Gibson M A, Kumaratilake J S, Cleary E G
Department of Pathology, University of Adelaide, South Australia.
J Biol Chem. 1989 Mar 15;264(8):4590-8.
A procedure has been developed which is much more specific for the solubilization of the elastin-associated microfibrils from fetal bovine nuchal ligament using treatment with reductive saline in place of reductive guanidine hydrochloride buffer. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reductive saline extracts were shown to contain only five major protein bands with Mrs of 340,000, 78,000, 70,000, 31,000, and 25,000. The 31-kDa species was identified immunologically as the previously described macromolecule named microfibril-associated glycoprotein (MAGP) (Gibson, M. A., Hughes, J. L., Fanning, J. C., and Cleary, E. G. (1986) J. Biol. Chem. 261, 11429-11436). The proteins were purified by gel permeation, ion exchange, and affinity chromatography. Amino acid analyses showed that each protein had a profile which was distinct from that of MAGP although each was also high in acidic amino acids and cystine. The 340- and 78-kDa species were each demonstrated by immunoelectron microscopy with affinity-purified antibodies to be derived from the elastin-associated microfibris, and these were provisionally named microfibrillar protein 340 (MP340) and microfibrillar protein 78 (MP78), respectively. Each of the above antibodies gave a tissue distribution identical to that of anti-MAGP antibodies, and thus MP340 and MP78 also were identified with the 12-nm microfibrils of nonelastic tissues. MP340 was shown to absorb out completely the microfibrillar immunoreactivity of anti-(reductive guanidine hydrochloride extract) antibodies, indicating that MP340 was (a) the major microfibrillar constituent in these extracts and (b) the second unidentified microfibrillar antigen described previously. The relationship of the 70- and 25-kDa proteins to microfibrils is yet to be established. Immunoblot and immunoabsorption studies showed that MAGP and MP78 were immunologically related to MP340 but not to each other. Cyanogen bromide peptide mapping indicated that MAGP was structurally related to MP340. It is postulated that MAGP and MP78 are constituents of MP340 which in turn is the subunit of which the 12-nm microfibrils are composed.
已开发出一种方法，该方法在用还原盐溶液代替还原盐酸胍缓冲液处理时，对从胎牛颈部韧带中溶解与弹性蛋白相关的微原纤维具有更高的特异性。当通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析时，还原盐溶液提取物显示仅含有五条主要蛋白带，其分子量分别为340,000、78,000、70,000、31,000和25,000。31 kDa的蛋白经免疫鉴定为先前描述的名为微原纤维相关糖蛋白（MAGP）的大分子（吉布森，M. A.，休斯，J. L.，范宁，J. C.，和克利里，E. G.（1986年）《生物化学杂志》261，11429 - 11436）。这些蛋白质通过凝胶渗透、离子交换和亲和色谱法进行纯化。氨基酸分析表明，每种蛋白质都有一个与MAGP不同的图谱，尽管每种蛋白质的酸性氨基酸和胱氨酸含量也很高。340 kDa和78 kDa的蛋白通过免疫电子显微镜用亲和纯化抗体证实均来自与弹性蛋白相关的微原纤维，它们分别被暂命名为微原纤维蛋白340（MP340）和微原纤维蛋白78（MP78）。上述每种抗体的组织分布与抗MAGP抗体相同，因此MP340和MP78也被鉴定为非弹性组织的12纳米微原纤维。结果表明，MP340能完全消除抗（还原盐酸胍提取物）抗体的微原纤维免疫反应性，这表明MP340是（a）这些提取物中的主要微原纤维成分，（b）先前描述的第二种未鉴定的微原纤维抗原。70 kDa和25 kDa蛋白与微原纤维的关系尚待确定。免疫印迹和免疫吸收研究表明，MAGP和MP78在免疫上与MP340相关，但彼此不相关。溴化氰肽图谱分析表明，MAGP在结构上与MP340相关。据推测，MAGP和MP78是MP340的组成成分，而MP340又是构成12纳米微原纤维的亚基。
