scholarly journals Purification and properties of neutral maltase from human granulocytes

1989 ◽  
Vol 263 (3) ◽  
pp. 647-652 ◽  
Author(s):  
P Delqué Bayer ◽  
C Vittori ◽  
P Sudaka ◽  
J Giudicelli
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Human Kidney ◽  
Ph Optimum ◽  
Major Band ◽  
Purification And Properties ◽  
A Minor ◽  
Triton X ◽  
Human Polymorphonuclear Leukocytes

A procedure for the purification of neutral maltase from human polymorphonuclear leukocytes is described, involving solubilization with Triton X-100, proteolytic attack and three chromatographic steps: DEAE ion exchange, AcA 22 gel filtration and a second DEAE chromatography. The enzyme was obtained with a final specific activity of 30 units/mg of protein, comparable with that of other neutral maltases previously purified. The Mr of the enzyme was 550,000 as determined by gel filtration. SDS/polyacrylamide-gel electrophoresis, under non-denaturing conditions, led to a major band of 500,000 and a minor one of 260,000, both active, suggesting a polymeric or aggregated form of the protein. The catalytic properties of the human granulocytic neutral maltase were investigated. The pH optimum was around 6. The enzyme exhibited a broad range of substrate specificity, hydrolysing di- and oligosaccharides with alpha (1→2), alpha (1→3) and alpha (1→4) glucosidic linkages. The highest activities were observed for alpha (1→4) glucose oligomers of three to five residues. It was also found to hydrolyse polysaccharides such as starch and glycogen. The results of the inhibition studies are interpreted in terms of the existence of a large site including several subsites. The enzyme properties are broadly similar to those observed for other purified neutral alpha-glucosidases, in particular that of human kidney origin.

The Purification and Properties of an Amidohydrolase from Soybean

1974 ◽  
Vol 52 (10) ◽  
pp. 903-910 ◽  
Author(s):  
Robert E. Hoagland ◽  
George Graf
Keyword(s):  
Arrhenius Plot ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hydrolysis Rate ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Hydrolysis Rates ◽  
Hydrolysis Of

An amidohydrolase (EC 3.5.1.13) was isolated from the roots of soybean (Glycine max Merril, var. Hawkeye) seedlings and purified 130-fold over the crude extract with 30% recovery. The purification steps entailed ammonium sulfate precipitation, gel filtration, cellulose ion-exchange chromatography, and polyacrylamide gel electrophoresis. The specific activity of the purified enzyme for the hydrolysis of Nα-benzoyl-DL-arginine p-nitroanilide (BAPA) was 810 mU/mg. The Km of the enzyme for this substrate was 5.78 × 10−6 M. The enzyme possessed a broad substrate specificity and catalyzed the hydrolysis of BAPA, glycine p-nitroanilide, L-leucine p-nitroanilide, and L-lysine p-nitroanilide. Specificity studies with a series of aminoacyl β-naphthylamides revealed a high hydrolysis rate on Nα-benzoyl-L-arginine β-naphthylamide, and lower hydrolysis rates on several other aminoacyl-substituted β-naphthylamides. The enzyme also displayed dipeptide hydrolase activity on several dipeptide substrates. The enzyme had a pH optimum of 8.0 in 0.05 M phosphate buffer with Nα-benzoyl-DL-arginine p-nitroanilide as substrate. The temperature optimum was 50 °C. The apparent activation energy determined from an Arrhenius plot was 6.3 kcal/mol (26 400 J/mol). The molecular weight estimated by gel filtration was approximately 63 000. Mercury (II) ion, silver (I) ion, p-benzoquinone, p-chloromercuribenzoate, and N-ethylmaleimide were effective inhibitors of the enzyme.

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

scholarly journals Purification, characterization and inhibition of human skin collagenase

1978 ◽  
Vol 169 (2) ◽  
pp. 265-276 ◽  
Author(s):  
David E. Woolley ◽  
Robert W. Glanville ◽  
Dennis R. Roberts ◽  
John M. Evanson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Serum Proteins ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Collagen Fibrils ◽  
Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

scholarly journals Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria

1984 ◽  
Vol 224 (1) ◽  
pp. 171-179 ◽  
Author(s):  
I R Cottingham ◽  
A L Moore
Keyword(s):  
Nadh Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Properties ◽  
Nadh Dehydrogenases ◽  
Arum Maculatum ◽  
A Minor ◽  
Considerable Loss

The external NADH dehydrogenase has been purified from Arum maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows, when the gel is silver-stained, that the purified enzyme contains two major bands of Mr 78 000 and 65 000 and a minor one of Mr about 76 000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly iron-sulphur centres, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme has been distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.

scholarly journals Purification and properties of l-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus

1987 ◽  
Vol 248 (3) ◽  
pp. 871-876 ◽  
Author(s):  
M E Hoey ◽  
N Allison ◽  
A J Scott ◽  
C A Fewson
Keyword(s):  
Lactate Dehydrogenase ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Potential Inhibitors ◽  
Triton X

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a ‘wall + membrane’ fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.

Purification and Properties of a Neutral Protease from Soil Bacterium WM 122

1977 ◽  
Vol 37 (03) ◽  
pp. 556-565 ◽  
Author(s):  
S. E Papaioannou ◽  
W. J Marsheck
Keyword(s):  
Methyl Ester ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Soil Bacterium ◽  
Ester Hydrochloride ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Hydrolysis Of

SummaryAn extracellular protease SN 687, secreted by the soil bacterium isolate WM 122, has been purified by means of gel filtration, ammonium sulfate precipitation, DEAE-Sephadex and hydroxylapatite chromatography. Apparent homogeneity was ascertained by Polyacrylamide gel electrophoresis. The protease was inactivated by ethylenediamine tetracetic acid (EDTA) but not by diisopropylfluorophosphate (DFP), and it was partially inhibited by serum inhibitors. SN 687 was shown to be of high specific activity against casein and fibrin, but it did not hydrolyze L- lysine -methyl ester dihydrochloride (LME), p-tosyl-L-arginine-methyl ester hydrochloride (TAME) and N-benzoyl-L-tyrosine-ethyl ester hydrochloride (BTEE) synthetic substrates. The optimum pH for hydrolysis of casein was 7.5 and the molecular weight, as determined by gel filtration, was 31,000.

scholarly journals Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans

1977 ◽  
Vol 161 (2) ◽  
pp. 357-370 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Cytochrome C ◽  
Electron Acceptor ◽  
Specific Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Transport Chain ◽  
Purification And Properties ◽  
Solubilized Enzyme ◽  
Triton X ◽  
Sepharose 4B

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.

Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase

1984 ◽  
Vol 247 (4) ◽  
pp. G385-G393 ◽  
Author(s):  
I. M. Roberts ◽  
R. K. Montgomery ◽  
M. C. Carey
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Pancreatic Lipase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Dodecyl Sulfate ◽  
Triton X

We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

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