[Isolation and certain properties of human brain glutamine synthetase].
A new effective procedure for purifying of glutamine synthetase (GS, EC 6.3.1.2) from human brain has been developed. The procedure includes homogenization of brain tissue, centrifugation, precipitation with (NH4)2SO4, ion-exchange chromatography on cellulose DE-32 and CM-32, gel-filtration on Sepharose CL-6B and chromatography on AH-Sepharose 4B. On SDS-PAAG electrophoresis purified GS gives a single protein band with a molecular mass of about 44 kDa. Two-dimensional electrophoresis of GS performed according to O'Farrell gives a protein spots multiplet (all the spots corresponding to M(r) approximately 44 kDa and pI 6.7-7.2), presumably as a result of covalent modification of GS subunits. The apparent Km value for L-Glu in the synthetase reaction is 14.3 mM; the Km value for L-Gln in the transferase reaction is 18.1 mM. The pH values of 6-7 and 7.2 were found to be optimal for the transferase and synthase GS activities in the imidazole buffer. The optimal concentrations of Mg2+ ions for the synthetase and transferase activities of GS were 10 and 15-20 mM, respectively; that of Mn2+ ions was 1 mM for the both reactions. Some biochemical properties (isoelectric point, subunit molecular masses, optimal concentrations of metal ions) differ essentially from those of animal GS. Rabbit polyclonal antibodies raised against the human brain GS detect GS in human brain homogenates, but not in rat or bovine brain homogenates.
Boksha
I S
IS
Tereshkina
E B
EB
Burbaeva
G Sh
GSh
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005974:Glutamate-Ammonia Ligase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D051381:Rats
D008345:Manganese; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium
Vydelenie i nekotorye svoĭstva glutaminsintetazy iz mozga cheloveka.