RESUMO
NADP-dependent, non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (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 structural properties were characterized. The purified enzyme exhibited a homotetrameric structure with a subunit molecular mass of 54 kDa. A high specificity of the enzyme for the substrates NADP(+) (K(m)=7 µM) and D-glyceraldehyde-3-phosphate (K(m)=127 µM) was observed. Maximal activity was determined at pH 8.5. The purified enzyme was highly unstable, requiring the addition of NADP(+) or conditions of high ionic strength in the medium. A hysteretic behavior, with a lag phase of minutes, was observed during activity measurement of the enzyme preincubated in the absence of substrates. The lag was inversely proportional to the protein concentration during preincubation. The hysteretic parameters were affected by the substrates, KCl and mannitol among other compounds. Distinctively, incubation with NADP(+) produced a near twofold activation of the enzyme. Results suggest that in alditol producing plants the enzyme plays a key role in the synthesis and partitioning of photoassimilates.
