STRUCTURAL INTEGRITY OF THE MEMBRANE DOMAINS IN EXTENSIVELY TRYPSINIZED NA,K-ATPASE FROM SHARK RECTAL GLANDS

Removal of extramembranous portions of the integral membrane protein N a,K-ATPase from shark salt glands by trypsin in the presence of Rb+ (a K+ congener) preserves the intramembranous association of the remaini ng membrane-spanning tryptic peptides. This is evidenced from comparis on of the rotational mobility of native and trypsinized Na,K-ATPase us ing saturation transfer electron spin resonance spectroscopy (ESR) and from study of the lipid-protein interactions using conventional ESR s pectroscopy. The interface between the lipids and the intramembranous domains is conserved on removal of the extramembranous parts of the pr otein, since the population of motionally restricted boundary lipids r emains essentially the same in the native and trypsinized preparations . The ability to occlude Rb+ is also retained by the trypsinized membr anes, as previously observed with pig kidney Na,K-ATPase. A 19-kDa fra gment remaining when Na,K-ATPase is trypsinized in the presence of Rb is degraded further when the trypsinization is carried out in the pre sence of Na+ instead of Rb+. The rotational mobility of the tryptic fr agments in the Na+ trypsinized membranes is lower than for the Rb+-try psinized membranes, indicating rearrangement of the peptides. In addit ion, occlusion capacity is lost when trypsinization is carried out in Na+, suggesting a correlation between structure and function in the tr ypsinized membranes. The sequences of four membrane-spanning tryptic f ragments of shark Na,K-ATPase are found to be almost identical to corr esponding sequences in pig kidney Na,K-ATPase.