Labeling of lysine 492 with pyridoxal 5'-phosphate in the sarcoplasmic reticulum Ca(2+)-ATPase. Lysine 492 residue is located outside the fluorescein 5-isothiocyanate-binding region in or near the ATP binding site.
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.
Yamagata
K
K
Department of Biochemistry, Asahikawa Medical College, Japan.
Daiho
T
T
Kanazawa
T
T
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism*; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000378:metabolism; D010449:Peptide Mapping; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D010755:Organophosphates; C011632:acetyl phosphate; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine