Reductive modification and nonreductive activation of purified spinach chloroplast NADP-dependent glyceraldehyde-3-phosphate dehydrogenase

Autor(en): Baalmann, E
Backhausen, JE
Rak, C
Vetter, S
Scheibe, R 
Stichwörter: Biochemistry & Molecular Biology; Biophysics; MODULATION; PEA; SEQUENCE; SUBUNITS; SYSTEM
Erscheinungsdatum: 1995
Volumen: 324
Ausgabe: 2
Startseite: 201
Seitenende: 208
Spinach chloroplast NAD(P)-glyceraldehyde-3-phosphate dehydrogenase (NAD(P)-GAPDH; EC, was purified as the 600-kDa oligomer of low specific activity. Incubation of the enzyme with either a reductant or a 1,3-bisphosphoglycerate (1,31bisPGA) 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 (A(2)B(2)). The higher activity of the tetramer is largely due to a decreased K-M value 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 (K-a) was 19.5 /- 4.1 mu M for the untreated enzyme and 2.0 /- 1.4 mu M for the thiol-pretreated enzyme. Thus, in vitro 1,3bisPGA, alone or - at much lower concentrations - together with a reductant can activate (and dissociate) NAD(P)-GAPDH. The enzyme exhibits similar K-a values in its reduced and its oxidized form for ATP (1-2 mM), NADP (50-200 mu M), and NADPH (0.3-0.5 mM) as positive effecters, but these effecters do not lead to any activation when present together with 0.14 mM NAD, Only 1,3bisPGA retained its characteristic effect in the presence of NAD. The dissociated enzyme reaggregates upon removal of the positive effecters, From these results it is concluded (i) that the role of the reduction of the NAD(P)-GAPDH in vivo is 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. (C) 1995 Academic Press, Inc.
ISSN: 00039861
DOI: 10.1006/abbi.1995.0031

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