ATPASE ACTIVITIES OF RABBIT AND BOVINE LENS EPITHELIAL MICROSOMES - ACONTINUOUS FLUOROMETRIC ASSAY STUDY

Purine nucleoside phosphorylase (EC 2.4.2.1) catalyzes the irreversibl e phosphorolysis of 7-methylguanosine (m(7)Guo), a fluorescent guanosi ne analogue. Using purine nucleoside phosphorylase and m(7)Guo, a cont inuous fluorimetric assay for microsomal ATPases from rabbit and bovin e lens is described. The decrease in m(7)Guo fluorescence intensity at 380 nm, which represents the hydrolysis of ATP, is linear with time u p to exhausting all m(7)Guo. The rate of the fluorescence decrease dep ends on the sample protein concentration. In the presence of ATPase in hibitors, ion-specific ATPase activities in the lens were determined f rom the difference of the fluorescence decay rates. Using the fluorime tric assay thapsigargin-sensitive Ca-ATPase in the bovine lens epithel ium has been characterized. The fluorimetric assay provides a number o f advantages over previous membrane ATPase assays.