Existence of a low-affinity ATP-binding site in the unphosphorylated Ca2(+)-ATPase of sarcoplasmic reticulum vesicles: evidence from binding of 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-[3H]AMP and -[3H]ATP.
ATP-binding sites in the unphosphorylated Ca2(+)-ATPase of sarcoplasmic reticulum vesicles were titrated with 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-[3H]AMP (TNP-AMP) or -[3H]ATP (TNP-ATP) in the absence of Ca2+ at pH 7.0 and 0 degrees C by using a centrifugation procedure. In some measurements, the bound TNP-nucleotides were chased with ATP. The data were analyzed by best-fit computer programs as well as by Scatchard plots. The results showed the existence of 1 mol of TNP-AMP binding sites with high affinity (Kd = 7.62 nM) per mole of phosphorylatable sites. The affinity of these sites for ATP (Kd = 10.1 microM) agreed with that of catalytic sites for ATP in the absence of Ca2+. The results further showed the existence of 2 mol of TNP-ATP binding sites with uniform affinity (Kd = 156 nM) per mole of phosphorylatable sites. Half of the bound TNP-ATP was fully chased by low concentrations of ATP. The affinity of this class of the sites for ATP (Kd = 8.9 microM) again agreed with that of catalytic sites for ATP. The other half of the bound TNP-ATP was fully chased only by much higher concentrations of ATP. Thus, the affinity of this class of the sites for ATP (Kd = 791 microM) was much lower than that of catalytic sites for ATP. Similar measurements were performed with sarcoplasmic reticulum vesicles pretreated by N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. Although the affinities for TNP-ATP and for ATP were appreciably altered by this pretreatment, the results were essentially the same as those obtained with native vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Suzuki
H
H
Department of Biochemistry, Asahikawa Medical College, Japan.
Kubota
T
T
Kubo
K
K
Kanazawa
T
T
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009282:Naphthalenesulfonates / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D009282:Naphthalenesulfonates; D000249:Adenosine Monophosphate; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; C036744:2',3'-O (2,4,6-trinitrocyclohexadienylidine)adenosine 5'-monophosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases