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.     

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 and characterization of arylsulfatase from<Emphasis Type="Italic"> Sphingomonas</Emphasis> sp. AS6330

Arylsulfatase was purified from Sphingomonas sp. AS6330 through ionic exchange, hydrophobic- and gel-chromatographies. The purity increased 12,800-fold with approximately 19.1% yield against cell homogenate. The enzyme was a monomeric protein with apparent molecular weight of 62 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 41 kDa as determined by gel filtration. The enzyme had optimum reaction conditions for hydrolysis of sulfate ester bonds in agar and p-nitrophenyl sulfate (NPS) at pH 7.0 and 45°C, with a specific activity of 3.93 and 97.2 U, respectively. The enzyme showed higher activity towards agar than other sulfated marine polysaccharides such as porphyran, fucoidan and carrageenan. The K _m and V _max of the enzyme for hydrolysis of NPS were 54.9 M and 113 mM/min, respectively. With reaction of 200 g agar with 100 U arylsulfatase for 8 h at 45°C, gel strength increased 2.44-fold, and 97.7% of the sulfate in the agar was hydrolyzed.     

Characterization of an inducible amidase from Pseudomonas acidovorans AE1.

The main molecular and catalytic properties of an acetanilide-hydrolyzing enzyme from Pseudomonas acidovorans AE 1, purified to a homogeneous state, were investigated. The molecular weight was 57 500 as determined by gel filtration and 55 300 as computed from the amino acid composition. By polyacrylamide gel electrophoresis in dodecylsulfate a polypeptide chain weight of 56 700 was obtained. Based on the reaction of the highly purufied enzyme with diethyl-4-nitrophenyl phosphate an equivalent weight of approximately 59 100 was found. From these results it was concluded that the enzyme consists of a single polypeptide chain and contains one active site per molecule. The enzyme hydrolyzed esters as well as certain aromatic amides. It also catalysed the transfer of acetyl groups to phenetidine yielding phenacetin. The activities towards aliphatic esters were much smaller. The enzyme was stable at pH values ranging from 7 to 9 and its pH-optimum was about 10. It was strongly inhibited by organophosphorous compounds, like diethyl-4-nitrophenyl phosphate or diisopropylphosphorofluoridate, as well as by physostigmine sulfate and -SH-blocking reagents, like HgCl-2 or 4-chloromercuribenzoic acid. o-Nitrophenol caused a competitive inhibition and phenetidine an uncompetitive inhibition.     

Purification and properties of asparagine synthetase from rat liver.

Asparagine synthetase (L-aspartate:ammonia ligase (AMP-forming, EC 6.3.1.1) activity in rat liver increased when the animals were put on a low casein diet. The enzyme was purified about 280-fold from the supernatant of rat liver homogenate by a procedure comprising ammonium sulfate fractionation. DEAE-Sepharose column chromatography, and Sephadex G-100 gel filtration. The optimal pH of the enzyme was in the range 7.4-7.6 with glutamine as an amide donor. The molecular weight was estimated to be approximately 110,000 by gel filtration. Chloride ion was required for the enzyme activity. The apparent Km values for L-aspartate, L-glutamine, ammonium chloride, ATP, and Cl- were calculated to be 0.76, 4.3, 10, 0.14, and 1.7 mM, respectively. The activity was inhibited by L-asparagine, nucleoside triphosphates except ATP, and sulfhydryl reagents. It has been observed that the properties of asparagine synthetase from rat liver are not so different from those of tumors such as Novikoff hepatoma and RADA 1.     

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 Some Properties of α-Galactosidase from Tubers of Stachys affinis

An α-galactosidase from tubers of S. affinis was purified about 130 fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The purified enzyme showed a single protein band on disc gel electrophoresis. The molecular weight of the enzyme was determined to be approximately 42,000 by gel filtration and 44,000 by SDS disc gel electrophoresis. The optimum reaction pH was 5.2. The enzyme hydrolyzed raffinose more rapidly than planteose. The activation energy of raffinose and planteose by the enzyme was estimated to be 7.89 and 11.4 kcal/mol, respectively. The enzyme activity was inhibited by various galactosides and structural analogs of d-galactose. Besides hydrolytic activity, the enzyme also catalyzed the transfer reaction of d-galactosyl residue from raffinose to methanol.     

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.     

Characterization of lysosomal acid lipase purified from rabbit liver

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.     

Purification and properties of mycodextranase from Streptomyces sp. J-13-3.

