Purification and properties of a beta-1,6-clucosidase from Flavobacterium.

An intracellular beta-1,6-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced semiconstitutively by Flavobacterium M64. This enzyme was purified 180-fold by fractionation with ammonium sulfate followed by chromatographies on carboxymethylcellulose, hydroxyapatite and Sephadex G-100. The final preparation appeared homogeneous on disc electrophoresis on polyacrylamide gel. The molecular weight of the enzyme was determined to be ca. 59 000 by Sephadex G-100 gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8 and the optimum temperature was 40 degrees C. The enzyme readily hydrolyzed oligomers with beta-a,6-glucosidic linkages, converting them to glucose. The Km values for gentio-biose, -triose, -tetraose and -pentaose were 2.8, 3.0, 4.2 and 4.6 times 10- minus 4 M, respectively. The rates of their hydrolyses decreased with increase in their chain lengths. The enzyme was concluded to be a beta-1,6-glucosidase from its substrate specificity, production of glucose, transferring ability and inhibition by glucono-delta-lactone. The enzyme activity was inhibited by Hg-2+, Cu-2+, Ag-+, Fe-3+, p-chloromercuribenzoate, N-ethylmaleimide, glucose and trishydroxyaminomethane (Tris) but not by ethylenediaminetetraacetic acid.     

Purification and characterization of arylamidase from monkey brain.

Arylamidase [EC3.4.11.2] was isolated from monkey brain extract and purified about 2100-fold in approximately 11% yield by a six-step procedure comprising extraction from monkey brain homogenate, ammonium sulfate fractionation, first hydroxylapatite chromatography, DEAE-cellulose chromatography, Sephadex G-200 gell filtration and second hydroxylapatite chromatography. The enzyme showed a single band on polyacrylamide disc electrophoresis and consisted of a single polypeptide chain, as judged by disc electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was strongly inhibited by PCMB, TPCK, and puromycin. Puromycin competitively inhibited the enzyme and the Ii value was about 5 x 10(-7)M. Treatment with EDTA resulted in a loss of enzyme activity. The enzyme activity was restored by addition of Zn2+, Co2+, Mn2+. Among various amino acid beta-naphthylamides, L-alanine beta-naphthylamide was most rapidly hydrolyzed and N-carbobenzoxyl-L-leucine beta-naphthylamide was not hydrolyzed by this enzyme preparation. The molecular weight of the enzyme was 92,000 as determined by gel filtration on Sephadex G-200.     

Purification of RNAase II by preparative polyacrylamide gel electrophoresis.

Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913--922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 +/- 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 +/- 2000, 33 000 +/- 2000, and 26 000 +/- 1000. The enzyme thus appears to consist of three dissimilar subunits.     

Brain pyridoxal kinase. Purification and characterization

Pyridoxal kinase has been purified 9000-fold from sheep brain. The purification procedure involves ammonium sulphate fractionation, DEAE-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration. The final chromatography step yields a homogeneous preparation of high specific activity with a pI of 5. The molecular mass of the native enzyme was estimated to be approximately 80 kDa by 10-25% gradient polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration. The subunit molecular mass was determined by sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis to be 40 kDa compared with a series of molecular mass standards. This indicates that pyridoxal kinase is a dimeric enzyme. Further results obtained from electron microscopy, using a negative staining technique, provide evidence that pyridoxal kinase exists as a dispherical subunit structure.     

Purification and properties of oxytocinase (cystine amino-peptidase) from monkey placenta.

Oxytocinase (cystyl-aminopeptidase) [EC 3.4.11.3] was isolated from monkey placenta in a purified form by a six-step prodedure comprising extraction from monkey placenta homogenate, ammonium sulfate fractionation, repeated chromatography on hydroxylapatite, chromatography on a column of DEAE-cellulose and gel filtration on a column of Sephadex G-200. The purified enzyme showed a single band on polyacrylamide disc electrophoresis. Oxytocin was inactivated by this enzyme preparation. The enzyme hydrolyzed several aminoacyl-beta-naphthylamides. A terminal amino group was required for enzyme activity. The molecular weight of the purified enzyme was estimated to be 87,000 by gel filtration and 83,000 by sodium dodecyl sulfate gel electrophoresis. Other properties of the enzyme, the effects of metal ions and various chemical reagents on the enzyme activity, the pH optimum, and Km values for a number of aminoacyl-beta-naphthylamides were also examined.     

Isolation of phospholipase D from cotton seeds]

A method of isolating homogenous phospholipase D from cotton seeds is described. The homogeneity of the enzyme preparation is demonstrated by means of polyacrylamide disc electrophoresis, gel filtration through Sephadex G-200 and determination of N-terminal amino acid residue, which turned to be glutamic acid. Molecular weight of the enzyme is found to be 71000 +/- 3000. Several equilibrium forms of the enzyme are observed.     

Purification and Properties of Wall-bound α-Glucosidase from Suspension-cultured Rice Cells

Wall-bound α-glucosidase (EC 3.2.1.20) has been solubilized from suspension-cultured rice cells with Sumyzyme C and Pectolyase Y-23 and isolated by a procedure including fractionation with ammonium sulfate, Sephadex G-100 column chromatography, CM-cellulose column chroma-tography, Sephadex G-200 column chromatography, and preparative disc gel electrophoresis. The molecular weight of the enzyme was 64,000. The enzyme readily hydrolyzed maltose, maltotriose, and amylose, but hydrolyzed isomaltose and soluble starch more slowly. The Michaelis constant for maltose of the enzyme was estimated to be 0.272 mm. The enzyme produced panose as the main α- glucosyltransferred product from maltose.     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.     

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.     

Purification and properties of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600

A preparative scale method for isolation of highly purified phenylalanyl-tRNA synthetase from E. coli MRE-600 was developed. It consists of cell destroying, nucleic acid precipitation with streptomycine sulfate, fractionation with ammonium sulfate followed by chromatography on different carriers (Sephadex G-200, DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite). The mode of cell destroying was found to affect the process of the further enzyme purification. The phenylalanyl-tRNA synthetase was purified 540-fold, with recovery being 20.6% and the specific activity - 540 units per mg protein. The enzyme content in the purified preparation was 80-90% judging by electrophoresis in PAAG. The molecular weights of the subunits determined by electrophoresis under denaturative conditions were found to be 102,000 +/- 4000 (beta) and 42,000 +/- 2000 (alpha). The molecular weight of the native enzyme determined by gel filtration through Sephadex G-200 and electrophoresis at varied concentrations of polyacrylamide was found to be 340,000 +/- 20,000. The Km values for tRNA, ATP and phenylalanine in the aminoacylation reaction are equal to 5.4 X 10(-7) M, 1,9 X 10(-4) M, and 3.7 X 10(-6) M, respectively.     

Human blood group glycosyltransferase. II. Purification of galactosyltransferase

A galactosyltransferase, which converts blood group O red bloodcells to B-cells, was purfied to homogeneity from plasma of blood group B subjects. The stepwise purification procedures include: (a) column chromatography with CM-Sephadex, followed by ammonium sulfate fractionation; (b) Sephadex G-200 gel filtration; (c) column chromatogr,phy with DEAE-Sephadex; and (d) column chromatography with hydroxylapatite. The procedures provided about a 400,000-fold increase of specific activity with a 40 to 50% yield. Further purification of the enzyme was performed by small scale preparative acrylamide gel electrophoresis at pH 4.3. The final enzyme preparation showed a single protein band which coincided with enzyme activity, in acrylamide gel electrophoresis, and revealed a single protein band in sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight, which was estimated by Sephadex gel filtration, and subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ for its activity and had a pH optimum at 7.0 to 7.5.     

A simple purification method for the preparation of solubilized chlorophyllase from Chlorella protothecoides

A simple and rapid purification procedure is described for the routine preparation of large quantities of purified chlorophyllase (chlorophyll chlorophyllido-hydrolase, EC 3.1.1.14) from Chlorella protothecoides. The enzyme with specific activity of 960 nmol chlorophyll a hydrolyzed (mg protein)^?1 min^?1 was prepared by treating the homogenate with n-butanol, ammonium sulfate fractionations and gel filtration through Sephadex G-200 and Sepharose CL-6B, with a yield of 53% of activity based on the butanol extract. The enzyme preparation showed apparent homogeneity as judged by polyacrylamide gel electrophoresis. The procedures take only 4 days and can be operated routinely without column repacking.     

