Protein chemistry of the Neurospora crassa plasma membrane H+-ATPase.
Protein chemistry of the Neurospora crassa plasma membrane H+-ATPase.
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粗糙脉孢菌质膜 H-ATP 酶的蛋白质化学。
DOI：
10.1016/0003-2697(88)90187-x
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发表时间：
1988
影响因子：
2.9
通讯作者：
Scarborough,GA
中科院分区：
文献类型：
--
作者：
SubrahmanyeswaraRao,U;HennesseyJr,JP;Scarborough,GA
本文报道了一种高效的粗糙脉孢菌质膜H ~+-ATP酶片段化和纯化的方法。用胰蛋白酶切割酶，形成含有疏水性和亲水性肽的极限消化物，然后通过用碳酸氢铵水溶液萃取分离疏水性和亲水性肽。通过Sephadex G-25柱色谱将亲水性肽分级分离成三个池，并且通过高效液相色谱纯化每个池中的单个肽。将疏水肽溶于纯三氟乙酸（TFA）中，用氯仿-甲醇（1：1）稀释，然后通过Sephadex LH-60柱色谱法在含有0.1%TFA的氯仿-甲醇（1：1）中分级分离由此获得的疏水肽溶液。上述方法的回收率均大于90%。通过该方法纯化的三种疏水H+-ATP酶肽的N-末端氨基酸序列已经确定，其通过参考公开的基因序列确定了这些肽在100，000 Da多肽链中的位置，并记录了以这种方式纯化的疏水肽的可测序性。这种方法应有利于鉴定的各种氨基酸残基的H+-ATP酶分子的结构和功能的重要。此外，与H+-ATP酶的蛋白质化学工作的总体策略也应该适用于其他两亲性整合膜蛋白。
A highly effective procedure for fragmenting the Neurospora crassa plasma membrane H+-ATPase and purifying the resulting peptides is described. The enzyme is cleaved with trypsin to form a limit digest containing both hydrophobic and hydrophilic peptides, and the hydrophobic and hydrophilic peptides are then separated by extraction with an aqueous ammonium bicarbonate solution. The hydrophilic peptides are fractionated by Sephadex G-25 column chromatography into three pools, and the individual peptides in each pool are purified by high-performance liquid chromatography. The hydrophobic peptides are dissolved in neat trifluoroacetic acid (TFA), diluted with chloroform-methanol (1:1), and the hydrophobic peptide solution thus obtained is then fractionated by Sephadex LH-60 column chromatography in chloroform-methanol (1:1) containing 0.1% TFA. The recoveries in all of the above procedures are greater than 90%. The N-terminal amino acid sequences of three of the hydrophobic H+-ATPase peptides purified by this methodology have been determined, which establishes the position of these peptides in the 100,000 Da polypeptide chain by reference to the published gene sequence, and documents the sequencability of the hydrophobic peptides purified in this way. This methodology should facilitate the identification of a variety of amino acid residues important for the structure and function of the H+-ATPase molecule. Moreover, the overall strategy for working with the protein chemistry of the H+-ATPase should be applicable to other amphiphilic integral membrane proteins as well.