Mechanism of an active transport of calcium. Ethoxyformylation of sarcoplasmic reticulum vesicles.
Ethoxyformylation of sarcoplasmic reticulum vesicles is performed to study a possible role of histidine residues in the calcium translocation process. The influence of the chemical modification is evaluated on the Ca2+-dependent ATPase activity, and on the Ca2+ uptake parameters: VCa (initial rate of calcium uptake) and CCa (amount of cation accumulated at the steady state). The substitution of the amino acids is monitored by three different techniques: (a) by amino acid analysis of the ethoxyformylated material further submitted to modification by diazonium-1-H-tetrazole, or by sulfhydryl titration using 5-5'-dithiobis (2-nitrobenzoic acid); (b) by 14C labeling followed by the removing of labels after NH2OH or imidazole treatment at pH 7; (c) by spectrophotometric measurements at 230 nm. The ethoxyformylation reaction is not specific for histidine at pH 6.1 and 10 degrees. About 1 lysyl group/mol of ATPase is first modified. Then 1 (with a pseudo-first order rate constant of 240 (+/- 20) 10(-3) min-1) or 2 histidines are modified. No substitution of tyrosine or sulfhydryl groups can be detected under our experimental conditions. A decrease of the Ca2+-dependent ATPase activity correlated with the inhibition of both VCa and Cca corresponds to the chemical substitution of the histidine. No direct correlation between the decrease of the activities and the modification of the lysine can be found. After removing the ethoxyformyl group from the histidine, only the Ca2+-dependent ATPase activity is restored to its initial value. No protection is found when the reaction is performed in the presence of ATP or p-nitrophenylphosphate. These results can be explained if one assumes that the ethoxyformylation of the histidine residue(s) induces a conformational change modifying the affinity of the membrane for nucleotides.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000812:Anhydrides; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D003545:Cysteine / Q000031:analogs & derivatives; D005019:Ethyl Ethers / Q000378:metabolism*; D006639:Histidine; D007700:Kinetics; D008433:Mathematics; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D000812:Anhydrides; D005019:Ethyl Ethers; D006639:Histidine; D000251:Adenosine Triphosphatases; D003545:Cysteine; D002118:Calcium