Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide.

The incubation of chloroplast fructose-1,6-bisphosphatase with both dithiothreitol and protein denaturants made sulfhydryl groups available for reaction with [1-14C]iodoacetamide (10-12 mol iodoacetamide incorporated/mol enzyme). Digestion of S-carboxyamidomethylated enzyme with trypsin and polyacry...

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Autores principales:	Stein, M., Lazaro, J.J., Wolosiuk, R.A.
Formato:	JOUR
Lenguaje:	English
Materias:	bacterial protein dithiothreitol fructose bisphosphatase iodoacetamide iodoacetic acid peptide fragment solvent thiol derivative thioredoxin article chloroplast drug effect enzyme activation enzymology light metabolism methylation oxidation reduction reaction polyacrylamide gel electrophoresis protein conformation protein denaturation Bacterial Proteins Chloroplasts Dithiothreitol Electrophoresis, Polyacrylamide Gel Enzyme Activation Fructose-Bisphosphatase Iodoacetamide Iodoacetates Light Methylation Oxidation-Reduction Peptide Fragments Protein Conformation Protein Denaturation Solvents Sulfhydryl Compounds Support, Non-U.S. Gov't Thioredoxin
Acceso en línea:	http://hdl.handle.net/20.500.12110/paper_00142956_v185_n2_p425_Stein
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

