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 NADPHactive 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 laM). It also causes the enzyme to dissociate into 160-kDa protomers. The K m of BPGA both with NADPH or NADH as coenzyme is 4 7 laM. 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 NADPHactivity and conformational state of spinach NAD(P)-GAPDH, all characterized by K a values three-to tenfold higher than the K m. Since NADP(H) is largely converted to NAD(H) in darkened chloroplasts , 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 concentra-Abbreviations: BPGA=glycerate 1,3-bisphosphate; Chl=chlorophyll; DTT= dithiothreitol; FPLC=fast protein liquid chromatography; NAD(P)-GAPDH=glyceraldehyde 3-phosphate dehydrogenase, NAD(P)-dependent; 3-PGA=glyerate 3-phosphate; PGK =phosphoglycerate kinase; Prt = protein; Tricine =N-tris (hydroxymethyl) methyl-glycine Correspondence to: V. Valenti; FAX: 39(75)5856425 tion increases during photosynthesis, in addition to reduced thioredoxin.