Purification of phosphate-dependent glutaminase from isolated mitochondria of Ehrlich ascites-tumour cells
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A R Quesada1, F Sanchez-Jimenez, J Perez-Rodriguez
1Department of Biochemistry and Molecular Biology, Facultad de Ciencias, Universidad de Málaga, Spain.
Abstract:
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.
Insights
Phosphate-dependent glutaminase was purified from tumor cell mitochondria. This enzyme exhibits sigmoidal kinetics and a pH optimum of 9, suggesting conformational changes upon extraction.
Area of Science:
- Biochemistry
- Enzymology
- Cancer Research
Background:
- Mitochondria play a crucial role in cellular metabolism.
- Phosphate-dependent glutaminase is an enzyme involved in amino acid metabolism.
- Understanding enzyme kinetics is vital for cancer research.
Purpose of the Study:
- To purify and characterize phosphate-dependent glutaminase from Ehrlich ascites-tumour cell mitochondria.
- To investigate the kinetic properties and subunit composition of the purified enzyme.
- To explore potential reasons for differing kinetic behavior between purified enzyme and mitochondrial preparations.
Main Methods:
- Enzyme purification to homogeneity from isolated mitochondria.
- Size exclusion chromatography (Sephacryl S-300) for molecular weight determination.
- SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) for subunit analysis.
- Immunoblot analysis using anti-(rat kidney glutaminase) antibodies.
- Kinetic studies to determine substrate concentration dependence (glutamine, phosphate), pH optimum, and activation energy.
Main Results:
- Purified phosphate-dependent glutaminase with an Mr of 135,000.
- SDS-PAGE revealed two subunits with Mr 64,000 and 56,000.
- Immunoblot analysis identified immunoreactive peptides of Mr 65,000 and 57,000.
- Sigmoidal kinetics observed for both glutamine and phosphate dependence (S0.5 values: 7.6 mM and 48 mM; Hill coefficients: 1.5 and 1.6).
- Enzyme exhibited a pH optimum of 9 and an activation energy of 58 kJ/mol.
- High specificity towards glutamine was noted.
Conclusions:
- The purified enzyme consists of two distinct subunits.
- The sigmoidal kinetics suggest cooperative substrate binding.
- A conformational change likely occurs during enzyme extraction from the mitochondrial membrane, explaining kinetic differences.
- This characterization provides insights into glutaminase function in tumor cell metabolism.