Matthews I, Sharma R P, Lee A G, East J M
Department of Biochemistry, University of Southampton, United Kingdom.
J Biol Chem. 1990 Nov 5;265(31):18737-40.
An antipeptide antibody was produced against a peptide corresponding to residues 877-888 of fast twitch rabbit sarcoplasmic reticulum ATPase. This antipeptide antibody bound strongly to the ATPase in sarcoplasmic reticulum vesicles only after the vesicles had been solubilized with the detergent C12E8 indicating that its epitope was located in the lumen of the sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum or purified (Ca2(+)-MG2+)-ATPase by proteinase K for up to 1 h resulted in a stable ATPase fragment of 30 kDa containing the epitope for the above antibody and the epitope for an antibody directed against the C terminus. Further proteolysis revealed smaller fragments (Mr 19,000 and 13,000) containing both epitopes. By contrast, small fragments of the ATPase (less than 29 kDa) containing the N-terminal epitope were not observed even after short exposures to proteinase K. These data support the view that the (Ca2(+)-MG2+)-ATPase has 10 transmembranous helices.
针对与快速抽搐兔肌浆网ATP酶877 - 888位残基对应的肽段制备了一种抗肽抗体。只有在用去污剂C12E8溶解肌浆网小泡后，这种抗肽抗体才会与肌浆网小泡中的ATP酶强烈结合，这表明其表位位于肌浆网腔中。用蛋白酶K对肌浆网或纯化的（Ca2 + - Mg2 +）-ATP酶消化长达1小时，会产生一个30 kDa的稳定ATP酶片段，该片段含有上述抗体的表位和针对C末端的抗体的表位。进一步的蛋白水解显示出含有两个表位的较小片段（Mr 19,000和13,000）。相比之下，即使短暂暴露于蛋白酶K后，也未观察到含有N末端表位的ATP酶小片段（小于29 kDa）。这些数据支持了（Ca2 + - Mg2 +）-ATP酶具有10个跨膜螺旋的观点。
