Purification of β-Glucosidase from Saccharomyces lactis Strains Y-14 and Y-1057A

Two strains of Saccharomyces lactis (Y-14 and Y-1057A), medium (B(m)) and low (B(1)) constitutive producers of beta-glucosidase, were grown in enriched medium. beta-Glucosidase was extracted by autolysis and purified by ammonium sulfate precipitation, gel filtration, and calcium phosphate gel adsorption-elution. The kinetics of release, purification, and stability of beta-glucosidase from strains Y-14 and Y-1057A were compared with the enzyme from strain Y-123. The ability of glycerol, sorbitol, and mannitol to stabilize the beta-glucosidases is presented. A lower molecular weight, labile form of the Y-14 enzyme is demonstrated. Differences in the initial specific activites of beta-glucosidase among the three strains are discussed.     

Soluble guanylate cyclase from rat lung exists as a heterodimer

The soluble form of guanylate cyclase (EC 4.6.1.2) from rat lung has been purified to homogeneity by a one-step immunoaffinity chromatographic procedure. The purified soluble guanylate cyclase has specific activities of 432 and 49.1 nmol of cyclic GMP formed per min/mg protein with manganese and magnesium ions as a cofactor, respectively. This represents a purification of approximately 2,000-fold with a 50% recovery. The native enzyme has a molecular weight of 150,000 and a Stokes radius of 4.8 nm as determined on Spherogel TSK-G3000SW gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with molecular weights of 82,000 and 70,000. The purified soluble guanylate cyclase was also subjected to native polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, ion exchange chromatography, and GTP-agarose affinity chromatography. These additional purification procedures confirmed the presence of a single protein peak coincident with enzyme activity. The two subunits separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were shown to have different primary structures by immunoblotting with monoclonal and polyclonal antibodies prepared against purified soluble guanylate cyclase and by peptide mapping with papain or Staphylococcus aureus V8 protease treatment. These data demonstrate that soluble guanylate cyclase purified from rat lung is a heterodimer composed of 82,000- and 70,000-dalton subunits with different primary structures.     

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.     

Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.     

Purification of soluble guanylate cyclase from rat lung.

The soluble form of guanylate cyclase from rat lung has been purified approximately 23,000-fold to homogeneity by isoelectric precipitation, GTP-Sepharose chromatography, and preparative gel electrophoresis. A single protein-staining band is observed after analytical gel electrophoresis on either 4 or 7.5% polyacrylamide gels. The final purified enzyme has a specific activity of about 700 nmol of cyclic GMP formed/min/mg of protein at 37 degrees C in the presence of 4.8 mM MnCl2 and 100 micrometer GTP. Bovine serum albumin appears to slightly increase guanylate cyclase activity, but mainly stabilizes the purified enzyme; in its presence, specific activities in excess of 1 mumol of cyclic GMP formed/min/mg of enzyme protein can be obtained. When Mg2+ or Ca2+ are substituted for Mn2+, specific activities decrease to approximately 21 and 40 nmol of cyclic GMP formed/min/mg of protein, respectively. The apparent Michaelis constant for MnGTP in the presence of 4.8 mM MnCl2 is 10.2 micrometer. Kinetic patterns on double reciprocal plots as a function of free Mn2+ are concave downward. The native enzyme has a molecular weight of approximately 151,000 as determined on Sephacryl S-200; sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with approximate molecular weights of 79,400 and 74,000. Thus, it appears that the soluble form of guanylate cyclase from rat lung exists as a dimer.     

Affinity purification and characterization of a key enzyme responsible for circadian rhythmic control of nyctinasty in Lespedeza cuneata L

The synthesis of an affinity gel aimed at leaf-opening factor beta-glucosidase (LOFG) and affinity purification of LOFG is presented. A gluconamidine-based beta-glucosidase inhibitor was used as the ligand of the affinity gel. beta-Glucosidase exhibiting an activity shift throughout the day was selectively purified from Lespedeza cuneata Don by the affinity gel. The resulting LOFG exhibited high substrate specificity toward the leaf-opening factor.     

