Miller R P, Farley R A
Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles 90033.
Biochemistry. 1990 Feb 13;29(6):1524-32. doi: 10.1021/bi00458a025.
Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., & Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, beta-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native beta subunit: 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the beta subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the beta subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the beta subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the beta subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the beta subunit from the titration experiments.(ABSTRACT TRUNCATED AT 250 WORDS)
先前对可滴定的（Na⁺ + K⁺）-ATP酶巯基的研究表明，每摩尔全酶存在一个二硫键。基于每个亚基滴定的“游离”巯基数量与滴定的巯基总数之间的差异，这个单一的二硫交联被归属于β亚基[埃斯曼，M.（1982年）《生物化学与生物物理学报》688卷，251页；川村，M.，& 长野，K.（1984年）《生物化学与生物物理学报》694卷，27页]。在本研究中，通过高效液相色谱法鉴定并纯化了含有二硫交联的β亚基胰蛋白酶肽段。通过用二硫苏糖醇还原，从每个二硫键连接的肽段产生了两个新肽段，并测定了这些还原肽段的氨基酸组成。数据表明，天然β亚基中有三个二硫键：125位半胱氨酸-148位半胱氨酸、158位半胱氨酸-174位半胱氨酸和212位半胱氨酸-275位半胱氨酸。β亚基中二硫键的数量也通过用[¹⁴C]碘乙酰胺滴定巯基来估算。鉴定出六个巯基：两个巯基在未预先还原的情况下被滴定，四个仅在蛋白质用二硫苏糖醇还原后被鉴定出来。这些表明β亚基含有两个二硫键的数据与肽段分离实验不一致，肽段分离实验直接鉴定出β亚基中有三个二硫键。通过证明在实验开始前β亚基中每个二硫键约20%被还原，从而导致滴定实验低估了β亚基中二硫键连接的巯基数量，解决了这种不一致性。（摘要截短于250字）
