5′-NUCLEOTIDASE OF ISOLATED HEPATIC CELL MEMBRANE FROM RAT LIVER

It has been shown from cytochemical study that the isolated hepatic cell membrane has 5'-nucleotidase(Ku and Liu, 1963). This paper deals with some properties of this enzyme. Basing on the analysis of various factors which affect enzyme activity, e.g. substrate concentration, enzyme concentration, pH and incubation time, the condition for the optimal determination of enzyme activity was determined as: 5'-adenosine phosphate (99.5% pure) 0.2 μmoles, Tris-maleate buffer, pH 7.6, 150 μmoles, cell membrane suspension 0.2ml, water to 2.0 ml and incubation at 37℃ for 30 min. This enzyme does not split phosphate from 2'-, 3'-(mixed isomer) adenosine, guanosine, cytidine and uridine monophosphates and β-glycerophosphate, but split 5'-uridine monophosphate at a rate of 70% of 5'-adenosine monophosphate. The effect of different ions on enzyme activity was studied and the results are shown in table 1. The activation of enzyme activity by Mn~(++), Ca~(++), and Mg~(++) and its inhibition by ethylenediamine tetraacetate may indicate that the divalent cation is strongly bound to the enzyme. 20 mM phosphate does not inhibit the enzyme activity either in Tris-maleate or in Tris-HC1 buffer at pH 7.6. Segal and Brenner(1960) showed that the rat liver microsome might contain two different kinds of 5'-nucleotidases, one can be inhibited by phosphate at 20mM and the other not. According to our experience, fragmentation of cell membrane may give rise to particles which can pass into the microsome fraction during differential centrifugation(Ku and Liu, 1963). It is highly probable that phosphate-noninhibited 5'-nucleotidase as noticed by the above authors may have its origin, wholly or partly, from cell membrane. Zn~(++), at a concentration of 1 mM, inhibits the enzyme activity almost completely and at 0.1mM, which corresponds to one-tenth of substrate concentration, inhibits 70%. This suggests that Zn~(++) affect the enzyme directly. Both deoxycholate(having 2 OH groups) and cholate(3 OH groups) can activate the enzyme, and the former has stronger effect than the latter. Since the effective concentration of cholate is very near to the concentration of cholate(184 mg/ml) in bile duct of rat as reported by Friedman and Byers(1953), it is tempting to suggest that the bile may regulate the activity of 5'-nucleotidase of liver cell membrane in vivo. The cell membrane has no adenosine deaminase as determined by the method of Kornberg(1951) and contains only negligible amount of non-specific phosphatases determined at pH 5.4 and 9.8 respectively (About 0—3% of the activity of 5'-nucleotidase).