Synthesis of bound adenosine triphosphate from bound adenosine diphosphate by the purified coupling factor 1 of chloroplasts. Evidence for direct involvement of the coupling factor in this "adenylate kinase-like" reaction

Abstract

Electrophoretically homogeneous coupling factor 1 from spinach chloroplasts binds ADP and converts the bound ADP to bound ATP and AMP. That this transphosphorylation of enzyme-bound ADP is catalyzed by the coupling factor itself, and not be a conventional adenylate kinase which might possibly contaminate preparations of the coupling factor, is supported by the following evidence. 1. The procedure for isolatio of the coupling factor is designed to separate this large (approximately 13 S) enzyme from the smaller (4.2 S) conventional adenylate kinase of spinach chloroplasts. The conventional adenylate kinase cannot be detected in purified preparations of the coupling factor by biochemical assay or by polyacrylamide gel electrophoresis. 2. The activity of spinach adenylate kinase is completely dependent upon magnesium ions. However, the production of bound ATP and AMP from bound ADP by the coupling factor can be assayed in the total absence of added magnesium ions or even in the presence of added EDTA. 3. Comparative studies with inhibitors show that the coupling factor can produce bound ATP from ADP under conditions where the activity of adenylate kinase is strongly inhibited. Conversely, the coupling factor is prevented from synthesizing bound ATP from ADP under other conditions where the conventional adenylate kinase has high levels of activity. 4. AMP, when added in solution to the coupling factor, does not bind to this enzyme, even in the presence of APT. Thus, it is unlikely that the appearance of AMP bound to the coupling factor after its incubation with ADP is due to the production of free AMP by contaminating adenylate kinase. These results demonstrate that the isolated, homogeneous coupling factor from spinach chloroplasts has the intrinsic capacity to perform a phosphoryl group transfer between two bound ADP molecules and thus to synthesize ATP. This reaction may have an important role in the photosynthetic production of ATP by the chloroplast, as is discussed in this communication.