Evidence for the de novo synthesis of poly(A) polymerase in germinated wheat embryos

Abstract

A linear rise in the activity of poly(A) polymerase occurs following germination of excised wheat embryos. A roughly three-fold increase in poly(A) polymerase activity was paralleled by a rise (2.4-fold) in the relative abundance of poly(A) +RNA isolated from 72 hr germinated embryos over that at six hr after imbibition. Administration of cycloheximide (20 μg/ml) to germinated embryos significantly inhibited (80%) poly(A) polymerase activity with a dramatic decline (77 %) in the levels of total poly(A) +RNA. This indicated that the relative abundance of poly(A) +RNA is regulated by the modulation of poly(A) polymerase activity. The enhancement of poly(A) polymerase activity following embryo germination is primarily achieved through de novo synthesis of the enzyme. This has been conclusively shown by in vivo labelling of the newly synthesized total proteins with 35SO 4 2- in wheat embryos and ultimately recovering the label in the purified preparation of poly(A) polymerase. Fractionation of purified poly(A) polymerase on native polyacrylamide gels revealed a single protein band, thereby establishing the electrophoretic homogeneity of the enzyme preparation. Autoradiography of this gel showed a single radioactive band which corresponded with the protein band of the purified poly(A) polymerase. Further characterization of the purified labelled poly(A) polymerase was obtained by acid hydrolysis of the enzyme protein followed by the chromatographic separation of the 35S labelled amino acids. Autoradiography of the chromatogram revealed the presence of the label in the cysteine residues of poly(A) polymerase. These studies clearly indicated that de novo synthesis of poly(A) polymerase indeed occurs during growth of wheat embryos.