Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Purification and properties of a DNA ligase from sea urchin embryos

DNA ligase was purified about 2,000-fold from blastulae of sea urchin, Hemicentrotus pulcherrimus, by means of 1 M KCl-extraction, phosphocellulose, DEAE-cellulose, Sepharose CL-6B, and double-stranded DNA cellulose column chromatography. The purified DNA ligase had a molecular weight of 80,000 (determined by Sephadex G-150) and a sedimentation coefficient of 4.1S (by glycerol gradient centrifugation). The purified enzyme required ATP and Mg2+ (or Mn2+) as cofactors for activity, and was inhibited by N-ethylmaleimide. Apparent Km values for ATP, Mg2+, and Mn2+ were 4 microM, 2.7 mM, and 0.3 mM, respectively.     

A galactokinase of Mycobacterium sp. 279 galactose mutant

A galactokinase and the other enzymes of a galactose catabolic pathway were found in Mycobacterium sp. 279 galactose mutant. The galactokinase was partially purified in a procedure involving ammonium sulfate precipitation, Sephadex G-100 filtration and DEAE-cellulose chromatography. The enzyme was 170-fold purified with 25% of recovery. It was most active at pH 7.8-8.0 in the presence of Mg2+, CO2+, Mn2+ or Fe2+ ions. The molecular weight of the enzyme as determined by Sephadex G-100 filtration amounted to 41,700. The apparent Michaelis constants for galactose and ATP in spectrophotometric test were 1.0 mM and 0.29 mM, respectively. Mercuric compounds at concentration of 0.4 mM completely blocked the enzyme. The galactokinase was quite stable during storage at moderatory temperatures and neutral pH but underwent rapid inactivation on heating above 50 degrees C.     

BamHI DNA甲基化酶的提纯及物理化学性质

 通过硫酸铵盐析,DEAE-纤维素柱层析,磷酸纤维素亲和层析及SephadexG-100凝胶过滤法,从噬淀粉芽孢杆菌HI(Bacillus amyloliguefaciens HI)提纯了DNA甲基化酶。用聚丙烯酰胺凝胶电泳检查,已达电泳均一,比活力提高了326倍。并用聚丙烯酰胺梯度凝胶电泳和Sephadex G-100凝胶过滤法测得其天然酶的分子量为273000,又用SDS聚丙烯酰胺凝胶电泳测得它的亚基分子量为34500,故该酶有8个分子量相同的亚基。用凝胶电聚焦法测得其pI_(22 c)=9.0。     

Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

In vitro effects of some antibiotics on glutathione reductase from sheep liver

The effects of gentamicin sulphate, thiamphenicol, ofloxacin, levofloxacin, cefepime, and cefazolin were investigated on the in vitro enzyme activity of glutathione reductase. The enzyme was purified 1,850-fold with a yield 18.76% from sheep liver using ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). The enzyme activity was measured spectrophotometrically at 340 nm, according to the method of Carlberg and Mannervik. From these six antibiotics, Ofloxacin, levofloxacin, cefepime, and cefazolin inhibited the activity of the purified enzyme; gentamicin sulphate and thiamphenicol showed little effect on the enzyme activity. The I50 values for these four antibiotics were 0.150 mM, 0.154 mM, 3.395 mM, and 18.629 mM, respectively. The Ki constants were 0.047 +/- 0.034 mM, 0.066 +/- 0.038 mM, 4.885 +/- 3.624 mM, and 6.511 +/- 1.894 mM, respectively and they were competitive inhibitors.     

Multiple proteins related to the soluble galactose-binding animal lectin revealed by a monoclonal anti-lectin antibody.

