Permeation of long-chain fatty acid into adipocytes. Kinetics, specificity, and evidence for involvement of a membrane protein.
Permeation of long-chain fatty acid into adipocytes. Kinetics, specificity, and evidence for involvement of a membrane protein.
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DOI：
10.1016/s0021-9258(17)47247-4
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发表时间：
1984-07
期刊：
影响因子：
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通讯作者：
N. Abumrad;Jane H. Park;Charles R. Park
中科院分区：
文献类型：
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作者：
N. Abumrad;Jane H. Park;Charles R. Park
这项研究扩展了我们早期的工作(Abumrad，N.A.，Perkins，R.C.，Park，J.H.和Park，C.R.J.Biol)。化学。256,9183-9191)，表明油酸主要通过促进扩散的转运过程进入大鼠脂肪细胞质膜。在目前的研究中，脂肪酸(FA)转运的特点是其特异性和对蛋白质修饰剂抑制的敏感性。竞争抑制油酸和硬脂酸运输的动力学是在竞争FAs与介质中白蛋白结合的并发症最小化的条件下显示的。硬脂酸盐用与其Km接近的KI抑制示踪剂油酸盐的流入，反过来，油酸盐也同样抑制示踪剂硬脂酸盐的流入。在使用不同链长的各种天然FA或FA类似物的研究中，显示了FA转运系统的特异性。油酸(Km=0.06微米)、硬脂酸(Km=0.16微米)、亚油酸(Km=0.22微米)、棕榈酸(Km=0.2微米)和月桂酸(Km=1.5微米)是较好的底物，但辛酸不能运输。硬脂酸盐的C-5上的恶唑烷环而不是C-16上的恶唑烷环阻断了与转运体的结合。羧基功能的甲基化而不是α-溴化会抑制转运。这些研究表明，FA必须具有至少九个碳的碳氢链和自由羧基功能，才能被转运蛋白识别。FA转运不需要钠或三磷酸腺苷。对完整细胞进行Pronase处理，而不是胰酶处理，可以减少脂肪酸流入。交通工具对马来酰亚胺不敏感。用二苯乙烯类化合物、4，4‘-二异硫氰基二苯乙烯-2，2’-二磺酸和4-acetamido-4‘-isothiocyanostilbene-2，2’-disulfonic酸处理细胞可强烈且不可逆地阻断该作用，但双嘧达莫仅轻微抑制该作用。经[~3H]-4，4‘-二异硫氰基二苯乙烯-2，2’-二磺酸处理的细胞质膜蛋白经聚丙烯酰胺凝胶电泳法测定，在MR=85,000处有一个放射性峰。当细胞在不同浓度的该试剂中孵育时，在峰中恢复的计数达到最大值，与最大限度地抑制运输相一致。我们得出结论，生理浓度的长链脂肪酸对脂肪细胞质膜的渗透是由一种具有明显特异性要求的蛋白质转运体介导的。
This study extends our earlier work (Abumrad, N. A., Perkins, R.C., Park, J.H., and Park, C.R. J. Biol. Chem. 256, 9183-9191) which showed that oleate permeates the plasma membrane of the rat adipocyte principally by a transport process with the characteristics of facilitated diffusion. In the present study, fatty acid (FA) transport is characterized with regard to its specificity and susceptibility to inhibition by protein modifiers. The kinetics of competitive inhibition for transport of oleate and stearate are shown under conditions where complications due to competition for binding of FAs to the albumin in the medium are minimized. Stearate inhibits influx of tracer oleate with a Ki that closely approximates its Km and, conversely, oleate inhibits similarly the influx of tracer stearate. Specificity of the FA transport system is shown in studies using a variety of natural FAs of different chain length, or FA analogues. Oleate (Km = 0.06 microM), stearate (Km = 0.16 microM), linoleate (Km = 0.22 microM), palmitate, (Km = 0.2 microM), and laurate (Km = 1.5 microM) are good substrates, but octanoate is not transported. An oxazolidine ring on C-5 but not on C-16 of stearate blocks binding to the transporter. Methylation of the carboxyl function but not alpha-bromination inhibits transport. These studies suggest that a FA must have a hydrocarbon chain of at least nine carbons and a free carboxyl function to be recognized by the transporter. FA transport does not require Na or ATP. Pronase but not trypsin treatment of intact cells reduces fatty acid influx. Transport is insensitive to maleimides. It is strongly and irreversibly blocked by pretreatment of the cells with the stilbene compounds, 4,4'-diisothiocyanostilbene-2,2'-disulfonate and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, but only slightly inhibited by dipyridamole. Polyacrylamide gel electrophoresis of plasma membrane proteins from cells treated with [3H] 4,4'-diisothiocyanostilbene-2,2'-disulfonate shows a peak of radioactivity at about Mr = 85,000. When cells are incubated in various concentrations of this agent, the counts recovered in the peak reach a maximum coincident with maximum inhibition of transport. We conclude that permeation of the plasma membrane of the adipocyte by long-chain FAs at physiological concentrations is mediated by a protein transporter with distinct specificity requirements.