Identification of tryptic cleavage sites for two conformational states of the Neurospora plasma membrane H+-ATPase.

摘要: Previous work has shown that the tryptic degradation pattern of Neurospora plasma membrane H+-ATPase varies with presence and absence ligands, thus providing information about conformational states enzyme (Addison, R., Scarborough, G. A. (1982) J. Biol. Chem. 257, 10421-10426; Brooker, R. J., Slayman, C. W. (1983) 258, 8827-8832). In present study, sites cleavage have been mapped by immunoblotting N- C-terminal specific antibodies direct sequencing proteolytic products after electro-transfer to polyvinylidene difluoride filters. ligands (likely represent E1 conformation), trypsin cleaved 100-kDa ATPase polypeptide at three very near N terminus: Lys-24, Lys-36, Arg-73. Removal first 36 amino acid residues only slightly affected activity, but removal subsequent 37 inactivated completely. vanadate Mg2+ (E2 rate trypsinolysis Arg-73 was greatly reduced, activity protected. addition, a new site C terminus (Arg-900) became accessible trypsin. Both effects occurred micromolar concentrations, well within range previously measured for inhibition activity. Taken together, these results suggest undergoes significant changes both termini during its reaction cycle.