Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles.
Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles.
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DOI：
10.1016/j.bbalip.2013.02.009
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发表时间：
2013-06
期刊：
影响因子：
--
通讯作者：
Lowe ME
中科院分区：
文献类型：
--
作者：
Ross LE;Xiao X;Lowe ME
辅脂酶的吸附对于胰甘油三酯脂肪酶的活性和有效的膳食脂肪消化是必需的。然而，很少有人知道哪些特定的氨基酸在疏水表面的辅脂酶的影响吸附。在这项研究中，我们系统地取代丙氨酸或色氨酸的残基牵连吸附的辅脂酶的接口。我们表达，纯化重组辅脂酶突变体，并表征了每个丙氨酸突变体在胆盐存在下恢复脂肪酶活性的能力。L16 A、Y55 A、I79 A和F84 A辅脂酶的功能受损最严重，活性范围为野生型辅脂酶的20 - 60%。接下来，我们表征了色氨酸突变体在胆盐-油酸混合胶束的存在和不存在下的荧光性质。我们进行了稳态发射光谱，以确定峰位移和I330/I350比和丙烯酰胺淬灭曲线，以表征残留物的环境。该分析支持吸附模型，包括第二环上的残基Leu 34和Leu 36、第三环上的Tyr 55和Tyr 59以及第四环上的Ile 75和Ile 79。分析证实，Phe 84不是吸附表面的一部分，并且可能稳定辅脂酶的构象。与计算机模拟的预测相反，结果为Tyr 55在辅脂酶吸附到混合胶束中的重要作用提供了强有力的支持。结果表明，辅脂酶的混合胶束的吸附介导的特定残基驻留在一个确定的表面的辅脂酶。
The adsorption of colipase is essential for pancreatic triglyceride lipase activity and efficient dietary fat digestion. Yet, little is known about which specific amino acids in the hydrophobic surface of colipase influence adsorption. In this study, we systematically substituted alanine or tryptophan at residues implicated in adsorption of colipase to an interface. We expressed, purified recombinant colipase mutants and characterized the ability of each alanine mutant to restore activity to lipase in the presence of bile salts. The functions of L16A, Y55A, I79A and F84A colipase were most impaired with activities ranging from 20 to 60% of wild-type colipase. We next characterized the fluorescence properties of the tryptophan mutants in the absence and presence of bile-salt-oleic acid mixed micelles. We performed steady-state emission spectra to determine peak shift and I330/I350 ratio and acrylamide quenching curves to characterize the environment of the residues. The analysis supports a model of adsorption that includes residues Leu 34 and Leu 36 on the 2nd loop, Tyr 55 and Tyr 59 on the 3rd loop and Ile 75 and Ile 79 on the 4th loop. The analysis confirms that Phe 84 is not part of the adsorption surface and likely stabilizes the conformation of colipase. Contrary to the predictions of computer modeling, the results provide strong support for an essential role of Tyr 55 in colipase adsorption to mixed micelles. The results indicate that the adsorption of colipase to mixed micelles is mediated by specific residues residing in a defined surface of colipase.
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影响因子：
64.8
作者：
VANTILBEURGH, H;EGLOFF, MP;CAMBILLAU, C
通讯作者：
CAMBILLAU, C
影响因子：
2.9
作者：
Dahim, M;Brockman, H
通讯作者：
Brockman, H
DOI：
10.1016/0167-4781(91)90173-j
发表时间：
1991-07-23
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
ERNST, EG;BEHNKE, WD
通讯作者：
BEHNKE, WD
影响因子：
11.4
作者：
Hermoso, J;Pignol, D;FontecillaCamps, JC
通讯作者：
FontecillaCamps, JC
影响因子：
2.9
作者：
EFTINK, MR;GHIRON, CA
通讯作者：
GHIRON, CA