Purification and characterization of polynucleotide phosphorylase from cucumber

Abstract

Polynucleotide phosphorylase (polyribonucleotide:orthophosphate nucleotidyltransferase, EC 2.7.7.8) activity has been found in many prokaryotes and studied in detail since 1955. Such enzymes have been detected also in plants. We now describe the purification of polynucleotide phosphorylase from cucumber cotyledons and leaves. This enzyme is a complex of three subunits, possibly not identical, of about Mr 50,000. Its enzymatic properties are similar to those of the tobacco enzyme. Unlike the prokaryotic enzymes, the plant enzyme shows activity in the absence of primer but is to various extents stimulated by various ribopolynucleotides or RNAs. RNA-dependent RNA polymerase, not previously shown to exist in non-virus-infected cucumber, has been found to be present at a low level and was separated from the much greater amount of polynucleotide phosphorylase, although some of the physical properties of the two enzymes are rather similar.In the course of a continuing study of the RNA-dependent RNA polymerase of cucumber cotyledons and leaves, it became evident that these plants, be they virus-infected or not, contained much of another enzyme, polynucleotide phosphorylase (polyribonucleotide:orthophosphate nucleotidyltransferase, EC 2.7.7.8). Polynucleotide phosphorylase was first discovered and purified from Lactobacillus and has since been isolated from many other aerobic and anaerobic bacteria (1)(2)(3)(4). It has proved to be a most useful tool in many molecular biological studies requiring polynucleotides of known composition, with one of the first important applications serving in the establishment of the genetic code. However, the role of these enzymes in prokaryotes has not yet been identified. It has been suggested that they could serve as alternate means to nucleases for the degradation of unwanted RNA, but only in systems in which the inorganic phosphate concentration is sufficiently high to reverse the reaction.Polynucleotide phosphorylase was detected, characterized, and partially purified from tobacco by Brishammar and Juntti (5). Its presence in animal cells is scanty and dubious (5-7). Del'vig and co-workers (8, 9) have studied the particular association of such enzymatic activity with polysomes.We now have isolated an almost pure enzyme of properties similar to those of tobacco (5)