Taylor H C, Lightner V A, Beyer W F, McCaslin D, Briscoe G, Erickson H P
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
J Cell Biochem. 1989 Oct;41(2):71-90. doi: 10.1002/jcb.240410204.
Tenascin is a large, disulfide-bonded glycoprotein of the extracellular matrix. The predominant form of tenascin observed by electron microscopy is a six-armed oligomer, termed a hexabrachion. We have determined the molecular mass of the native human hexabrachion to be 1.9 x 10(6) Da by sedimentation equilibrium analysis and by electrophoresis on non-reducing agarose gels. On reducing polyacrylamide gel electrophoresis (SDS-PAGE), human tenascin showed a single prominent band at 320 kDa and minor bands of 220 and 230 kDa. The molecular weight of the native human hexabrachion is thus consistent with a disulfide-bonded hexamer of the 320 kDa subunits. Upon treatment with neuraminidase, the apparent molecular weights of all human and chicken tenascin subunits on reducing SDS-PAGE were decreased by about 10 kDa. Prolonged incubation with alpha-mannosidase, however, caused no apparent change in the apparent molecular weight of tenascin subunits. Sedimentation in a cesium chloride gradient gave a higher buoyant density for human tenascin than for fibronectin, suggesting that it has a higher degree of glycosylation. The far-UV circular dichroism spectrum indicates a predominance of beta-structure and a lack of collagen-like or alpha-helical structure. When human hexabrachions were reduced and acetylated, the resulting fragments were single arms which sedimented at 6 S in glycerol gradients and migrated at 320 kDa on non-reducing gels. Treatment of tenascin with trypsin and alpha-chymotrypsin also produced large fragments which were fractionated by gradient sedimentation and analyzed by non-reducing SDS-PAGE and electron microscopy. We present a structural model for the assembly of the observed fragments into the elaborate native hexabrachion.
腱生蛋白是细胞外基质中一种由二硫键连接的大型糖蛋白。通过电子显微镜观察到的腱生蛋白的主要形式是一种六臂寡聚体，称为六臂体。我们通过沉降平衡分析和在非还原琼脂糖凝胶上进行电泳，确定天然人六臂体的分子量为1.9×10⁶道尔顿。在还原聚丙烯酰胺凝胶电泳（SDS-PAGE）中，人腱生蛋白在320 kDa处显示出一条单一的突出条带，以及220 kDa和230 kDa的次要条带。因此，天然人六臂体的分子量与320 kDa亚基的二硫键连接六聚体一致。用神经氨酸酶处理后，在还原SDS-PAGE上所有人和鸡腱生蛋白亚基的表观分子量降低了约10 kDa。然而，用α-甘露糖苷酶长时间孵育并没有导致腱生蛋白亚基的表观分子量出现明显变化。在氯化铯梯度中沉降显示，人腱生蛋白的浮力密度高于纤连蛋白，这表明它具有更高程度的糖基化。远紫外圆二色光谱表明β-结构占主导，且缺乏胶原样或α-螺旋结构。当人六臂体被还原和乙酰化时，产生的片段是单臂，在甘油梯度中以6 S沉降，在非还原凝胶上以320 kDa迁移。用胰蛋白酶和α-糜蛋白酶处理腱生蛋白也产生了大片段，这些片段通过梯度沉降分级，并通过非还原SDS-PAGE和电子显微镜进行分析。我们提出了一个将观察到的片段组装成精细的天然六臂体的结构模型。
