Purification of phosphate-dependent glutaminase from isolated mitochondria of Ehrlich ascites-tumour cells.
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.
Quesada
A R
AR
Department of Biochemistry and Molecular Biology, Facultad de Ciencias, Universidad de Málaga, Spain.
Sanchez-Jimenez
F
F
Perez-Rodriguez
J
J
Marquez
J
J
Medina
M A
MA
Nuñez de Castro
I
I
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D005972:Glutaminase / Q000302:isolation & purification*; D005973:Glutamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007680:Kidney Neoplasms / Q000201:enzymology; D007700:Kinetics; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D010710:Phosphates / Q000494:pharmacology; D051381:Rats
D010710:Phosphates; D005973:Glutamine; D005972:Glutaminase