Nonreversible d-Glyceraldehyde 3-Phosphate Dehydrogenase of Plant Tissues

Abstract

Preparations of TPN-linked nonreversible D-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.9), free of TPN-linked reversible D-glyceraldehyde 3-phosphate dehydrogenase, have been obtained from green shoots, etiolated shoots, and cotyledons of pea (Pisum sativum), cotyledons of peanut (Arachis hypogea), and leaves of maize (Zea mays). The Plant tissues contain three enzymes for the oxidation of D-G3P' (12). Two of these catalyze a Pi-dependent, reversible oxidation of D-G3P to 1,3-diP-glycerate; one enzyme is DPNspecific and found in both photosynthetic and nonphotosynthetic tissues, while the other uses TPN and is most probably confined to the chloroplast. Both enzymes have been extensively studied (29). The third enzyme, which is TPN-specific, oxidizes D-G3P to glycerate-3-P in a reaction which does not require Pi and is not reversible. Evidence for this enzyme activity in green leaves was reported by leaves. Recently, Jacob and d'Auzac (17) have purified the enzyme from the latex of Hevea brasiliensis. In this laboratory, the partially purified enzyme from pea shoots was inhibited strongly by L-G3P (19). It is possible that the scarcity of reports on the enzyme is related to the presence of this inhibitor in commercial preparations of the substrate D-G3P.In the present investigation, the significance of this nonreversible D-G3P dehydrogenase to plant carbohydrate metabolism has been evaluated. A preparation of pea shoot enzyme, free of reversible Pi-dependent D-G3P dehydrogenase activity, has now been obtained, and the properties have been compared with those of the same enzyme partially purified from several other plant tissues. In addition, a relatively specific assay for the determination of the enzyme activity in cell-free extracts is described. Changes in the enzyme activity during the germination of peas and castor beans and the distribution of the enzyme in representative tissues were examined.The properties and in vivo localization of the three D-G3P dehydrogenases were compared, and the results indicate that, in some tissues, a major portion of triose-P oxidation may be mediated by the nonreversible enzyme.MATERIALS AND METHODS Materials. Pea (Pisum sativum L. var. Progress No. 9), maize (Zea mays L. var. Early Fortune), peanuts (Arachis hypogea L. var. Jumbo Virginia), and castor bean (Ricinus communis L.) were grown under natural lighting in a glasshouse at 16 to 18 C or in the dark at 22 C.The following chemicals and enzymes were obtained from the Sigma Chemical Company: ATP, dihydroxyacetone-P, DPN, D-erythrose-4-P, D-fructose-1 ,6-diP, DL-G3P, D-glycerate-3-P, P-glycolate, P-hydroxypyruvate, D-ribose-5-P, TPN, TPNH, G3P dehydrogenase, 3-P-glycerate kinase, and triose-P isomerase. Chloroacetol-P was a gift of Dr. F. C. Hartman through the courtesy of Dr. Louise Anderson. All chemicals were prepared as sodium salts at the recommended pH before use.L-G3P was prepared according to the method of Venkataraman and Racker (38). The D-G3P present in 0.65 mmole of DL-G3P was converted to glyce...

Glyceraldehyde 3-Phosphate:NADP+ Reductase of Spinach Leaves

“…7). No activity was observed when 3PGA (0.1-50 mM) and NADPH (10-300 ,aM) were assayed together in the absence of substrates of the forward reaction, considered to be irreversible ( 19).…”

“…This is scarcely compatible with other major metabolisms such as synthesis of sucrose, and it is expected that GNR activity is modulated by metabolites. Some effectors have been detected (17)(18)(19).…”

“…GNR affinity for the substrate G3P is high (16,19,22), and the enzyme could act as an effective scavenger of triose phosphate competitive to other cytosolic enzymes including fructose bisphosphate aldolase and NAD-triose phosphate dehydrogenase. This is scarcely compatible with other major metabolisms such as synthesis of sucrose, and it is expected that GNR activity is modulated by metabolites.…”

“…GNR specific activity increases during leafgreening, e.g. to 20 to 30 nmol min-' mg-' protein in spinach (19,27).…”

“…The effects of reaction products and several possible modulators Most of the carbon assimilated during plant photosynthesis is exported from chloroplasts as triose phosphate by the phosphate carrier (15) to be converted to sucrose in the cytoplasm. Some triose phosphate, however, may be oxidized to 3-phosphoglycerate by two cytosolic enzyme systems acting in parallel (19,23) have also been examined. It is suggested that leaf GNR is often inhibited in vivo by cellular metabolites, and de-inhibited under photosynthetic conditions.…”