Preparation, proteolysis and reversible oxidation of highly purified Azotobacter vinelandii polynucleotide phosphorylase

Abstract

1. A new method has been developed for the preparation in good yield of highly purified Azotobacter vinelandii polynucleotide phosphorylase in its reduced form. 2. Aging or digestion with trypsin causes the enzyme to develop a primer requirement that is not eliminated by β-mercaptoethanol. 3. The development of a primer requirement is accompanied by marked changes of the electrophoretic mobility of the enzyme in polyacrylamide gels. 4. The enzyme is inactivated by aerial oxidation or thiol-specific reagents. The lost activity is restored by β-mercaptoethanol, but not by oligonucleotide primers.