Extraction and Analysis of Phosphate Activated Glutaminase From Isolated Mitochondria of Sarcoma- 180 Tumour Cells
Sunit Kumar Chakraborty
Department of Zoology, Raja Rammohun Roy Mahavidyalaya, Radhanagar, Nangulpara, Hooghly- 712406 (India) E-mail: sunitc2001@gmail.com
ABSTRACT
Phosphate-dependent glutaminase purified to homogeneity from isolated mitochondria of Sarcoma 180 tumour cells. The enzyme was purified 113 fold over the original cell free extract with a specific activity of 125µM/mg protein. The enzyme had an Mr of 64 KD as judged by chromatography on DEAE Sepharose ion exchange and Sephacryl S-300 column cgromatography. Two major immunoreactive peptides of Mr values of 64000 and 56000 were found by immunoblot analysis using anti-( rat kidney glutaminase) antibodies. The activity of the purified tumour glutaminase was maximal at pH 8.0. The concentration dependence for both phosphate and glutamine were sigmoid when assayed at pH 8.0. Glutaminase enzyme from S-180 tumour gives its maximum activity at 100mM potassium phosphate buffer which corresponds with the phosphate dependence of glutaminase purified from EAC tumour cells. Glutamine concentration optima of the purified glutaminase is 0.2mM. The Km value of S-180 cell glutaminase enzyme is found to be low i.e. 0.83 than rat liver glutaminase (17mM).