Glutaraldehyde cross-links Lys-492 and Arg-678 at the active site of sarcoplasmic reticulum Ca(2+)-ATPase.
It has been shown previously that glutaraldehyde cross-links the Ca(2+)-ATPase of sarcoplasmic reticulum intramolecularly at the active site, involving residues participating in nucleotide binding and the conformational change that results in Ca2+ release to the vesicle lumen and formation of ADP-insensitive E2-P (Ross, D. C., Davidson, G. A., and McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621). This study shows that 10 nmol of [14C]glutaraldehyde/mg of protein attached irreversibly to the ATPase under conditions optimal for formation of the intramolecular cross-link. Half of this amount (i.e. 1 mol/mol ATPase) was inhibited by nucleotide binding. Thermolysin digestion of derivatized vesicles released two nucleotide-sensitive 14C-labeled species, which were isolated and identified as FSRDR*S AND FSRDR*S FA* FA*VEPS where the missing residues are Lys-492 and Arg-678. The majority of the 14C label was released in the sixth cycle of both Edman degradations, confirming the cross-link position. Lys-492 and Arg-678 are evidently close together in the active site, but their distance apart in the linear sequence suggests that they may arise from separate domains, which together constitute an ATP binding cleft. Residues in both regions, and Lys-492 in particular (McIntosh, D.B., Woolley, D.G., and Berman, M.C. (1992) J. Biol. Chem. 267, 5301-5309), have been derivatized by nucleotide-based affinity probes. Mutations of both of these residues in some of the bacterial P-type ATPases suggest that they do not play an essential catalytic role, and the inability of the cross-linked ATPase to form E2-P and to release Ca2+ to the lumen is probably because an essential tertiary structural movement at the active site is blocked.
McIntosh
D B
DB
Department of Chemical Pathology, University of Cape Town Medical School, Observatory, South Africa.
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents; D005976:Glutaral / Q000737:chemistry*; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D016415:Sequence Alignment; D013820:Thermolysin / Q000494:pharmacology
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D001120:Arginine; D013820:Thermolysin; D000252:Calcium-Transporting ATPases; D008239:Lysine; D005976:Glutaral