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ATPase activities of rabbit and bovine lens epithelial microsomes: a continuous fluorimetric assay study
J Zeng1, D Borchman, C A Paterson
1Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Research Institute, University of Louisville School of Medicine 40292, USA.
Abstract:
Purine nucleoside phosphorylase (EC 2.4.2.1) catalyzes the irreversible phosphorolysis of 7-methylguanosine (m7Guo), a fluorescent guanosine analogue. Using purine nucleoside phosphorylase and m7Guo, a continuous fluorimetric assay for microsomal ATPases from rabbit and bovine lens is described. The decrease in m7Guo fluorescence intensity at 380 nm, which represents the hydrolysis of ATP, is linear with time up to exhausting all m7Guo. The rate of the fluorescence decrease depends on the sample protein concentration. In the presence of ATPase inhibitors, ion-specific ATPase activities in the lens were determined from the difference of the fluorescence decay rates. Using the fluorimetric assay thapsigargin-sensitive Ca-ATPase in the bovine lens epithelium has been characterized. The fluorimetric assay provides a number of advantages over previous membrane ATPase assays.