I. Isolierung, Reinigung und Charakterisierung der Polynucleotid-Phosphorylase aus der Blaualge Anacystis nidulans

Abstract

A method for the isolation and purification of polynucleotide phosphorylase from the blue-green alga Anacystis nidulans is described. It consists in an initial fractional centrifugation which yields up to 70% of the enzyme associated with the ribosomal fraction while the RNA-polymerase together with the bulk DNA is mainly confined to the supernatant. The ribosomal fraction is then subjected to column chromatography (DEAE-cellulose) and gel filtration (Sephadex G-200) to obtain a 120 — 150 fold increase in specific activity over the crude extract. The enzyme is essentially free from nucleic acids, phosphatases, kinases and RNA-polymerase. With ADP, UDP or CDP as substrate the corresponding homopolymers poly-A, poly-U and poly-C, respectively, are synthesized with good yields; no lag phase occurs. Equimolar mixtures of these nucleoside diphosphates give rise to heteropolymers. The optimal formation of poly-G and of AGUC polymer, both of which have rather low yields under standard conditions is achieved by incubating at higher temperatures (45° and 60°) and by replacing Mg2⊕ by Mg2⊕. In the presence of inorganic phosphate the highly purified enzyme also catalyzes the cleavage, to nucleoside diphosphates, of synthetic polynucleotides and of native high molecular RNA from various organisms. The possible association of polynucleotide phosphorylase with the ribosomes and its function is discussed.