ABSTRACT
The mechanism of activation of thioredoxin-linked NADP-malate dehydrogenase was investigated by using 14C-iodoacetate and 14C-dansylated thioredoxin m, and Sepharose affinity columns (thioredoxin m, NADP-malate dehydrogenase) as probes to monitor enzyme sulfhydryl status and enzyme-thioredoxin interaction. The data indicate that NADP-malate dehydrogenase, purified to homogeneity from corn leaves, is activated by a net transfer of reducing equivalents from thioredoxin m, reduced by dithiothreitol, to enzyme disulfide groups, thereby yielding oxidized thioredoxin m and reduced enzyme. The appearance of new sulfhydryl groups that accompanies the activation of NADP-malate dehydrogenase appears to involve a structural change that is independent of the formation of a stable complex between the enzyme and reduced thioredoxin m. The data are consistent with the conclusion that oxygen promotes deactivation of NADP-malate dehydrogenase through oxidation of SH groups on reduced thioredoxin and on the reduced (activated) enzyme.
Subject(s)
Bacterial Proteins/pharmacology , Malate Dehydrogenase/metabolism , Photosynthesis , Plants/enzymology , Thioredoxins/pharmacology , Chromatography, Affinity , Dansyl Compounds , Dithiothreitol/pharmacology , Enzyme Activation/drug effects , Oxidation-Reduction , Sulfhydryl Compounds/metabolism , Zea maysABSTRACT
Phosphofructokinase has been partially purified from spinach (Spinacia oleracea) chloroplasts and studied from the standpoint of light/dark regulation. At concentrations reported to occur physiologically, NADPH effected a sharp inhibition of the enzyme by: (a) lowering its affinity (increasing the apparent K(m)) for both of its substrates, ATP and fructose 6-phosphate; and (b) lowering its V(max). Inhibition by NADPH was independent of pH and was observed both at pH 7.9 (pH of chloroplast stroma in the light) and pH 7.0 (stromal pH in the dark). The results are consistent with the conclusion that NADPH provides a mechanism for linking light to the modulation of phosphofructokinase activity and thereby to the regulation of glycolysis in chloroplasts.
Subject(s)
Bacterial Proteins/pharmacology , Fructose-Bisphosphatase/metabolism , Phosphoric Monoester Hydrolases/metabolism , Photosynthesis , Plants/enzymology , Thioredoxins/pharmacology , Animals , Enzyme Activation/drug effects , Fructose-Bisphosphatase/isolation & purification , Heptoses/isolation & purification , Heptoses/metabolism , Immunodiffusion , Magnesium/pharmacology , Phosphoric Monoester Hydrolases/isolation & purification , Substrate Specificity , Sugar Phosphates/isolation & purification , Sugar Phosphates/metabolism , Swine , Zea mays/enzymologyABSTRACT
Phenylalanine ammonia-lyase (PAL) from spinach (Spinacia oleracea L.) leaves was resolved into three forms by diethyl-aminoethyl(DEAE)-cellulose chromatography. Two forms were found in isolated chloroplasts, and the third form (the major component) was located outside of the chloroplasts. One of the chloroplast forms of the enzyme (designated the regulatory form) was activated by reduced thioredoxin. Neither the other chloroplast form nor the extra-chloroplast form showed a response to thioredoxin. After further purification by hydroxyapatite column chromatography and gel filtration, the regulatory form of chloroplast PAL was stimulated approximately 3-fold by thioredoxin reduced either photochemically by chloroplast membranes, via ferredoxin and ferredoxin-thioredoxin reductase, or chemically by dithiothreitol. Once activated, the enzyme required an added oxidant for deactivation. Physiological oxidants-oxidized glutathione (GSSG) and dehydroascorbate-as well as nonphysiological oxidants-sodium tetrathionate and diamide-were effective in deactivation. The results indicate that chloroplast PAL is regulated by light via the ferredoxin/thioredoxin system in a manner similar to that described for regulatory enzymes of CO2 assimilation. The extra-chloroplast form of the enzyme, by contrast, appears to be regulated by light via the earlier-described phytochrome-linked system.
