Photoaffinity labeling of the major nucleoside triphosphatase of rat liver nuclear envelope

Abstract

The photoaffinity probe 8-azidoadenosine 5''-triphosphate (aATP) was employed to identify the nuclear envelope (NE) nucleoside triphosphatase activity (NTPase) implicated in control of RNA transport. The photoprobe was hydrolyzed at rates comparable to those for ATP, with a Km of 0.225 mM. Photolabeling was dependent upon UV irradiation (300-nm max) and was not affected by quercetin. Unlabeled ATP or GTP competed with [32P]aATP in photolabeling experiments, and UTP was a less effective competitor, paralleling the substrate specificity of the NTPase. Incubation of NE with aATP led to a UV, time and concentration dependent irreversible inactivation of NTPase. The inactivation could be blocked by ATP or GTP. Polyacrylamide gel electrophoresis and autoradiography of photolabeled NE showed selective, UV-dependent labeling of a 46-kDa [kilodalton] protein with both [.gamma.-32P]aATP and [.gamma.-32P]aATP. This band was not labeled with [.gamma.-32P]ATP. Since the NE NTPase implicated in RNA transport is modulated by RNA, the effects of RNA on the labeling process were examined. Removal of RNA from the NE preparations (by RNase/DNase digestion) reduced NTPase by 30-40% and eliminated photolabeling of the 46-kDa band. Addition of yeast RNA to such preparations increased NTPase activity to control levels and selectively reinstated photolabeling of the 46-kDa band. The 46-kDa protein may represent the major NTPase implicated in RNA transport.