An enzyme catalysing the O-methylation of isobutyraldoxime by S-adenosyl-L-methionine was isolated from Pseudomonas sp. N.C.I.B. 11652. The enzyme was purified 220-fold by DEAE-cellulose chromatography, (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and chromatography on calcium phosphate gel. Homogeneity of the enzyme preparation was confirmed by isoelectric focusing on polyacrylamide gel and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme showed a narrow pH optimum at 10.25, required thiol-protecting agents for activity and was rapidly denatured at temperatures above 35 degrees C. The Km values for isobutyraldoxime and S-adenosyl-L-methionine were respectively 0.24 mM and 0.15 mM. Studies on substrate specificity indicated that attack was mainly restricted to oximes of C4-C6 aldehydes, with preference being shown for those with branching in the 2- or 3-position. Ketoximes were not substrates for the enzyme. Gel filtration on Sephadex G-100 gave an Mr of 84 000 for the intact enzyme, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated an Mr of 37 500, suggesting the presence of two subunits in the intact enzyme. S-Adenosylhomocysteine was a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 0.027 mM. The enzyme was also susceptible to inhibition by thiol-blocking reagents and heavy-metal ions. Mg2+ was not required for maximum activity.     

Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of gamma-glutamyl kinase.

A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.     

A novel enzyme, tagatose kinase, from Mycobacterium butyricum

A novel enzyme catalyzing the phosphorylation of D-tagatose to D-tagatose 6-phosphate with ATP has been identified in extracts of dulcitol-grown Mycobacterium butyricum. The enzyme was purified 100-fold with 29% recovery. It required Mg2+, Mn2+ or Fe2+ and showed maximum activity at pH 7.5. The molecular weight as determined by Sephadex G-100 filtration amounted to 63 000. The apparent Michaelis constants for D-tagatose and ATP were 0.8 and 1.0 mM, respectively. The enzyme preparations were not very sensitive to SH group inhibitors and heavy metals but rapidly lost activity on heating above 50 degrees C.     

A soluble beta-cyanoalanine synthase from the gut of the variegated grasshopper Zonocerus variegatus (L.)

Beta-cyanoalanine synthase (beta-cyano-l-alanine synthase; l-cysteine: hydrogen sulphide lyase (adding hydrogen cyanide (HCN)); EC 4. 4.1.9) was purified from the cytosolic fraction of the gut of grasshopper Zonocerus variegatus (L.) by ion-exchange chromatography on DEAE-Cellulose and gel filtration on Sephadex G-100 columns. The crude enzyme had a specific activity of 2.16nmol H2S/min/mg. A purified enzyme with a specific activity, which was seventeen times higher than that of the crude extract, was obtained. A molecular weight of about 55.23+/-1.00Kd was estimated from its elution volume on Sephadex G-100. The fraction when subjected to sodium dodecyl sulphate-polyacrylamide elel electrophoresis revealed the presence of a protein band with Mr of 23.25+/-0.25Kd. The enzyme exhibited Michaelis-Menten kinetics having Km of 0.38mM for l-cysteine and Km of 6.25mM for cyanide. The optimum temperature and pH for activity were determined to be at 30 degrees C and pH 9.0, respectively. This enzyme might be responsible for the ability to detoxify cyanide in this insect pest and hence its tolerance of the cyanogenic cassava plant. Biophysical, biochemical and kinetic properties of this enzyme, which will reveal how this ability can possibly be compromised by enzyme inhibition, may lead, in the long term, to the potential use of this enzyme as drug target for pest control.     

In Vitro Effects of Some Antibiotics on Glutathione Reductase from Sheep Liver

The effects of gentamicin sulphate, thiamphenicol, ofloxacin, levofloxacin, cefepime, and cefazolin were investigated on the in vitro enzyme activity of glutathione reductase. The enzyme was purified 1,850-fold with a yield 18.76% from sheep liver using ammonium sulphate precipitation, 2′, 5′-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). The enzyme activity was measured spectrophotometrically at 340?nm, according to the method of Carlberg and Mannervik. From these six antibiotics, Ofloxacin, levofloxacin, cefepime, and cefazolin inhibited the activity of the purified enzyme; gentamicin sulphate and thiamphenicol showed little effect on the enzyme activity. The I50 values for these four antibiotics were 0.150?mM, 0.154?mM, 3.395?mM, and 18.629?mM, respectively. The Ki constants were 0.047±0.034?mM, 0.066±0.038?mM, 4.885±3.624?mM, and 6.511±1.894?mM, respectively and they were competitive inhibitors.     

