Purification and properties of dextransucrase and invertase from Streptococcus mutans

Invertase (beta-d-fructofuranoside fructohydrolase, EC 3.2.1.26) and dextransucrase (alpha-1, 6-glucan: d-fructose 2-glucosyltransferase, EC 2.4.1.5) were purified from the culture fluids of Streptococcus mutans by chromatography on Sepharose 6B and diethylaminoethyl-cellulose followed by treatment with hydroxyapatite. Each of the enzyme preparations gave a single band when analyzed by either polyacrylamide gel electrophoresis or immunodiffusion. The antigenic determinant of invertase was different from that of dextransucrase on immunodiffusion. The pH optima were 5.25 for invertase and 5.75 for dextransucrase, and the K(m) values were 20 mM for invertase and 2.0 mM for dextransucrase. The molecular weights determined by sodium dodecyl sulfate gel electrophoresis were 160,000 for invertase and 170,000 for dextransucrase. The data obtained suggest that the dextransucrase had dextran-synthesizing activity and invertase-like activity.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Isolation and Some Properties of a beta-d-Xylosidase from Clostridium acetobutylicum ATCC 824

A beta-d-xylosidase from C. acetobutylicum ATCC 824 was purified by column chromatography on CM-Sepharose, hydroxylapatite, Phenyl Sepharose, and Sephadex G-200. The enzyme had an apparent molecular weight of 224,000 as estimated by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme consisted of two subunits of 85,000 and one subunit of 63,000 daltons. It exhibited optimal activity at pH 6.0 to 6.5 and 45 degrees C. the enzyme had an isoelectric point of 5.85. It hydrolyzed p-nitrophenylxyloside readily with a K(m) of 3.7 mM. The enzyme hydrolyzed xylo-oligosaccharides with chain lengths of 2 to 6 units by cleaving a single xylose from the chain end. It showed little or no activity against xylan, carboxymethyl cellulose, and other p-nitrophenylglycosides.     

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.     

Separation and Some Properties of Two Intracellular beta-Glucosidases of Sporotrichum (Chrysosporium) thermophile

Intracellular, inducible beta-glucosidase from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile (ATCC 42464) was fractionated by gel chromatography or isoelectric focusing into components A and B. Enzyme A (molecular weight 440,000) had only aryl-beta-glucosidase activity, whereas enzyme B (molecular weight 40,000) hydrolyzed several beta-glucosides but had only low activity against o-nitrophenyl-beta-d-glucopyranoside (ONPG). Both enzymes had temperature optima of about 50 degrees C. The pH optimum was 5.6 for enzyme A and 6.3 for enzyme B, respectively. The K(m) (ONPG) value for enzyme A was 0.5 mM, and the corresponding values for enzyme B were 0.18 mM (ONPG) and 0.28 mM (cellobiose). Enzyme B, when tested with ONPG, showed substrate inhibition at a substrate concentration above 0.4 mM which could be released by cellobiitol and other alditols. Enzyme A was isoelectric at pH 4.48, and enzyme B was isoelectric at pH 4.64. Several inhibitors were tested for their action on the activity of enzymes A and B. Both enzymes were found to be concomitantly induced in cultures with either cellobiose or cellulose as carbon source.     

Ornithine carbamoyltransferase of Streptomyces fradiae: purification and properties

Abstract The enzyme ornithine carbamoyltransferase was purified from Streptomyces fradiae . A 1200-fold increase in specific activity was achieved by ammonium sulphate precipitation, DEAE-cellulose and aminohexyl-agarose chromatography and gel filtration. The purified enzyme has a M _r of 87 000. Its isoelectric point is 5.3 as determined by isoelectric focusing. Apparent K _m values at pH 7.7 for ornithine and carbamoyl phosphate are 1.8 and 1.2 mM, respectively.     

Purification and characterization of a glycosyltransferase complex from the culture broth of Streptococcus mutans FA1.

The extracellular glycosyltransferases from Streptococcus mutans FA1 were purified by using the following procedures: ammonium sulfate precipitation, poly-(acrylamide) gel filtration, DEAE-cellulose chromatography, and agarose-gel filtration. The dextransucrase and levansucrase activities were purified 350- and 500-fold, repsectively, and the ratio of the two activities remained almost constant throughout the purification. Both enzymes have a pH optimum of 6.0, a Km for sucrose of 55mM, and isoelectric points of 3.7 and 4.6. The enzymes are inactivated by repeated freezing and thawing, but retain partial activity even after heating at 100 degrees. The enzyme preparation contains a carbohydrate moiety which does not appear to be either bound levan or dextran.     

Purification and properties of polyphosphoinositide phosphomonoesterase from rat brain.

