67] Assays and characterization of Mg2+-ATPase in the rod outer segment of vertebrate photoreceptors

Abstract

This chapter discusses the assays and characterization of Mg 2+ - adenosine triphosphatase (ATPase) in the rod outer segment (ROS) of vertebrate photoreceptors. It reports the existence of a specific ATPase system that undoubtedly resides in the photoreceptor outer segment and whose presence can be assayed by three distinct techniques: (1) conventional assay of ATPase activity measuring the amount of inorganic phosphate liberated, (2) light-scattering: “A D .” A large decrease in turbidity occurs when rod outer segments (ROS) are incubated in the presence of Mg 2+ and ATP in the dark, and (3) light-scattering: “A L .” A rapid further decrease in turditity occurs on flash illumination when ROS had been preincubated in the presence of Mg 2+ and ATP. ATP hydrolysis by ROS membranes was measured as release of inorganic phosphate, employing the method of Taussky and Schorr. When ROS fragments are incubated with both Mg 2+ and ATP in the dark, their turbidity is rapidly reduced.