Regulation of Glyoxysomal Enzymes during Germination of Cucumber: 2. Isolation and Immunological Detection of Isocitrate Lyase and Catalase

Abstract

The glyoxysomal enzymes isocitrate lyase and catalase have been isolated from etiolated cucumber (Cucumis sativus) cotyledons. The enzymes co-purified through polyethyleneimine precipitation and (NH(4))(2)SO(4) precipitation, and were resolved by gel filtration on Sepharose 6B followed by chromatography on diethylaminoethyl-cellulose (isocitrate lyase) or hydroxylapatite (catalase). Purity of the isolated enzymes was assessed by sodium dodecyl sulfate-polyacrylamide electrophoresis, isoelectric focusing, and immunoelectrophoresis. Antibodies raised to both enzymes in rabbits and in tumor-bearing mice were shown to be monospecific by immunoelectrophoresis against total homogenate protein. Isocitrate lyase and catalase represent about 0.56% and 0.1%, respectively, of total extractable cotyledonary protein. Both enzymes appear to be present in a single form. Molecular weights of the native enzymes and its subunits are 225,000 and 54,500 for catalase, and 325,000 and 63,500 for isocitrate lyase. The pH optimum for isocitrate lyase is about 6.75 in morpholinopropane sulfonic acid buffer, but varies significantly with buffer used. The K(m) for d-isocitrate is 39 micromolar. A double antibody technique (rabbit anti-isocitrate lyase followed by (125)I-labeled goat anti-rabbit immunoglobulin G) has been used to visualize isocitrate lyase subunit protein on sodium dodecyl sulfate-polyacrylamide with high specificity and sensitivity.