Chen C H, Hansma H G
Department of Physics, University of California, Santa Barbara, California 93106, USA.
J Struct Biol. 2000 Jul;131(1):44-55. doi: 10.1006/jsbi.2000.4252.
The major macromolecules of basement membranes-collagen IV, laminin-1, and heparan sulfate proteoglycan (HSPG)-have been analyzed by atomic force microscopy (AFM), both individually and in combination with each other. The positions of laminin binding to collagen IV were mapped and compared with the positions of imperfections in the amino acid sequence of collagen IV; the apparent molecular volumes of the HSPG proteoglycans were measured and used to estimate the corresponding molecular weights. Even the thin, thread-like strands of the polyanion heparan sulfate can be visualized with AFM without staining, coating, or fixation. These strands are single polysaccharide chains and are thus thinner than single-stranded DNA. The heparan sulfate strands in HSPG are necessary for protein filtration in kidney basement membranes. We propose that these thin strands filter proteins by functioning as an entropic brush-i.e., that they filter proteins by their constant thermally driven motion in the basement membrane. These AFM analyses in air are a step toward AFM analyses under fluid of basement membrane macromolecules interacting with each other.
已通过原子力显微镜（AFM）对基底膜的主要大分子——IV型胶原蛋白、层粘连蛋白-1和硫酸乙酰肝素蛋白聚糖（HSPG）进行了单独分析以及相互组合分析。绘制了层粘连蛋白与IV型胶原蛋白结合的位置，并与IV型胶原蛋白氨基酸序列中的缺陷位置进行了比较；测量了HSPG蛋白聚糖的表观分子体积，并用于估算相应的分子量。即使是带负电的硫酸乙酰肝素的细线状链，也可以在不进行染色、包被或固定的情况下用AFM观察到。这些链是单多糖链，因此比单链DNA更细。HSPG中的硫酸乙酰肝素链对于肾基底膜中的蛋白质过滤是必需的。我们提出，这些细链通过作为熵刷发挥作用来过滤蛋白质——也就是说，它们通过在基底膜中持续的热驱动运动来过滤蛋白质。这些在空气中进行的AFM分析是朝着在流体环境下对相互作用的基底膜大分子进行AFM分析迈出的一步。
