FUNCTIONAL-STUDIES OF CHLOROPLAST GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE SUBUNIT-A AND SUBUBIT-B EXPRESSED IN ESCHERICHIA-COLI - FORMATION OF HIGHLY-ACTIVE A(4) AND B-4 HOMOTETRAMERS AND EVIDENCE THAT AGGREGATION OF THE B-4 COMPLEX IS MEDIATED BY THE B-SUBUNIT CARBOXY-TERMINUS

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C. 1.2.1.13) (GAPDH) of higher plants exists as an A(2)B(2) heterot etramer that catalyses the reductive step of the Calvin cycle. In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form. We have expressed in Escherichia coli p roteins corresponding to the mature A and B subunits of spinach chloro plast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE). One mg of each of the three proteins so expressed was purified to ele ctrophoretic homogeneity with conventional methods. Spinach GapA purif ied from E. coli is shown to be a highly active homotetramer (50-70 U/ mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca. 600 kDa. Since Bq forms of the enzyme have not been described from any source, we were surprised to f ind that spinach GapB purified from E. coil was active (15-35 U/mg). S pinach GapB lacking the CTE purified from E. coli is more highly activ e (130 U/mg) than GapB with the CTE. Under aggregating conditions, Gap B lacking the CTE is a tetramer that does not associate to HMM forms w hereas GapB with the CTE occurs exclusively as an aggregated HMM form. The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.