Phosphate-dependent
glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich
ascites-tumour cells. The
enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/
polyacrylamide-gel electrophoresis displayed two
protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive
peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney
glutaminase)
antibodies. The concentration-dependences for both
glutamine and
phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The
glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The
enzyme showed a high specificity towards
glutamine. A possible explanation for the different kinetic behaviour found for purified
enzyme and for isolated mitochondria [Kovacević (1974)
Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the
enzyme is extracted from the mitochondrial inner membrane.