Plant Microbody Proteins

Abstract
1 Isocitrate lyase from cotyledons of cucumber seedlings (Cucumis sativus) has been purified 100-fold. Two methods of preparing the soluble glyoxylate cycle enzyme are described: an elaborated method which used crude extracts of cucumber cotyledons, and another procedure which started with purified glyoxysomes from 4-day-old cotyledons and included a separation of glyoxysomal matrix enzymes by zonal centrifugation. The product behaved as a single species when tested by (a) polyacrylamide gel electrophoresis in the presence of dodecyl sulfate, (b) zonal centrifugation, and (c) double immunodiffusion against rabbit antibody to isocitrate lyase. 2 Isocitrate lyase of cucumber glyoxysomes exhibited a molecular weight of 255 000 and was composed of four apparently identical subunits of Mr 64 000. An isoelectric point of 5.9 was determined. 3 It was shown that isocitrate lyase is a glycoprotein, (a) by Schiff stain on polyacrylamide gels, (b) by periodate oxidation of the enzyme, subsequent reduction with NaB[3H]4 and electrophoretic analysis of the labelled glycoprotein, and (c) by incorporation of [3H]glucosamine in vivo into a protein which could be precipitated with antibodies to isocitrate lyase and revealed a 64 000-Mr band upon electrophresis.

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