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 transporter s. 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 proteoliposome s. In the presence of symmetrical solutions of urate and KCI, but abse nce of uricase, no current was generated when the voltage was ramped b etween +/-100 mV. Following fusion of uricase with the bilayer, single channel activity was evident: the reconstituted channel rectified wit h a mean slope conductance of 8 pS, displayed voltage sensitivity, and demonstrated a marked selectivity for urate relative to K+ and Cl-. T he channel was more selective to oxonate, an inhibitor of bath enzymat ic 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 de pendent organic anion channel is formed when a purified preparation of uricase is reconstituted in lipid bilayers.