Butkowski R J, Todd P, Grantham J J, Hudson B G
J Biol Chem. 1979 Oct 25;254(20):10503-13.
Renal tubules from rabbit kidneys were isolated from thin shavings of the kidney surface. Basement membrane was then prepared following sonication of the isolated tubules. To insure preservation of the integrity of the basement membrane polypeptides, the protease inhibitors, diisopropyl fluorophosphate, ethylenediaminetetraacetic acid, N-ethylmaleimide, and epsilon-amino-caproic acid were used at all stages of the preparations. The optimal conditions of sonication and centrifugation were established and the chemical composition of basement membrane prepared under these conditions was examined in detail. Glycine, hydroxyproline, and hydroxylysine were found in concentrations of 206, 65, and 18 residues per thousand, respectively, in basement membrane from young kidneys. About 38% of the basement membrane was found to be soluble in sodium dodecyl sulfate upon incubation at 90 degrees C, and to possess relatively low amounts of the amino acids characteristic of collagen. Electrophoretic analysis of this fraction revealed that the major subunits ranged in approximate molecular weight from 18,500 to greater than 10(6). When analyzed with disulfide bonds reduced, a molecular weight range from 31,000 to 275,000 was observed for this fraction. The sodium dodecyl sulfate-insoluble fraction could be dissolved upon reduction and alkylation and its composition was enriched in the amino acids characteristic of collagen. Polypeptides from this fraction were analyzed by electrophoresis in agarose and in agarose-acrylamide gels. The approximate molecular weight of the smallest component was 164,000. Additional polypeptides were observed whose molecular weights occurred in multimers of this component, up to 1.1 x 10(6), possibly indicating covalent cross-linked multimers of a basic collagen-like polypeptide(s).
从兔肾肾表面的薄片中分离出肾小管。然后对分离出的肾小管进行超声处理，制备基底膜。为确保基底膜多肽的完整性得以保留，在制备的各个阶段都使用了蛋白酶抑制剂二异丙基氟磷酸、乙二胺四乙酸、N - 乙基马来酰亚胺和ε - 氨基己酸。确定了超声处理和离心的最佳条件，并详细研究了在这些条件下制备的基底膜的化学成分。在幼龄兔肾的基底膜中，甘氨酸、羟脯氨酸和羟赖氨酸的浓度分别为每千个残基206、65和18个。发现约38%的基底膜在90℃孵育时可溶于十二烷基硫酸钠，且具有相对少量的胶原蛋白特征性氨基酸。对该部分进行电泳分析表明，主要亚基的近似分子量范围为18,500至大于10⁶。当在二硫键还原的情况下进行分析时，该部分观察到的分子量范围为31,000至275,000。十二烷基硫酸钠不溶性部分在还原和烷基化后可溶解，其组成富含胶原蛋白特征性氨基酸。通过在琼脂糖和琼脂糖 - 丙烯酰胺凝胶中进行电泳对该部分的多肽进行分析。最小成分的近似分子量为164,000。还观察到其他多肽，其分子量以该成分的多聚体形式出现，高达1.1×10⁶，这可能表明一种基本的类胶原蛋白多肽存在共价交联的多聚体。