1. On subcellular fractionation of rat brain homogenate, polyphosphoinositide phosphomonoesterase activity was greater in the cytosol than the membranous fractions. 2. The enzyme was purified from the cytosol by column chromatography on DEAE-cellulose, calcium phosphate gel and Sephadex G-100. 3. The final preparation of the enzyme showed a 430-fold purification over the whole homogenate and appeared to be homogeneous since it gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on isoelectric focusing. The enzyme has a relatively low molecular weight and an isoelectric point of 6.8. 4. The phosphatase showed a high affinity for triphosphoinositide. Without added Mg2+, the Km was 25 muM and V was 33 mumol Pi released/min/mg protein. 5. The enzyme hydrolysed diphosphoinositide at a slower rate than triphosphoinositide. In the presence of 10 mM Mg2+, the Km values for triphosphoinositide and diphosphoinositide were 5 muM and 25 muM respectively and V was the same for each substrate. 6. Both Mg2+ and Ca2+ activated the enzyme. While Ca2+ produced maximum activation at 100 muM, a much higher concentration of Mg2+ (10 mM) was required to elicit comparable activation. The enzyme did not show an absolute requirement for Mg2+ or Ca2+ as it exhibited low activity in the presence of 0.5 mM EDTA or EGTA. 7. The phosphatase showed maximum activity between 7.4 and 7.6. A drop in pH to 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity. 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity.     

Purification and comparative properties of the glycoprotein nicotinamide adenine dinucleotide glycohydrolase from rat liver microsomal and plasma membranes.

NAD glycohydrolase, or NADase (NAD+ glycohydrolase, EC 3.2.2.5) was solubilized with porcine pancreatic lipase from isolated fractions of microsomes and plasma membranes obtained from rat livers. The enzyme from each organelle was further purified by DEAE-cellulose chromatography, gel filtration and isoelectric focusing. The solubilized, partially purified enzymes had similar molecular weights, pH-activity profiles and Km values. Marked charge heterogeneity was observed for the microsomal enzyme on isoelectric focusing between pH 6 and 8 with maximum activity focusing at pH 8.0. Plasma membrane NADase displayed a single peak at pH 6.7. Treatment of the partially purified microsomal or plasma membrane enzyme with neuraminidase resulted in a single peak of activity on isoelectric focusing (pH 3.5--10) with a pI of 9.2. Polyacrylamide gel electrophoresis of either NADase revealed a periodate-Schiff positive band which was coincident with enzyme activity. Compositional analyses of the microsomal enzyme focusing at pH 8.0 confirmed the presence of hexoses, hexosamines and sialic acid. Differences in carbohydrate composition might be important in determining the subcellular distribution of this enzyme.     

Characterization and purification of thermostable beta-glucosidase from Clostridium thermocellum.

A β-glucosidase was isolated from $\text{Clostridium thermocellum}$; the enzyme was localized in the periplasmic space.It was purified in a five-step procedure including ion-exchange chromatography on DEAE-Cellulose, chromatography on HA-Ultrogel and DEAE-Sephadex, gel filtration on AcA 34 Ultrogel and isoelectric focusing.The final preparation was purified 944-fold with a recovery of about 5% of the initial enzyme activity.Polyacrylamide disc electrophoresis of the purified enzyme gave a single band at pH 8.3. The enzyme is active towards cellobiose and p-nitrophenyl-β-D-glucoside(PNPG) and developed maximum activities at pH 6.0 and 65°C. A molecular weight of 50,000 daltons was estimated by gel filtration and the enzyme was isoelectric at pH 4.68.     

Resolution and purification of taurine- and GABA-synthesizing decarboxylases from calf brain

The present work describes a procedure for the co-purification of cysteine sulfinate decarboxylase (CSAD) and glutamate decarboxylase (GAD) from calf brain. A crude enzyme preparation was first made from brain homogenate by acid precipitation and ammonium sulphate fractionation. Subsequent fractionation of the decarboxylase preparation by cation exchange chromatography on CM-Sepharose CL-6B revealed the existence of a specific CSAD enzyme, which has no GAD activity. The GAD activity peak was found to possess CSAD activity. Further fractionation by gel filtration on Sephacryl S-200 separated the specific CSAD activity into two enzyme forms, one of them having a molecular weight of 150,000 and the other of 71,000. GAD activity was eluted from the gel filtration column in a single peak (mol wt 330,000) and showed CSAD activity. The purification of the specific CSAD enzyme was 920-fold and that of GAD activity 850-fold as compared with the starting material, whole calf brain. SDS gel electrophoresis indicated that the purified CSAD and GAD enzymes consisted of two or more subunits. The crude decarboxylase preparation was analysed by isoelectric focusing in ultra-thin polyacrylamide gel in the pH range 3.5-10.0. The most active fraction of CSAD indicated an isoelectric point of 6.5 and that of GAD 6.8. The pH optimum for CSAD activity in the crude preparation was 7.2 and that for GAD activity 7.9.     

