Activation of spinach chloroplast glyceraldehyde 3-phosphate dehydrogenase: effect of glycerate 1-3-bisphosphate

• Published in: Planta 1993-06, Vol.190 (3), p.320-326
• Main Authors: Trost, P. (Bologna Univ. (Italy). Dipt. di Biologia), Scagliarini, S, Valentiv, V, Pupillo, P
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Biological and medical sciences; Calvin-Zyklus; Chloroplast; CHLOROPLASTE; CHLOROPLASTS; CLOROPLASTO; Coenzymes; Dehydrogenases; Enzymaktivitaet; Enzymes; ENZYMIC ACTIVITY; FOTOSINTESIS; Fundamental and applied biological sciences. Psychology; GLICERALDEHIDO 3 FOSF DESHIDROG; GLYCERALDEHYDE 3 PHOSPHATE DEHYDROG; GLYCERALDEHYDE 3 PHOSPHATE DESHYDRO; Kinetics; Metabolism; Phosphates; PHOTOSYNTHESE; PHOTOSYNTHESIS; Photosynthesis, respiration. Anabolism, catabolism; Plant physiology and development; Plants; Protein subunits; Spinach; SPINACIA OLERACEA; Spinat
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/bf00196960

Spinach (Spinacia oleracea L.) chloroplast NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) was purified. The association state of the protein was monitored by fast protein liquid chromatography-Superose 12 gel filtration. Protein chromatographed in the presence of NADP+ and dithiothreitol consisted of highly NADPH-active protomers of 160 kDa; otherwise, it always consisted of a 600-kDa oligomer (regulatory form) favoured by the addition of NAD+ in buffers and with low NADPH-dependent activity (ratio of activities with NADPH versus NADH of 0.2—0.4). Glycerate 1,3-bisphosphate (BPGA) was prepared enzymatically using rabbit-muscle NAD-GAPDH, and purified. Among known modulators of spinach NAD(P)-GAPDH, BPGA is the most effective on a molar basis in stimulating NADPH-activity of "dark" chloroplast extracts and purified NAD(P)-GAPDH (activation constant, Ka = 12 μM). It also causes the enzyme to dissociate into 160-kDa protomers. The Km of BPGA both with NADPH or NADH as coenzyme is 4—7 μM. NAD+ and NADH are inhibitory to the activation process induced by BPGA. This compound, together with NADP(H) and ATP belongs to a group of substrate-modifiers of the NADPH-activity and conformational state of spinach NAD(P)-GAPDH, all characterized by Ka values three- to tenfold higher than the Km. Since NADP(H) is largely converted to NAD(H) in darkened chloroplasts (Heineke et al. 1991, Plant Physiol. 95, 1131—1137, it is proposed that NAD+ promotes NAD(P)—GAPDH association into a regulatory conformer with low NADPH-activity during dark deactivation. The process is reversed in the light by BPGA and other substrate-modifiers whose concentration increases during photosynthesis, in addition to reduced thioredoxin.