An enzyme hydrolyzing nigeran (alternating alpha-1,3- and alpha-1,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by percipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-100. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS-PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50 degrees C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50 degrees C. The enzyme activity was inhibited significantly by Hg+, Hg2+, and p-chloromercuribenzoic acid. The Km (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the alpha-1,4-glucosidic linkages in nigeran.     

Purification and properties of cyclomaltodextrinase from alkalophilic Bacillus sp.

An alkalophilic strain isolated from soil produced intracellular cyclomaltodextrinase on the culture medium at an initial pH of 10.6. The strain was identified as closely resembling Bacillus circulans. The enzyme was purified 252-fold from the cell extract by chitosan treatment, ammonium sulfate fractionation, DEAE-Toyopearl column chromatography, and gel filtration. The pH and temperature optima of the purified enzyme were 6.0 and 50°C. The molecular weight of the enzyme was 126,000, with two subunits of 67,000. The isoelectric point was pH 4.2. Enzyme activity was inhibited by Ag⁺, Hg²⁺, Cu²⁺, and p-chloromercuribenzoate. The enzyme hydrolyzed α-, β-, and γ-cyclodextrins, as well as linear maltodextrins, to yield maltooligosaccharides. Starch and maltose were not degraded by the enzyme.     

Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone

Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.     

Purification and characterization of an aminopeptidase from the edible basidiomycete Grifola frondosa

An aminopeptidase was purified 178-fold from an extract of Grifola frondosa by ammonium sulfate precipitation and a series of column chromatographies on phenyl-Toyopearl, Sephadex G-25, and Mono-Q. The molecular mass of the enzyme was estimated to be 27 kDa and 30 kDa by gel filtration and SDS-PAGE, respectively. The enzyme had an optimum pH of 8.5 and was stable between pH 6.0 and pH 10.5, and it also had a high level of heat stability. The enzyme was inactivated by EDTA and o-phenanthroline, and it was also strongly inhibited by bestatin, but no inhibitory effect of DFP was observed. The enzyme preferentially hydrolyzed peptides containing hydrophobic residues in the N-terminal position.     

Proline iminopeptidase from Bacillus megaterium: purification and characterization

Proline iminopeptidase [EC 3.4.11.5] was purified about 1,700-fold from cell free extract of Bacillus megaterium by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose and hydroxyapatite, and gel filtration on Toyopearl FW-55. The purified enzyme still contained a minor contaminant as judged by disc gel electrophoresis. The enzyme was most active at pH 7.0 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminal. The enzyme was completely inactivated by p-chloromercuribenzoate (PCMB), but was not inhibited by metal chelators, diisopropylphosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by adding 2-mercaptoethanol. From this result and the chromatographic profile on PCMB-T-Sepharose, the enzyme seems to be a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0. The molecular weight of the enzyme was estimated to be 58,000 by gel filtration on Toyopearl and 60,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is a monomer.     

Purification and properties of pig liver kynureninase.

Kynureninase [L-kynurenine hydrolase, EC 3.7.1.3] was purified from pig liver by a procedure including DEAE-cellulose chromatography, hydroxyapatite chromatography, ammonium sulfate fractionation, DEAE-Bio Gel chromatography, Sephacryl S-200 gel filtration, kynurenine-Sepharose affinity chromatography, and Sephadex G-200 gel filtration. The enzyme was found to be homogeneous by the criterion of disc-gel electrophoresis. The enzyme has a molecular weight of about 100,000 and exhibits absorption maxima at 280 and 420 nm. The optimum pH and the isoelectric point of the enzyme are 8.5 and 5.0, respectively. The Michaelis constants were determined to be as follows: L-kynurenine, 7.7 X 10(-4) M; L-3-hydroxykynurenine, 1.3 X 10(-5) M; and pyridoxal 5'-phosphate, 1.8 X 10(-6) M. L-3-Hydroxykynurenine is hydrolyzed more rapidly than L-kynurenine; the liver enzyme can be regarded as a 3-hydroxy-kynureninase.     

Proline iminopeptidase from Bacillus coagulans: purification and enzymatic properties

Proline iminopeptidase [EC 3.4.11.5] was purified about 2,700-fold from cell-free extract of Bacillus coagulans by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose, and hydroxyapatite, and gel filtration on Sephadex G-150. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.3 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid including proline, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminus. Pro-D-amino acid bonds were also susceptible to the enzyme. The enzyme was completely inhibited by p-chloromercuribenzoate (PCMB) and partially by proline but not by metal chelators, diisopropylphosphorofluoridate (DFP), or phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by incubation with 2-mercaptoethanol. These results and the chromatographic profile on PCMB-T-Sepharose suggest that the enzyme is a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0, and the molecular weight of the enzyme was estimated to be 40,000 by gel filtration on Sephadex G-100 and 35,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, indicating that the enzyme exists as a monomer.     

