In situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels.
Ornithine transcarbamylase deficiency is a human genetic disease potentially susceptible to gene therapy. A murine model system exists for the disease in the sparse-fur (spf) mouse. Before gene therapy studies can be performed it is necessary to have practical methods which could detect successful gene transfer. Therefore we have developed an in situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels. Following electrophoretic separation under nondenaturing conditions inorganic phosphate cleaved from carbamyl phosphate in gels as a result of enzymatic activity was precipitated as phosphomolybdic acid and visualized by reduction with ascorbic acid. Results from the procedure correlated with ornithine transcarbamylase activity as measured by solution assay for citrulline, the other product of the reaction. This procedure readily distinguished mutant forms of ornithine transcarbamylase as exemplified by the murine spf mutation and resolved ornithine transcarbamylases of all animals tested into multiple forms. The procedure further distinguished ornithine transcarbamylases of animals of several different genera while yielding virtually identical patterns of the enzyme from species within the same genus. This procedure also suggested that the human enzyme was more labile than murine ornithine transcarbamylase; direct thermolability studies confirmed this finding.
D000818:Animals; D001205:Ascorbic Acid; D011232:Chemical Precipitation; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008930:Mitochondria, Liver / Q000201:enzymology; D008982:Molybdenum; D009154:Mutation; D009954:Ornithine Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D010710:Phosphates; D010756:Phosphoric Acids; D011817:Rabbits; D051381:Rats; D013045:Species Specificity; D013194:Staining and Labeling / Q000379:methods*
D007527:Isoenzymes; D010710:Phosphates; D010756:Phosphoric Acids; D008982:Molybdenum; D009954:Ornithine Carbamoyltransferase; D001205:Ascorbic Acid; C003125:phosphomolybdic acid