Phosphorylation of adenosine in anoxic hepatocytes by an exchange reaction catalysed by adenosine kinase.
The elevation of adenosine levels induced by anoxia in isolated rat hepatocytes has been shown to result mainly from an arrest of the recycling of the nucleoside by adenosine kinase [Bontemps, Vincent and Van den Berghe (1993) Biochem. J. 290, 671-677]. To assess the activity of the latter enzyme in intact hepatocytes, incorporation of radioactive adenosine into the cells' adenine nucleotides was measured. Unexpectedly, despite the near-absence of ATP in anoxic cells, 40% of 50 microM [8-14C]adenosine was still incorporated into adenylates over 5 min. Moreover, whereas unlabelled and labelled adenosine were utilized in parallel in normoxic cells, uptake of [8-14C]adenosine did not correspond to a net disappearance of adenosine in anoxic cells. Addition of 1 mM unlabelled adenosine to anoxic hepatocytes in which the adenine nucleotides had been prelabelled with [U-14C]adenine induced an immediate loss of their radioactivity. The latter was recovered in the form of adenosine, but the size of the adenylate pool was not modified. Taken together, these results suggest the occurrence of an exchange reaction between AMP and adenosine. Incubation of Sephadex G-25-filtered high-speed supernatants of rat liver with 20 microM [8-14C]adenosine, 10 mM MgCl2 and 1 mM AMP resulted in the labelling of AMP in the total absence of ATP. This labelling was influenced by effectors of both adenosine kinase and cytosolic IMP-GMP 5'-nucleotidase; the latter is known to catalyse an exchange reaction [Worku and Newby (1982) Biochem. J. 205, 503-510]. Chromatography of cytosolic fractions of rat liver on DEAE-Sepharose, followed by Sephacryl S-200 and AMP-Sepharose, demonstrated that the exchange reaction between adenosine and AMP co-purified with adenosine kinase. It is concluded that incorporation of labelled adenosine into adenine nucleotides should not be considered to be proof of adenosine kinase activity in anoxia.
Bontemps
F
F
Laboratory of Physiological Chemistry, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Mimouni
M
M
Van den Berghe
G
G
D015720:5'-Nucleotidase / Q000378:metabolism; D000241:Adenosine / Q000378:metabolism* / Q000494:pharmacology; D000248:Adenosine Kinase / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D015636:Magnesium Chloride / Q000378:metabolism; D008297:Male; D010100:Oxygen / Q000008:administration & dosage* / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar
D015636:Magnesium Chloride; D000249:Adenosine Monophosphate; D000248:Adenosine Kinase; D015720:5'-Nucleotidase; D000241:Adenosine; D010100:Oxygen