Caenorhabditis elegans: Purification of isocitrate lyase and the isolation and cell-free translation of poly(A+)RNA

Abstract

Isocitrate lyase (EC 4.1.3.1) from mixed larval populations of Caenorhabditis elegans was stabilized in crude extracts by centrifugation over a 0.2–0.6 M sucrose gradient for 2.5 hr in a vertical rotor (VTi 50) at 210,000g.The peak fractions from this sucrose gradient showed a half-life of 33 hr at 30 C and 225 hr at 4 C in contrast to 2.5 and 52 hr, respectively, for the crude extract. A purification scheme involving (NH 4) 2SO 4 precipitation and chromatography on Sepharose 6B and diethylaminoethyl-cellulose yielded isocitrate lyase that gave one band after electrophoresis in a sodium dodecyl sulfate-gel polymerized from 12% acrylamide. The purified enzyme with a molecular weight of 250,000 and subunit molecular weight of 61,600, had a specific activity of 2 μmoles glyoxylate formed min −1 mg protein −1, and was obtained in a 4% yield. Isocitrate lyase from C. elegans lost 80–85% of its activity in the precipitation by 33–55% (NH 4) 2SO 4, but this step appeared to be necessary for purification to homogeneity. The use of fast protein liquid chromatography appeared to be promising in that it provided an enzyme preparation that was about 50% pure with a specific activity as high as 3 μmoles glyoxylate formed min −1 mg protein −1. Poly(A+)RNA was isolated from C. elegans and translated in wheat germ cell-free system. Analysis on a 10% sodium dodecyl sulfate-polyacrylamide gel showed varied translation products including one or more 60,000-Da polypeptides.