Identification of tryptic cleavage sites for two conformational states of the Neurospora plasma membrane H+-ATPase.
Previous work has shown that the tryptic degradation pattern of the Neurospora plasma membrane H+-ATPase varies with the presence and absence of ligands, thus providing information about conformational states of the enzyme (Addison, R., and Scarborough, G. A. (1982) J. Biol. Chem. 257, 10421-10426; Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 8827-8832). In the present study, sites of tryptic cleavage have been mapped by immunoblotting with N- and C-terminal specific antibodies and by direct sequencing of proteolytic products after electro-transfer to polyvinylidene difluoride filters. In the absence of ligands (likely to represent the E1 conformation), trypsin cleaved the 100-kDa ATPase polypeptide at three sites very near the N terminus: Lys-24, Lys-36, and Arg-73. Removal of the first 36 amino acid residues only slightly affected ATPase activity, but removal of the subsequent 37 residues inactivated the enzyme completely. In the presence of vanadate and Mg2+ (E2 conformation), the rate of trypsinolysis at Arg-73 was greatly reduced, and enzyme activity was protected. In addition, a new cleavage site near the C terminus (Arg-900) became accessible to trypsin. Both effects of vanadate occurred at micromolar concentrations, well within the range previously measured for vanadate inhibition of ATPase activity. Taken together, these results suggest that the Neurospora ATPase undergoes significant conformational changes at both termini of the polypeptide during its reaction cycle.
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D010446:Peptide Fragments; D014638:Vanadates; D014357:Trypsin; D006180:Proton-Translocating ATPases