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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1989
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00142956_v185_n2_p425_Stein http://hdl.handle.net/20.500.12110/paper_00142956_v185_n2_p425_Stein |
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paper:paper_00142956_v185_n2_p425_Stein2023-06-08T14:36:41Z Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide. Stein, Mariana Wolosiuk, Ricardo Alejandro 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. 1989 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00142956_v185_n2_p425_Stein 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) |
| 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, Mariana Wolosiuk, Ricardo Alejandro 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. |
| author |
Stein, Mariana Wolosiuk, Ricardo Alejandro |
| author_facet |
Stein, Mariana Wolosiuk, Ricardo Alejandro |
| author_sort |
Stein, Mariana |
| 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. |
| publishDate |
1989 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00142956_v185_n2_p425_Stein http://hdl.handle.net/20.500.12110/paper_00142956_v185_n2_p425_Stein |
| work_keys_str_mv |
AT steinmariana concertedactionofcosolventschaotropicanionsandthioredoxinonchloroplastfructose16bisphosphatasereactivitytoiodoacetamide AT wolosiukricardoalejandro concertedactionofcosolventschaotropicanionsandthioredoxinonchloroplastfructose16bisphosphatasereactivitytoiodoacetamide |
| _version_ |
1768541828110352384 |