic activity on [Mg2+]. Ca2+ inhibited the enzyme at nonphysiological concentrations with 50% inhibition observed at a free [Ca2+] of approximately 300 microM. The purified enzyme efficiently utilized both ATP and 2'-deoxy-ATP as phosphoryl donors and specifically phosphorylated phosphatidylinositol on the fourth position. No phosphatidylinositol-4-phosphate 5-kinase activity was observed in the purified enzyme preparations. To our knowledge, this is the first reported purification of a phosphatidylinositol-specific phosphatidylinositol 4-kinase.     

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.     

Purification and characterization of a protein tyrosine kinase from bovine spleen

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.     

Purification and characterization of a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands. Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 300 mM, phosphorylated only phosvitin and was not retained on phosphocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhibited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent Km of 100 micrograms/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent Km of 25 and 28 microM, respectively. It had an absolute requirement for Mg2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecular weight of 35000 suggesting a polymeric structure of the enzyme.     

Purification and characterization of a 15-ketoprostaglandin delta 13-reductase from bovine lung.

15-Ketoprostaglandin delta 13-reductase from bovine lung has been purified using affinity chromatography to apparent homogeneity, as judged from polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate. Valine was identified as tne N-terminal aumino acid, and the isoelectric point was estimated at pH 7.8. Molecular weights of 56,000 and 39,500 were found by the use of gel filtration and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme was found to be specific for the 15-keto group, thus 15-ketoprostaglandin E4 (apparent Km = microM) is a substrate, in contrast to prostaglandin E1. The enzyme was active with both NADH (apparent Km = 88--94 microM) and NADH (apparent Km = 5--9 microM) as coenzyme, but the V max with NADH was more than twice that obtained with NADPH. The enzyme did not catalyze the reversed reaction: 13,14-dihydro-15-keto-prostaglandin E1 to 15-ketoprostaglandin E1. The turnover number of the enzyme was determined to be either 60 or 42 min-1. The low value of the turnover number is compensated by a high concentration (96.4 mU/g tissue) of the enzyme in lung tissue, resulting in a high metabolic capacity. Thus, 15-ketoprostaglandin delta 13-reductase together with 15-hydroxyprostaglandin dehydrogenase ensures an irreversible catabolism of prostaglandins.     

Purification and characterization of a unique osteoinductive factor from bovine bone

A unique protein that promotes ectopic osteoinduction in the rat has been isolated and characterized. Osteoinductive factor (OIF) was extracted from the organic matrix of bovine bone with 4 M guanidine HCl and purified by gel filtration, ion-exchange chromatography, affinity chromatography, and reversed phase high performance liquid chromatography. OIF is a glycoprotein with an apparent molecular mass of 22-28 kDa based on sodium dodecyl sulfate gel electrophoresis. Enzymatic or chemical deglycosylation of OIF reduces its mass to about 12 kDa with apparent loss of activity. OIF activity in the model used is substantially increased by addition of transforming growth factor (TGF)-beta 1 or TGF-beta 2, suggesting an important role for TGF-beta 1 and -2 in bone regeneration and repair. The N-terminal sequence of OIF has no homology to other reported proteins.     

Purification and characterization of L-asparaginase with anti-lymphoma activity from Vibrio succinogenes.

Homogeneols L-asparaginase with anti-lymphoma activity was prepared from Vibrio succinogenes, an anaerobic bacterium from the bovine rumen. An overall yield of pure L-asparaginase of 40 to 45% and a specific activity of 200 +/- 2 IU per mg of protein was obtained. The pure enzyme can be stored at -20 degrees for at least 3 months with no loss of activity. The isoelectric point of the L-asparaginase is 8.74. No carbohydrate, phosphorus, tryptophan, disulfide, or sulfhydryl groups were detected. The enzyme has a molecular weight of 146,000 and a subunit weight of approximately 37,000. The Km of the enzyme for L-asparagine is 4.78 X 10(-5) M and the pH optimum of the L-asparaginase reaction is 7.3. D-Asparagine was hydrolyzed at 6.5% of the rate found with the L isomer. L-Glutamine and a variety of other amides were not hydrolyzed at significant rates; the activity of the enzyme for L-glutamine was 130- to 600-fold less than that of other therapeutically effective L-asparaginases of bacterial origin. The L-asparaginase from V. succinogenes is immunologically distinct from the L-asparaginase (EC-2) of Escherichia coli.     

Purification and characterization fo a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands/ Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 3000 mM, phosphorylated only phosvitin and was not retained on phophocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhbiited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent K_m of 100 μg/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent K_m of 25 and 28 μM, respectively. It had an absolute requirement for Mg²⁺ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecualr weight of 35 000 suggesting a polymeric structure of the enzyme.