Subunit structure of three glyceraldehyde 3-phosphate dehydrogenases of some flowering plants

Abstract

A procedure is described for the purification of three glyceraldehyde phosphate dehydrogenases from a batch of beet leaves. Glyceraldehyde 3-phosphate:NADP + reductase, nonphosphorylating (EC 1.2.1.9) has been purified over 1500-fold. The M r of this enzyme is 190,000 and its subunits have an M r of 53,000, suggesting a tetramer as the active form. Its p I is 6.0. Cytosolic glyceraldehyde 3-phosphate dehydrogenase, NAD dependent (EC 1.2.1.12), has an M r of 145,000 and subunits of M r 37,000. It is dissociated to inactive dimers by ATP, whereas NAD + in the presence of reductant promotes its reactivation. The amino acid composition is related to glyceraldehyde 3-phosphate dehydrogenases from animal sources and is most similar to pea seed glyceraldehyde 3-phosphate dehydrogenase. The enzyme exhibits a range of p I values from 5 to 7, but a second electrofocusing in the presence of dithioerythritol results in a single main form with p I 5.33, consistent with the behavior in polyacrylamide and cellulose acetate gel electrophoresis. Chloroplast NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) has been obtained from beet, pea, Ranunculus, Arum, and maize leaves. The stable form is an oligomer of about 800,000 M r (±10%), while a minor, possibly damaged fraction elutes as a retarded peak from agarose columns. The M r 800,000 form is reversibly dissociated to protomers of M r 160,000 by NADP +, with increase of apparent NADP-dependent activity. Two subunits are present in similar amounts in all association states and after all treatments: α with M r 36,000, and β with M r 41,000. The form found in density gradient ultracentrifugation has an M r of 390,000. Isoelectric points of the various forms lie between pH 4.1 and 4.7 for all species, with a main peak usually at p I 4.45. The amino acid composition of beet chloroplast glyceraldehyde phosphate dehydrogenase is not closely related to that of beet leaf NAD-dependent glyceraldehyde 3-phosphate dehydrogenase.