ZYMOGRAPHIC ANALYSIS OF WATER-SOLUBLE PROTEASES OF VIBRIO CHOLERAE O1 AND О139 SEROGROUPS

Aim. Zymographic analysis of water-soluble proteases of Vibrio cholerae O1 и О139 serogroups. Materials and methods. Bacteria cultivated on a casein-yeast agar (рН 7,6) at 37 ° with during one twenty-four hours and washed off physiological solution. Bacterial mass (by the concentration of 109 cell./...

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Autores principales:	S. N. Kozlov, V. B. Nikolaev, E. Yu. Markov, L. Ya. Urbanovich
Formato:	article
Lenguaje:	RU
Publicado:	Scientific Сentre for Family Health and Human Reproduction Problems 2013
Materias:	substrate electrophoresis ureal extracts proteases vibrio cholerae zymography Science Q
Acceso en línea:	https://doaj.org/article/e939d7396bbf4fc987c517f2d177cdae
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spelling	oai:doaj.org-article:e939d7396bbf4fc987c517f2d177cdae2021-11-23T06:14:26ZZYMOGRAPHIC ANALYSIS OF WATER-SOLUBLE PROTEASES OF VIBRIO CHOLERAE O1 AND О139 SEROGROUPS2541-94202587-9596https://doaj.org/article/e939d7396bbf4fc987c517f2d177cdae2013-04-01T00:00:00Zhttps://www.actabiomedica.ru/jour/article/view/1463https://doaj.org/toc/2541-9420https://doaj.org/toc/2587-9596Aim. Zymographic analysis of water-soluble proteases of Vibrio cholerae O1 и О139 serogroups. Materials and methods. Bacteria cultivated on a casein-yeast agar (рН 7,6) at 37 ° with during one twenty-four hours and washed off physiological solution. Bacterial mass (by the concentration of 109 cell./ml) was treatment by sterile solution of urea in an eventual concentration 4,5 М. After day's display and determination of sterility of got lysate insoluble in urea material (cell walls) was deleted by high-speed centrifugation. Supernatant liquid was exposed to the dialysis, released from insoluble in water sediment centrifugation and freeze dried out. Protease activity was determined in diffusion test in 1 % agarose gel, containing 0,5 % gelatin or 0,5% casein. The spectrum of proteases was analysed by a substrate electrophoresis in the blocks of 8% polyacrylamide gel, impregnated in the process of polymerization gelatin or casein (in an eventual concentration 0,1 %), in presence the dodecylsulphate of sodium. Results. After differential centrifugation of ureal lysate of cells and dialysis there are mainly intracellular water-soluble proteases in the investigated extracts. Diffusion tests showed that all preparations of the investigated strains of Vibrio cholerae possessed protease activity of different intensity. Changing of substrate in diffusion test from gelatin to the casein resulted in the general diminishing of the registered areas of hydrolysis, specifying that a casein fissions less actively by comparison to gelatin. Substrate electrophoresis showed that proteins, making the spectrum of water-soluble pro teases of Vibrio cholerae are extracted by urea, possessed molecular mass, varying within from less than 30 кйа to more than 120 кDа. Strains distinctions are marked in quantitative and high-quality description of spectrums of soluble proteases. Dependence of description of type of water-soluble proteases is educed on used substrate. At the estimation of spectrums of proteases clear confirmation is got, that on gels are impregnated by gelatin electrophoretic mobility of proteases is higher, than in gels, containing a casein. Conclusion. Substrate electrophoresis of preparations of cell-free lysates of Vibrio cholerae showed the presence of a few water-soluble proteases, quantitative and high-quality interstrain distinctions, dependence of spectrum of active proteases from of used substrate.S. N. KozlovV. B. NikolaevE. Yu. MarkovL. Ya. UrbanovichScientific Сentre for Family Health and Human Reproduction Problemsarticlesubstrate electrophoresisureal extractsproteasesvibrio choleraezymographyScienceQRUActa Biomedica Scientifica, Vol 0, Iss 2(2), Pp 139-143 (2013)
institution	DOAJ