Isolation and characterization of angiotensin converting enzyme from human kidney

Angiotensin converting enzyme [EC 3.4.15.1] was solubilized from the membrane fraction of human kidney cortex using trypsin and purified to homogeneity by DEAE-cellulose, hydroxylapatite and DEAE-Sephadex A-50 column chromatographies, preparative isoelectric focusing, and Sephadex G-200 gel filtration. The final recovery of the enzyme was 13.9%. The molecular weight of the enzyme was estimated to be 199,000 by a sedimentation equilibrium method. A value of 170,000 was obtained for the reduced and denatured enzyme by dodecylsulfate-polyacrylamide gel electrophoresis. The enzyme was a glycoprotein consisting of a single polypeptide chain with an isoelectric point of 5.10. Neutral sugar accounted for 13% per weight of the enzyme. The purified enzyme had a specific activity of 96.9 mumol/min/mg protein for hippurylhistidylleucine. The Km value, Kcat value and hydrolytic coefficient (Kcat/Km) of the enzyme for hippurylhistidylleucine were 2.0 mM, 545 s-1 and 273 mM-1 . s-1, respectively. Rabbit antibody against the human kidney converting enzyme inhibited the activities of the enzymes from human lung and serum as equally as that from human kidney, but not those from sheep, dog, or rat sera. The human kidney and lung converting enzymes were immunologically identical on double immunodiffusion analysis.     

Purification and characterization of basic glucosyltransferase from Streptococcus mutans serotype c

Streptococcus mutans Ingbritt (serotype c) was found to secrete basic glucosyltransferase (sucrose: 1,6-alpha-D-glucan 3-alpha and 6-alpha-glucosyltransferase). The enzyme preparation obtained by ethanol fractionation, DEAE Bio-Gel A chromatography, chromatofocusing and preparative isoelectric focusing was composed of three isozymes with slightly different isoelectric points (pI 8.1-8.4). The molecular weight was estimated to be 151000 by SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 9.8 IU per mg of protein and the optimum pH was 6.5. The enzyme was activated 2.4-fold by commercial dextran T10, and had Km values of 7.1 micro M for the dextran and 4.3 mM for sucrose. Glucan was de novo synthesized from sucrose by the enzyme and found to be 1,6-alpha-D-glucan with 17.7% of 1,3,6-branching structure by a gas-liquid chromatography-mass spectroscopy.     

Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.     

Purification and characterization of an alkaline invertase from shoots of etiolated rice seedlings

One alkaline invertase and two acid invertase activities were detected in the shoots of etiolated rice ( Oryza sativa ) seedlings. The alkaline invertase (AIT) was purified to homogeneity through steps of ammonium sulphate fractionation, concanavalin A-Sepharose affinity chromatography (non-retained), DEAE-Sephacel chromatography and preparative electrophoresis. The pH optimum of AIT was 7.0 and the molecular mass, determined by gel filtration, was 240 kDa. It is apparently a homotetrameric enzyme (subunit molecular mass 60 kDa). The isoelectric point was 4.4 by isoelectric focusing. The best substrate of the enzyme was sucrose, with a K _m of 2.53 mM. The enzyme also hydrolysed raffinose, but not maltose or lactose, so it is a β-D-fructofuranosidase. It gave negative glycoprotein staining. Of the hydrolysis products, fructose was a competitive inhibitor and glucose was a non-competitive inhibitor. Treatment with an alkaline phosphatase could activate AIT, whereas other proteins such as BSA, concanavalin A and urease had no effect on the enzyme activity. The enzyme activity was inhibited by Tris, thiol reagents and heavy metal ions.     

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.     

Purification and characterization of a novel glutathione S-transferase from Atactodea striata

A novel GST isoenzyme was purified from hepatopancreas cytosol of Atactodea striata with a combination of affinity chromatography and reverse-phase HPLC. The molecular weight of the enzyme was determined to be 24 kDa by SDS-PAGE electrophoresis and 48 kDa by gel chromatography, in combination with GST information from literature revealed that the native enzyme was homodimeric with a subunit of M(r) 24 kDa. The purified enzyme, exhibited high activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with respect to CDNB as substrate revealed a K(m) of 0.43 mM and V(max) of 0.24 micromol/min/mg and a specific activity of 108.9 micromol/min/mg. The isoelectric point of the enzyme was 5.5 by isoelectric focusing and its optimum temperature was 38 degrees C and the enzyme had a maximum activity at approximately pH 8.0. The amino acid composition was also determined for the purified enzyme.     

