5?-Nucleotidase from bovine brain cortex: effect of solubilization on enzyme kinetics and modulation

The kinetic characteristics and the EDTA inhibition of microsomal 5?-nucleotidase from bovine brain cortex were studied and compared with the properties of the enzyme solubilized with Lubrol WX. The K(m) value after enzyme solubilization was not significantly different from that of the membrane-bound enzyme. Likewise, di- and trinucleotides performed a similar competitive inhibition of the two forms of the enzyme. In contrast, divalent cations inhibited the intact microsomal enzyme activity at the same concentrations in which they increased the soluble-enzyme activity. The solubilization of microsomal 5?-nucleotidase did not change the progressive and irreversible character of the EDTA inhibition, but the mechanism of the irreversible inhibition was different. The addition of divalent metal cations did not affect the irreversibility of either inhibition, even though the effect on the residual activities was different. The Arrhenius plot of the 5?-nucleotidase activity in intact microsomal fraction exhibited a well-defined break at 31 +/- 0.1 degrees C, whereas that of the solubilized enzyme was a straight line. It is concluded then that microsomal 5?-nucleotidase from bovine brain cortex does not require the membrane environment to express its activity, although the influence of this lipidic environment was evident in the differences observed in the enzyme activity modulation by EDTA, cations and temperature.