Polygalacturonase from Rhizopus stolonifer, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings

Apparently homogeneous polygalacturonase-elicitor purified from the filtrates of Rhizopus stolonifer cultures stimulates germinating castor bean seedlings to produce greatly increased levels of casbene synthetase activity. The purification procedure involved gel-filtration chromatography on Sephadex G-25 and G-75 columns followed by cation-exchange chromatography on a Sephadex CM C-50 column. Homogeneity of the purified preparation was indicated by the results of cationic polyacrylamide disc gel electrophoresis and isoelectric focusing (pI = 8.0). The identity of the casbene elicitor activity and polygalacturonase were indicated by the coincidence of the two activities at all stages of purification, the coincidence of both activities with the single protein-staining band detected on a cationic polyacrylamide disc gel and an isoelectric focusing gel, and the identical behavior of both activities on an agarose gel affinity column. The purified polygalacturonase-elicitor is a glycoprotein with approximately 20% carbohydrate content and an estimated molecular weight of 32,000 by polyacrylamide disc gel electrophoresis.     

Cathepsin D of rat spleen. Affinity purification and properties of two types of cathepsin D.

Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin--Sepharose 4B and concanavalin-A--Sepharose 4B, and chromatography on Sephadex G-100 and DEAE-Sephacel. The purified major enzyme (85% of the cathepsin D activity after DEAE-Sephacel chromatography), termed cathepsin D-I, represented about a 1000-fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15%), termed cathepsin D-II, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pI: 4.2, 4.9, 6.1 and 6.5) and three (pI: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corresponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D-I contained 6.6% carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8:2:1:1:5 with a trace of sialic acid. The properties of purified enzymes were also compared.     

Two affinity chromatography methods for the purification of glyoxalase I from rabbit liver

The purification of Glyoxalase I from rabbit liver using Blue Dextran-Sepharose-4B and S-hexyl Glutathione Sepharose-6B is described. Elution of Glyoxalase I from both the columns was accomplished with S-hexyl glutathione, a competitive inhibitor of the enzyme. The purified enzyme gave two bands on disc electrophoresis. After treatment with glutathione, only one band was found. Except for these interconvertible forms, the purified enzyme was homogeneous as shown by disc electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Purification and characterization of UDP-N-acetyl-D-glucosamine:dolichol phosphate N-acetyl-D-glucosamine-1-phosphate transferase involved in the biosynthesis of asparagine-linked glycoproteins in the mammary gland

The GlcNAc-1-P-transferase that initiates the dolichol cycle for the biosynthesis of asparagine-linked glycoproteins has been purified from the lactating bovine mammary gland. After solubilization from microsomes with 0.25% Nonidet P-40, the enzyme activity was stabilized with 20% glycerol, 20 micrograms/ml phosphatidylglycerol, 5 microM dolichol phosphate, and 2.5 microM UDP-GlcNAc. The purification protocol involved (NH4)2SO4 precipitation, gel filtration on Sephacryl S-300, DEAE-TSK, and hydroxylapatite chromatography. The purified enzyme was devoid of several readily detectable glycosyltransferases of the dolichol cycle. It showed two bands (A, 50 kDa and B, 46 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after either Coomassie Blue or silver staining. Antisera (anti-A and anti-B) raised against individual bands A and B inhibited the enzyme activity in solubilized microsomes. Each of the partially purified antibodies recognizes both bands A and B on Western blots of the enzyme; with the solubilized microsomes, the antibodies also recognize an additional polypeptide of approximately 70 kDa. When radioiodinated microsomes were immunoprecipitated with anti-B and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, again bands of 46, 50, and 70 kDa were observed. The peptide mapping of 50 and 46 kDa bands of the purified enzyme by chemical cleavage with N-chlorosuccinimide gave similar fragmentation patterns. The results indicate that either 70 kDa band is a precursor form of the enzyme or this polypeptide, representing the native enzyme or its subunit, is proteolyzed to smaller, enzymatically active peptide(s) of 50 and 46 kDa during purification despite the inclusion of several inhibitors against serine-proteases in all buffers used for tissue homogenization and enzyme purification. A number of properties of the purified enzyme, including its specific activation by Man-P-Dol were also characterized.     

