Chemical modification of an arginine residue in the ATP-binding site of Ca2+ -transporting ATPase of sarcoplasmic reticulum by phenylglyoxal.
Phenylglyoxal (PGO) was used as a reagent for chemical modification of the ATP-binding site of Ca2+ -transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum (SR-ATPase). When 1 mM PGO was reacted with SR-ATPase at 30 degrees C at pH 8.5, PGO was bound to the ATPase molecule in two-to-one stoichiometry with concomitant loss of activity of the ATPase to form the phosphorylated intermediate (E-P). ATP and ADP prevented the binding of PGO and thereby protected the enzyme from inactivation. The SR membranes were labeled with [14C]PGO and then digested with pepsin to identify the attachment site of PGO. A 14C-labeled peptide (402Ile-Arg*-Ser-Gly-Gln406) was purified to homogeneity by C18-reversed phase HPLC (Arg* denotes the binding site of [14C]PGO). These results indicate that Arg403 is located in the ATP binding site of the SR-ATPase.
Yamamoto
H
H
Department of Physiological Chemistry, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Japan.
Kawakita
M
M
D000227:Adenine Nucleotides / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D007202:Indicators and Reagents / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology; D010658:Phenylglyoxal / Q000737:chemistry*; D010710:Phosphates / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology*
D000227:Adenine Nucleotides; D007202:Indicators and Reagents; D010710:Phosphates; D000255:Adenosine Triphosphate; D001120:Arginine; D000252:Calcium-Transporting ATPases; D010658:Phenylglyoxal