scholarly journals The purification and properties of yeast proteinase B from Candida albicans

1986 ◽  
Vol 236 (1) ◽  
pp. 177-184 ◽  
Author(s):  
P C Farley ◽  
M G Shepherd ◽  
P A Sullivan
Keyword(s):  
Candida Albicans ◽  
Polyacrylamide Gel Electrophoresis ◽  
Serine Proteinase ◽  
Aromatic Amino Acids ◽  
Peptide Bond ◽  
Amino Acid Ester ◽  
Heat Stable ◽  
Purification And Properties ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.

scholarly journals Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

1988 ◽  
Vol 250 (2) ◽  
pp. 453-458 ◽  
Author(s):  
H Sobek ◽  
H Görisch
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sulfolobus Acidocaldarius ◽  
Sedimentation Equilibrium ◽  
Prolonged Storage ◽  
Heat Stable ◽  
Equilibrium Data ◽  
Poly Ethylene ◽  
Hydrolysis Of

A heat-stable esterase has been purified 1080-fold to electrophoretic homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium; 20% of the starting activity is recovered. The purified enzyme shows a specific activity of 158 units/mg, based on the hydrolysis of p-nitrophenyl acetate. The esterase hydrolyses short-chain p-nitrophenyl esters, aliphatic esters and triacylglycerols. It is strongly inhibited by paraoxon and phenylmethanesulphonyl fluoride, but only weakly by eserine. From sedimentation-equilibrium data and molecular sieving in polyacrylamide gels, the Mr of the esterase is estimated to be 117000-128000. SDS/polyacrylamide-gel electrophoresis reveals a single band of protein, of Mr 32000. The purified esterase crystallizes in the presence of poly(ethylene glycol) in short rods. The enzyme is inactivated only on prolonged storage at temperature above 90 degrees C.

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 Purification and properties of cytochrome P-450-dependent 14 α-sterol demethylase from Candida albicans

1989 ◽  
Vol 263 (2) ◽  
pp. 573-579 ◽  
Author(s):  
C A Hitchcock ◽  
K Dickinson ◽  
S B Brown ◽  
E G V Evans ◽  
D J Adams
Keyword(s):  
Candida Albicans ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane System ◽  
Sodium Cholate ◽  
Blue Staining ◽  
Purification And Properties ◽  
Opportunistic Fungal Pathogen ◽  
High Degree ◽  
Cytochrome P 450 ◽  
Coomassie Brilliant

The purification of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) from the important opportunistic fungal pathogen, Candida albicans, is described. Optimal purification (875-fold) was achieved by extracting the cytochrome from microsomes with sodium cholate followed by hydroxyapatite, octyl-Sepharose and CM-Sepharose chromatographies, giving a cytochrome preparation of 17.5 nmol/mg of protein. By the use of SDS/polyacrylamide-gel electrophoresis the cytochrome was judged to be highly purified on the basis of Coomassie Brilliant Blue staining of protein. The Mr of P-450DM was estimated to be 51,000. The absorption spectrum of oxidized P-450DM was characteristic of a low-spin cytochrome, and its reduced CO complex had a Soret absorption peak at 447 nm. When reconstituted in a model membrane system of dilauroylphosphatidylcholine with NADPH and O2, P-450DM catalysed the complete 14 alpha-demethylation of lanosterol, which was inhibited by CO. The cytochrome appeared to have a high degree of substrate specificity; it was unable to oxidize a number of xenobiotic compounds in the reconstituted assay.

scholarly journals Elicitors of Plant Defense Responses from Biocontrol Strains of Trichoderma viren

2004 ◽  
Vol 94 (2) ◽  
pp. 171-176 ◽  
Author(s):  
L. E. Hanson ◽  
C. R. Howell
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Active Material ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Defense Responses ◽  
Proteinase K ◽  
Homology Search ◽  
Amino Terminal ◽  
Heat Stable ◽  
Significant Stimulation

Effective biocontrol strains of Trichoderma virens can induce the production of defense-related compounds in the roots of cotton. Ineffective strains do not induce these compounds to significant levels. This elicittation was found to be heat stable, insoluble in chloroform, passed through a 5K molecular weight cut-off (MWCO) filter, but not a 3K MWCO filter, and was sensitive to treatment by proteinase K. When the active material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several bands were present in the material from biocontrol-active strains that were lacking in inactive strains. When eluted and tested for elicitation activity, with or without renaturation, four bands stimulated cotton terpenoid production. One band showed cross-reaction with an antibody to the ethylene-inducing xylanase from T. viride. Another band of approximately 18 kDa, gave significant stimulation of cotton terpenoid production and increased peroxidase activity in cotton radicles in all tests, with or without renaturation. The 18-kDa protein was subjected to amino-terminal sequence analysis, and the first 19 amino acids at the amino terminus were determined to be DTVSYDTGYDNGSRSLNDV. A database homology search using the BLASTp algorithm showed the highest similarity to a serine proteinase from Fusarium sporotrichioides.

scholarly journals Purification and properties of myo-inositol-1-phosphatase from bovine brain

1988 ◽  
Vol 253 (2) ◽  
pp. 387-394 ◽  
Author(s):  
P V Attwood ◽  
J B Ducep ◽  
M C Chanal
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Bovine Brain ◽  
Native Enzyme ◽  
Hydroxyl Groups ◽  
Inositol 1 ◽  
Substrate Structure ◽  
Purification And Properties ◽  
Phosphorylated Compounds ◽  
Hydrolysis Of

myo-Inositol-1-phosphatase from bovine brain was purified over 2000-fold. The native enzyme has a Mr of 59,000, and on SDS/polyacrylamide-gel electrophoresis the subunit Mr was 31,000. Thus the native enzyme is a dimer of two apparently identical subunits. The enzyme, purified to a specific activity of more than 300 units/mg of protein (1 unit of enzyme activity corresponds to the release of 1 mumol of Pi/h at 37 degrees C), catalysed the hydrolysis of a variety of phosphorylated compounds, the best one, in terms of V/Km, being D-myo-inositol 1-phosphate. Kinetic constants of compounds tested, including both isomers of glycerophosphate and two deoxy forms of beta-glycerophosphate, were measured. They show the importance of the two hydroxyl groups which are adjacent to the phosphate in myo-inositol 1-phosphate. With a wide variety of substrates Li+ was found to be an uncompetitive inhibitor whose Ki varied with substrate structure.

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.

scholarly journals Proteins of the kidney microvillar membrane. Purification and properties of the phosphoramidon-insensitive endopeptidase (‘endopeptidase-2’) from rat kidney

1987 ◽  
Vol 245 (2) ◽  
pp. 515-524 ◽  
Author(s):  
A J Kenny ◽  
J Ingram
Keyword(s):  
Rat Kidney ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Staining Intensity ◽  
Aminopeptidase Activity ◽  
Ph Optimum ◽  
Reducing Conditions ◽  
Purification And Properties ◽  
Relationship Of ◽  
Hydrolysis Of

A second endopeptidase is present in the renal microvillar membrane of rats that can be distinguished from endopeptidase-24.11 by its insensitivity to inhibition by phosphoramidon. The purification of this enzyme, referred to as endopeptidase-2, is described. The enzyme was efficiently released from the membrane by treatment with papain. The subsequent four steps depended on ion-exchange and gel-filtration chromatography. These steps were monitored by the hydrolysis of various substrates: 125I-insulin B chain (the normal assay substrate), benzoyl-L-tyrosyl-p-aminobenzoate (Bz-Tyr-pAB), azocasein and benzyloxycarbonyl-L-phenylalanyl-L-arginine 7-amino-4-methylcoumarylamide (Z-Phe-Arg-NMec). All four assays revealed comparable stepwise increases in activity in the main stages of the purification, although it was apparent that the last-named fluorogenic assay depended on traces of aminopeptidase activity present in the preparation. The Km for 125I-insulin B chain was 16 microM and that for Bz-Tyr-pAB was 4.7 mM. Several experimental approaches confirmed that both peptides were hydrolysed by the same enzyme. The pH optimum was 7.3. Phosphate buffers were inhibitory and shifted the optimum to above pH 9. Zinc was detected in the purified enzyme; EDTA and 1,10-phenanthroline were strongly inhibitory. SDS/polyacrylamide-gel electrophoresis revealed polypeptides of equal staining intensity of Mr 80,000 and 74,000 in reducing conditions. In non-reducing conditions a single band of apparent Mr 220,000 was seen. Gel filtration yielded an Mr of 436,000. These results are consistent with an oligomeric structure in which the alpha and beta chains are linked by disulphide bridges. Endopeptidase-2 hydrolysed a number of neuropeptides. Enkephalins resisted attack, only the heptapeptide [Met]enkephalin-Arg6-Phe7 being susceptible to slow hydrolysis. Luliberin (luteinizing-hormone-releasing hormone) and bradykinin were rapidly hydrolysed. Neurotensin was shown to be slowly attacked at the Tyr3-Glu4 bond. Thus the specificity appears to be limited to the hydrolysis of bonds involving the carboxy group of aromatic residues, provided that this P1 residue is extended by additional residues, at least to the P3′ position. The relationship of this membrane metalloendopeptidase to mouse meprin and human ‘PABA peptidase’ is discussed.

scholarly journals The specificity of the S1′ subsite of papain

1974 ◽  
Vol 141 (2) ◽  
pp. 495-501 ◽  
Author(s):  
M. Robert Alecio ◽  
Malcolm L. Dann ◽  
Gordon Lowe
Keyword(s):  
Amino Acid ◽  
Kinetic Parameters ◽  
Proteolytic Enzyme ◽  
Peptide Bond ◽  
Amide Group ◽  
Hydrophobic Residues ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester

The specificity of the S1′ subsite of the proteolytic enzyme papain was investigated by studying the effect of l-α-amino acid amides on the enzyme-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester and by determining the kinetic parameters for the enzyme-catalysed hydrolysis of some N-benzyloxycarbonylglycyl-l-amino acid amides. These studies showed that the S1′ subsite has a predilection for hydrophobic residues, in particular l-leucine and l-tryptophan. The specificity for these residues is manifest in both the binding and acylation steps. N-Benzyloxycarbonylglycine amide is not hydrolysed under comparable conditions, indicating that the amide group adjacent to and on the C-terminal side of the peptide bond about to be cleaved makes an important contribution to the rate of the papain-catalysed hydrolysis of peptides.

Plasminogen Activator and Plasminogen Activator Inhibitor Associated with Granulomatous Inflammation: A Study with Murine Leprosy

1984 ◽  
Vol 52 (03) ◽  
pp. 243-249 ◽  
Author(s):  
S Izaki ◽  
T Hibino ◽  
Y Isozaki ◽  
P S Hsu ◽  
M Izaki ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Soluble Fraction ◽  
Proteinase Inhibitors ◽  
Granulomatous Inflammation ◽  
Serine Proteinase ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Weakly Alkaline ◽  
Heat Stable ◽  
Type Plasminogen Activator

SummaryPlasminogen activator that is associated with the development of hypersensitivity granulomas (gPA) was partially purified from a saline soluble fraction of murine lepromas elicited in “resistant” mice, C57BL/6N. The gPA was shown to consist of two subspecies (23,000 and 48,000 in molecular weight) with essentially identical enzymologic properties. The gPA was found to be a relatively heat stable weakly alkaline serine proteinase with trypsin-like characteristics in the specificity for synthetic substrates and proteinase inhibitors. It showed a high affinity for H- D-Ile-Pro-Arg-pNA (Km = 1.4 × 10-4 M) H-D-Val-Leu-Lys- pNA (Km = 5.2 × 10-4 M), and L-pyroGlu-Gly-Arg-pNA (Km = 9.3 × 10-4 M). The gPA did not demonstrate antigenic cross reaction with urokinase-type or tissue-type plasminogen activator.Two distinct enzymatic regulators of the gPA were also demonstrated in the saline soluble fraction of the hypersensitivity granulomas. The gPA and its regulation are assumed to be correlated with macrophage activation in the hypersensitivity granulomas

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