collection	DOAJ
language	RU
topic	substrate electrophoresis ureal extracts proteases vibrio cholerae zymography Science Q
spellingShingle	substrate electrophoresis ureal extracts proteases vibrio cholerae zymography Science Q S. N. Kozlov V. B. Nikolaev E. Yu. Markov L. Ya. Urbanovich ZYMOGRAPHIC ANALYSIS OF WATER-SOLUBLE PROTEASES OF VIBRIO CHOLERAE O1 AND О139 SEROGROUPS
description	Aim. Zymographic analysis of water-soluble proteases of Vibrio cholerae O1 и О139 serogroups. Materials and methods. Bacteria cultivated on a casein-yeast agar (рН 7,6) at 37 ° with during one twenty-four hours and washed off physiological solution. Bacterial mass (by the concentration of 109 cell./ml) was treatment by sterile solution of urea in an eventual concentration 4,5 М. After day's display and determination of sterility of got lysate insoluble in urea material (cell walls) was deleted by high-speed centrifugation. Supernatant liquid was exposed to the dialysis, released from insoluble in water sediment centrifugation and freeze dried out. Protease activity was determined in diffusion test in 1 % agarose gel, containing 0,5 % gelatin or 0,5% casein. The spectrum of proteases was analysed by a substrate electrophoresis in the blocks of 8% polyacrylamide gel, impregnated in the process of polymerization gelatin or casein (in an eventual concentration 0,1 %), in presence the dodecylsulphate of sodium. Results. After differential centrifugation of ureal lysate of cells and dialysis there are mainly intracellular water-soluble proteases in the investigated extracts. Diffusion tests showed that all preparations of the investigated strains of Vibrio cholerae possessed protease activity of different intensity. Changing of substrate in diffusion test from gelatin to the casein resulted in the general diminishing of the registered areas of hydrolysis, specifying that a casein fissions less actively by comparison to gelatin. Substrate electrophoresis showed that proteins, making the spectrum of water-soluble pro teases of Vibrio cholerae are extracted by urea, possessed molecular mass, varying within from less than 30 кйа to more than 120 кDа. Strains distinctions are marked in quantitative and high-quality description of spectrums of soluble proteases. Dependence of description of type of water-soluble proteases is educed on used substrate. At the estimation of spectrums of proteases clear confirmation is got, that on gels are impregnated by gelatin electrophoretic mobility of proteases is higher, than in gels, containing a casein. Conclusion. Substrate electrophoresis of preparations of cell-free lysates of Vibrio cholerae showed the presence of a few water-soluble proteases, quantitative and high-quality interstrain distinctions, dependence of spectrum of active proteases from of used substrate.
format	article
author	S. N. Kozlov V. B. Nikolaev E. Yu. Markov L. Ya. Urbanovich
author_facet	S. N. Kozlov V. B. Nikolaev E. Yu. Markov L. Ya. Urbanovich
author_sort	S. N. Kozlov
title	ZYMOGRAPHIC ANALYSIS OF WATER-SOLUBLE PROTEASES OF VIBRIO CHOLERAE O1 AND О139 SEROGROUPS
title_short	ZYMOGRAPHIC ANALYSIS OF WATER-SOLUBLE PROTEASES OF VIBRIO CHOLERAE O1 AND О139 SEROGROUPS
title_full	ZYMOGRAPHIC ANALYSIS OF WATER-SOLUBLE PROTEASES OF VIBRIO CHOLERAE O1 AND О139 SEROGROUPS
title_fullStr	ZYMOGRAPHIC ANALYSIS OF WATER-SOLUBLE PROTEASES OF VIBRIO CHOLERAE O1 AND О139 SEROGROUPS
title_full_unstemmed	ZYMOGRAPHIC ANALYSIS OF WATER-SOLUBLE PROTEASES OF VIBRIO CHOLERAE O1 AND О139 SEROGROUPS
title_sort	zymographic analysis of water-soluble proteases of vibrio cholerae o1 and о139 serogroups
publisher	Scientific Сentre for Family Health and Human Reproduction Problems
publishDate	2013
url	https://doaj.org/article/e939d7396bbf4fc987c517f2d177cdae
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ZYMOGRAPHIC ANALYSIS OF WATER-SOLUBLE PROTEASES OF VIBRIO CHOLERAE O1 AND О139 SEROGROUPS

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