Regulation of chloride transport in parotid secretory granules by membrane fluidity.
Regulation of chloride transport in parotid secretory granules by membrane fluidity.
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通过膜流动性调节腮腺分泌颗粒中的氯离子转运。
DOI：
10.1021/bi00483a018
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Hopfer,U
中科院分区：
文献类型：
--
作者：
Gasser,KW;Goldsmith,A;Hopfer,U
材料和方法颗粒和膜分离。通过先前详细描述的方法，从 Sprague-Dawley 大鼠（150-200 g）的腮腺或胰腺，或从新西兰白兔的胃中分离出分泌颗粒（Gasser 等人，1988b；Sharma 等人，1989）。简而言之，该方法采用低离子强度缓冲液中的氮空化破坏细胞，然后使用 Percoll 密度梯度离心分离。收获的颗粒呈高密度带 (1.11-1.13 g/mL)。通过用蒸馏水渗透裂解和通过在50000g离心1小时收集膜来从纯化的颗粒制备颗粒膜。 Percoll 梯度还产生了富含质膜的低密度级分 (1.03-1.06 g/mL)。将低密度级分在由 250 mM 蔗糖、40 mMMOPS、1 用 NaOH、0.1 mM EGTA 和 0.1 mM MgSO4 滴定至 pH 7.0 的溶液中进行 1:20 稀释后，通过差速离心进一步富集腮腺质膜。丢弃2000g沉淀，并将随后的30000g沉淀重悬于相同缓冲液中并用作粗制质膜级分。大多数颗粒制剂均使用单一动物。如 Hopfer 等人（1983）所述，从大鼠小肠和肾近端小管等非外分泌组织中分离出高纯度刷状缘膜。
Materials and MethodsGranule and Membrane Isolation. Secretory granules were isolated from the parotid glands or the pancreas of Sprague-Dawley rats (150-200 g), or from the stomach of New Zea-land white rabbits, by a methodology which has been previ-ously described in detail (Gasser et al., 1988b; Sharma et al., 1989). Briefly, the method employs cell disruption by nitrogen cavitation in a low ionic strength buffer followed by separation using Percoll density gradientcentrifugation. The granules were harvested as a high-density band (1.11-1.13 g/mL). Granule membranes were prepared from purified granules by osmotic lysis with distilledwater and collection of the mem-branes by centrifugation at 50000gfor 1 h. The Percoll gradient also yielded a low-density fraction (1.03-1.06 g/mL), rich in plasma membranes. The plasma membranes from the parotids were further enriched by dif-ferential centrifugation after a 1: 20 dilution of the low-density fraction in a solution consisting of 250 mM sucrose, 40 mMMOPS, 1 titrated to pH 7.0 with NaOH, 0.1 mM EGTA, and 0.1 mM MgS04. A 2000g pellet was discarded, and a subsequent 30000g pellet was resuspended in the same buffer and used as a crude plasma membrane fraction. A single animal was used for most granule preparations. High-purity brush border membranes from nonexocrine tissues such as rat small intestine and kidney proximal tubule were isolated as described by Hopfer et al.(1983).