Development of an immobilized brain glutamine synthetase liquid chromatographic stationary phase for on-line biochemical studies.
Glutamine synthetase (GS) plays a key role in the regulation of glutamate availability to neurons. In the present study glutamine synthetase was immobilized on a silica-based immobilized artificial membrane liquid chromatographic stationary phase (IAM-SP) to create the GS-IAM. The stability of GS was improved by immobilization, but the enzyme's affinity for the substrates L-glutamate and D-glutamate was significantly decreased. In contrast, immobilization significantly increased GS sensitivity to inhibition by methionine sulfoximine. The GS-IAM was packed into a chromatography column to create an immobilized enzyme reactor (GS-IMER). On-line experiments with the GS-IMER demonstrated that the immobilized enzyme was comparable to the non-immobilized enzyme with regards to retention of activity and selectivity toward substrates and inhibitors and was reusable for several weeks.
Cloix
J F
JF
Department of Pharmacology, Georgetown University School of Medicine, Washington, DC 20007, USA.
Wainer
I W
IW
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005974:Glutamate-Ammonia Ligase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D012756:Sheep; D013379:Substrate Specificity
D018698:Glutamic Acid; D005974:Glutamate-Ammonia Ligase