Isolation of L-dynurenine 3-hydroxylase from the mitochondrial outer membrane of rat liver.

Outer membrane preparations of rat liver mitochondria were isolated, after the mitochondria had been prepared by mild digitonin treatment under isotonic conditions. L-Kynurenine 3-hydroxylase [EC 1.14.13.9] was solubilized on a large scale from outer membrane by mixing with 1% digitonin or 1% Triton X-100, followed by fractionation into a minor fraction I and a major fraction II by DEAE-cellulose column chromatography. The distribution of total L-Dynurenine 3-hydroxylase was roughly 20 and 80% in fraction I and II, respectively. Fraction I consisted of crude enzyme loosely bound to anion exchanger. In the present investigation, fraction I was not used because of its low activity and rapid inactivation. In contrast, fraction II consisted of crude enzyme with high activity, excluded from DEAE-cellulose column chromatography in the presence of 1 M KC1. In addition, fraction II was purified by Sephadex G-200 gel filtration and DEAE-Sephadex A-50 column chromatography with linear gradient elution, adding 1 M KC1 and 1% Triton X-100 to 0.05 M Tris-acetate buffer, pH 8.1. After isoelectric focusing, the purified enzyme preparation was proved to be homogeneous, since the L-kynurenine 3-hydroxylase fraction gave a single band on disc gel electrophoresis. The molecular weight of this enzyme was estimated to be approximately 200,000 or more by SDS-polyacrylamide gel electrophoresis and from the elution pattern on Sephadex G-200 gel filtration. A 16-Fold increase of the enzyme activity was obtained compared with that of the mitochondrial outer membrane. The isoelectric point of the enzyme was determined to be pH 5.4 by Ampholine isoelectric focusing.     

Purification and characterization of phosphoinositide 3-kinase from rat liver

Phosphoinositide 3-kinase was purified 27,000-fold from rat liver. The enzyme was purified by acid precipitation of the cytosol followed by chromatography on DEAE-Sepharose, S-Sepharose, hydroxylapatite, Mono-Q, and Mono-S columns. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified phosphoinositide 3-kinase preparation contained an 85-kDa protein and a protein doublet of approximately 110 kDa. The 85- and 110-kDa proteins focus together on native isoelectric focusing gels and are cross-linked by dithiobis(succinylamide propionate), showing that the 110- and 85-kDa proteins are a complex. The apparent size of the native enzyme, as determined by gel filtration, is 190 kDa. The 85-kDa subunit is the same protein previously shown to associate with polyoma virus middle T antigen and the platelet-derived growth factor receptor (Kaplan, D. R., Whitman, M., Schaffhausen, B., Pallas, D. C., White, M., Cantley, L., and Roberts, T. M. (1987) Cell 50, 1021-1029). The two proteins co-migrate on two-dimensional gels; and, using a Western blotting procedure, 32P-labeled middle T antigen specifically blots the 85-kDa protein. The purified enzyme phosphorylates phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. The apparent Km values for ATP were found to be 60 microM with phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate as the substrate. The apparent Km for phosphatidyinositol is 60 microM, for phosphatidylinositol 4-phosphate is 9 microM, and for phosphatidylinositol 4,5-bisphosphate is 4 microM. The maximum specific activity using phosphatidylinositol as the substrate is 0.8 mumol/mg/min. The enzyme requires Mg2+ with an optimum of 5 mM. Substitution of Mn2+ for Mg2+ results in only approximately 10% of the Mg2(+)-dependent activity. Physiological calcium concentrations have no effect on the enzyme activity. Phosphoinositide 3-kinase has a broad pH optimum around 7.     

Purification and general properties of δ-aminolaevulate dehydratase from cow liver

1. delta-Aminolaevulate dehydratase, the enzyme catalysing the condensation of delta-aminolaevulic acid to porphobilinogen, has been prepared from cow liver and its properties have been studied. The enzyme has been purified 310-fold. 2. The purified preparation behaves as a single protein under gel filtration on Sephadex and Bio-Gel columns; it migrates as a single band in disk and starch-gel electro-phoresis at different pH values and it sediments as a single symmetrical peak in the ultracentrifuge. 3. The pH optimum for the pure enzyme was 6.8, the K(m) at pH 6.8 and 38 degrees was 1.5x10(-4)m, the isoelectric point was about pH 4.9 and the molecular weight was 140000+/-14000 by the gel-filtration method. Maximal enzyme activity was observed at 65 degrees . 4. The presence of thiol groups in the enzyme system, essential for its activity, was indicated and the total number of thiol groups was determined. 5. After the first steps of purification the enzyme required cysteine or reduced glutathione for activity.