Journal of Experimental Botany, Vol 49, 1307-1315, Copyright © 1998 by Oxford University Press
S Scagliarini, P Trost and P Pupillo
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 A4 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 150kDa 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
Km(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 × 10-2 M,
the experimentally derived value being Keq=16
× 10-2 M. The kinetic responses of GAPDHR
in the aggregated state (600 kDa) were identical to those of GAPDHN, except
that Vmax with NADP(H) 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.Keywords: Glyceraldehyde-3-phosphate
dehydrogenase, C3 cycle, NADPH, isoenzymes, chloroplast
ARTICLES
The non-regulatory isoform of NAD(P)-glyceraldehyde-3-phosphate dehydrogenase from spinach chloroplasts
Dipartimento di Biologia, Università di Bologna, Via Imerio 42, I-40126 Bologna, Italy; Corresponding author
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