Ishii T, Aoki N, Noda A, Adachi T, Nakamura R, Matsuda T
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Japan.
Biochim Biophys Acta. 1995 Dec 14;1245(3):285-92. doi: 10.1016/0304-4165(95)00102-6.
A cDNA encoding mouse butyrophilin was obtained by reverse transcriptase-coupled polymerase chain reaction (RT-PCR) using poly (A)+ RNA from lactating mouse mammary gland as a template and screening a cDNA library with the RT-PCR-amplified fragment as a probe. DNA sequencing and computer analysis revealed that it has a rather long 3'-untranslated sequence and that the carboxy-terminal cytoplasmic domain was well conserved between mouse and bovine butyrophilins. To elucidate the biological function of butyrophilin, the cytoplasmic region expressed as fusion protein with glutathione S-transferase (GST) was purified and incubated with the cell lysate of mouse mammary epithelial cell lines, COMMA-ID and HC11. A 150-kDa protein was shown to specifically associate with the cytoplasmic domain and the protein increased in amount when the cells were treated with basal medium supplemented with lactogenic hormones such as prolactin, insulin and glucocorticoid. N-terminal amino acid sequencing indicated that the protein is xanthine dehydrogenase/oxidase which has been cloned from mouse liver. Further, the cytoplasmic domain also bound xanthine dehydrogenase/oxidase from bovine milk fat globule membrane. These results suggest that butyrophilin might be physiologically associated with xanthine dehydrogenase/oxidase and might function in a complex form in milk fat secretion.
以泌乳期小鼠乳腺的聚腺苷酸加尾RNA（poly (A)+ RNA）为模板，通过逆转录酶偶联聚合酶链反应（RT-PCR）获得编码小鼠嗜乳脂蛋白的cDNA，并以RT-PCR扩增片段为探针筛选cDNA文库。DNA测序和计算机分析表明，它具有相当长的3'非翻译序列，并且小鼠和牛嗜乳脂蛋白的羧基末端胞质结构域高度保守。为了阐明嗜乳脂蛋白的生物学功能，将与谷胱甘肽S-转移酶（GST）融合表达的胞质区域纯化，并与小鼠乳腺上皮细胞系COMMA-ID和HC11的细胞裂解物一起孵育。结果显示，一种150 kDa的蛋白质与胞质结构域特异性结合，并且当细胞用补充了催乳素、胰岛素和糖皮质激素等泌乳激素的基础培养基处理时，该蛋白质的量会增加。N端氨基酸测序表明该蛋白质是从小鼠肝脏克隆的黄嘌呤脱氢酶/氧化酶。此外，胞质结构域还与牛乳脂肪球膜中的黄嘌呤脱氢酶/氧化酶结合。这些结果表明，嗜乳脂蛋白可能在生理上与黄嘌呤脱氢酶/氧化酶相关联，并可能以复合物形式在乳脂肪分泌中发挥作用。
