Differential phosphorylation by GTP and ATP in isolated rod outer segments of the retina

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Abstract
Isolated bovine rod outer segment protein was phosphorylated with GTP-.gamma.-32P and ATP-.gamma.-32P and to a much lesser extent by CTP-.gamma.-32P and UTP-.gamma.-32P. Phosphorylation with both GTP (GTP-kinase activity) and ATP (ATP-kinase activity) was markedly stimulated by light; phosphorylation with GTP was lower in dark-adapted and higher in light-adapted rod outer segments than was phosphorylation with ATP. Km values of 20 and 200 .mu.M and Vmax values of 2.1 and 5.9 nmol/(mg min-1) were calculated using ATP and GTP, respectively, in light-adapted outer segments. When outer segments were incubated with GTP .gamma.-32P under the usual conditions employed in these experiments, no formation of ATP-.gamma.-32P was detected by the techniques of high-pressure liquid chromatography and TLC. In intact, light-bleached outer segments, GTP appeared to specifically phosphorylate rhodopsin. Histone and phosvitin were not phosphorylated to any appreciable extent by GTP. Histone appeared to block rhodopsin phosphorylation by GTP, while histone and, to some extent, phosvitin both acted as substrates for ATP-kinase activity. Cyclic AMP and other adenine derivates had a marked inhibitory effect on GTP-kinase activity. Phosphate also inhibited GTP-kinase activity but stimulated ATP-kinase activity. Such differences in phosphorylation with GTP and ATP indicated that these activities were either due to separate enzyme systems or, if only 1 enzyme is involved, the activities are under separate physiological control in the photoreceptor unit.

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