Abstract
Sarcoplasmic reticulum vesicles were labeled with 40 microM 3'-O-(5-fluoro-2,4-dinitrophenyl)-ATP (FDNP-ATP) at 25 degrees C and pH 7.0 for 4 h. The Ca(2+)-ATPase was inhibited strongly. The enzyme was almost completely protected either by 20 mM Mg.ATP or by 50 mM acetyl phosphate against this inhibition. Pi gave no protection. There was a linear relationship between the extent of this inhibition and the Mg.ATP-sensitive part of the content of bound FDNP-ATP. Extrapolation showed that the enzyme is completely inhibited by Mg.ATP-sensitive binding of 3.6 nmol of FDNP-ATP/mg of the vesicle protein. This value is in good agreement with the content of the phosphorylation site (3.3 nmol/mg of the vesicle protein) in the vesicles used. These findings indicate that binding of 1 mol of FDNP-ATP per mol of the active sites leads to a complete inhibition of the enzyme. The acetylphosphatase activity and phosphorylation with ATP were also strongly inhibited by this labeling, whereas phosphorylation with Pi was not inhibited. The labeled vesicles were solubilized in SDS, and the Ca(2+)-ATPase was purified by size exclusion high performance liquid chromatography. Mapping the labeled peptides in the tryptic digest by reversed-phase high performance liquid chromatography and sequencing showed that Lys-492 was exclusively labeled with FDNP-ATP. These results show that Lys-492 is located in or near the ATP binding site and apart from the phosphorylation site and Pi binding site. Molecular modeling of FDNP-ATP suggests that this Lys-492 residue is situated on the 3'-OH side of the ribose moiety of bound ATP and is close to the alpha-phosphoryl group.
Highlights
AND DISCUSSIONFDNP-[a-32P]ATPin the presence of 7.6 mM Mg2+for various FIG.. Inhibition of the CaB+-ATPase blyabeling withFDNP-
Sarcoplasmic reticulum vesicles were labeled with40 351 (3-6) to form an EP intermediate (7, 8).Acetyl phosphate pm 3’-0-(5-fluoro-2,4-dinitrophenyl)-(AFTDPNP-ATP)at can serveas a substrate throughEP formation (9, 10)
There was have been identified by affinity labeling
Summary
FDNP-[a-32P]ATPin the presence of 7.6 mM Mg2+for various FIG.. Inhibition of the CaB+-ATPase blyabeling withFDNP-. A, an aliquot of the eluate was assayed for protein concentration and Ca2+-ATPase activity.The Ca2+-ATPase activity at zero time (1.4-1.6 pmollrngmin) was set at100%.B , the congave 3.6 nmol of bound D N P - [ C Z - ~ ~ P ] AoTfPth/ ~e ~vesicle protein This value is in good agreement with the content of the phosphorylation site (3.3 nmoVmg of the vesicle protein)in the SR vesicles used. The SR vesicles were treated with 0 or 40 p~ FDNP-ATP in the absence or presence of 20 m~ MgATP for 4 h, otherwise as described under "Experimental Procedures." The reaction mixtuwreas centrifuged at 541,000x g for 5 min at 0 "C, and the pellet was washed with a solution containing 0.1 m~ CaCl,, 0.1M KC], 0.1 M sucrose, and 5 m~ MOPWs (pH 7.0).The Ca2+-ATPase activityE, P formation from ATP or Pi, and acetylphosphatase activitywere determined. The Ca2+-independent acetylphosphatase activity determinintheedpresence of 5 m~ EGTA without added CaC12was 0-0.03pmol/mgmin
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