Tsilibary E C, Koliakos G G, Charonis A S, Vogel A M, Reger L A, Furcht L T
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455.
J Biol Chem. 1988 Dec 15;263(35):19112-8.
Interactions between type IV collagen and heparin were examined under equilibrium conditions with rotary shadowing, solid-phase binding assays, and affinity chromatography. With the technique of rotary shadowing and electron microscopy, heparin appeared as thin, short strands and bound to the following three sites: the NC1 domain, and in the helix, at 100 and 300 nm from the NC1 domain. By solid-phase binding assays the binding of [3H]heparin in solution to type IV collagen immobilized on a solid surface was found to be specific, since it was saturable and could be displaced by an excess of unlabeled heparin. Scatchard analysis indicated three classes of binding sites for heparin-type IV collagen interactions with dissociation constants of 3, 30, and 100 nM, respectively. Furthermore, by the solid-phase binding assays, the binding of tritiated heparin could be competed almost to the same extent by unlabeled heparin and chondroitin sulfate side chains. This finding indicates that chondroitin sulfate should also bind to type IV collagen. By affinity chromatography, [3H]heparin bound to a type IV collagen affinity column and was eluted with a linear salt gradient, with a profile exhibiting three distinct peaks at 0.18, 0.22, and 0.24 M KCl, respectively. This suggested that heparin-type IV collagen binding was of an electrostatic nature. Finally, the effect of the binding of heparin to type IV collagen on the process of self-assembly of this basement membrane glycoprotein was studied by turbidimetry and rotary shadowing. In turbidity experiments, the presence of heparin, even in small concentrations, drastically reduced maximal aggregation of type IV collagen which was prewarmed to 37 degrees C. By using the morphological approach of rotary shadowing, lateral associations and network formation by prewarmed type IV collagen were inhibited in the presence of heparin. Thus, the binding of heparin resulted in hindrance of assembly of type IV collagen, a process previously described for interactions between various glycosaminoglycans and interstitial collagens. Such regulation may influence the assembly of basement membranes and possibly modify functions. Furthermore, qualitative and quantitative changes of proteoglycans which occur in certain pathological conditions, such as diabetes mellitus, may alter molecular assembly and possibly permeability functions of several basement membranes.
在平衡条件下，采用旋转阴影法、固相结合测定法和亲和色谱法研究了IV型胶原与肝素之间的相互作用。运用旋转阴影技术和电子显微镜观察，肝素呈现为细短链状，并结合于以下三个位点：NC1结构域，以及在螺旋结构中，距离NC1结构域100和300纳米处。通过固相结合测定法发现，溶液中的[3H]肝素与固定在固体表面的IV型胶原的结合具有特异性，因为它具有饱和性，且可被过量的未标记肝素取代。Scatchard分析表明，肝素与IV型胶原相互作用存在三类结合位点，解离常数分别为3、30和100 nM。此外，通过固相结合测定法，未标记肝素和硫酸软骨素侧链对氚标记肝素结合的竞争程度几乎相同。这一发现表明硫酸软骨素也应与IV型胶原结合。通过亲和色谱法，[3H]肝素结合到IV型胶原亲和柱上，并用线性盐梯度洗脱，洗脱图谱分别在0.18、0.22和0.24 M KCl处呈现三个明显的峰。这表明肝素与IV型胶原的结合具有静电性质。最后，通过比浊法和旋转阴影法研究了肝素与IV型胶原结合对这种基底膜糖蛋白自组装过程的影响。在比浊实验中，即使是低浓度的肝素存在，也会显著降低预热至37摄氏度的IV型胶原的最大聚集量。通过旋转阴影的形态学方法，在肝素存在下，预热的IV型胶原的侧向缔合和网络形成受到抑制。因此，肝素的结合导致IV型胶原组装受阻，这一过程先前已在各种糖胺聚糖与间质胶原的相互作用中描述过。这种调节可能会影响基底膜的组装，并可能改变其功能。此外，在某些病理状况（如糖尿病）下发生的蛋白聚糖的定性和定量变化，可能会改变几种基底膜的分子组装及可能的通透性功能。
