Photoaffinity labeling of the major nucleosidetriphosphatase of rat liver nuclear envelope.
We employed the photoaffinity probe 8-azido-adenosine 5'-triphosphate (aATP) to identify the nuclear envelope (NE) nucleosidetriphosphatase activity (NTPase) implicated in control of RNA transport. The photoprobe was hydrolyzed at rates comparable to those for ATP, with a Michaelis constant 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 protein with both [gamma-32P]aATP and [alpha-32P]aATP. This band was not labeled with [gamma-32P]ATP. Since the NE NTPase implicated in RNA transport is modulated by RNA, we examined the effects of RNA on the labeling process. 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. These results suggest that the 46-kDa protein represents the major NTPase implicated in RNA transport.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D009685:Nuclear Envelope / Q000201:enzymology*; D043583:Nucleoside-Triphosphatase; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D051381:Rats; D011919:Rats, Inbred Strains; D013056:Spectrophotometry, Ultraviolet
D000345:Affinity Labels; D001386:Azides; D010761:Phosphorus Radioisotopes; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D043583:Nucleoside-Triphosphatase