Interaction of spin-labeled nucleotides with sarcoplasmic reticulum adenosinetriphosphatase.
Spin-labeled derivatives of AMP-PCP, ATP, and 2'-deoxy-ATP, with a nitroxide moiety attached to the ribose ring [3'-O-(1-oxy-2,2,5,5-tetramethylpyrroline-3-carbonyl)nucleotide], are used to study the nucleotide binding site stoichiometry of sarcoplasmic reticulum (SR) ATPase. With all derivatives, a maximal binding of 4.5 nmol/mg of SR protein is found, a value close to the number of phosphorylation sites obtained with ATP. The spin-labeled nucleotides cannot be utilized by the enzyme as substrates. Binding of spin-labeled nucleotides is inhibited by labeling the ATPase with fluorescein 5'-isothiocyanate, indicating that all the labeled nucleotide is located at the catalytic site. Additions of spin-labeled ATP to vesicle suspensions during steady turnover demonstrate competitive inhibition of both catalysis and the regulatory effect normally exhibited by ATP. As secondary binding of spin-labeled ATP is not detected at pertinent concentrations, it is suggested that both functions of ATP may be effected through a single site.
Oliveira
C R
CR
Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Brazil.
Coan
C
C
Verjovski-Almeida
S
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013113:Spin Labels
D003838:Deoxyadenine Nucleotides; D009711:Nucleotides; D013113:Spin Labels; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C026600:2'-deoxyadenosine triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate