Synthesis and decomposition of alcohol esters of 5′-AMP by rat liver plasma membrane

Abstract

1. 1. Recent data in the literature indicated that particulate fractions prepared from rat liver and adipose tissue were capable of synthesizing an unknown nucleotide from ATP and glycerol. In the present study, we provide direct evidence that the enzyme responsible for this activity is present in purified plasma membranes from rat liver, and that the nucleotide can be characterized as glycerol ester of adenosine 5-monophosphate. The synthetic activity was of similar extent with intact hepatocytes and with broken hepatocyte cell preparations, indicating an external location of alcohol-AMP-synthesizing enzyme in plasma membrane. 2. 2. Glycerol-AMP synthesis by rat liver plasma membranes was inhibited by EDTA; among the divalent cations tested, only Zn 2+ could effectively restore its synthesis. 3. 3. The enzymic reaction, studied with both ATP and glycerol as varying substrates, followed a sequential mechanism. The competitive inhibition of ATP by pyrophosphate was consistent with this mechanism. 4. 4. The K m values for ATP and glycerol were 0.30–0.80 mM and 5 M, respectively, at pH 9.5, with different preparations of plasma membrane. 5. 5. The synthesis of glycerol-AMP was competitively inhibited by 5′-AMP, but non-competitively (partial competition) by NADH. A number of substrates of the nucleotide pyrophosphatase were also inhibitory. Unlike 5′-AMP, the inhibitory effect of NADH was dependent on the concentration of glycerol. These data suggest that the catalytic site of nucleotide pyrophosphatase (EC 3.6.1.9) was not involved in the synthesis of glycerol AMP. 6. 6. Glycerol-AMP was decomposed by a liver plasma membrane enzyme, resulting in the formation of 5′-AMP. Such hydrolytic activity was inhibited by the presence of 5′-AMP.