Phosphate dependent
glutaminase was purified from
ascites fluid of
ovarian cancer patients. The purified
enzyme showed a final specific activity of 110 unit / mg
protein with 72 fold purification and 21% yield. Purified
enzyme gives one dark band of Mr approximately 65.5 KD and two light bands of Mr approximately 47.5 KD and approximately 45 KD respectively on 10% SDS-PAGE. One major immunoreactive band was found in trans-immunoblot analysis using
antibodies against rat kidney and
ascites fluid
glutaminase raised in rabbit and mice respectively.
Phosphate dependent
glutaminase enzyme purified from mitochondria of malignant and non malignant ovarian tissue also showed bands of same molecular weight on 10% SDS-PAGE and gave same immunoreactive bands in trans-immunoblot like the purified
glutaminase from
ascites fluid. This result was confirmed by using the specific activity
stain for
glutaminase, which indicates that same
enzyme activity is probably due to leakage of the same
enzyme from malignant tissue into the
ascites fluid. The purified
enzyme from human peritoneal fluid showed a high specificity toward
glutamine, therefore is a true
glutaminase. Moreover,
ascites fluid taken from patients of different age group with different stages of ovarian
carcinoma revealed the presence of same
glutaminase on 10% SDS-PAGE, and exhibited immunoreaction on ELISA, trans-immunoblot and dot immunoblot analysis.