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

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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 μM). 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 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 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.

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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

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Authors and Affiliations

  1. Dipartimento di Biologia, Università di Bologna, Via Irnerio 42, I-40126, Bologna, Italy

    Paolo Trost, Sandra Scagliarini & Paolo Pupillo

  2. Dipartimento di Biologia Vegetale, Università di Perugia, Borgo XX Giugno 74, I-06121, Perugia, Italy

    Vincenzo Valenti

Authors
  1. Paolo Trost
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  2. Sandra Scagliarini
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  3. Vincenzo Valenti
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  4. Paolo Pupillo
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Additional information

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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Trost, P., Scagliarini, S., Valenti, V. et al. Activation of spinach chloroplast glyceraldehyde 3-phosphate dehydrogenase: effect of glycerate 1,3-bisphosphate. Planta 190, 320–326 (1993). https://doi.org/10.1007/BF00196960

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