Nakazato K, Muraoka M, Adachi E, Hayashi T
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo.
J Biochem. 1996 Nov;120(5):889-94. doi: 10.1093/oxfordjournals.jbchem.a021502.
It is known that the type IV collagen extracted from EHS tumor assembles under a physiological condition, but not in a gel form. The EHS type IV collagen requires the other basement membrane components, laminin1, heparansulfate proteoglycan, and/or nidogen for gelation. On the other hand, Muraoka et al. reported that the bovine lens capsule type IV collagen alone gelated under a unique and unexpected condition of 2 M guanidine-HCl and 50 mM dithiothreitol, a condition which is thought to be dissociative for most biological macromolecules, including extracellular matrix [Muraoka, M. et al. (1996) J. Biochem. 119, 167-172]. The present report shows that the bovine lens capsule type IV collagen formed a gel under physiological conditions of pH and ionic environment, though the apparent rigidity of the gel was weaker than that of the gel formed in 2 M guanidine-HCl and dithiothreitol. The rigidity depended greatly on the incubation temperature and NaCl concentration of the type IV collagen solution, as observed in terms of the contractility of gel volume under centrifugal force. the gel formed in 150 mM NaCl and 20 mM phosphate, pH 7.3, at 28 degrees C contracted to 20% of the original volume on centrifugation of 1,800 x g for 10 min, while the gel formed at 4 degrees C, where type I collagen did not gelate at all, retained 90% of the original volume at the same centrifugal force. NaCl concentration was another important factor influencing the mechanical properties of type IV collagen gel. The gel formed at 150 mM showed maximal rigidity in the range of 0 to 300 mM in terms of the contractility on centrifugation. An image of a Pt/C replica of the gelated type IV collagen reconstituted at 4 or 28 degrees C in 20 mM phosphate, pH 7.3, containing 150 mM NaCl showed fine meshworks consisting of rather homogeneous pore sizes, resembling the skeletal structure of basal lamina. Since the condition where the type IV collagen alone formed gels was physiological in terms of ionic strength and pH, the aggregate structure and gel properties might reflect the in vivo type IV collagen supramolecular structure and the property.
已知从EHS肿瘤中提取的IV型胶原在生理条件下会组装，但不会形成凝胶形式。EHS IV型胶原需要其他基底膜成分，如层粘连蛋白1、硫酸乙酰肝素蛋白聚糖和/或巢蛋白才能形成凝胶。另一方面，村冈等人报道，牛晶状体囊IV型胶原在2M盐酸胍和50mM二硫苏糖醇这种独特且意想不到的条件下单独形成凝胶，而这种条件被认为对包括细胞外基质在内的大多数生物大分子具有解离作用[村冈，M.等人（1996年）《生物化学杂志》119，167 - 172]。本报告表明，牛晶状体囊IV型胶原在生理pH和离子环境条件下形成了凝胶，尽管该凝胶的表观硬度比在2M盐酸胍和二硫苏糖醇中形成的凝胶弱。从离心力作用下凝胶体积的收缩情况来看，硬度很大程度上取决于IV型胶原溶液的孵育温度和NaCl浓度。在150mM NaCl和20mM磷酸盐（pH 7.3）中于28℃形成的凝胶，在1800×g离心10分钟后收缩至原始体积的20%，而在4℃形成的凝胶（I型胶原在该温度下根本不会形成凝胶）在相同离心力下保留了90%的原始体积。NaCl浓度是影响IV型胶原凝胶力学性能的另一个重要因素。就离心收缩性而言，在150mM形成的凝胶在0至300mM范围内显示出最大硬度。在含有150mM NaCl的20mM磷酸盐（pH 7.3）中于4℃或28℃重构的凝胶化IV型胶原的Pt/C复制品图像显示，由相当均匀孔径组成的精细网络，类似于基底层的骨架结构。由于IV型胶原单独形成凝胶的条件在离子强度和pH方面是生理性的，其聚集结构和凝胶特性可能反映了体内IV型胶原的超分子结构和性质。
