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Purification and Comparative Properties of Microsomal and Glyoxysomal Malate Synthase from Castor Bean Endosperm ^1,2

Postgraduate School of Biological Sciences, University of Bradford, Yorkshire, BD7 1DP, England

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 µM 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 µM) and glycolate (K_i 150 µM) 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 KCI extracts of both microsomes and glyoxysomes isolated from [³⁵S]methionine-labeled endosperm tissue.

² The term "microsomes" is used in this and the accompanying paper to define the material recovered as a discrete band at a density of 1.12 g/ml after sucrose density gradient centrifugation of castor bean endosperm homogenates. This fraction is comprised of smooth surfaced membrane-bound segments derived from the endoplasmic reticulum. To the best of our knowledge this fraction is not contaminated with other membranous components which would be expected constituents of "microsomal" fractions obtained by high speed centrifugation of postmitochondrial supernatants.