Partial purification and properties of long-chain acyl-CoA hydrolase from rat brain cytosol

Long-chain acyl-CoA hydrolase (EC 3.1.2.2.) has been partially purified from the 100,000 × g supernatant fraction of rat brain tissue. The purification procedure included chromatography on gel filtration media, DEAE-cellulose, CM-cellulose, and hydroxyapatite. The partially purified enzyme had a specific activity of 7.1 mol/min-mg, and when analyzed by polyacrylamide gel electrophoresis, revealed one major and three minor bands of protein in the presence of dodecyl sulfate and two major bands of protein in the absence of dodecyl sulfate. The enzyme had a molecular weight of 65,000 and showed no evidence of aggregated or dissociated forms. The highest catalytic activity was exhibited with palmitoyl-CoA and oleoyl-CoA as substrates. Lower activity was found with decanoyl-CoA as the substrate and little or no activity was found with acetyl-CoA, malonyl-CoA, butyryl-CoA, or acetoacetyl-CoA. The enzyme was inhibited by CoA, various metal ions, including Mn²⁺, Mg²⁺ and Ca²⁺, and by bovine serum albumin. Heating the enzyme produced a loss of activity which corresponded to a first-order kinetic process, the rate of which was independent of the choice of substrate used to measure enzyme activity. This finding supports the idea that the purification procedure yields a single species of long-chain acyl-CoA hydrolase.     

Comparison of the catalytic and immunological properties of beta-glucosidases from three strains of Saccharomyces lactis

beta-Glucosidase from Saccharomyces lactis strains Y-123 (B(h)), Y-14 (B(m)), and Y-1057A (B(1)) was partially purified. The pH optima, Michaelis constants, and activation energies were determined for the hydrolysis of p-nitro-phenyl-beta-d-glucoside by each of the enzymes. Differences among these constants were not enough to account for the low specific activity of beta-glucosidase in strains Y-14 and Y-1057A. Enzyme-inhibitor constants were measured for a series of alkyl and aryl glucosides. In general, the three enzymes are arylglucosidases. Tris(hydroxymethyl)-aminomethane inhibited all three enzymes in an uncompetitive fashion. The inhibition was antagonized by Mg(++). An antiserum was prepared to the highly purified (200-fold) beta-glucosidase from strain Y-123. The nature and degree of cross-reaction between the three beta-glucosidases was investigated by double diffusion in agar and neutralization tests. Spur formation in the immunodiffusion tests and similar equivalence points in the neutralization tests indicated a strong degree of cross-reaction between the three enzymes. The ratio of enzyme activity to antigenicity was used to compare the relative molecular activity of beta-glucosidase in the three strains. Each strain produced the same amount of beta-glucosidase per milligram of cell protein. The results are consistent either with a lower turnover number for the beta-glucosidase in strains Y-14 and Y-1057A or with the production of beta-glucosidase with a "normal" turnover number and enough cross-reacting material to effectively reduce the specific activity to the observed levels.     

Purification and characterization of an extracellular beta-glucosidase from Monascus purpureus

An extracellular beta-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A 22 central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at 50 degrees and pH 5.5. The beta-glucosidase showed moderate thermostability, was inhibited by HgCl2, K2CrO4, and K2Cr2O7, whereas other reagents including beta- mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-beta-D-glucopyranoside, and maltose indicates that the beta-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-beta-Dcellobiose. beta-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

This paper describes a rapid purification procedure for 3-hydroxy-3-methylglutaryl coenzyme A reductase, the major regulatory enzyme in hepatic cholesterol biosynthesis. A freeze-thaw technique is used for solubilizing the enzyme from rat liver microsomal membranes. No detergents or other stringent conditions are required. The purification procedure employs Blue Dextran-Sepharose-4B affinity chromatography, and purification can be carried out from microsomal membranes to purified enzyme in 8 to 10 hours. The purified enzyme has a specific activity of 517 nmoles/min/mg protein, and it is 975-fold purified with respect to the original microsomal membrane suspension. SDS polyacrylamide gel electrophoresis of the purified enzyme shows only trace impurities; the subunit molecular weight for the enzyme measured by this technique is 47,000.     

Purification and characterization of phytase from cotyledons of germinating soybean seeds

Soybean phytase (myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 X 10(4) M-1 cm-1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250-300 mM. The enzyme possessed a high affinity for phytic acid (apparent Km = 48 microM), and was strongly inhibited by phosphate (apparent Ki = 18 microM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5-4.8 and 55 degrees C, respectively.     

