2',3'-Dialdehyde ATP analog labels the Ca(2+)-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 Ca(2+)-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 Ca(2+)-ATPase, as indicated by Pi formation (hydrolysis) and Ca(2+)-uptake ability. oATP binding to the intact light sarcoplasmic reticulum is observed in the absence and presence of the competitive adenosine nucleotide inhibitor, fluorescein isothiocyanate with apparent affinity constants of 1.2 mM and 2.2 mM, respectively. Autoradiography of tryptic fragments from partially purified Ca(2+)-ATPase labeled with [alpha-32P]oATP or [gamma-32P]oATP locates the covalent binding site to the A1 fragment, even in the fluorescein-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.
Hohenegger
M
M
Pharmacological Institute, Vienna, Austria.
Makinose
M
M
D000241:Adenosine / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002384:Catalysis; D002413:Cations, Divalent; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D009711:Nucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin
D000345:Affinity Labels; D002413:Cations, Divalent; D009711:Nucleotides; C017199:2',3'-dialdehyde ATP; D000255:Adenosine Triphosphate; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D000241:Adenosine; D002118:Calcium