Kyte J, Xu K Y, Bayer R
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Biochemistry. 1987 Dec 15;26(25):8350-60. doi: 10.1021/bi00399a049.
Evidence that the peptide HLLVMKGAPER, which can be released from intact sodium and potassium ion activated adenosinetriphosphatase by tryptic digestion, is located on the cytoplasmic surface of the native enzyme has been obtained. An immunoadsorbent directed against the carboxy-terminal sequence of this tryptic peptide has been constructed. The peptide KGAPER was synthesized by solid-phase techniques. Antibodies against the sequence -GAPER were purified by immunoadsorption, using the synthetic peptide attached to agarose beads. These antibodies, in turn, were coupled to agarose beads to produce an immunoadsorbent. Sealed, right-side-out vesicles, prepared from canine kidneys, were labeled with pyridoxal phosphate and sodium [3H]borohydride in the absence or presence of saponin, respectively. A tryptic digest of these labeled vesicles was passed over the immunoadsorbent. Large increases in the incorporation of radioactivity into the peptides bound by the immunoadsorbent were observed in the digests obtained from the vesicles exposed to saponin. From the results of several control experiments examining the labeling reaction as applied to these vesicles, it could be concluded that this increase in incorporation resulted only from the access that the reagents gained to the inside of the vesicles in the presence of saponin and that the increase in the extent of modification was due to the cytoplasmic disposition of this segment in the native enzyme.
有证据表明，肽HLLVMKGAPER可通过胰蛋白酶消化从完整的钠钾离子激活的三磷酸腺苷酶中释放出来，且该肽位于天然酶的细胞质表面。已构建了一种针对该胰蛋白酶肽羧基末端序列的免疫吸附剂。肽KGAPER通过固相技术合成。针对序列-GAPER的抗体利用附着在琼脂糖珠上的合成肽通过免疫吸附进行纯化。这些抗体继而与琼脂糖珠偶联以制备免疫吸附剂。分别在不存在或存在皂角苷的情况下，用磷酸吡哆醛和硼氢化钠[3H]对从犬肾制备的密封的外翻囊泡进行标记。这些标记囊泡的胰蛋白酶消化产物通过免疫吸附剂。在从暴露于皂角苷的囊泡获得的消化产物中，观察到与免疫吸附剂结合的肽中放射性掺入量大幅增加。从针对这些囊泡应用的标记反应进行的若干对照实验结果可以得出结论，这种掺入量的增加仅源于在存在皂角苷的情况下试剂能够进入囊泡内部，并且修饰程度的增加是由于该片段在天然酶中位于细胞质一侧。
