Comparative Enzymology of the Glyceraldehyde 3-Phosphate Dehydrogenases from Pisum sativum

Abstract

Glyceraldehyde 3-phosphate dehydrogenases (EC 1.2.1.12 and 1.2.1.13) have been purified from the seed, root, etiolated, and green shoot of peas (Pisum sativum). These enzymes are tetramers of 140,000 daltons, with subunits of 35,000 daltons. The enzymes differ in isoelectric point. The seed enzyme has a pl of 5.1, and the root enzyme has a pI of 4.5. The cytoplasmic enzyme from etiolated shoots is slightly acidic with a pI of 5.7 to 6.1 and is found in two separable forms. The chloroplast enzyme (from green shoots) is most basic with a pI of 8.0.In immunodiffusion experiments, the seed, root, and cytoplasmic enzymes of the etiolated shoot share antigenic homology, while the chloroplast enzyme does not cross react antigenically with the extra-chloroplast enzymes. The antiserum to the pea chloroplast enzyme did, however, cross react with glyceraldehyde 3-phosphate dehydrogenase purified from the spinach chloroplast. Therefore, the chloroplast enzyme is significantly different from the extra-chloroplast enzymes with respect to prinary sequence.The NADP analog phosphoadenosine diphosphoribose showed competitive inhibition to the chloroplast enzyme with either pyridine nucleotide. The NAD analog pyridine 3-aldehyde NAD was competitive with respect to the NAD activity but was hyperbolic competitive in the presence of NADP, indicating a complexity in the binding of pyridine nucleotide to the chloroplast enzyme.Five glyceraldehyde-3-P dehydrogenases have been recognized in higher plants. Of these, three are NAD-associated enzymes, located in the seed (14), root (12). and cytoplasm of the leaf (20). A fourth is the chloroplast enzyme linked with either pyridine nucleotide (12). Finally, there is the nonphosphaterequiring, irreversible enzyme located in the cytoplasm of the leaf which utilizes NADP (4, 18).The structural relationship of the cytoplasmic enzyme, which is NAD specific, to the chloroplast enzyme, which uses both pyridine nucleotides, is unknown. Based on their studies in 1This research was supported by Atomic Energy Commission Grant AT (1 1-1 Incorporation of the cytoplasmic enzyme into the chloroplast enzyme would mean that the purified chloroplast and cytoplasmic enzymes should have similar structural properties. In order to evaluate the structural similarity or dissimilarity of the chloroplast and cytoplasmic glyceraldehyde-3-P dehydrogenases, it is necessary to characterize the purified enzymes. Yonuschot et al. (31) have purified the spinach chloroplast enzyme and shown it to be of a higher mol wt (600,000) than any previously studied glyceraldehyde-3-P dehydrogenase, including the various muscle and yeast enzymes (1, 2, 7). Another characteristic of the spinach chloroplast enzyme is a reversible polymerization between a 600,000 and a 140,000 mol wt form affected by NADP (25).These reports clearly indicate the need for a comparative study of the purified chloroplast and cytoplasmic glyceraldehyde-3-P dehydrogenases so that the structural similarity or dissimilarity of the molecules might be inves...

Activation of Chloroplast NADP-linked Glyceraldehyde-3-Phosphate Dehydrogenase by the Ferredoxin/Thioredoxin System

“…The relation of the partially purified regulatory form of the enzyme used in the present investigation to the pure preparations of a form of the enzyme that did not require activation (16,29) remains to be determined.…”

“…Anderson (1) previously observed an enhancement of the NADP-glyceraldehyde-3-P dehydrogenase activity in extracts of etiolated peas when the assay was executed in the presence of DTT. 16 ,ug of Chl or partially purified regulatory enzyme (9 ,ug of protein) was preincubated for 3 min in 0.1 ml of a solution containing 10 umol of Tricine-NaOH buffer (pH 8.4) and, as indicated, I ,umol of DTT and 50 pAg of thioredoxin. After preincubation, the mixture was injected into a 1-cm cuvette of l-ml capacity that, in a final volume of 0.9 ml, contained 2 units of glycerate-3-P phosphokinase and the following (umol): TricineNaOH buffer (pH 8.4), 40; MgSO4, 10; Na-glycerate-3-P, 5 pmol of Tricine NaOH buffer (pH 8.4).…”

Glyceraldehyde-3-Phosphate Dehydrogenase in Greening Zea mays L. Leaves

“…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 glyceraldehyde-3-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.…”

Algal Glyceraldehyde‐3‐Phosphate Dehydrogenase

“…A cytoplasmic enzyme (EC 1.2.1.12), which absolutely requires NAD' as coenzyme, has been isolated from seeds, roots and etiolated leaves [1,2]. All these cytoplasmic enzymes have very similar properties to the enzymes purified from animal muscle.…”