Effects of some antibiotics on glutathione reductase activities from human erythrocytes in vitro and from rat erythrocytes in vivo

The effects of streptomycin sulfate, gentamicin sulfate, thiamphenicol, penicillin G, teicoplanin, ampicillin, cefotaxime, and cefodizime on the enzyme activity of glutathione reductase (GR) were studied using human and rat erythrocyte GR enzymes in in vitro and in vivo studies, respectively. The enzyme was purified 5,342-fold from human erythrocytes in a yield of 29% with 50.75 U/mg. The purification procedure involved the preparation of hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies, and rat erythrocyte hemolysate was used in the in vivo studies. In the in vitro studies, I50 and K(i) values were 12.179 mM and 6.5123 +/- 4.1139 mM for cefotaxime, and 1.682 mM and 0.7446 +/- 0.2216 mM for cefodizime, respectively, showing the inhibition effects on the purified enzyme. Inhibition types were noncompetitive for cefotaxime and competitive for cefodizime. In the in vivo studies, 300 mg/kg cefotaxime and 1000 mg/kg cefodizime when administered to rats inhibited enzyme activity during the first 2h (p < 0.01). Cefotaxime led to increased enzyme activity at 4h (p < 0.05), but neither cefotaxime nor cefodizime had any significant inhibition or activation effects over 6 h (p > 0.05).     

Purification and Characterization of a Soluble Phosphatidylinositol 4-Kinase from Carrot Suspension Culture Cells

Previously we reported the presence of a soluble phosphatidylinositol 4-kinase (PI 4-Kinase) in carrot (Daucus carota L.) suspension culture cells (C.M. Okpodu, W. Gross, W.F. Boss [1990] Plant Physiol 93: S-63). We have purified the enzyme over 1000-fold using Q-Sepharose ion exchange, hydroxylapatite, and G-100 gel filtration column chromatography. The Mr of the enzyme was estimated to be 83,000 by gel filtration. PI 4-kinase activity was recovered after renaturation of the 80-kD region of polyacrylamide gels, and an 80-kD peptide cross-reacted with antibodies to the yeast 55-kD membrane-associated PI 4-kinase on western blots. The isolated lipid kinase phosphorylated PI but not lysophosphatidylinositol or phosphatidylinositol monophosphate. Maximal PI kinase activity occurred when the substrate was added as Triton X-100/PI mixed micelles at pH 8. The enzyme required divalent cations. At low concentrations (1-5 mM), Mn2+ was more effective than Mg2+ in increasing enzyme activity; however, maximal activity occurred at 25 to 40 mM Mg2+. Calcium from 0.01 [mu]M to 1 mM had no effect on the enzyme activity. The Km of the enzyme for ATP was estimated to be between 400 and 463 [mu]M. The enzyme was inhibited by adenosine (100 [mu]M); however, ADP (up to 100 [mu]M) had no effect on the activity. The biochemical characteristics of the carrot soluble PI 4-kinase are compared with the previously reported PI 4-kinases from animals and yeast.     

Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

In vitro effects of some anaesthetic drugs on lactoperoxidase enzyme activity

In vitro effects of ketamine and bupivacaine drugs on bovine lactoperoxidase (LPO; E.C. 1.11.1.7) enzyme activity were investigated. Lactoperoxidase was purified with Amberlite CG 50 resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from skimmed bovine milk. Rz(A412/A280) value for the purified LPO was found to be 0.8. Inhibition or activation effects of the drugs on LPO enzyme were determined using 2,2(1)-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a chromogenic substrate at pH = 6.0. The I50 values of ketamine and bupivacaine were 0.29 mM and 0.155 mM, respectively and the K(i) constants for ketamine and bupivacaine were 0.019 +/- 0.031 and 0.015 +/- 0.021 mM, respectively; they were non-competitive inhibitors.     

Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver. Purification, characterization and subcellular localization

1. Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver was purified to homogeneity. 2. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity. 3. Gel filtration on Sephadex G-200 gave an apparent molecular weight of 110,000 with two apparent identical subunits of 54,000-56,000 as determined by sodium dodecyl sulphate gel electrophoresis. 4. The maximum of enzyme activity was obtained in the presence of 3-5 mM ZnCl2 at pH 6-6.2, however, higher concentrations of metal are inhibitory. The enzyme hydrolyses p-nitrophenylphosphate, o-carboxyphenylphosphate and phenylphosphate, was insensitive to NaF and was inhibited by phosphate and ATP. The Km for p-nitrophenylphosphate was 0.28 x 10(-3)M at pH 6 in 50 mM sodium acetate/100 mM NaCl. 5. Phosphate is a competitive inhibitor (Ki = 0.5 x 10(-3)M) whereas ATP seems to be a non-competitive inhibitor (Ki = 0.35 x 10(-3)M). The isoelectric point determined by isoelectric focusing on polyacrylamide gel is 7.5. 6. Cell fractionation studies indicate that the Zn2+-dependent acid p-nitrophenylphosphatase of chicken liver is a soluble enzyme form.     

