Purification and biochemical characterization of gamma-glutamylcysteine synthetase from a human malignant astrocytoma cell line.
gamma-Glutamylcysteine synthetase (EC 6.3.2.2.) the key regulatory enzyme in glutathione biosynthesis was purified from a human malignant astrocytoma cell line using a combination of ammonium sulfate fractionation, DE-52 cellulose chromatography and ATP-agarose affinity chromatography. The purified protein had a specific activity of 1725 units/mg protein, which represented an 86-fold purification and a 22% yield. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major subunits with apparent molecular sizes of 72 kDa and 32 kDa. The Km values for L-glutamate and L-alpha-aminobutyrate were 0.03 mM and 0.14 mM respectively. These molecular and catalytic properties of gamma-glutamylcysteine synthetase from astrocytoma cell line are similar, but not identical to those purified from rat kidney.
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Related Subject Headings
- Neutralization Tests
- Molecular Weight
- Methionine Sulfoximine
- Immunohistochemistry
- Humans
- Glutathione
- Glutamate-Cysteine Ligase
- Electrophoresis, Polyacrylamide Gel
- Chromatography, Affinity
- Chemical Fractionation
Citation
Published In
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Neutralization Tests
- Molecular Weight
- Methionine Sulfoximine
- Immunohistochemistry
- Humans
- Glutathione
- Glutamate-Cysteine Ligase
- Electrophoresis, Polyacrylamide Gel
- Chromatography, Affinity
- Chemical Fractionation