PHOSPHORYLATION COUPLED TO OXIDATION OF DIHYDRODIPHOSPHOPYRIDINE NUCLEOTIDE

Abstract

It has been shown in this laboratory that aerobic incubation of dihydrodiphosphopyridine nucleotide (DPNHZ) with a properly supplemented suspension of particulate elements of rat liver caused extensive incorporation of inorganic phosphate labeled with P32 into a form having the properties of the acid-labile group of adenosinetriphosphate (ATP) (1, 2). Such incorporation did not occur under nitrogen, in the absence of Mg++ or ATP, or when the oxidized form of the nucleotide (DPN) was substituted for DPNH,. It was concluded that oxidative phosphorylation had occurred coupled to the passage of electrons from DPNH, to oxygen via the cytochrome system, but no conclusion could be drawn from these data concerning the quantitative relationship between phosphorylation and oxidation. These findings were substantiated by subsequent work in which the DPN-linked P-hydroxybutyric dehydrogenase was used to generate DPNHt continuously from DPN and the substrate (3, 4). In the latter case it was not only possible to observe P32 uptake coupled to this DPNlinked oxidation, but it was also found possible to measure the P:O ratio directly in non-isotopic experiments with adenylic acid as phosphate acceptor (4). A sufficiently large proportion of the measurements yielded values over 2.0 (uncorrected for dephosphorylation losses of known and relatively large magnitude) to warrant the tentative conclusion that the actual ratio is 3.0. In this paper experiments are described in which highly purified DPNH, was used as the test substrate to determine directly in non-isotopic experiments the amount of phosphorylation occurring when electrons pass from DPNHz to oxygen by measurement of phosphate uptake. These experiments show in a direct way that most of the phosphorylation observed during the DPN-linked oxidation of /3-hydroxybutyrate to acetoacetate can be accounted for as being coupled to the oxidation of DPNH,. In addition, a number of observations were made which have a bearing on the question of the existence of permeability or “availability” barriers in the