1. Two enzymes (A and B) which catalyse the sulphation of p-nitrophenol and l-tyrosine methyl ester have been isolated from female rat livers. One of these enzymes (A) also catalyses the sulphation of dehydroepiandrosterone. 2. The K(m) values for the sulphation of p-nitrophenol and l-tyrosine methyl ester by enzyme B at pH7.5 are 1.5mum and 2.9mm respectively. 3. Enzyme B is oxidized on keeping at 0 degrees C when the K(m) and V(max.) values for the sulphation of p-nitrophenol are increased approx. 200-fold and fourfold respectively. This oxidized preparation of enzyme B fails to catalyse the sulphation of l-tyrosine methyl ester. 4. When the oxidized form of enzyme B is kept at 0 degrees C and low ionic strength then further forms of p-nitrophenol sulphotransferase are produced having even lower affinities for the sulphate acceptor. 5. The K(m) value for adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate is not affected during storage of the enzyme under these conditions. 6. Prolonged storage of enzyme B at low ionic strength leads to a considerable degree of polymerization of p-nitrophenol sulphotransferase and l-tyrosine methyl ester sulphotransferase. 7. The changes in the kinetic properties and molecular size of enzyme B during storage are reversed by dithiothreitol.

1. The kinetics of the enzymic transfer of sulphate from adenosine 3'-phosphate 5'[35S]-sulphatophosphate to derivatives of L-tyrosine were investigated with a partially purified enzyme preparation from rat liver. 2. At pH 7.5 and 370C the Km values for L-tyrosine methyl ester and adenosine 3'-phosphate 5'[35S]-sulphatophosphate are 0.3mM and 8nM respectively. The Km value for either substrate is independent of the concentration of the other. The available data are consistent with the sulphation reaction proceeding according to a rapid-equilibrium random Bi Bi mechanism. 3. From the effect of pH on the Km and Vmax. values for L-tyrosine methyl ester, tyramine and N-acetyl-L-tyrosine ethyl ester it is concluded that the enzyme is specific for substrate molecules with a free and unprotonated amino group and an un-ionized hydroxyl group. 4. The only ionizing group that can be positively attributed to the enzyme appears to influence the binding of adenosine 3'-phosphate 5'[35S]-sulphatophosphate and has an apparent pK value of approx. 9.5. It is suggested that this group may be an essential thiol. 5. The enzyme is inhibited by iodoacetamide at pH 7.5 and 30°C and this inhibition is prevented by the presence of adenosine 3'-phosphate 5'[35S]-sulphatophosphate but not by L-tyrosine methyl ester.The preceding paper (Mattock & Jones, 1970) contrasted some of the properties of the enzymes responsible for the sulphation of L-tyrosine methyl ester and tyramine on the one hand and p-nitrophenol on the other. From the evidence quoted, it appears that the two enzymes are distinct from one another. Hence it is not likely that the appearance of L-tyrosine 0-sulphate in mammalian urines (John, Rose, Wusteman & Dodgson, 1966) is an artifact of a detoxication type of reaction. However, whether the enzyme that catalyses the sulphation of simple derivatives of L-tyrosine is responsible for the appearance of sulphated tyrosine residues in proteins is unknown, and an investigation of the substrate requirements of this enzyme may throw light on its possible metabolic function. In this connexion, Segal & Mologne (1959) have shown that, with a rat liver enzyme preparation, the rate of transfer of sulphate from p-nitrophenyl sulphate via adenosine 3',5'-diphosphate to carboxyl-substituted derivatives of L-tyrosine was higher at pH 9.3 than at pH 7.8. These authors suggested that these observations reflected the ionization of the amino group of the substrates in

identical with the elongation factors of the supernatant, are present in the ribosomal fraction. These factor(s), presumably, are involved in initiation: however, further investigation is required to elucidate this point. Moreover, preliminary results indicate that a fraction with stimulatory activity similar to that described above can be removed from ribosomes with 0.25M-armmonium chloride. The nature of this fraction is also currently under investigation. In order to investigate the factors that control the reaccumulation of hepatic glycogen after starvation we have measured net rates of glycogen deposition in perfused livers from starved rats. Livers from 170-190g male rats, starved for 48h, were perfused with bicarbonate-albumin-saline, essentially as described by R. Hems, Ross, Berry & Krebs (1966), except that washed erythrocytes from fresh defibrinated rat blood (Baron & Roberts, 1963) were employed, so that haemoglobin was 4% (w/v). Liver samples (0.5g) were taken at 20min (ventral notched lobe) and 50min (large main lobe).When livers from 48h-starved rats were perfused with glucose alone (30mM) glycogen deposition was 0-0.4,umol/min per g wet wt. of liver, but if pyruvate, glycerol and serine (each 5mM) were added after 15min perfusion with 30mM-glucose the glucose concentration in the medium was stabilized and the rate of glycogen accumulation was 0.68+0.05 (10),umol/min per g wet wt. of liver.Unless the glucose concentration in the medium was 25-30mM, glycogen accumulation was less than 0.5,umol/min per g wet wt. of liver and glucose was released into the medium.When liver was perfused under the present conditions with glycerol, pyruvate and serine (each 5mM) in the absence of glucose gluconeogenesis was 1.4,umol/min per g wet wt. of liver for 20-35min, after which the rate decreased. In this experiment average glucose formation between 20-50min was 0.76p mol/min per g wet liver. Rapid

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