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 beta-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 beta-mercaptoethanol, but not by oligonucleotide primers.
Olmstead & Lowe (1959) showed that trypsintreated Micrococcus lysodeikticus polynucleotide phosphorylase was inhibited by iodoacetate or, less reproducibly, PCMB*. These authors suggested that free thiol groups in the protein molecule were required in the polymerization reaction. Their results have recently been confirmed and extended (Klee & Singer, 1968). Further, the preferential loss of the CDP-polymerization activity during carefully limited trypsin degradation (Fitt & Fitt, 1967), or the general inactivation and increase in primer requirement caused by more extensive proteolysis (
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