Multifunctional glucose-6-phosphatase studied in permeable isolated hepatocytes.

With isolated liver microsomes, both synthetic and hydrolytic activities of glucose-6-phosphatase (D-ghcase-6-phosphate phosphohydrolase, EC 3.1.3.9) are characterized by considerable latency (ie activity manifest only in the presence of detergent). Believing that latency may relate to the morphological state of the cellular structure to which the enzyme is bound, we have performed studies to determine the behavior of this complex catalyst in situ. To this end, intact isolated rat hepatocytes were rendered permeable to charged substrates with filipin, a polyene agent which is selective for the cell membrane. Phosphohydrolase, measured on the basis of Pi release in the presence of phosphorylative inhibitors, and phosphotransferase, assayed on the basis of D-[2-3H]glucose detritiation or accumulation of D-glucose-6-P or 3-O-methyl-D-[U“‘Cl-glucose-6-P, were studied at pH 7.4 with these preparations. Latencies of both phosphohydrolase and phosphotransferase of cells were considerably lower than those observed with microsomes isolated either from liver homogenates or from hepatocytes. For example, only 27% of glucose-6-P phosphohydrolase was found to be latent in hepatocytes compared with 45% in isolated microsomes. Corresponding values for carbamoyl-P-dependent phosphotransferase activities were 55% for hepatocytes and 80 to 85% for microsomes. Significant amounts of PPi-dependent phosphotransferase with but 18% latency also were observed at pH 7.4 with hepatocytes. Latencies calculated for endoplasmic reticulum of the hepatocytes were 22% for glucose-6-P phosphohydrolase and 60% for carbamoylP:glucose phosphotransferase. Michaelis constant values determined with hepatocyte preparations corresponded reasonably well with those determined with detergent-activated microsomes. It is concluded that, when provided with adequate levels of substrates, a significant proportion of total phosphotransferase as well as glucose-6-P phosphohydrolase activity may be manifested in the cell.