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3'-O-(5-fluoro-2,4-dinitrophenyl)-ATP exclusively labels Lys-492 at the active site of the sarcoplasmic reticulum Ca(2+)-ATPase. 1994

K Yamasaki, and T Daiho, and T Kanazawa
Department of Biochemistry, Asahikawa Medical College, Japan.

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.

UI MeSH Term Description Entries
D008239 Lysine An essential amino acid. It is often added to animal feed. Enisyl,L-Lysine,Lysine Acetate,Lysine Hydrochloride,Acetate, Lysine,L Lysine
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010446 Peptide Fragments Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques. Peptide Fragment,Fragment, Peptide,Fragments, Peptide
D010449 Peptide Mapping Analysis of PEPTIDES that are generated from the digestion or fragmentation of a protein or mixture of PROTEINS, by ELECTROPHORESIS; CHROMATOGRAPHY; or MASS SPECTROMETRY. The resulting peptide fingerprints are analyzed for a variety of purposes including the identification of the proteins in a sample, GENETIC POLYMORPHISMS, patterns of gene expression, and patterns diagnostic for diseases. Fingerprints, Peptide,Peptide Fingerprinting,Protein Fingerprinting,Fingerprints, Protein,Fingerprint, Peptide,Fingerprint, Protein,Fingerprinting, Peptide,Fingerprinting, Protein,Mapping, Peptide,Peptide Fingerprint,Peptide Fingerprints,Protein Fingerprint,Protein Fingerprints
D011817 Rabbits A burrowing plant-eating mammal with hind limbs that are longer than its fore limbs. It belongs to the family Leporidae of the order Lagomorpha, and in contrast to hares, possesses 22 instead of 24 pairs of chromosomes. Belgian Hare,New Zealand Rabbit,New Zealand Rabbits,New Zealand White Rabbit,Rabbit,Rabbit, Domestic,Chinchilla Rabbits,NZW Rabbits,New Zealand White Rabbits,Oryctolagus cuniculus,Chinchilla Rabbit,Domestic Rabbit,Domestic Rabbits,Hare, Belgian,NZW Rabbit,Rabbit, Chinchilla,Rabbit, NZW,Rabbit, New Zealand,Rabbits, Chinchilla,Rabbits, Domestic,Rabbits, NZW,Rabbits, New Zealand,Zealand Rabbit, New,Zealand Rabbits, New,cuniculus, Oryctolagus
D000252 Calcium-Transporting ATPases Cation-transporting proteins that utilize the energy of ATP hydrolysis for the transport of CALCIUM. They differ from CALCIUM CHANNELS which allow calcium to pass through a membrane without the use of energy. ATPase, Calcium,Adenosinetriphosphatase, Calcium,Ca(2+)-Transporting ATPase,Calcium ATPase,Calcium Adenosinetriphosphatase,Adenosine Triphosphatase, Calcium,Ca2+ ATPase,Calcium-ATPase,ATPase, Ca2+,ATPases, Calcium-Transporting,Calcium Adenosine Triphosphatase,Calcium Transporting ATPases,Triphosphatase, Calcium Adenosine
D000255 Adenosine Triphosphate An adenine nucleotide containing three phosphate groups esterified to the sugar moiety. In addition to its crucial roles in metabolism adenosine triphosphate is a neurotransmitter. ATP,Adenosine Triphosphate, Calcium Salt,Adenosine Triphosphate, Chromium Salt,Adenosine Triphosphate, Magnesium Salt,Adenosine Triphosphate, Manganese Salt,Adenylpyrophosphate,CaATP,CrATP,Manganese Adenosine Triphosphate,MgATP,MnATP,ATP-MgCl2,Adenosine Triphosphate, Chromium Ammonium Salt,Adenosine Triphosphate, Magnesium Chloride,Atriphos,Chromium Adenosine Triphosphate,Cr(H2O)4 ATP,Magnesium Adenosine Triphosphate,Striadyne,ATP MgCl2
D000345 Affinity Labels Analogs of those substrates or compounds which bind naturally at the active sites of proteins, enzymes, antibodies, steroids, or physiological receptors. These analogs form a stable covalent bond at the binding site, thereby acting as inhibitors of the proteins or steroids. Affinity Labeling Reagents,Labeling Reagents, Affinity,Labels, Affinity,Reagents, Affinity Labeling
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia

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