HIGH-CAPACITY BINDING OF ALPHA-CRYSTALLINS TO VARIOUS BOVINE LENS MEMBRANE PREPARATIONS
HIGH-CAPACITY BINDING OF ALPHA-CRYSTALLINS TO VARIOUS BOVINE LENS MEMBRANE PREPARATIONS
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DOI：
10.3109/02713689309029230
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发表时间：
1993-11-01
影响因子：
2
通讯作者：
CHANDRASEKHER, G
中科院分区：
文献类型：
--
作者：
CENEDELLA, RJ;CHANDRASEKHER, G
本研究检查了完整和羧基末端截短的α A（α A）晶体蛋白与两种类型的透镜膜制剂的高容量结合;通过尿素和碱提取去除外源蛋白和一些脂质的膜，以及通过在蔗糖梯度上离心总水不溶性蛋白分离的未提取膜（天然膜）。一旦alphaA底物浓度达到约1 mg/ml培养基，就可以看到alphaA晶状体蛋白与尿素处理的膜的高容量结合。该膜在5 mg alphaA/ml培养基的浓度下结合高达1 mg alphaA/mg内在蛋白（MP26），结合比在高亲和力但低容量结合位点饱和时由其他人观察到的结合大5至10倍。羧基末端截短的α A（胰蛋白酶）和完整的α A的高容量结合之间没有明显的差异，虽然每个晶体蛋白可以拮抗其他的结合。然而，一旦膜结合，无论是晶体蛋白出现严重取代其他。使用羧基末端截短的α晶体蛋白作为模型底物，天然膜被认为具有更高的能力，结合截短的α晶体蛋白比尿素提取的膜和结合更好地与预先存在的α A含量的天然膜比其MP26含量。通过将截短的α A预结合到尿素提取的膜来制备人工天然膜。该制剂比不含预结合晶状体蛋白的尿素提取膜结合更完整的α A。我们的结论是，透镜天然膜具有很高的能力，结合α晶体蛋白，这种结合可以通过蛋白质-蛋白质相互作用介导的α晶体蛋白结合在原位的膜作为外源性蛋白。
This study examines the high capacity binding of intact and carboxyl-terminal-truncated alpha A(alphaA) crystallin to two types of lens membrane preparations; membrane stripped of extrinsic protein and some lipid by extraction with urea and alkali and unextracted membrane isolated by centrifugation of total water insoluble protein on a sucrose gradient (native membrane). High capacity binding of alphaA crystallin to the urea-treated membrane was seen once the alphaA substrate concentration reached about 1 mg/ml of media. The membrane bound up to one mg of alphaA per mg of intrinsic protein (MP26) at a concentration of 5 mg alphaA/ml media, binding 5 to 10 times greater than that seen by others at saturation of the high affinity but low capacity binding sites. No apparent differences were seen between high capacity binding of carboxyl terminal--truncated alphaA (by trypsin) and intact alphaA, although each crystallin could antagonize binding of the other. However, once membrane bound, neither crystallin appeared to grossly displace the other.Using the carboxyl terminal-truncated alpha crystallin as a model substrate, native membrane was seen to have a higher capacity to bind the truncated alpha crystallin than urea-extracted membrane and binding was better correlated with the preexisting alphaA content of the native membrane than its MP26 content. An artificial native membrane was prepared by prebinding the truncated alphaA to urea-extracted membrane. This preparation bound more intact alphaA than urea-extracted membrane bearing no prebound crystallin. We conclude that lens native membrane possesses a high capacity to bind alpha crystallins and that this binding could be mediated through protein-protein interactions with alpha crystallin bound in situ to the membrane as extrinsic protein.