Niyibizi C, Fietzek P P, van der Rest M
J Biol Chem. 1984 Nov 25;259(22):14170-4.
Human type V collagen was purified from placenta and found to contain alpha 1(V), alpha 2(V), and alpha 3(V) chains in varying ratios. Using any of three independent nondenaturing methods (phosphocellulose chromatography, high-performance ion-exchange chromatography on IEX-540 DEAE, and ammonium sulfate precipitation), this preparation could be resolved into two fractions. Analysis of the two fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that one fraction contained alpha 1(V) and alpha 2(V) in a 2:1 ratio and the other contained alpha 1(V), alpha 2(V), and alpha 3(V) in a 1:1:1 ratio. When the crude placental type V collagen was electrophoresed under nondenaturing conditions, two bands were observed, one co-migrating with purified (alpha 1(V]2 alpha 2(V) and the other co-migrating with the fractions containing alpha 1(V), alpha 2(V), and alpha 3(V) chains in a 1:1:1 ratio. Electrophoresis in a second dimension under denaturing conditions confirmed that the fast-migrating band contained (alpha 1(V]2 alpha 2(V) and that the slow-migrating band contained the three chains in equimolar ratio. CD spectra of the two fractions and resistance to trypsin-chymotrypsin digestion confirmed that the two fractions contain triple helical collagen. Thermal denaturations were monitored by the changes in CD signal at 221 nm. The two fractions purified by ammonium sulfate precipitation melted at 39.1 and 36.4 degrees C for the (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) fractions, respectively. Trypsin cleavage of these two native fractions at temperatures near melting produced completely different fragmentation patterns, indicating different partial unwinding sites of the alpha 1(V) and alpha 2(V) chains in the two preparations and thus different molecular assemblies. Our data demonstrate the existence of two different molecular assemblies of type V collagen in human placenta consisting of (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) heterotrimers.
人V型胶原从胎盘中纯化出来，发现含有比例各异的α1(V)、α2(V)和α3(V)链。使用三种独立的非变性方法中的任何一种（磷酸纤维素色谱法、IEX - 540 DEAE上的高效离子交换色谱法和硫酸铵沉淀法），该制剂可被分离成两个组分。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳对这两个组分进行分析表明，一个组分含有比例为2:1的α1(V)和α2(V)，另一个组分含有比例为1:1:1的α1(V)、α2(V)和α3(V)。当粗制的胎盘V型胶原在非变性条件下进行电泳时，观察到两条带，一条与纯化的(α1(V)2α2(V)共迁移，另一条与含有比例为1:1:1的α1(V)、α2(V)和α3(V)链的组分共迁移。在变性条件下进行的二维电泳证实，快速迁移的带含有(α1(V)2α2(V)，而慢速迁移的带含有等摩尔比例的三条链。这两个组分的圆二色光谱以及对胰蛋白酶 - 糜蛋白酶消化的抗性证实这两个组分都含有三螺旋胶原。通过监测221 nm处圆二色信号的变化来监测热变性。通过硫酸铵沉淀法纯化的两个组分，(α1(V)2α2(V)组分和α1(V)α2(V)α3(V)组分分别在39.1℃和36.4℃时发生熔解。在接近熔解温度下对这两个天然组分进行胰蛋白酶切割产生了完全不同的片段化模式，表明两种制剂中α1(V)和α2(V)链的部分解旋位点不同，因此分子组装也不同。我们的数据证明，人胎盘中存在由(α1(V)2α2(V)和α1(V)α2(V)α3(V)异源三聚体组成的两种不同的V型胶原分子组装形式。
