钠钾ATP酶中游离巯基和二硫键的分析

Analysis of free sulfhydryl groups and disulfide bonds in Na+,K+-ATPase.

作者信息

Gevondyan N M, Gevondyan V S, Gavrilyeva E E, Modyanov N N

机构信息

Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow.

出版信息

FEBS Lett. 1989 Sep 25;255(2):265-8. doi: 10.1016/0014-5793(89)81103-2.

DOI:10.1016/0014-5793(89)81103-2

PMID:2551733

Abstract

The content of free SH groups and disulfide bonds in the purified pig kidney Na+,K+-ATPase was determined by ammetric titration with silver nitrate. In the native enzyme, most of the free SH groups are masked due to their location in the polypeptide chain regions poorly accessible to SH reagents. Denaturation with 5% SDS and 8 M urea makes these regions accessible thus revealing 22 free SH groups/mol of the protein. After complete blocking of free SH groups with silver ions, 8 SH groups/mol of the protein are being released upon sulfitolysis which indicates the presence of four disulfide bonds in the enzyme. At least one disulfide bridge is located in the alpha-subunit whereas the beta-subunit contains three disulfide bonds.

摘要

采用硝酸银安培滴定法测定纯化猪肾钠钾ATP酶中游离巯基和二硫键的含量。在天然酶中，大多数游离巯基由于位于巯基试剂难以接近的多肽链区域而被掩盖。用5%十二烷基硫酸钠（SDS）和8M尿素变性后，这些区域变得可及，从而揭示出每摩尔蛋白质有22个游离巯基。在用银离子完全封闭游离巯基后，在亚硫酸解时每摩尔蛋白质释放出8个巯基，这表明该酶中存在四个二硫键。至少有一个二硫桥位于α亚基中，而β亚基含有三个二硫键。