Glyceraldehyde-3-phosphate dehydrogenase (NADP) from Sinapis alba L. NAD(P)-induced conformation changes of the enzyme.

Abstract

Partially purified glyceraldehyde‐3‐phosphate dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings was filtered on Sephadex G‐200 in the presence of NADP(H) and NAD(H). With NADP(H) the enzyme elutes as a dissociated species (Mr∼ 170 000), whereas with NAD(H) it travels as an aggregate in the void volume (Mr≥ 800 000). The NAD‐specific enzyme (EC 1.2.1.12) elutes with an Mr of about 160 000, regardless of whether NADP(H) or NAD(H) is present. If the NADP‐dependent enzyme is first filtered with NADP+ on Sephadex G‐200 and then with NAD+ on Sepharose 6B, only part of the enzyme aggregates during the second filtration. The other part (fraction S) travels as a narrow band slightly ahead of the marker cytochrome c. Refiltration of fraction S with NADP+ and with increasing [NADP+]/[NAD+]ratios on Sepharose 6B demonstrates that this transport retardation is NAD‐dependent and counteracted by NADP. The effect is also eliminated by inorganic cations and is not observed on Sephadex G‐200. Dodecylsulfate/polyacrylamide gel electrophoresis of fraction S before and after affinity chromatography on NADP‐agarose shows that the enzyme is pure after transport retardation on Sepharose 6B. It is active with both NADP and NAD and contains at least three different polypeptide chains with apparent Mr of 39 000, 42 000, and 43 000. The NAD‐specific enzyme is shown to be a tetramer of four 38 000‐Mr subunits. Kinetic investigations show that the binding of NADP(H) and NAD(H) with glyceraldehyde‐3‐phosphate dehydrogenase (NADP) is mutually exclusive. The present data indicate that the NADP‐dependent enzyme, a species structurally distinct from the NAD‐specific enzyme, occurs in two different conformations in the presence of NADP(H) and NAD(H) respectively. The existence of a ‘sticky’ NAD conformer is suggested, which interacts with Sepharose 6B and/or aggregates with other molecules in crude or partially purified extracts.