Nucleotide-binding sites in the functional unit of sarcoplasmic reticulum Ca2+-ATPase as studied by photoaffinity spin-labeled 2-N3-SL-ATP.
2-N3-SL-ATP [2-azido-2',3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl-pyrroline) adenosine triphosphate], a photoaffinity spin-labeled derivative of ATP with a nitroxide moiety attached to the ribose ring and an azido group attached to C2 of the adenine ring, was used to study the nucleotide-binding site stoichiometry of sarcoplasmic reticulum (SR) Ca2+-ATPase. The label was shown to bind at the catalytic site of the enzyme, even though the rate of hydrolysis was poor. A maximal binding ratio of 1 mol/mol of ATPase was found. The ESR spectra showed signals from spin-spin interactions between two radicals corresponding to a distance of about 15 A between labels bound to adjacent sites on the enzyme. This indicates that the minimal functional unit of the Ca2+-ATPase is a dimer with the nucleotide-binding sites in close proximity.
Palm
T
T
Fachbereich Chemie, Universität Kaiserslautern, Germany.
Coan
C
C
Trommer
W E
WE
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000737:chemistry*; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D004578:Electron Spin Resonance Spectroscopy; D008872:Microwaves; D009711:Nucleotides / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013113:Spin Labels
D000345:Affinity Labels; D001386:Azides; D009711:Nucleotides; D013113:Spin Labels; C075134:2-azido-2'-3'-(2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester)ATP; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases