Purification and characterization of an extracellular proteinase from Brevibacterium linens

1990 ◽  
Vol 36 (7) ◽  
pp. 510-512 ◽  
Author(s):  
Ondrej Juhasz ◽  
Bohumil Škárka
Keyword(s):  
Gel Electrophoresis ◽  
Serine Proteinase ◽  
Purification And Characterization ◽  
Extracellular Proteinase ◽  
Brevibacterium Linens ◽  
Caseinolytic Activity ◽  
Inhibition Studies ◽  
Substrate Proteins ◽  
Optimal Ph

The alkaline proteinase of Brevibacterium linens was partially purified through ultrafiltration and chromatography on Sephacryl S-200. The molecular weight of this enzyme was determined by gel electrophoresis in polyacrylamide to be 52 000 – 55 000. The proteinase hydrolyses natural polypeptides such as casein, haemoglobin, bovine serum albumin, and ovalbumin. An apparent Km for casein was determined to be 1.3 mg∙mL−1. The optimal pH for caseinolytic activity was between 7.0 and 8.5 at the optimal temperature of 45 °C. The isolated enzyme is thermolabile: incubation at 50 °C destroyed proteolytic activity. Substrate proteins have a stabilizing effect. Our inhibition studies revealed that the B. linens proteinase belongs to the serine proteinase class. Key words: Brevibacterium linens, proteinase, purification.

Purification and characterization of extracellular proteinase produced by Brevibacterium linens ATCC 9172

1998 ◽  
Vol 63 (4) ◽  
pp. 499-503 ◽  
Author(s):  
Jarmila Tomaschova ◽  
Wolfgang Buchinger ◽  
Werner Hampel ◽  
Jaroslav Zemanovic
Keyword(s):  
Purification And Characterization ◽  
Extracellular Proteinase ◽  
Brevibacterium Linens

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M Ribieto ◽  
J Elion ◽  
D Labie ◽  
F Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

Purification and Characterization of Serine Proteinase from a Halophilic Bacterium,Filobacillussp. RF2-5

2005 ◽  
Vol 69 (1) ◽  
pp. 38-44 ◽  
Author(s):  
Kazumi HIRAGA ◽  
Yasushi NISHIKATA ◽  
Sirilak NAMWONG ◽  
Somboon TANASUPAWAT ◽  
Katsumi TAKADA ◽  
...  
Keyword(s):  
Serine Proteinase ◽  
Halophilic Bacterium ◽  
Purification And Characterization

Purification and characterization of an extracellular proteinase from hendersonula toruloidea

1990 ◽  
Vol 1 (2) ◽  
pp. 114 ◽  
Author(s):  
V. Rojanavanich ◽  
T. Yoshiike ◽  
R. Tsuboi ◽  
K. Takamori ◽  
H. Ogawa
Keyword(s):  
Purification And Characterization ◽  
Extracellular Proteinase

Purification and Characterization of a 3,17 β-Hydroxysteroid Dehydrogenase from Streptomyces hydrogenans

1979 ◽  
Vol 34 (7-8) ◽  
pp. 533-540 ◽  
Author(s):  
Helmut Duchmann ◽  
Lothar Träger
Keyword(s):  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Sedimentation Coefficient ◽  
Hydroxysteroid Dehydrogenase ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization

3,17 β-Hydroxysteroid dehydrogenase has been enriched and purified from cytosol of Streptomyces hydrogenans. After ammonium sulfate precipitation and filtration on Sephadex G-100 the enzyme was finally purified by preparative gel electrophoresis and DEAE-Sephadex A-50 chro­matography. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate gave a single band of mobility corresponding to molecular weight of 70 200 ± 2 500. 3 β-. 17 β- as well as 20 β-hydroxy steroids were dehydrogenated by the enzyme in the presence of NAD+. The dehydrogenation proceeded faster than the reduction of the corresponding ketosteroids in the presence of NADH. The enzyme does not accent NADP+ or NADPH as co-substrates. The apparent Km values were calculated to be 11 μᴍ for 5 α-dihydrotestosterone, 20 μᴍ for testosterone ana 68 μᴍ for epiandrosterone in the NAD+-driven reaction, 1.8 x 10-4 m for NADH+ and 1.9 x 10-4 ᴍ for NADH. The catalytic activity was influenced by the ratio of NAD+/ATP. The inhibition by ATP appears to be of a competitive type with respect to NAD+ (Ki 1.15 x 10-3 ᴍ).After sucrose gradient centrifugation in a preparative ultracentrifuge the enzyme sediments with 4.1 ± 0.1 S as estimated in comparison to other proteins of known sedimentation coefficient. The isoelectric point was determined to be 3.9 with the LKB preparative isoelectric focusing col­umn (pH 2-11) and 4.1 with the analytical flat bed polyacrylamide isofocusing (pH 3 - 5). The number of SH groups was determined to be 2 mol/mol enzyme. In the presence of 6 M urea the fig­ure inceases to 3 mol SH/mol enzyme. In the presence of an excess of p-chloromercuribenzoate the enzyme activity decreases only partially.

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