Subcellular localization and isolation of gamma-glutamyltransferase from rat hepatoma cells.
Cultured rat hepatoma cells were homogenized and subjected to subcellular fractionation by analytical sucrose density centrifugation to determine the localization of gamma-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 gamma-glutamyltransferase activity was observed. At least 12% of the total cell complement of gamma-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 Vapp 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.
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D004072:Digitonin / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008297:Male; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013425:Sulfanilic Acids / Q000494:pharmacology; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification / Q000378:metabolism*
D013425:Sulfanilic Acids; D005723:gamma-Glutamyltransferase; D004072:Digitonin