Phosphorylation of deoxyguanosine in intact and fractured mitochondria

Abstract

The phosphorylation of deoxyguanosine was measured in fractured and intact mitochondria and an apparent K_m of 16 μM for deoxyguanosine was calculated using fractured mitochondria. The effects of various deoxynucleotides on the phosphorylating activity in fractured organelles was tested at both a high and low ratio of NXP/ATP and at two pH values, 7.0 and 5.5. Exogenous dGTP, dGDP or dITP were inhibitory under all conditions tested. With a NXP/ATP ratio of 0.08 at pH 7.0, TTP, TDP, dADP, ADP, UTP and UDP were stimulatory, but at pH 5.5 only TTP elicited that response. When the NXP/ATP ratio was 10 at pH 5.5, TTP and UTP increased the activity more than 10-fold, whereas, at pH 7.0 TTP, TDP, dADP, ADP, UTP, UDP caused stimulation, but to a much lesser extent. When exogenous Mg²⁺, Mn²⁺ or Ca²⁺ were added to intact mitochondria, the rates of phosphorylation were lowered. In fractured mitochondria in the absence of exogenous ATP, little phosphorylation occurs, hence these metal ions caused little change. ATP-Mg, ATP-Mn and ATP-Ca, each at 0.05 mM caused a small inhibition with intact mitochondria, whereas, these compounds supported phosphorylation with fractured organelles. ATP-Mn (10 mM) or ATP-Ca (10 mM) stimulated phosphorylation in both intact and fractured mitochondria. Intact mitochondria synthesized dGMP, dGDP and dGTP when metal ion or ATP-Me concentrations were low (0.05 mM) or when Mg²⁺ concentration was high (10 mM). Additions of ATP-Ca, ATP-Mn, ATP-Mg, Mn²⁺ or Ca²⁺ at 10 mM cause the loss of dGDP and dGTP formation and, in most cases, an increase in the synthesis of dGMP. Fractured mitochondria make only dGMP and the levels of its synthesis are greater than that observed for intact mitochondria. These data suggest that intact mitochondria are required for the synthesis of dGTP and that its synthesis is regulated by mitochondria nucleotides.