EXPRESSION AND CHARACTERIZATION OF PEA CHLOROPLASTIC GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE COMPOSED OF ONLY THE B-SUBUNIT

A cDNA fragment coding for the pea (Pisum sativum L.) chloroplastic gl yceraldehyde-3-P dehydrogenase (EC 1.2.1.13) B-subunit and a truncated form corresponding in length to the A-subunit have been cloned into a n expression vector, expressed in the absence of the A-subunit in a ga p(-) Escherichia coli strain, purified, and studied. Like the isolated enzyme from higher plant chloroplasts, the recombinant enzymes have d ual specificity for NADPH and NADH. The recombinant glyceraldehyde-3-P dehydrogenases have the same optimal pH as the enzyme isolated from p ea chloroplasts. Like the native chloroplast enzyme, the recombinant B -subunit has a marked tendency to form large aggregates, whereas the t runcated B-subunit exists as the tetramer. The recombinant B-subunit g lyceraldehyde 3-P dehydrogenase is more sensitive to dithiothreitol th an its truncated form. It seems likely that a different pair of cystei nes is responsible for the redox sensitivity of the activity of the en zyme composed of B-subunits than the cysteine residues implicated in t he modulation of the activity of the enzyme composed of A-subunits by previous work in this laboratory.