Purification and comparative properties of microsomal and glyoxysomal malate synthase from castor bean endosperm.

Sucrose density gradient centrifugation was employed to separate microsomes, mitochondria, and glyoxysomes from homogenates prepared from castor bean (Ricinus communis) endosperm. In the case of tissue removed from young seedlings, a significant proportion of the characteristic glyoxysomal enzyme malate synthase was recovered in the microsomal fraction. Malate synthase was purified from both isolated microsomes and glyoxysomes by a procedure involving osmotic shock, KCI solubilization, and sucrose density gradient centrifugation. All physical and catalytic properties examined were identical for the enzyme isolated from both organelle fractions. These properties include a molecular weight of 575,000, with a single subunit type of molecular weight 64,000, a pH optimum of 8, apparent K(m) for acetyl-CoA of 10 mum and glyoxylate of 2 mm. Microsomal and glyoxysomal malate synthases showed identical responses to various inhibitors. Adenine nucleotides were competitive inhibitors with respect to acetyl-CoA, and oxalate (K(i) 110 mum) and glycolate (K(i) 150 mum) were competitive inhibitors with respect to glyoxylate. Antiserum raised in rabbits against purified glyoxysomal malate synthase was used to confirm serological identity between the microsomal and glyoxysomal enzymes, and was capable of specifically precipitating (35)S-labeled malate synthase from KCI extracts of both microsomes and glyoxysomes isolated from [(35)S]methionine-labeled endosperm tissue.