Konishi Y
Department of Ophthalmology, School of Medicine, University of Tokushima, Japan.
Nippon Ganka Gakkai Zasshi. 1990 Dec;94(12):1148-56.
The abundant soluble protein of bovine cornea (CSP) was partially purified by chromatographies on Sephacryl S-200, DEAE cellulose and agarose to which mouse anti-CSP monoclonal antibody produced by the hybridoma technique was conjugated. SDS-polyacrylamide gel electrophoresis of the purified CSP yielded a single band with a molecular weight of 54,000 daltons. Isoelectric focusing of the CSP yielded four bands of pI 6.41, 6.32, 6.22 and 6.16. Chromatofocusing with PBE 94 (Pharmacia Uppsala Sweden) allowed the separation of these four components of CSP with iso-electric points of 6.28, 5.89, 5.85 and 5.68. In order to biochemically differentiate the four components, we attempted ammonium sulfate fractionation and chromatographies on heparin-sepharose and hydroxylapatite. However, these methods failed to separate the four CSP components. We examined whether the CSP was a glycoprotein or not by Western-blotting with various kinds of biotinylated rectins. Con A and UEA-I appeared to slightly bind the CSP. But CSP was not bound in chromatography on Con A-sepharose. These results suggested that the CSP was not a glycoprotein.
牛角膜丰富的可溶性蛋白（CSP）通过在Sephacryl S - 200、DEAE纤维素和琼脂糖上进行色谱分离进行部分纯化，其中琼脂糖偶联了通过杂交瘤技术产生的小鼠抗CSP单克隆抗体。纯化后的CSP进行SDS - 聚丙烯酰胺凝胶电泳产生一条分子量为54,000道尔顿的条带。CSP的等电聚焦产生了pI为6.41、6.32、6.22和6.16的四条带。使用PBE 94（瑞典乌普萨拉法玛西亚公司）进行色谱聚焦可以分离出CSP的这四个组分，其等电点分别为6.28、5.89、5.85和5.68。为了从生物化学角度区分这四个组分，我们尝试了硫酸铵分级分离以及在肝素 - 琼脂糖和羟基磷灰石上进行色谱分离。然而，这些方法未能分离出CSP的四个组分。我们通过用各种生物素化凝集素进行蛋白质印迹来检测CSP是否为糖蛋白。伴刀豆球蛋白A（Con A）和荆豆凝集素I（UEA - I）似乎与CSP有轻微结合。但CSP在Con A - 琼脂糖色谱中未结合。这些结果表明CSP不是糖蛋白。