慈菇（Sagittaria safittifolia）α－半乳糖苷酶的纯化与性质

以对硝基苯糖苷基为底物,测定了慈菇的１２种糖苷酶,其中α－甘露糖苷酶、α－和β－半乳糖苷酶活力较高;经硫酸铵分级沉淀,ＳｅｐｈａｄｅｘＧ－１５０分子筛层析,ＣｏｎＡＳｅｐｈａｒｏｓｅ４Ｂ亲和层析,ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ离子交换层析,从慈菇抽提液纯化了α－半乳糖苷酶。纯化酶的比活提高１０７２倍,活力回收１５．６％,在圆盘聚丙烯酰胺凝胶电泳和ＳＤＳ－ＰＡＧＥ上均显示１条蛋白质带,在α－半乳糖苷酶浓度为１５０ｍＵ／ｍｌ的溶液中测不到其他糖苷酶的活力。慈菇α－半乳糖苷酶的分子量用ＳｅｐｈａｄｅｘＧ－１００凝胶过滤柱测定或在ＳＤＳ－ＰＡＧＥ上测定均为６０ｋＤ,酶反应的最适ｐＨ在５．８附近,最适温度为６０℃。该酶分解对硝基苯基－α－半乳糖苷的Ｋ＿ｍ值为３．７×１０￣（－４）ｍｏｌ／Ｌ,Ｖ＿ｍ值为２．１×１０￣（－４）ｍｏｌ／Ｌ。银离子、汞离子显著抑制酶活力,Ｄ－半乳糖和密二糖均竞争性地抑制该酶水解对硝基苯基α－Ｄ－半乳糖苷的活力,根据Ｄｉｘｏｎ作图求得其Ｋ＿ｉ值分别为０．９２×１０￣（－３）ｍｏｌ／Ｌ和１．９８×１０￣（－３）ｍｏｌ／Ｌ。２－脱氧－Ｄ－半乳糖和Ｌ－岩藻糖为酶活力的非竞争性抑制剂。化学修饰     

华丽曲霉Z58有机磷农药降解酶的纯化和性质

华丽曲霉(Aspergillus ornatus)Z58有机磷农药降解酶经硫酸铵分级沉淀、Sephadex G100凝胶过滤、DEAE52离子交换层析得到了分离纯化,用聚丙烯酰胺凝胶电泳(PAGE)鉴定为单一组分。凝胶过滤法测得分子量为67 000,提纯倍数为34.2,收率为17.8%。该酶的最适反应温度45℃,最适反应pH72,对热较稳定,并且能在pH6～10范围保持活性。重金属Cu2+对该酶具有明显的促进作用,而SDS对酶具有抑制作用。此酶对所试的有机磷农药都有较好降解作用。     

Purification and properties of a second enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62.

An enzyme (splitting enzyme 2) which catalyzes the splitting of carbon-mercury linkage of arylmercury compounds was found in extracts of mercury-resistant Pseudomonas K-62. This enzyme was purified about 725-fold by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-75 and diethylaminoethyl-cellulose. A purified preparation of the enzyme showed a single band in electrophoresis either on polyacrylamide or sodium dodecyl sulfate-containing polyacrylamide gels. The molecular weight of the enzyme was estimated to be 20,000 (determined by Sephadex G-75 gel filtration) 17,000 (determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis). The enzyme showed a Km of 180 micron and a Vmax of 3.1 mumol/min per mg for p-chloromercuribenzoic acid and a Km of 250 micron and a Vmax of 20 mumol/min per mg for phenylmercuric acetate. The optimum temperature and pH for the reaction were 40 degrees C and 5.0, respectively.