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spelling	todo:paper_00142956_v185_n2_p425_Stein2023-10-03T14:11:52Z Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide. Stein, M. Lazaro, J.J. Wolosiuk, R.A. bacterial protein dithiothreitol fructose bisphosphatase iodoacetamide iodoacetic acid peptide fragment solvent thiol derivative thioredoxin article chloroplast drug effect enzyme activation enzymology light metabolism methylation oxidation reduction reaction polyacrylamide gel electrophoresis protein conformation protein denaturation Bacterial Proteins Chloroplasts Dithiothreitol Electrophoresis, Polyacrylamide Gel Enzyme Activation Fructose-Bisphosphatase Iodoacetamide Iodoacetates Light Methylation Oxidation-Reduction Peptide Fragments Protein Conformation Protein Denaturation Solvents Sulfhydryl Compounds Support, Non-U.S. Gov't Thioredoxin The incubation of chloroplast fructose-1,6-bisphosphatase with both dithiothreitol and protein denaturants made sulfhydryl groups available for reaction with [1-14C]iodoacetamide (10-12 mol iodoacetamide incorporated/mol enzyme). Digestion of S-carboxyamidomethylated enzyme with trypsin and polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, yielded two 14C-labeled fragments whose apparent molecular mass were 10 kDa and 16 kDa. In the absence of either dithiothreitol or protein denaturants the incorporation of iodoacetamide to the enzyme was lower than 4 mol. When chloroplast fructose-1,6-bisphosphatase was initially incubated with dithiothreitol (2.5 mM) and (a) high concentrations of both fructose 1,6-bisphosphate (4 mM) and Ca2+ (0.3 mM) or (b) low concentrations of both fructose 1,6-bisphosphate (0.8 mM) and Ca2+ (0.05 mM) in the presence of either 2-propanol (15%, by vol.), trichloroacetate (0.15 M) or chloroplast thioredoxin-f (0.5 microM) and subsequently subjected to proteolysis and electrophoresis, S-carboxyamidomethylated tryptic fragments had similar molecular masses. Thus, conditions that stimulated the specific activity of chloroplast fructose-1,6-bisphosphatase caused conformational changes which favoured both the reduction of disulfide bridges and the exposure of sulfhydryl groups. In this aspect, thioredoxin exerted structural and kinetic effects similar to compounds not involved in redox reactions (organic solvents, chaotropic anions). These results indicated that the modification of hydrophobic (intramolecular) interactions in chloroplast fructose-1,6-bisphosphatase constituted the underlying mechanism in light-activation by the ferredoxin-thioredoxin system. Fil:Stein, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Wolosiuk, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR English info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00142956_v185_n2_p425_Stein
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
language	English
orig_language_str_mv	English
topic	bacterial protein dithiothreitol fructose bisphosphatase iodoacetamide iodoacetic acid peptide fragment solvent thiol derivative thioredoxin article chloroplast drug effect enzyme activation enzymology light metabolism methylation oxidation reduction reaction polyacrylamide gel electrophoresis protein conformation protein denaturation Bacterial Proteins Chloroplasts Dithiothreitol Electrophoresis, Polyacrylamide Gel Enzyme Activation Fructose-Bisphosphatase Iodoacetamide Iodoacetates Light Methylation Oxidation-Reduction Peptide Fragments Protein Conformation Protein Denaturation Solvents Sulfhydryl Compounds Support, Non-U.S. Gov't Thioredoxin
spellingShingle	bacterial protein dithiothreitol fructose bisphosphatase iodoacetamide iodoacetic acid peptide fragment solvent thiol derivative thioredoxin article chloroplast drug effect enzyme activation enzymology light metabolism methylation oxidation reduction reaction polyacrylamide gel electrophoresis protein conformation protein denaturation Bacterial Proteins Chloroplasts Dithiothreitol Electrophoresis, Polyacrylamide Gel Enzyme Activation Fructose-Bisphosphatase Iodoacetamide Iodoacetates Light Methylation Oxidation-Reduction Peptide Fragments Protein Conformation Protein Denaturation Solvents Sulfhydryl Compounds Support, Non-U.S. Gov't Thioredoxin Stein, M. Lazaro, J.J. Wolosiuk, R.A. Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide.
topic_facet	bacterial protein dithiothreitol fructose bisphosphatase iodoacetamide iodoacetic acid peptide fragment solvent thiol derivative thioredoxin article chloroplast drug effect enzyme activation enzymology light metabolism methylation oxidation reduction reaction polyacrylamide gel electrophoresis protein conformation protein denaturation Bacterial Proteins Chloroplasts Dithiothreitol Electrophoresis, Polyacrylamide Gel Enzyme Activation Fructose-Bisphosphatase Iodoacetamide Iodoacetates Light Methylation Oxidation-Reduction Peptide Fragments Protein Conformation Protein Denaturation Solvents Sulfhydryl Compounds Support, Non-U.S. Gov't Thioredoxin
description	The incubation of chloroplast fructose-1,6-bisphosphatase with both dithiothreitol and protein denaturants made sulfhydryl groups available for reaction with [1-14C]iodoacetamide (10-12 mol iodoacetamide incorporated/mol enzyme). Digestion of S-carboxyamidomethylated enzyme with trypsin and polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, yielded two 14C-labeled fragments whose apparent molecular mass were 10 kDa and 16 kDa. In the absence of either dithiothreitol or protein denaturants the incorporation of iodoacetamide to the enzyme was lower than 4 mol. When chloroplast fructose-1,6-bisphosphatase was initially incubated with dithiothreitol (2.5 mM) and (a) high concentrations of both fructose 1,6-bisphosphate (4 mM) and Ca2+ (0.3 mM) or (b) low concentrations of both fructose 1,6-bisphosphate (0.8 mM) and Ca2+ (0.05 mM) in the presence of either 2-propanol (15%, by vol.), trichloroacetate (0.15 M) or chloroplast thioredoxin-f (0.5 microM) and subsequently subjected to proteolysis and electrophoresis, S-carboxyamidomethylated tryptic fragments had similar molecular masses. Thus, conditions that stimulated the specific activity of chloroplast fructose-1,6-bisphosphatase caused conformational changes which favoured both the reduction of disulfide bridges and the exposure of sulfhydryl groups. In this aspect, thioredoxin exerted structural and kinetic effects similar to compounds not involved in redox reactions (organic solvents, chaotropic anions). These results indicated that the modification of hydrophobic (intramolecular) interactions in chloroplast fructose-1,6-bisphosphatase constituted the underlying mechanism in light-activation by the ferredoxin-thioredoxin system.
format	JOUR
author	Stein, M. Lazaro, J.J. Wolosiuk, R.A.
author_facet	Stein, M. Lazaro, J.J. Wolosiuk, R.A.
author_sort	Stein, M.
title	Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide.
title_short	Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide.
title_full	Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide.
title_fullStr	Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide.
title_full_unstemmed	Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide.
title_sort	concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. reactivity to iodoacetamide.
url	http://hdl.handle.net/20.500.12110/paper_00142956_v185_n2_p425_Stein
work_keys_str_mv	AT steinm concertedactionofcosolventschaotropicanionsandthioredoxinonchloroplastfructose16bisphosphatasereactivitytoiodoacetamide AT lazarojj concertedactionofcosolventschaotropicanionsandthioredoxinonchloroplastfructose16bisphosphatasereactivitytoiodoacetamide AT wolosiukra concertedactionofcosolventschaotropicanionsandthioredoxinonchloroplastfructose16bisphosphatasereactivitytoiodoacetamide
_version_	1807314821768544256

Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide.

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