Calcium transport activity of rat cardiac sarcoplasmic reticulum isolated in the presence of dithiothreitol

Journal of pharmacological methods Pub Date : 1991-03-01 DOI:10.1016/0160-5402(91)90022-W

Shawn C. Black, John H. McNeill, Sidney Katz

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引用次数: 2

Abstract

We examined the effect of dithiothreitol (DTT) on the isolation and calcium transport properties of rat cardiac sarcoplasmic reticulum (SR). DTT at a concentration of 4 mM was included in the homogenization/initial isolation and the high-salt (KCI) buffers used in the preparation of the SR fraction. The inclusion of DTT in the SR isolation media did not affect the total amount of SR protein isolated from ventricular tissue. However, SR isolated in the presence of DTT exhibited a significantly increased (p < 0.05) rate of calcium transport at free calcium concentrations ranging from 0.1 μm up to 2.0 μm, and a 26% increase in the apparent affinity of the SR calcium transport system for Ca²⁺. DTT included in the calcium-transport reaction medium did not significantly alter the rate of SR calcium transport in SR preparations isolated in the absence or presence of DTT. These results demonstrate that functionally important sulfhydryl groups of the SR calcium transport pump may be oxidized during the isolation of the membrane fraction and that such oxidation can be reduced by including DTT in the isolation buffers.

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二硫苏糖醇存在下大鼠心肌肌浆网钙转运活性的研究

研究了二硫苏糖醇(DTT)对大鼠心肌肌浆网(SR)分离及钙转运特性的影响。匀浆/初始分离中加入浓度为4 mM的DTT，制备SR馏分时使用高盐缓冲液(KCI)。在SR分离培养基中加入DTT不影响从心室组织分离的SR蛋白总量。然而，在DTT存在下分离的SR表现出显著增加(p <在0.1 μm ~ 2.0 μm的游离钙浓度范围内，SR钙转运系统对Ca2+的表观亲和力提高了26%。在没有或存在DTT的情况下，钙转运反应介质中加入DTT对分离的SR制剂中SR钙转运的速率没有显著影响。这些结果表明，在分离膜组分的过程中，SR钙转运泵的重要功能巯基可能被氧化，而在分离缓冲液中加入DTT可以减少这种氧化。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Journal of pharmacological methods

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