5-[I-125]IODONAPHTHYL AZIDE, A REAGENT TO DETERMINE PENETRATION OF PROTEINS INTO LIPID BILAYER OF BIOLOGICAL-MEMBRANES
5-[I-125]IODONAPHTHYL AZIDE, A REAGENT TO DETERMINE PENETRATION OF PROTEINS INTO LIPID BILAYER OF BIOLOGICAL-MEMBRANES
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DOI：
10.1021/bi00601a020
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发表时间：
1978-01-01
期刊：
影响因子：
2.9
通讯作者：
GITLER, C
中科院分区：
文献类型：
--
作者：
BERCOVICI, T;GITLER, C
5-[125 I]碘萘基1-叠氮是一种用于测定蛋白质渗透到生物膜脂双层中程度的有用试剂。该标记可以很容易地制成高放射性的，并储存合理的时间或重新纯化，然后使用。在黑暗中，它对膜脂的分配系数很高。对于叠氮化物到反应性氮宾的光介导转化，它具有高消光系数。它可以被短时间的光激活，这些光的波长是膜蛋白和脂质不能吸收的，因此它们的辐射损伤是最小的。光产生的氮宾以非常高的效率共价插入膜组分中。对于不同的膜制备物，20-55%的添加的标记插入膜蛋白和脂质中。它似乎主要从脂质双层内插入到内在膜蛋白中;很少，如果有任何插入到外在蛋白中。在兔骨骼肌肌浆网中，氮烯的主要插入发生在Ca 2+敏感的ATP酶中。ATP酶活性不受标记程序的影响。100，000 MW Ca 2 +-ATP酶的温和胰蛋白酶裂解导致形成MW 52，000和46，000的2个片段。2个片段被同样标记，表明蛋白质通过其多肽链的至少2个片段与双层接触。在完整的红细胞和无血红蛋白的红细胞膜中，很少（如果有的话）标记插入条带1、2或5（分别为血影蛋白和红细胞肌动蛋白）。标记发生在谱带3、高碘酸-希夫1、2和3区域以及谱带7中。
5-[125I]Iodonaphthyl 1-azide is a useful reagent for the determination of the extent of penetration of proteins into the lipid bilayer of biological membranes. The label can readily be made highly radioactive and stored for reasonable times or repurified and then used. In the dark it has a high partition coefficient into membrane lipids. It has a high extinction coefficient for the light-mediated conversion of the azide into the reactive nitrene. It can be activated by short periods of light at wavelengths which membrane proteins and lipids do not absorb so that their radiation damage is minimal. The light-generated nitrene inserts covalently with very high efficiencies into the membrane components. With different membrane preparations, 20-55% of the added label inserts into the membrane proteins and lipids. It appears to insert from within the lipid bilayer mainly into intrinsic membrane proteins; very little, if any insertion occurs into extrinsic proteins. In rabbit skeletal muscle sarcoplasmic reticulum, the main insertion of the nitrene occurs into the Ca2+-sensitive ATPase. The ATPase activity is not affected by the labeling procedure. Mild tryptic cleavage of the 100,000 MW Ca2+-ATPase results in the formation of 2 fragments of MW 52,000 and 46,000. The 2 fragments are equally labeled, suggesting that the protein is in contact with the bilayer by at least 2 segments of its polypeptide chain. In intact erythrocytes and hemoglobin-free erythrocyte membranes, little, if any, label is inserted into bands 1, 2 or 5 (spectrin and erythrocyte actin, respectively). Label occurs in the region of bands 3, periodic acid-Schiff 1, 2 and 3 and in band 7.