Structural and kinetic characterization of NADP-dependent, non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from celery leaves

NADP-dependent, non-phosphorylating glyceraldehyde-3-phosphate dehydrogenas e (EC 1.2.1.9) from celery leaves was purified over 1200-fold to a specific activity of 35 units/mg protein, and its kinetic, regulatory and structura l properties were characterized. The purified enzyme exhibited a homotetram eric structure with a subunit molecular mass of 54 kDa. A high specificity of the enzyme for the substrates NADP(+) (K-m = 7 mu M) and D-glyceraldehyd e-3-phosphate (K-m = 127 mu M) was observed. Maximal activity was determine d at pH 8.5. The purified enzyme was highly unstable, requiring the additio n of NADP(+) or conditions of high ionic strength in the medium. A hysteret ic behavior, with a lag phase of minutes, was observed during activity meas urement of the enzyme preincubated in the absence of substrates. The lag wa s inversely proportional to the protein concentration during preincubation. The hysteretic parameters were affected by the substrates, KCI and mannito l among other compounds. Distinctively, incubation with NADP(+) produced a near twofold activation of the enzyme. Results suggest that in alditol prod ucing plants the enzyme plays a key role in the synthesis and partitioning of photoassimilates. (C) 2000 Published by Elsevier Science Ireland Ltd. Al l rights reserved.