Glyoxylate cycle enzymes of the glyoxysomal membrane from cucumber cotyledons

Abstract

Glyoxysomes were isolated from etiolated cotyledons of cucumber seedlings. After separation of matrix proteins from the glyoxysomal membranes, enzymes were solubilized from the membranes by 100 m m MgCl 2 and purified by sedimentation velocity centrifugation, ion exchange chromatography, and separation on hydroxylapatite. Malate synthase, citrate synthase, and malate dehydrogenase the three enzymes of the glyoxylate cycle which were primarily membrane bound in this type of microbody-were thus obtained in a homogeneous form, as judged by sodium dodecyl sulfate-gel electrophoresis. Enzymatically active malate synthase, as obtained by solubilization of membrane proteins, behaved on Sepharose 6B columns as a protein with a molecular weight of about 70,000 and is characterized by an acidic isoelectric point. Malate synthase aggregates in the presence of Mg 2+ and glyoxylate, yielding an active octamer with an alkaline isoelectric point and a molecular weight of about 540,000. Upon sodium dodecyl sulfate-gel electrophoresis, a subunit molecular weight of 63,000 was estimated. Citrate synthase exists as a dimer (molecular weight of 100,000) and tetramer (molecular weight of 200,000) and exhibits the same subunit molecular weight as the liver enzyme (46,000). Malate dehydrogenase was found to have a molecular weight similar to the microbody catalase (about 225,000), while for the single peptide chain a value of approximately 34,000 was determined.