Purification of NADPH-cytochrome c reductase from swine testis microsomes by chromatofocusing and characterization of the purified reductase

A purified NADPH-cytochrome c reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was prepared from swine testis microsomes by detergent solubilization followed by a procedure including chromatofocusing. The reductase was eluted at an isoelectric point of 4.8 from the chromatofocusing column. 730-fold purification was achieved with an overall yield of 1.2%. The preparation was found to be homogeneous upon polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (SDS). Upon SDS-polyacrylamide gel electrophoresis, however, the purified preparation resolved into one major band (Mr 78 000) and two minor bands (Mr 60 000 and 15 000). The enzyme contained about 1 mol each of FMN and FAD, which were both extractable with trichloroacetic acid and also boiling water. The oxidized form of the enzyme showed the absorption spectrum of a typical flavoprotein. Aerobic reduction with NADPH resulted in conversion of the spectrum into one of an air-stable semiquinone form. The activity of the purified preparation was 26 mumol cytochrome c reduced/min per mg protein under the standard assay conditions at 22 degrees C. The enzyme catalyzed the reaction through a ping-pong mechanism.     

A beta-glucosidase from lodgepole pine xylem specific for the lignin precursor coniferin.

Coniferin, the glucoside of the monolignol coniferyl alcohol, accumulates to high levels in gymnosperms during spring-cambial reactivation. A cinnamyl alcohol glucoside/beta-glucosidase system is thought to play a key role in lignification by releasing the monolignol aglycones. Investigation of such an enzyme system in the xylem of Pinus contorta var latifolia Engelm. revealed two major beta-glucosidases. One efficiently hydrolyzed the native substrate, coniferin, and the other was more active against synthetic glucosides. The coniferin beta-glucosidase was purified to apparent homogeneity using anion exchange, hydrophobic interaction, and size-exclusion chromatography. The apparent native molecular weight was estimated to be 60,000. A dominant 28-kD protein and a minor 24-kD protein were detected in the purified preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunological evidence from polyclonal antibodies directed against the synthetic N-terminal peptide of the 24-kD protein suggested that the native protein is a dimer of 28-kD subunit size. The N-terminal sequence showed that coniferin beta-glucosidase has high homology to known plant beta-glucosidases. Coniferin, syringin, and a synthetic coniferin analog were preferred substrates for the coniferin beta-glucosidase. In situ localization using the chromogenic coniferin analog showed the exclusive presence of beta-glucosidase activity in the differentiating xylem, similar to peroxidase activity.     

Simple, high-yield purification of xanthine oxidase from bovine milk.

Xanthine oxidase, a commercially important enzyme with a wide area of application, was extracted from fresh milk, without added preservatives, using toluene and heat. The short purification procedure, with high yield, consisted of extraction, ammonium sulfate fractionation, and DEAE-Sepharose (fast flow) column chromatography. Xanthine oxidase was eluted as a single activity peak from the column using a buffer gradient. The purification fold, specific activity and yield for the purified xanthine oxidase were 328, 10.161 U/mg and 69%, respectively. The enzyme was concentrated by ultrafiltration, although 31% of the activity was lost during concentration, no change in specific activity was observed. Activity and protein gave coincident staining bands on native polyacrylamide gels. The intensity and the number of bands were dependent on the oxidative state(s) of the enzyme; reduction by 2-mercaptoethanol decreased the intensity of the slow-moving bands and increased the intensity of the fastest-moving band. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands (molecular masses of 152 and 131 kDa) were observed, accounting for > or = 95% of xanthine oxidase. Native- and SDS-PAGE showed that the purified xanthine oxidase becomes a heterodimer due to endogenous proteases.     

Purification, properties, and isozyme pattern of galactose-1-phosphate uridyl transferase from calf liver.

Galactose-1-phosphate uridyl transferase was purified approximately 2000-fold from calf liver with a yield of 15%. The purification procedure involved ammonium sulfate fractionation, calcium phosphate-gel adsorption, and chromatography on DEAE-cellulose, hydroxylapatite, and Sephadex columns. The purified product demonstrated five protein bands on polyacrylamide-gel electrophoresis. Each band had transferase activity as five peaks of activity were observed on preparative polyacrylamide-gel electrophoresis. Galactose-1-phosphate uridyl transferase showed no requirement for divalent metals for activity. In contrast, it was inhibited by Mg²⁺ and other divalent metals. The purified enzyme but not the crude preparation was stimulated by sulfhydryl compounds. The enzyme was completely inhibited by low concentrations of p-hydroxymercuribenzoate.