Purification and characterization of a cell wall peptidase from Lactococcus lactis subsp. cremoris IMN-C12.

A peptidase from the cell wall fraction of Lactococcus lactis subsp. cremoris IMN-C12 has been purified to homogeneity by hydrophobic interaction chromatography, two steps of anion-exchange chromatography, and gel filtration. The molecular mass of the purified enzyme was estimated to be 72 kDa by gel filtration and 23 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pI of 4.0, and it has the following N-terminal sequence from the 2nd to the 17th amino acid residues: -Arg-Leu-Arg-Arg-Leu-?-Val-Pro-Gly-Glu-Ileu-Val-Glu-Glu-Leu-Leu. The peptidase is most active at pH 5.8 and at 33 degrees C with trileucine as the substrate. Reducing agents such as dithiothreitol, beta-mercaptoethanol, and cysteine strongly stimulated enzyme activity, while p-chloromercuribenzoate had an inhibitory effect. Also, metal chelators lowered the peptidase activity, which could not be restored with Ca2+ and Mg2+. The divalent cations Cu2+, Zn2+, Fe2+, and Hg2+ completely inhibited peptidase activity. The peptidase is capable of hydrolyzing tripeptides and some dipeptides, with a preference for peptides containing leucine and with the highest activity towards the tripeptides Leu-Leu-Leu, Leu-Trp-Leu, and Ala-Leu-Leu, which were hydrolyzed with Kms of 0.37, 0.18, and 0.61 mM, respectively.     

A Proteinase from Germinating Barley : I. Purification and Some Physical Properties of a 30 kD Cysteine Endoproteinase from Green Malt

A proteinase was purified from germinated barley (green malt from Hordeum vulgare L. cv Morex) by acidic extraction, ammonium sulfate fractionation and successive chromatographies on CM-cellulose, hemoglobin sepharose, Sephadex G-75 and organomercurial agarose columns. The overall purification and final recovery were 290-fold and 7.5%, respectively. The purified enzyme was homogeneous on analytical gel electrophoresis, yielding a single protein associated with protease activity. An apparent molecular weight of about 20 kilodaltons was estimated for the native enzyme from gel filtration. SDS-gel electrophoresis revealed a single polypeptide of about 30 kilodaltons. The optimum pH for the hydrolysis of hemoglobin was around 3.8. The enzyme was strongly inhibited by leupeptin but was insensitive to phenylmethylsulfonyl fluoride, indicating that it was a cysteine proteinase. It hydrolyzed several large proteins from various origins. The ability of the enzyme to digest barley storage proteins in vitro was examined using SDS-gel electrophoresis. The hydrolysis patterns obtained showed that the enzyme rapidly hydrolyzed the large hordein polypeptides into relatively small fragments. The results of this study suggest that this 30 kilodalton enzyme is one of the predominant cysteine proteinases secreted into the starchy endosperm during barley germination and that it plays a major role in the mobilization of storage proteins.     

Characterizations of acylagmatine amidohydrolase and carboxypeptidase from Fusarium anguioides.

Previously an enzyme, named acylagmatine amidohydrolase, hydrolyzing bleomycin B2 to bleomycinic acid and agmatine was found in the mycelia of Fusarium anguioides Sherbakoff. In this work the enzyme was purified further, but not completely. The crude enzyme preparation hydrolyzed various acylagmatines and also peptidyl arginine, but the latter activity could be separated from acylagmatine amidohydrolase activity by gel filtration on Sephadex G-100. The enzyme was inhibited by PCMB and its molecular weight was estimated as 65,000 by gel filtration. It showed substrate specificity with respect to the alkyl-chain length of the amine moiety. The other hydrolase fraction with activity toward Bz-Gly-Arg was found to be of a sort of carboxypeptidase, which preferentially hydrolyzed peptides with arginine or lysine at the carboxyl terminus, including bradykinin, but liberated neutral amino acids as well from the terminus when the penultimate residue of the substrates was phenylalanine. With Bz-Gly-Arg as substrate Fusarium carboxypeptidase was sensitive to chelating agents but not to diisopropyfluorophosphate, and its molecular weight was estimated to be 145,000.     

A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.