Activation of spinach chloroplast glyceraldehyde 3-phosphate dehydrogenase: effect of glycerate 1,3-bisphosphate |
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Authors: | Paolo Trost Sandra Scagliarini Vincenzo Valenti Paolo Pupillo |
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Institution: | (1) Dipartimento di Biologia, Università di Bologna, Via Irnerio 42, I-40126 Bologna, Italy;(2) Dipartimento di Biologia Vegetale, Università di Perugia, Borgo XX Giugno 74, I-06121 Perugia, Italy |
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Abstract: | 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, K
a= 12 M). 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 M. 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 K
a values three- to tenfold higher than the K
m. 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.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
This work was supported by grants from the Ministero dell'Università e della Ricerca Scientifica e Technologica in years 1990–1991. We are grateful to Dr. G. Branlant (Laboratoire d'Enzymologie et de Génie Génétique, Vandoeuvre les Nancy, France) for introducing us to the BPGA purification procedure. |
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Keywords: | Calvin cycle Glyceraldehyde 3-phosphate dehydrogenase NAD(P)-dependent Glycerate 1 3-bisphosphate Photosynthesis Spinacia |
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