Optimization and application of glutamate cysteine ligase measurement in wildlife species.

ABSTRACT

Glutamate cysteine ligase (GCL), synthesizing gamma-glutamylcysteine from glutamate and cysteine, is the rate-limiting enzyme in glutathione (GSH) biosynthesis. GCL activity measurement was optimized in tissues from deer mice, Sprague Dawley rats, and mallard ducks. Varying glutamic acid concentrations from 5 to 80 mM did not affect GCL activities markedly, whereas cysteine concentrations from 2.5 to 40 mM influenced GCL activities substantially. Optimal cysteine concentrations for deer mouse, Sprague Dawley rat, and mallard duck (respectively) were 30, 30, and 20 mM in liver, 10, 10, and 20 mM in kidney, 20, 20, and 30 mM in brain, and 30 mM in heart for all three species. Responses of mallard duck GCL activity to acid metalliferous water were evaluated. After subacute exposure, low doses increased GCL activity and GSH content in liver by 48.3% and 54.4%, respectively. High doses reduced GCL activities significantly in liver and kidney to 31.2% and 43.0% of the control, respectively.