The non-regulatory isoform of NAD(P)-glyceraldehyde-3-phosphate dehydrogenase from spinach chloroplasts

Isoforms of NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.13) have been separated from spinach chloroplast extracts by FPLC-anion exchange chromatography in phosphate buffer and purified to homogeneity. Peak I from Q-Sepharose corresponds to a tetramer of A-subunits of 36 kDa showing a constant ratio of NADPH- to NADH-activity of 2 (insensitive to substrate-modulators), and is defined as A, or non-regulatory isoform (GAPDHN). GAPDHN always amounts to 15–20% of total enzyme regardless of the purification procedure. A small peak II in Q-Sepharose eluates gives rise to 300 kDa and 150 kDa species. Peak III isoform from Q-Sepharose corresponds to the well-known regulatory NAD(P)-glyceraldehyde-3-phosphate dehydrogenase oligomer (GAPDHR) and contains equimolar quantities of 36 kDa (A) and 39 kDa (B) subunits. Following storage of GAPDHR under reducing conditions, partial degradation of B-subunits occurred, affecting the quaternary structure of the active enzyme. Steady-state kinetics of GAPDHN have been studied at pH 7.5. The patterns are consistent with the general reaction mechanism of glyceraldehyde-3-phosphate dehydrogenases and feature high K m(G3P) and substrate inhibition responses with increasing glyceraldehyde-3-phosphate or phosphate. The Vmax values of reactions with either NADP+ or NADPH at saturating concentrations of all substrates are similar, and 2.5-fold higher than for reactions using NAD+ or NADH. Haldane relationships result in Keq=4.6 x 10−2 M, the experimentally derived value being Keq=16 x 10−2 M. The kinetic responses of GAPDHR in the aggregated state (600 kDa) were identical to those of GAPDHN, except that Vmax with NADPH) was 8-fold lower on a protein basis. The kinetic data are consistent with a GAPDHR model where B-subunits are mostly responsible for regulatory effects and A-subunits for catalysis.