The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves

Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins. Materials and methods. Chloro...

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Autores principales:	Andriy V. Semenikhin, Volodymyr V. Sukhovieiev, Mykola V. Patyka, Vasyl S. Lukach
Formato:	article
Lenguaje:	EN RU UK
Publicado:	National University of Pharmacy (Kharkiv, Ukraine) 2021
Materias:	chloroplasts rubisco atp synthase carbonic anhydrase esterase activity two-dimensional electrophoresis Chemistry QD1-999
Acceso en línea:	https://doi.org/10.24959/ophcj.21.240779 https://doaj.org/article/1bf29949a5a8491dba34a887d5e08bcc
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spelling	oai:doaj.org-article:1bf29949a5a8491dba34a887d5e08bcc2021-11-14T22:16:35ZThe effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaveshttps://doi.org/10.24959/ophcj.21.2407792308-83032518-1548https://doaj.org/article/1bf29949a5a8491dba34a887d5e08bcc2021-09-01T00:00:00Zhttp://ophcj.nuph.edu.ua/article/view/240779https://doaj.org/toc/2308-8303https://doaj.org/toc/2518-1548Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins. Materials and methods. Chloroplasts were isolated from the leaves of two-week-old pea sprouts, protein complexes of purified thylakoid membranes were solubilized with digitonin (10 mg of digitonin per 1 mg of protein), the protein concentration was determined according to Lowry. Native electrophoresis with displacement of the charge of the soluble protein fraction from the chloroplast stroma, as well as membrane proteins, was carried out in the modified system of Anderson et al., Kolisnichenko et al. A modified Lemmley system was applied to the protein electrophoresis in the polyacrylamide gel in the presence of sodium dodecyl sulfate. The methods of Alain and Hintsik, as well as Gomorrah were used to determine the ATPase activity in the polyacrylamide gel. Visualization of the carbonic anhydrase activity in the polyacrylamide gel was performed by the method of Edwards and Petton. Results and discussion. Using physicochemical methods of potentiometry, spectrophotometry the ATPase, carbonic anhydrase and esterase activities of the enzymes were studied. The results obtained indicate that specific carbonic anhydrase inhibitors (acetazolamide and ethoxyzolamide) also block the esterase and ATPase activity of the enzyme complexes. “Extracon” (a multifunctional microbiological preparation) almost 1.5 times increases the activity of the enzymes, showing a complex activating effect of the fertilizer on both light and dark reactions of photosynthesis. Conclusions. The method of identification and isolation of RuBisCO and ATP synthase on the basis of two-dimensional electrophoresis and electrophoretic elution has been proposed. It allows determining the presence of certain enzyme activity of complexes at first in SDS plates (express analysis) and further to study the effect of various factors of endogenous and exogenous origin on the enzymatic properties of electrophoretically pure enzymes. The use of two-dimensional electrophoresis as a tool for assessing the impact of various factors of endogenous and exogenous origin on the plant cell and the plant as a whole through constant monitoring of the work and activity of enzyme systems of the plant cell is promising.Andriy V. SemenikhinVolodymyr V. SukhovieievMykola V. PatykaVasyl S. LukachNational University of Pharmacy (Kharkiv, Ukraine)articlechloroplastsrubiscoatp synthasecarbonic anhydraseesterase activitytwo-dimensional electrophoresisChemistryQD1-999ENRUUKЖурнал органічної та фармацевтичної хімії, Vol 19, Iss 3, Pp 21-27 (2021)
institution	DOAJ
collection	DOAJ
language	EN RU UK
topic	chloroplasts rubisco atp synthase carbonic anhydrase esterase activity two-dimensional electrophoresis Chemistry QD1-999
spellingShingle	chloroplasts rubisco atp synthase carbonic anhydrase esterase activity two-dimensional electrophoresis Chemistry QD1-999 Andriy V. Semenikhin Volodymyr V. Sukhovieiev Mykola V. Patyka Vasyl S. Lukach The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves
description	Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins. Materials and methods. Chloroplasts were isolated from the leaves of two-week-old pea sprouts, protein complexes of purified thylakoid membranes were solubilized with digitonin (10 mg of digitonin per 1 mg of protein), the protein concentration was determined according to Lowry. Native electrophoresis with displacement of the charge of the soluble protein fraction from the chloroplast stroma, as well as membrane proteins, was carried out in the modified system of Anderson et al., Kolisnichenko et al. A modified Lemmley system was applied to the protein electrophoresis in the polyacrylamide gel in the presence of sodium dodecyl sulfate. The methods of Alain and Hintsik, as well as Gomorrah were used to determine the ATPase activity in the polyacrylamide gel. Visualization of the carbonic anhydrase activity in the polyacrylamide gel was performed by the method of Edwards and Petton. Results and discussion. Using physicochemical methods of potentiometry, spectrophotometry the ATPase, carbonic anhydrase and esterase activities of the enzymes were studied. The results obtained indicate that specific carbonic anhydrase inhibitors (acetazolamide and ethoxyzolamide) also block the esterase and ATPase activity of the enzyme complexes. “Extracon” (a multifunctional microbiological preparation) almost 1.5 times increases the activity of the enzymes, showing a complex activating effect of the fertilizer on both light and dark reactions of photosynthesis. Conclusions. The method of identification and isolation of RuBisCO and ATP synthase on the basis of two-dimensional electrophoresis and electrophoretic elution has been proposed. It allows determining the presence of certain enzyme activity of complexes at first in SDS plates (express analysis) and further to study the effect of various factors of endogenous and exogenous origin on the enzymatic properties of electrophoretically pure enzymes. The use of two-dimensional electrophoresis as a tool for assessing the impact of various factors of endogenous and exogenous origin on the plant cell and the plant as a whole through constant monitoring of the work and activity of enzyme systems of the plant cell is promising.
format	article
author	Andriy V. Semenikhin Volodymyr V. Sukhovieiev Mykola V. Patyka Vasyl S. Lukach
author_facet	Andriy V. Semenikhin Volodymyr V. Sukhovieiev Mykola V. Patyka Vasyl S. Lukach
author_sort	Andriy V. Semenikhin
title	The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves
title_short	The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves
title_full	The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves
title_fullStr	The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves
title_full_unstemmed	The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves
title_sort	effect of exogenous factors on the polyenzymatic activity of rubisco and atp synthase of chloroplasts from pea leaves
publisher	National University of Pharmacy (Kharkiv, Ukraine)
publishDate	2021
url	https://doi.org/10.24959/ophcj.21.240779 https://doaj.org/article/1bf29949a5a8491dba34a887d5e08bcc
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The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves

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