Abstract
1. A novel technique for the subfractionation of rat liver smooth and rough microsomal fractions according to their content of glucose 6-phosphatase is described. This technique, based on the Gomori lead histochemical procedure, involves incubation of smooth and rough microsomal fractions with low concentrations of Pb(NO(3))(2) and glucose 6-phosphate. Control experiments, in which enzyme was assayed in the presence of various amounts of Pb(NO(3))(2) or in which microsomal fractions were reisolated after incubation with low concentrations of Pb(NO(3))(2) and glucose 6-phosphate, showed that lead does not interfere with glucose 6-phosphatase activity. 2. Discontinuous sucrose-density-gradient centrifugation of microsomal fractions which had previously been incubated with various amounts of Pb(NO(3))(2) and glucose 6-phosphate showed that it is possible to subfractionate both smooth- and rough-microsomal fractions into several bands, owing to a differential modification of the density of the microsomal vesicles by the trapping of lead phosphate within them. 3. When the material in the bands obtained by density-gradient centrifugation of incubated microsomal fractions was assayed for glucose 6-phosphatase activity, it was found that the modification of the density of the microsomal fractions was directly related to their relative enrichment in glucose 6-phosphatase activity. Control experiments, in which microsomal fractions were incubated with Pb(NO(3))(2) and glucose 6-phosphate and then treated with EDTA, showed that the subfractionation was not due to aggregation of microsomal vesicles, lead and glucose 6-phosphate. Thus the resolution of microsomal preparations into subfractions with different glucose 6-phosphatase activities is interpreted as indicating heterogeneity of glucose 6-phosphatase distribution in the microsomal vesicles. 4. Electron micrographs of both smooth- and rough-microsomal subfractions show deposits of lead phosphate within the microsomal vesicles. The frequency and extent of these deposits correlate with the different amounts of glucose 6-phosphatase activity measured biochemically. 5. The nature of the heterogeneous distribution of glucose 6-phosphatase is discussed and the more general applicability of the technique for studying membrane fractions containing a heterogeneous distribution of phosphatases is indicated.