2',3'-DIALDEHYDE ATP ANALOG LABELS THE CA2+-ATPASE OF SARCOPLASMIC-RETICULUM VIA THE CATALYTIC ADENOSINE-NUCLEOTIDE-BINDING SITE

The 2',3'-dialdehyde ATP analog (oATP) was synthesized and its ability to activate the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum via the adenosine-nucleotide-binding site was investigated. After reduction by sodium borohydride, oATP binds covalently to the catalytic adenosine-nucleotide-binding site of the enzyme, resulting in 85% loss of acetyl-phosphate-driven Ca2+ uptake and ATP-hydrolysing ability. In the absence of a reducing agent, oATP serves as a substrate for the Ca2+-ATPase, as indicated by P(i) formation (hydrolysis) and Ca2+-uptake ability. oATP binding to the intact light sarcoplasmic reticulum is observed in the absence and presence of the competitive adenosine nucleotide inhibitor, fluorescine isothiocyanate with apparent affinity constants of 1.2 mM and 2.2 mM, respectively. Autoradiography of tryptic fragments from partially purified Ca2+-ATPase labeled with [alpha-P-32]oATP or [gamma-P-32]oATP locates the covalent binding site to the A1 fragment, even in the fluorescine-isothiocyanate-labeled pump protein. With high probability, a lysine residue in the tryptic A1 fragment is labeled by the ribose-modified ATP analog close to the phosphorylation site at Asp351.