Enzymatic activities in thylakoid membranes, which form medium [32P]NDP and [32P]ATP from 32Pi. Polynucleotide phosphorylase and adenylate kinase.

Soluble chloroplast coupling factor 1 (CF1) and the ATP synthase complex, under uncoupled conditions, can form bound ATP from tightly bound ADP and medium Pi. This partial reaction is a powerful probe of the mechanism of ATP synthesis. During our study of the synthesis of bound ATP by CF1 other enzyme activities, which generate [32P]nucleotides from 32Pi, were characterized and controlled. Two enzymes present at significant levels in the preparations are polynucleotide phosphorylase and adenylate kinase. Polynucleotide phosphorylase (PNPase) was found both in thylakoid and CF1 preparations and catalyzed the formation of [beta-32P]ADP via its Pi----ADP exchange activity. The formation of [beta-32P]ADP during net photophosphorylation is attributable to adenylate kinase action on the [32P]ATP formed since hexokinase and glucose effectively block its production. In addition, PNPase also degraded RNA present in thylakoid preparations yielding all four [32P]nucleoside diphosphates. PNPase was also shown to catalyze a Pi----ATP exchange that is dependent on RNA primers and other cofactors.