Mechanism of an active transport of calcium. Ethoxyformylation of sarcoplasmic reticulum vesicles.
Mechanism of an active transport of calcium. Ethoxyformylation of sarcoplasmic reticulum vesicles.
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钙的主动转运机制。
DOI：
10.1016/s0021-9258(17)33299-4
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发表时间：
1976
期刊：
影响因子：
--
通讯作者：
J. Carrette
中科院分区：
文献类型：
--
作者：
J. Tenu;C. Chelis;D. Leger;J. Carrette
对肌浆网囊泡进行乙氧基化，以研究组氨酸残基在钙转运过程中的可能作用。评价化学修饰对Ca 2+依赖性ATP酶活性和对Ca 2+摄取参数的影响：VCa（钙摄取的初始速率）和CCa（稳态时累积的阳离子量）。通过三种不同的技术监测氨基酸的取代：（a）通过对乙氧基甲酰化物质进行氨基酸分析，进一步用重氮-1-H-四唑进行修饰，或通过巯基滴定，使用5- 5 ′-二硫代双（2-硝基苯甲酸）;（B）通过14 C标记，随后在pH 7下NH 2 OH或咪唑处理后除去标记;（c）在230 nm处进行分光光度测量。在pH 6.1和10 ℃下，乙氧基化反应对组氨酸无特异性。约1个赖氨酰基团/mol ATP酶首先被修饰。然后修饰1个（具有240（+/-20）10（-3）min-1的伪一级速率常数）或2个组氨酸。在我们的实验条件下，没有检测到酪氨酸或巯基的取代。与VCa和Cca的抑制相关的Ca 2+依赖性ATP酶活性的降低对应于组氨酸的化学取代。赖氨酸的修饰与ATP酶活性的降低无直接关系，组氨酸去乙氧甲酰基后，只有依赖Ca ~（2+）的ATP酶活性恢复到初始值。当反应在ATP或对硝基苯基磷酸盐存在下进行时，没有发现保护作用。如果假设组氨酸残基的乙氧基化诱导构象变化，改变膜对核苷酸的亲和力，则可以解释这些结果。
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.