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.
Sriram
R
R
Department of Experimental Pediatrics, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Ali-Osman
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F
D000645:Ammonium Sulfate; D000906:Antibodies / Q000276:immunology; D001254:Astrocytoma / Q000201:enzymology*; D015153:Blotting, Western; D019328:Buthionine Sulfoximine; D002460:Cell Line; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D008970:Molecular Weight; D009500:Neutralization Tests
D000906:Antibodies; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D000645:Ammonium Sulfate