Azuma M, Anderson R S, Sameshima S, Matsumoto T, David L L, Shearer T R
Department of Biochemistry, School of Dentistry, Oregon Health Sciences University, Portland 97201.
Invest Ophthalmol Vis Sci. 1992 Jul;33(8):2476-82.
This study was conducted to provide a description of calpain proteolytic enzyme (EC 3.4.22.17) in normal rat cornea and to document immunohistochemical changes in calpain distribution during maturation. Corneal soluble proteins were fractionated by diethylaminoethyl chromatography on high-performance liquid chromatography. Fractions were analyzed for calpain by enzyme-linked immunosorbent assay, immunoblotting, and caseinolytic enzyme activity with fluorescein isothiocyanate-labeled casein. Calpain II from the soluble fraction of 2-week-old and 3-month-old rat corneas eluted at a similar NaCl concentration (220-240 mmol/l) as calpain II from other tissues, was inhibited by E64, contained an 80-kilodalton subunit in immunoblots, and was present at specific activity of 473 units per gram of protein in 3-month-old rats and 801 units per gram of protein in 2-week-old rats. Calpain antigen also was present in the ethylenediaminetetraacetic acid and EGTA washed insoluble fraction of cornea. Calpain was found (by immunohistochemical analysis) in all layers of the epithelium but not in the stroma. Enzyme-linked immunsorbent assay, immunoblots, and immunohistochemical analysis showed that calpain in the whole cornea did not change with corneal maturation. It was hypothesized that calpain in the cornea may be involved in the turnover of epithelial cells during normal maturation.
本研究旨在描述正常大鼠角膜中的钙蛋白酶解酶（EC 3.4.22.17），并记录其在成熟过程中分布的免疫组织化学变化。通过在高效液相色谱上进行二乙氨基乙基色谱法对角膜可溶性蛋白进行分级分离。通过酶联免疫吸附测定、免疫印迹以及用异硫氰酸荧光素标记的酪蛋白进行酪蛋白分解酶活性分析来检测各组分中的钙蛋白酶。2周龄和3月龄大鼠角膜可溶性组分中的钙蛋白酶II在与其他组织中的钙蛋白酶II相似的NaCl浓度（220 - 240 mmol/l）下洗脱，被E64抑制，在免疫印迹中含有一个80千道尔顿的亚基，在3月龄大鼠中的比活性为每克蛋白质473单位，在2周龄大鼠中的比活性为每克蛋白质801单位。钙蛋白酶抗原也存在于角膜经乙二胺四乙酸和乙二醇双四乙酸洗涤后的不溶性组分中。（通过免疫组织化学分析）发现钙蛋白酶存在于上皮的所有层中，但不存在于基质中。酶联免疫吸附测定、免疫印迹和免疫组织化学分析表明，整个角膜中的钙蛋白酶不会随角膜成熟而变化。据推测，角膜中的钙蛋白酶可能在正常成熟过程中参与上皮细胞的更新。
