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 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.
Stein
M
M
Instituto de Investigaciones Bioquímicas, Fundación Campomar, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Lazaro
J J
JJ
Wolosiuk
R A
RA
D001426:Bacterial Proteins / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D007460:Iodoacetamide / Q000378:metabolism*; D007461:Iodoacetates / Q000378:metabolism*; D008027:Light; D008745:Methylation; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D012997:Solvents / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
D001426:Bacterial Proteins; D007461:Iodoacetates; D010446:Peptide Fragments; D012997:Solvents; D013438:Sulfhydryl Compounds; D013879:Thioredoxins; D006597:Fructose-Bisphosphatase; D004229:Dithiothreitol; D007460:Iodoacetamide