Abstract
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.
Highlights
The influence of the chemical modification is evaluated on the Ca2+-dependent ATPase activity, and on the Cal+ uptake parameters: V,. and calcium accumulated in the steady state (Ccs)
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 “C labeling followed by the removing of labels after NH,OH or imidazole treatment at pH 7; (c) by spectrophotometric measurements at 230 nm
Obtained by Chemical Modification of Histidine Measured by Spectroscopy (Figs. 3 to 6)-In Fig. 7 the relative decrease of the Ca*+-dependent ATPase activity and the calcium uptake parameters (Cc. and Vc.) are correlated with the amount of the fast modified histidine residues (10 + 2 nmol/mg of protein, reacting with a pseudo-first order rate constant of 240 10mSmix--‘)
Summary
ATP, p-nitrophenylphosphate, 5.5’.dithiobis(2.nitrobenzoic acid) and N-acetyl-L-cysteine were purchased from Sigma Chemical Co., Hepes’ from Calbiochem, sodium dodecyl sulfate (recrystallized before use) from Eastman Kodak, and ethoxyformic anhydride from Carlo. Anhydride-Prior to each experiment, the concentration of the reagent dissolved in ethanol was determined by its change of absorbance at 230 nm after reaction with lOm~imidazoleatpH7,5(Ae. The reaction of ethoxyformic anhydride with N-acetyl-L-cysteine was performed as follows. “ClEthoxyformic anhydride dissolved in ethanol was added to a sarcoplasmic reticulum solution (2 mg of protein/ml in 200 mM sodium succinate, pH 6.1) at 10” until a concentration of 2.5 mM was reached. On aliquots of treated or untreated sarcoplasmic reticulum solutions (0.5 mg/ml) solubilized in 1% SDS/l00 mM sodium phosphate, pH 8.0, with 0.17 mM final concentration of 5-5’-dithiobis(2-nitrobenzoic acid). Since ethoxyformylated amino acids were not stable under acid hydrolysis, the determination of a possible modification of lysine, tyrosine, and histidine residues was done as follows: the ethoxyformylated preparations were treated by the diazonium-1-H-tetrazole [21] in the presence of 1% SDS. Controls performed without ethoxyformic anhydride and in presence of NH,OH showed that the preparations were stable under the conditions used for these experiments
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