Reductive Modification and Nonreductive Activation of Purified Spinach Chloroplast NADP-Dependent Glyceraldehyde-3-phosphate Dehydrogenase

• Published in: Archives of biochemistry and biophysics 1995-12, Vol.324 (2), p.201-208
• Main Authors: Baalmann, Elisabeth, Backhausen, Jan E., Rak, Christiane, Vetter, Susanne, Scheibe, Renate
• Format: Article
• Language: English
• Subjects: Chloroplasts - enzymology; Diphosphoglyceric Acids - pharmacology; Enzyme Activation; Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism; Kinetics; Models, Biological; Oxidation-Reduction; Protein Conformation; Spinacia oleracea - enzymology; Sulfhydryl Compounds - pharmacology
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1995.0031

Spinach chloroplast NAD(P)-glyceraldehyde-3-phosphate dehydrogenase (NAD(P)-GAPDH; EC, 1.2.1.13) was purified as the 600-kDa oligomer of low specific activity. Incubation of the enzyme with either a reductant or a 1,3-bisphosphoglycerate (1,3bisPGA) generating system, but most effectively with both, resulted in an increase of the apparent NADPH-dependent activity. Only the 1,3bisPGA treatment caused dissociation and yielded the 150-kDa heterotetramer (A2B2). The higher activity of the tetramer is largely due to a decreasedKMvalue for the substrate 1,3bisPGA. Reductive treatment alone does not dissociate the enzyme. Reduction was equally effective with glutathione as with dithiothreitol or with reduced thioredoxin f. The concentration of 1,3bisPGA required to obtain 50% activity (Ka) was 19.5 ± 4.1 μMfor the untreated enzyme and 2.0 ± 1.4 μMfor the thiol-pretreated enzyme. Thus,in vitro1,3bisPGA, alone or—at much lower concentrations—together with a reductant can activate (and dissociate) NAD(P)-GAPDH. The enzyme exhibits similarKavalues in its reduced and its oxidized form for ATP (1–2 mM), NADP (50–200 μM), and NADPH (0.3–0.5 mM) as positive effectors, but these effectors do not lead to any activation when present together with 0.14 mMNAD. Only 1,3bisPGA retained its characteristic effect in the presence of NAD. The dissociated enzyme reaggregates upon removal of the positive effectors. From these results it is concluded (i) that the role of the reduction of the NAD(P)-GAPDHin vivois to increase its sensitivity toward the activator 1,3bisPGA and (ii) that the actual activation (and aggregation) state of the enzyme in chloroplasts in the light is regulated by the concentration of 1,3bisPGA as activator in the stroma and its actual activity by the availability of 1,3bisPGA as substrate.