PURIFICATION AND SOME PROPERTIES OF GLUTAMINE-SYNTHETASE FROM HUMAN BRAIN

An effective new procedure for purifying glutamine synthetase (GS, EC 6.3. 1.2) from human brain has been developed. The procedure includes homogenization of brain tissue, centrifugation, precipitation with (NH 4)(2)SO4, ion exchange chromatography on celluloses DE-32 and CM-32, g el filtration on Sepharose CL-6B, and chromatography on AH-Sepharose 4 B. On SDS-PAGE, the purified GS gives a single protein band with a mol ecular mass of about 44 kD. Two-dimensional electrophoresis of the GS performed according to O'Farrell gives a series of protein spots (all spots corresponding to molecular mass 44 kD, with pI values ranging fr om 6.7 to 7.2), presumably the result of covalent modification of GS s ubunits. The apparent K-m value for L-glutamate (L-Glu) in the synthet ase reaction is 14.3 mM; the K-m value for L-glutamine (L-Gln) in the transferase reaction is 18.1 mM. The pH values of 6-7 and 7.2 were fou nd to be optimal for the GS transferase and synthetase activities in i midazole buffer. The optimal concentrations of Mg2+ for synthetase and transferase activities of the GS are 10 and 15-20 mM, respectively; t hat of Mn2+ was 1 mM for the both reactions. Some biochemical properti es (isoelectric point, subunit molecular masses, optimal concentration s of metal ions) differ significantly from those the GSs from animals. Rabbit polyclonal antibodies raised against human brain GS detect GS in human brain homogenates, but not in rat or bovine brain homogenates .