Stoichiometry of Phosphorylation to Fluorescein 5-Isothiocyanate Binding in the Ca2+-ATPase of Sarcoplasmic Reticulum Vesicles

Satoshi Nakamura,Hiroshi Suzuki,Tohru Kanazawa

doi:10.1074/jbc.272.10.6232

Satoshi Nakamura, Hiroshi Suzuki + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.10.6232

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Abstract

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.

Highlights

Mediate [4, 5]
In the subsequent study [10], we have determined the total content of the sulfhydryl group of the Ca2ϩ-ATPase in the sarcoplasmic reticulum (SR) vesicles after exhaustive reduction with dithiothreitol and found that the content of the phosphorylation site in the SR vesicles is again half that of the Ca2ϩ-ATPase molecule calculated from this sulfhydryl content
These findings lead to the conclusion that only half of the Ca2ϩ-ATPase molecules present in the SR vesicles can be phosphorylated

Summary

EXPERIMENTAL PROCEDURES

Preparation of SR Vesicles—SR vesicles were prepared from rabbit skeletal muscle as described previously [8] and stored in 0.3 M sucrose, 0.1 mM CaCl2, 0.1 M KCl, and 5 mM MOPS/Tris (pH 7.0) at Ϫ80 °C. The SR vesicles (0.5 mg of protein/ml) were labeled with 15 ␮M FITC at 25 °C in the dark in 2 mM EGTA, 5 mM MgCl2, 0.1 M KCl, 1% (v/v) N,N-dimethylformamide, and 50 mM Tris/ HCl (pH 8.0), unless otherwise stated. Method A: the labeling was stopped by adding 4.5 volumes of an ice-cold solution containing 12.2 mM ATP, 2 mM EGTA, 5 mM MgCl2, 0.1 M KCl, and 122 mM MOPS/Tris (pH 7.0). Tryptic Digestion of the FITC-labeled Ca2ϩ-ATPase and Reversed Phase HPLC—The fraction at the peak of protein concentration in the size exclusion HPLC was applied twice to centrifuge columns (5-ml disposable syringe filled with coarse Sephadex G-50) preequilibrated with 1 mM CaCl2 and 10 mM sodium phosphate (pH 7.0) at 4 °C according to Penefsky [17] in order to remove excess dodecyl sulfate. Protein concentrations were determined by the method of Lowry et al [20] with bovine serum albumin as a standard

RESULTS

From ATP

DISCUSSION