Reconstitution of hepatic uricase in planar lipid bilayer reveals a functional organic anion channel.
Rat renal proximal tubule cell membranes have been reported to contain uricase-like proteins that function as electrogenic urate transporters. Although uricase, per se, has only been detected within peroxisomes in rat liver (where it functions as an oxidative enzyme) this protein has been shown to function as a urate transport protein when inserted into liposomes. Since both the uricase-like renal protein and hepatic uricase can transport urate, reconstitution studies were performed to further characterize the mechanism by which uricase may function as a transport protein. Ion channel activity was evaluated in planar lipid bilayers before and after fusion of uricase-containing proteoliposomes. In the presence of symmetrical solutions of urate and KCl, but absence of uricase, no current was generated when the voltage was ramped between +/- 100 mV. Following fusion of uricase with the bilayer, single channel activity was evident: the reconstituted channel rectified with a mean slope conductance of 8 pS, displayed voltage sensitivity, and demonstrated a marked selectivity for urate relative to K+ and Cl-. The channel was more selective to oxonate, an inhibitor of both enzymatic uricase activity and urate transport, than urate and it was equally selective to urate and pyrazinoate, an inhibitor of urate transport. With time, pyrazinoate blocked both its own movement and the movement of urate through the channel. Channel activity was also blocked by the IgG fraction of a polyclonal antibody to affinity purified pig liver uricase. These studies demonstrate that a highly selective, voltage dependent organic anion channel is formed when a purified preparation of uricase is reconstituted in lipid bilayers.
D000818:Animals; D000838:Anions; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D008051:Lipid Bilayers; D008099:Liver / Q000201:enzymology*; D010094:Oxonic Acid / Q000494:pharmacology; D011718:Pyrazinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D013552:Swine; D014503:Urate Oxidase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*
D000838:Anions; D007473:Ion Channels; D008051:Lipid Bilayers; D011718:Pyrazinamide; C005296:pyrazinoic acid; D010094:Oxonic Acid; D014503:Urate Oxidase