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

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

Purification of lysophospholipase of Vibrio parahaemolyticus and its properties.

Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.     

A heterogeneous beta-D-glucosidase activator from monkey parotid gland and its use as an affinity ligand in the purification of human salivary beta-D-glucosidase

We have isolated a heat-stable, low molecular weight activator peptide(s) from monkey parotid gland that specifically activated human salivary beta-D-glucosidase. This activator appeared to be heterogeneous on Sephadex G-25 gel filtration and polyacrylamide gel electrophoresis under non-denaturing conditions. About 45% of the human salivary beta-glucosidase could bind to the activator immobilised on Sepharose and be eluted by Cutscum. The purified enzyme was nearly homogeneous, with a subunit Mr of 46,000 as revealed by SDS-gel electrophoresis and silver staining.     

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.     

Isolation and characterization of hyaluronidase from scorpion (Heterometrus fulvipes) venom

Hyaluronidase (Hyaluronate lyase, E.C. 3.2.1.35) has been isolated from Heterometrus fulvipes scorpion venom by a combination of gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE-cellulose. The enzyme preparation showed a single band on polyacrylamide gel electrophoresis and a molecular weight of 82,000. The final preparation was purified 27-fold. The optimum pH for enzyme activity was 4.0. No loss of activity was observed up to 30 degrees C and showed a sharp decrease in activity at 50 degrees C. Heparin inhibited the enzyme activity.     

Further studies on endo-beta-N-acetylglucosaminidase D1.

The purification procedure for endo-beta-N-acetylglucosaminidase D was improved to yield an enzyme preparation which was homogeneous upon gel electrophoresis. The molecular weight of the enzyme as estimated by Sephadex G-200 column chromatography was 280,000, while SDS-gel electrophoresis after reduction with 2-mercaptoethanol gave a value of 150,000. The purified enzyme did not show any chitinase, hyaluronidase or lysozyme activity. In the presence of exoglycosidases removing peripheral sugars, the endoglycosidase acted on serum glycoproteins such as transferrin and fetuin. The enzyme also hydrolyzed an oligosaccharide, (Man)5(GlcNAc)2, indicating that the peptide portion of substrates does not have much effect on susceptibility to the enzyme.     

Purification and properties of L-asparaginase from Mycobacterium phlei.

1. L-asparaginase from M. phlei was purified about 170-fold with an 11% yield. The purification procedure consisted of: fractionation with ammonium sulphate; adsorption of contaminating proteins on calcium phosphate gel; chromatography on Sephadex G-150 and DEAE-cellulose. The specific activity of the final preparation was 32.6 i.u./mg protein. 2. Molecular weight of the enzyme as determined by Sephadex G-100 filtration amounted to 126 000. Optimum pH was 8.8-9.2. The enzyme did not hydrolyse L-glutamine over the pH range 4-9, and was inhibited by D-asparagine. The apparent Michaelis constant for L-asparagine was 0.7 mM; energy of activation, 9800 cal/mole. 3. On polyacrylamide-gel electrophoresis the final preparation revealed two protein bands, one of which was coincident with the enzyme activity.     

Studies on lipase from Mucor javanicus. I. Purification and properties.

1. Lipase produced by a mold, Mucor javanicus, was purified about 180-fold from the ethanol precipitate of the culture filtrate. Purification was achieved by acid precipitation followed by gel filtrations on Sephadex G-200 (at low ionic strength) and Sephadex G-75 (at a high ionic strength). The purified enzyme preparation showed unusual behavior on polyacrylamide gel electrophoresis. The molecular weight was estimated to be 21 000. The enzyme had a positional specificity towards the position 1 and 3 of triacylglycerols. 2. Lipase in the crude preparation takes an aggregated form. aggregated form was achieved by raising the ionic strength of the medium. 3. The purified lipase preparation from Mucor javanicus exhibits phospholipase A1 activity, hydrolyzing the carboxyl ester at the 1-position of phosphatidylcholine. This activity seems to be due to the action of the lipase itself and not due to any other specific phospholipases.