Year-end Sale % OFF 
Abstract
Sarcoplasmic reticulum vesicles were treated with 2 mM pyridoxal 5'-phosphate (PLP) at 25 degrees C and pH 7.0 for 6 min and reduced by NaBH4. Both the activity of the Ca(2+)-ATPase and the capacity for high affinity Mg-ATP binding were greatly reduced. Acetyl phosphate hydrolysis or phosphoenzyme formation from Pi was not inhibited. The enzyme was protected by high affinity Mg-ATP binding against the PLP-induced inhibition. A similar protective effect was obtained by Mg-AMP as well as by Mg-ADP. Acetyl phosphate or Pi gave no protection. The PLP-treated vesicles were solubilized in SDS, and the Ca(2+)-ATPase was purified by size exclusion high performance liquid chromatography (HPLC). Mapping the fluorescently labeled peptides in the tryptic digest by reversed phase HPLC revealed a single fluorescent peak, which was protected by Mg-ATP against labeling. Sequencing showed that Lys-492 is the residue labeled with PLP. These results demonstrate that Lys-492 is located in or near the ATP binding site but not in the phosphorylation site or the Pi binding site. When Lys-515 was entirely prelabeled with fluorescein 5-isothiocyanate (FITC), the subsequent labeling of Lys-492 with PLP was not prevented. This finding demonstrates that Lys-492 is located outside the FITC-binding region. It has been widely accepted that FITC occupies the adenosine-binding region within the ATP binding site. In contrast to FITC, Mg-AMP strongly inhibited the labeling of Lys-492 with PLP. These findings lead to the conclusion that Lys-492 is located outside the adenosine-binding region, most probably in or near the region occupied by the alpha-phosphoryl group of Mg-ATP bound to the ATP binding site.
Highlights
From the $Department of Biochemistry and the §Third Departmentof Internal Medicine, Asahikawa Medical College, Asahikawa 078, Japan
The capacity for EP formation from ATP or Pi and the acetyl phosphatase activity remained constant during the 60-min treatment without pyridoxal 5”phosphate (PLP)
The present results show that labeling of Lys-492 with no appreciable labeling of Lys-515 in the 6-min treatment with PLP (Fig. 5A) is accompanied by a strong inhibition of the Ca2+-ATPase (Fig. 1 andTable I) andthatan exclusive prevention of the labeling of Lys-492 (Fig. 5B and E) is accompanied by a pronounced protection of the enzyme against the inhibition (Fig. 2A and Table I)
Summary
From the $Department of Biochemistry and the §Third Departmentof Internal Medicine, Asahikawa Medical College, Asahikawa 078, Japan. This value is about twice the content of the phosphorylation site (5.1 nmol/mg of the purified ATPase protein), which wasdetermined by purification of the phosphorylated Ca2+-ATPase asdescribed under "Experimental Procedures." This is consistent with the findings that two distinct treated either with PLP in the absence (trace 1) and presence (trace 2 ) of 20 mM Mg-ATP or without PLP in the absence of MgATP (trace 3 ) .Absorption spectra of the Ca2+-ATPasepurified from the vesicles weredetermined inthe buffer used for elution in the size exclusion HPLC.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
