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首页> 外文期刊>Biochemistry >Rhodopsin Activity Varies in Proteoliposomes Prepared by Different Techniques
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Rhodopsin Activity Varies in Proteoliposomes Prepared by Different Techniques

机译：不同技术制备的蛋白脂质体中视紫质活性的变化。

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A variety of techniques are currently in use for preparing protein-containing lipid vesicles known as proteoliposomes. However, the functionality of membrane protein in proteoliposomes prepared by various techniques has rarely been evaluated directly. We prepared rhodopsin-containing proteoliposomes consisting of asolectin or native retinal rod outer segment disk lipids using n-octyl β-d-glucopyranoside and the detergent dialysis (DD) and rapid dilution (RD) techniques and measured the activity of rhodopsin using equilibrium UV/vis and flash photolysis spectroscopy. A significant difference in rhodopsin activity was observed in proteoliposomes prepared by these techniques. The equilibrium constant of metarhodopsin I−metarhodopsin II is 30−45% higher, and the apparent rate constant of MII formation is up to 3-fold faster in proteoliposomes prepared by RD vs DD. The DD technique produced larger yet more heterogeneous vesicles, while the RD technique yielded smaller and more homogeneous vesicles, as determined by electron microscopy and isopicnic centrifugation. Both proteoliposomes and empty lipid vesicles lacking rhodopsin were formed in the DD preparation, while only proteoliposomes were formed in the RD preparation. Under identical conditions, proteoliposomes prepared by RD have a higher L/P ratio, which is consistent with the higher level of rhodopsin activity in RD proteoliposomes. Overall, the results presented here suggest that the RD technique has an advantage over the DD technique in terms of preserving optimal rhodopsin activity and controlling the lipid to protein ratio in the final proteoliposomes.
机译：当前有多种技术用于制备被称为蛋白脂质体的含蛋白质的脂质囊泡。然而，通过各种技术制备的蛋白脂质体中的膜蛋白的功能很少被直接评估。我们使用正辛基β-d-吡喃葡萄糖苷和去污剂透析（DD）和快速稀释（RD）技术制备了由视蛋白或天然视网膜视杆外段盘脂质组成的视紫质蛋白脂质体，并采用平衡UV /可见光和快速光解光谱。在通过这些技术制备的蛋白脂质体中观察到了视紫红质活性的显着差异。 RD和DD制备的蛋白脂质体中，视紫红质I-甲视紫红质II的平衡常数高30-45％，MII形成的表观速率常数快3倍。 DD技术产生的更大，但异质性的囊泡，而RD技术产生的更小，更均匀的囊泡，通过电子显微镜和等速离心测定。在DD制剂中形成了脂质体和缺乏视紫红质的空脂质囊泡，而在RD制剂中仅形成了脂质体。在相同条件下，通过RD制备的蛋白脂质体具有较高的L / P比，这与RD蛋白脂质体中视紫红质活性的较高水平相一致。总的来说，这里提出的结果表明，RD技术在保持最佳视紫红质活性和控制最终蛋白脂质体中脂蛋白比方面比DD技术更具优势。

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  • 来源
    《Biochemistry》 |2009年第1期|p.156-163|共8页
  • 作者

    Shui-Lin Niu Brian Doctrow and Drake C. Mitchell;

  • 作者单位

    Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892-9410;

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