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Specific vasopressin binding to rat hepatocytes and rat liver membranes was measured using biologically active (3H)-Tyr2-Lys8-vasopressin (8.5 Ci/mM). In both systems, vasopressin binding was found to be time-dependent, reversible, and saturable. The kinetic parameters for vasopressin binding were: apparent dissociation constants (Kd): 4.9 nM and 15 nM; maximal binding capacities: 0.83 pmoles/mg protein and 2.10(5) sites/Cell for purified membranes and intact cells respectively. The relative affinities of 19 vasopressin structural analogues were deduced from competition experiments and compared to the previously determined glycogenolytic (or antiglycogenolytic) potencies of these analogues. For both agonists and antagonists, a highly significant correlation was demonstrated between pKd and pKa (or pKi) values, suggesting that the detected binding sites are the physiological receptors involved in the glycogenolytic action of vasopressin on the rat liver. The affinity of antagonists for binding to these receptors is the same for both membranes and cells. In contrast, agonists which bind to vasopressin receptor sites have a higher affinity for purified membranes than for intact cells (Kd membranes/Kd cells = 8 +/- 1). GTP (0.1mM) reduced the affinity of agonists but not of antagonists for binding to membranes and abolished the differences between Kd values for binding to hepatocytes and membranes.

The use of anesthetized animals and of quick-freezing techniques has allowed to obtain a reliable estimation of the level of phosphorylase a in the liver. A striking inverse relationship was observed between the levels of phosphorylase a and of synthetase a in the liver of fed mice; this observation is in agreement with the hypothesis that the inactivation of phosphorylase is a prerequisite to the activation of glycogen synthetase. Accordingly, the administration of glucose to fed rats caused a n extensive inactivation of liver phosphorylase which was usually terminated within two minutes; glycogen synthetase became activated only when and if the level of phosphorylase a had been taken down below a threshold value equal to approximately loo/, of the total phosphorylase. This threshold is of the same magnitude in mice. Glucose also caused a slight decrease in the activity of liver phosphorylase when the animals had been previously treated with glucagon. These observations are adequately explained by the previously described stimulation of the phosphorylase phosphatase reaction by glucose and inhibition of synthetase phosphatase by phosphorylase a. I n some experiments, insulin caused a partial inactivation of phosphorylase in the liver of rats but more often was without effect, whereas a subsequent load of glucose caused the usual precipitous response, demonstrating that this effect of glucose was not mediated by insulin.Liver glycogen synthetase exists in two forms, one active, a, and the other inactive, b [I], and the rate of glycogen synthesis in the liver depends upon the amount of synthetase a [2]. I n fed mice, the enzyme is almost entirely b during day-time and is in great part converted into a within 5-10 min after an intravenous administration of glucose. This activation is preceded by a very characteristic latency period of 1-2 min [2]. These observations have initiated a series of investigations on the effect of glucose on the interconversion of glycogen phosphorylase and of glycogen synthetase in liver extracts and in purified enzymic systems. It has been shown that glucose stimulates the inactivation of liver phosphorylase Abbreviation. Cyclic AMP, adenosine 3': 5'-monophosphate.Enzymes. Glycogen phosphorylase or a-1,4-glucan: orthophosphate glucosyltransferase (EC 2.4.1.1) ; glycogen synthetase or UDPG: a-1,4-glucan a-4-glucosyltransferase (EC 2.4.1.11) ; phosphorylase phosphatase or phosphorylase phosphohydrolase (EC 3.1.3.17) ; phosphorylase kinase or ATP: phosphorylase phosphotransferase (EC 2.7.1.38).by its phosphatase [3] and that this effect is the result of the binding of glucose to phosphorylase a [4]. The activation of glycogen synthetase in vitro is preceded by a latency which is markedly shortened by glucose [5]. This latency as well as its shortening by glucose have been adequately explained by a strong inhibition of synthetase phosphatase by phosphorylase a, although not by phosphorylase b [6]. Due to this inhibition, the activation of glycogen synthetase requires the previous inactivat...