Fractionation and comparative studies of enzymes in aqueous extracts of spinach chloroplasts

Abstract

Water and dilute salt solutions were employed gor the hypotonic rupture and repeated extraction of proteins from spinach chloroplasts. The extracts were partially fractionated with ammonium sulfate, and the fractions so obtained were resolved by sucrose-gradient centrifugation and by electrophoresis on acrylamide gels. These techniques physically separated several enzymes of phosphate metabolism which are interdependent upon common substrates, co-factors, and reaction products, and which may function critically in photophosphorylation or its reversal. These enzymes are a Ca ++-dependent ATPase ( S 20 w 12.7; DTT stimulated; active as coupling factor in photophosphorylation; morphologically identical to quantasomes), a discretely different Mg ++-dependent ATPase ( S 20 w 6.0; entirely different from mitochondrial ATPases; does not require activation but is stabilized by reductants), and ATP-ADP exchange enzyme ( S 20 w 7.0), adenylate kinase ( S 20 w 4.2), and an alkaline inorganic pyrophosphatase (associated with fructose diphosphatase activity). In addition, a similar interacting system of CO 2-fixing enzymes, comprised of ribulose diphosphate carboxylase, ribose phosphate isomerase, and phosphoribulokinase was separated. The fractionation and isolation conditions described were chosen to preserve all of these activities, and to provide an environment suited to a uniform comparison of their physical and enzymatic properties. These properties are compared with those of enzymes having similar functions, purified from chloroplasts by other means.