Evidence for the participation of endogenous guanosine triphosphate in substrate level phosphate transfer in intact mitochondria

Abstract

Guanosine and inosine diphosphate (GDP end IDP) have been shown to be possible phosphate acceptors in the succinic thiokinase reaction in mammalian kidney and heart, ( Mazumder, Sanadi and Rodwell 1960 ): Succinyl CoA + XDP -+ Succinat + XTP + CoA (1) X = inosine or guanosine The nucleotide specifity of reaction (1) has been studied only in isolated enzyme preparations. The difference of Km between guanine and inosine nucleotides was shown to be small. Thus the question as to which nucleotide system,(the inosine or the guanine system) is the physiological participant in the phosphate transfer reactions (1) remained open. In order to answer this question, the role of guanine and inosine nucleotides in intact liver mitochondria was studied by following the incorporation of P 32 into phosphate compounds of mitochondria. The basis of the experiments was the ultramicro scale ion exchange chromatography for separation of phosphate compounds ( Schnitger et al. 1959, Heldt,.1963 ). This was combined with simultaneous chart recording of ultraviolet absorption, analysis of total phosphorous content and radioactivity. Exact data on radioactivity were obtained by digital recording. The PT2 labelled compounds were identified by comparing the chromatographic behaviour of the collected fractions with added components on thin layer radio electrophoresis and