Stoichiometry of phosphorylation to fluorescein 5-isothiocyanate binding in the Ca2+-ATPase of sarcoplasmic reticulum vesicles.
In an attempt to establish the stoichiometry of phosphorylation in the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles, phosphorylation by ATP (or Pi) or labeling by fluorescein 5-isothiocyanate (FITC) was performed with the SR vesicles under the conditions in which almost all the phosphorylation sites or FITC binding sites are phosphorylated or labeled. The resulting vesicles were solubilized in lithium dodecyl sulfate and then the Ca2+-ATPase was purified by size exclusion high performance liquid chromatography. Peptide mapping and sequencing of the tryptic digest of the purified enzyme showed that Lys-515 of the Ca2+-ATPase was exclusively labeled with FITC, in agreement with the previously reported findings. The content of the phosphoenzyme from ATP (4.57 nmol/mg of Ca2+-ATPase protein) or from Pi (4.94 nmol/mg of Ca2+-ATPase protein) in the purified enzyme was approximately half the content of the FITC binding site (8.17-8.25 nmol/mg of Ca2+-ATPase protein) and also half the content of the Ca2+-ATPase molecule (9.06 nmol/mg of Ca2+-ATPase protein) calculated from its molecular mass (110,331 Da). These results show that there is one specific FITC binding site per molecule of the Ca2+-ATPase (in agreement with the previously reported findings) and that the stoichiometry of phosphorylation to FITC binding is approximately 0. 5:1.0. All the above findings lead to the conclusion that only half of the Ca2+-ATPase molecules present in the SR vesicles can be phosphorylated. FITC binding completely inhibited the ATP-induced phosphorylation before the binding reached its maximum level. This finding indicates that FITC preferentially binds to a part of the Ca2+-ATPase molecules and that this binding is primarily responsible for the inhibition of phosphorylation, suggesting an intermolecular ATPase-ATPase interaction.
Nakamura
S
S
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan.
Suzuki
H
H
Kanazawa
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology; D010449:Peptide Mapping; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate