STRUCTURAL AND FUNCTIONAL-PROPERTIES OF A MULTIENZYME COMPLEX FROM SPINACH-CHLOROPLASTS .1. STOICHIOMETRY OF THE POLYPEPTIDE-CHAINS

Antibodies have been raised specifically against chloroplast phosphori bulokinase, glyceraldehyde-3-phosphate dehydrogenase and ribulose 1,5- bisphosphate carboxylase-oxygenase. Each of these antibodies recognize s the same macromolecular entity isolated and purified from chloroplas ts. This entity is a multi-enzyme complex, previously isolated and mad e up of ribose-phosphate isomerase, phosphoribulokinase, ribulose 1,5- bisphosphate carboxylase-oxygenase, phosphoglycerate kinase and glycer aldehyde-3-phosphate dehydrogenase. Under denaturing conditions the mu lti-enzyme complex contains two polypeptides of 54 kDa and 15 kDa corr esponding to the large and the small subunits of ribulose 1,5-bisphosp hate carboxylase-oxygenase, the two polypeptides of the glyceraldehyde -3-phosphate dehydrogenase of 39 kDa and 37 kDa, one polypeptide of 40 kDa pertaining to phosphoribulokinase and one polypeptide of 30 kDa v ery likely pertaining to ribose-phosphate isomerase. The combined use of immunochemical and densitometric techniques allows one to determine the number and the stoichiometry of the various types of polypeptide chains and to compare them with the quaternary structure of the corres ponding isolated enzymes. Ribulose 1,5-bisphosphate carboxylase-oxygen ase of higher plants consists of eight large and eight small subunits. Glyceraldehyde-3-phosphate dehydrogenase is made up of two types of p olypeptide chains called A and B and its simplest quaternary structure is A2B2. Finally, phosphoribulokinase is a dimer made up of two ident ical subunits. Therefore, for the three isolated enzymes, the stoichio metry of the polypeptide chains is always 1: 1. Within this multi-enzy me complex, there are two subunits of phosphoribulokinase, two A and B subunits of glyceraldehyde-3-phosphate dehydrogenase and two large an d four small subunits of ribulose 1,5-bisphosphate carboxylase-oxygena se. Therefore the number and the stoichiometry of the polypeptide chai ns of phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase are the same in the multi-enzyme complex and in the free enzymes, but those of ribulose 1,5-bisphosphate carboxylase-oxygenase are complete ly different. This conclusion that the multi-enzyme complex contains t wo active sites for ribulose 1,5-bisphosphate may be confirmed indepen dently by kinetic inhibition studies using 6-phosphogluconate.