Plant Microbody Proteins

Abstract

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. 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. 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.

A Bifunctional Enzyme from Glyoxysomes

“…All solutions were prepared in buffer B. Centrifugation was at 27000 rev./min for 4 h in a Beckman SW-27 rotor. Fractionation was as described earlier [4,7].…”

“…Rate zonal centrifugations were performed at 40000 rev./min using a SW 40-rotor for 46 h (Beckman) as described earlier [7,16]. Malate hydro-lyasc and liver citrate synthase were used as internal markers.…”

“…Thiolase activity was monitored by following the decrease of magnesium acetoacetyl-CoA at 303 nm [I 91. Catalase, malate hydro-lyase, isocitrate lyase, and citrate synthase were measured as described earlier [4,7].…”

“…Immunoelectrophoresis [7] was performed on agarose with a buffer consisting of 15 niM potassium phosphate, pH 7.5, 100 mM KCI, and 1 mM dithiothreitol. Antibodies were raised in rabbits according to [20].…”

Occurrence of several glycoproteins in glyoxysomal membranes of castor beans

“…Thus glyoxysomal malate synthase from castor beans [3] and from cucumber [4] and isocitrate lyase from cucumber [5] have been claimed to be glycoproteins.…”