Labeling the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum with maleimidylsalicylic acid.
Maleimidylsalicylic acid reacts with the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum with high affinity and inhibits the ATPase activity following a pseudo-first-order kinetic with a rate constant of 8.3 m(-1) s(-1). Calcium binding remains unaffected in the maleimide-inhibited ATPase. However, the presence of ATP, ADP, and, to a lesser extent, AMP protects the enzyme against inhibition. Furthermore, ATPase inhibition is accompanied by a concomitant decrease in ATP binding. The stoichiometry of the nucleotide-dependent maleimidylsalicylic acid binding is 6-10 nmol/mg ATPase, which corresponds to the binding of up to one molecule of maleimide/molecule of ATPase. The stoichiometry of maleimide binding is decreased in the presence of nucleotides and in the ATPase previously labeled with fluorescein-5'-isothiocyanate or N-ethylmaleimide A fluorescent peptide was isolated by high performance liquid chromatography after trypsin digestion of the maleimide-labeled ATPase. Analysis of the sequence and mass spectrometry of the peptide leads us to propose Cys(344) as the target for maleimidylsalicylic acid in the inhibition reaction. The effect of Cys(344) modification on the nucleotide site is discussed.
Velasco-Guillén
I
I
Departamento de Bioquimica y Biologia Molecular A, Facultad de Veterinaria, Campus de Espinardo, Universidad de Murcia, 30100 Murcia, Spain.
Guerrero
J R
JR
Gomez-Fernández
J C
JC
Teruel
J A
JA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry* / Q000494:pharmacology*; D018482:Muscle, Skeletal / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D012459:Salicylates / Q000737:chemistry* / Q000494:pharmacology*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008301:Maleimides; D012459:Salicylates; C419384:maleimidylsalicylic acid; D001224:Aspartic Acid; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D005033:Ethylmaleimide