Subcellular localization and isolation of γ-glutamyltransferase from rat hepatoma cells

Abstract

Cultured rat hepatoma cells were homogenized and subjected to subcellular fractionation by analytical sucrose density centrifugation to determine the localization of γ-glutamyltransferase ((5-glutamyl)-peptide : amino-acid 5-glutamyltransferase, EC 2.3.2.2). The activity was exclusively localized to the plasma membrane. Diazotized sulphanilic acid was used as a non-penetrant membrane reagent which inactivates ectoenzymes. With both intact and sonicated cells, only 70–75% inhibition of γ-glutamyltransferase activity was observed. At least 12% of the total cell complement of γ-glutamyltransferase activity is highly resistant to inactivation by diazotized sulphanilic acid even after Triton X-100 solubilization. The enzyme was purified from hepatoma cells and its properties compared with enzyme from normal liver. Apart from the striking increase in V^app there were only minor differences between the enzymes from the two sources. In contrast to the complete abolition of transpeptidase activity of the purified hepatoma enzyme by diazotized sulphanilic acid, the hydrolytic activity of this preparation was only slightly inhibited.