Identification of a cross-linked double-peptide from the catalytic site of the Ca2+-ATPase of sarcoplasmic reticulum formed by the Ca2+- and pH-dependent reaction with ATP γP-imidazolidate

• Published in: FEBS letters 1993-06, Vol.324 (3), p.314-318
• Main Authors: Gutowski-Eckel, Zeynep, Mann, Karlheinz, Bäumert, Hans G.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; AP3PL, adenosine 5'-triphosphopyridoxal; ATP γP-imidazolidate; Biological and medical sciences; Ca2+-ATPase; Catalytic site; Double-peptide; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Intramolecular cross-linking; Sarcoplasmic reticulum; SR, sarcoplasmic reticulum; TNP-8-N3,-ATP, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(93)80142-H

The Ca2+-ATPase from sarcoplasmic reticulum can be inhibited by the Ca2+- and pH-dependent reaction with ATP γP-imidazolidate. The chemically and monofunctionally activated inhibitor introduces an intramolecular cross-link between two neighbouring peptides of the active site. This can be followed by the reduced mobility of the ATPase upon SDS-PAGE analysis which becomes even more pronounced after limited trypsinolysis. After cleavage of the cross-linked ATPase molecule by cyanogen bromide and separation of the peptides a double-peptide can be detected which upon sequencing can be identified as part of the phosphorylation and the nucleotide binding site, respectively.