Purification and Comparative Properties of Microsomal and Glyoxysomal Malate Synthase from Castor Bean Endosperm

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Abstract
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, KCl 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 MW of 575,000, with a single subunit type of MW 64,000, a pH optimum of 8, apparent Km for acetyl-CoA of 10 .mu.M 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 (Ki 110 .mu.M) and glycolate (Ki 150 .mu.M) 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 35S-labeled malate synthase from KCl extracts of both microsomes and glyoxysomes isolated from [35S]methionine-labeled endosperm tissue.