Glyceraldehyde-3-Phosphate Dehydrogenase in Greening Zea

Nicotinamide adenine dinucleotide phosphate (NADP)-dependent glyceraldehyde-3-phosphate dehydrogenase (GPDH) (EC 1.2.1.13), a chloroplast enzyme, had low activity in etioplasts of maize leaves. A light dependent increase of enzyme activity of 7-day-old etiolated seedlings showed a lag period of about 2.5 hours followed by a rapid increase in activity during the next 10 hours. The chlorophyll content followed a similar pattern of increasing concentration, but its formation was not directly related to NADP-GPDH formation. The specific activity of NADP-GPDH was lowest in the morphologically youngest tissue near the base of the lamina. The increase in NADP-GPDH was inhibited by cycloheximide but not by chloramphenicol. This indicates that at least some of the enzyme polypeptides are synthesized by 80S ribosomes in the cytoplasm, transported into chloroplasts and become active in chloroplasts. In etiolated maize shoots subjected to a combination of both 3-(p-chlorophenyl)-1,1-dimethylurea, monuron at 7 x 10-i M and far red light treatment for 15 hours, the NADP-GPDH activity increased 42% over the dark control compared to 70% increase for the light control. It is concluded that NADPH is not absolutely required for the activation of NADP-GPDH in maize leaves under physiological conditions. In higher plants, NADPH-specific glyceraldehyde-3-P dehydrogenase (NADP-GPDH) (EC 1.2.1.13) occurs only in photosynthetic tissue (4, 11, 12, 14). Seedlings grown in the dark contain little NADPH-GPDH activity, but the enzyme activity increases markedly if the seedlings are exposed to the light (14, 19, 20). A comprehensive investigation of the specificity of glyceraldehyde3-P dehydrogenase was done by Pupillo (24) and Pupillo and Piccari (25) who concluded that in all green plants both the NADand NADP-dependent GPDH activities of chloroplasts are catalyzed by one enzyme which is different from the NAD-linked cytoplasmic enzyme. Schulman and Gibbs (26) were unable to separate the NADP and NAD linked GPDH activities from spinach leaves, and pea shoots. More recently McGowan and Gibbs (20) found that the enzyme purified from seeds, roots, and etiolated and green shoots of pea consisted of tetramers with subunits of 35,000 daltons. Based on isoelectric point and immunodiffusion experiments, they concluded that the primary sequences of amino acids in the chloroplast enzyme and in the cytosol enzyme are different. Muller et aL (21-23) found that incubation of homogenates of Viciafaba leaves and sonically ruptured spinach leaf chloroplasts 'This research was supported in part by National Science Foundation Grants BMS 72-02274 AOZ and PCM 77-12546. 2Present address: Department of Botany, National Chung Hsing University, Taichung, Taiwan. with 2.7 mm NADPH gave up to a 600%o increase of NADPGPDH activity over the control. This increase in the activity was accompanied by a decrease in the activity of the NAD-linked GPDH. The activation with NADPH was not a nonspecific reduction of disulfide linkages, since DTT could not substitute for NADPH in the activation by NADPH in the crude homogenate. NADPH was also reported to activate partially purified NADPGPDH (23). The concentration curve response to NADPH activation was sigmoidal in shape. It was postulated that in the light there is an increased concentration of NADPH and that the NADPH allosterically activates the GPDH causing a transition from a NAD-dependent to a NADP-dependent form of the enzyme (23). Pupillo and Piccari (25) were able to obtain an equilibrium mixture of different oligomers in several discrete aggregational states depending upon the experimental conditions. The protomer, which has a low Km for NADP+ and NAD+, reverts to tetramers in the absence ofNADP+ with considerable increase in the apparent Km for NADP+. NAD+ was unable to induce dissociation but appeared to stabilize the tetramer against the effects of NADP+ and to accelerate the decay of NADP-linked activity. Although they conclude that it is premature to extrapolate their in vitro findings to the in vivo situation, it is possible that the activity of the chloroplast GPDH is regulated by pyridine nucleotides acting as effectors as well as substrates. They point out that it is known that the ratios ofNADP-NADPH to NAD-NADH change during light-dark transitions, and they speculate that the chloroplast GPDH is mainly (or possibly only) in the protomer form when reducing NADP. ATP was similarly effective in activating the NADP-dependent dehydrogenase. The activation curve as a function of ATP concentration was also sigmoidal in shape, indicating a positive cooperative mechanism. A combination of ATP and NADPH activated the enzyme no more than did ATP or NADPH alone. MgCl2 at 50 mm activated the enzyme, while higher concentrations and AMP inhibited the NADP-dependent enzyme. In contrast to the situation with NADPH, the activation of the NADP-dependent GPDH by ATP in vitro is not accompanied by a decrease in the activity of the NAD-dependent form. Other than NADPH and ATP as effectors, activation ofNADPGPDH by strong reducing agents has also been reported for crude pea extracts. It was suggested that the divergent results reported by different laboratories are probably related to differences in the state of enzyme aggregation (subunit aggregation) rather than to different plants (1, 2). The relative importance of cytoplasmic ribosomes and of chloroplast ribosomes in the synthesis of NADP-GPDH is still in question. Ellis (9) and Ellis and Hartley (10) on the basis of inhibitor studies with lincomycin and 2-(4-methyl-2,6-dinitroaniline)-N-methylpropinoamide suggested that NADP-GPDH was synthesized on cytoplasmic ribosomes in greening detached shoots of pea. In contrast, Batt and Woolhouse (6) with Perillafrulescens, Ireland and Bradbeer (16) with Phaseolus vulgaris, and Berger and