SUBMITOCHONDRIAL LOCALIZATION AND MEMBRANE TOPOGRAPHY OF EHRLICH ASCITIC TUMOR-CELL GLUTAMINASE

The intramitocondrial localization of the phosphate-activated glutamin ase from Ehrlich cells has been examined by a combination of technique s, including: mitochondria subfractionation studies, chemical modifica tion with sulfhydryl group reagents of different permeability, enzymat ic digestion in both sides of the inner mitochondrial membrane, and im munological studies. Using alkaline extraction at high ionic strength, hypoosmotic shock and freezing-thawing cycle techniques, the enzyme w as found in the particulate fraction. On the contrary, glutaminase act ivity was labile when subfractionation was carried out by digitonin/lu brol method; Western blot analysis localized the inactive enzyme in th e matrix fraction. In addition, glutaminase was fully inactivated when mitoplasts were incubated with phospholipase A(2) and phospholipase C . The enzyme also showed a non-linear Arrhenius plot with a break at 2 4 degrees C. The membrane-impermeant thiol reagents mersalyl and p-chl oromercuriphenylsulfonic acid do not inhibit glutaminase activity in f reeze-thawed mitochondria and mitoplasts, but N-ethylmaleimide, which is membrane permeant, strongly inhibited the enzyme. However, mersalyl and p-chloromercuriphenylsulfonic acid were effective inhibitors when the alkylation was performed on the matrix side of mitoplasts or usin g detergent-solubilized enzyme. Furthermore, trypsin digestion of mito plasts was only effective inactivating glutaminase when the proteolysi s was carried out on the matrix side of the vesicles. Enzyme-linked im munosorbent assay of the soluble and membrane fractions obtained in th e preparation of submitochondrial particles, revealed that most of the enzyme was solubilized, but in the inactive form. Phase separation wi th Triton X-114 rendered most of the protein in the aqueous phase. The se results taken together discard a transmembrane localization for the protein, whereas they are consistent with anchorage of glutaminase on the matrix side of the inner mitochondrial membrane, the matrix porti on of the enzyme being relevant for its function.