Sulphydryl groups of (Na+ + K+)-ATPase from rectal glands of Squalus acanthias. Titrations and classification

Abstract

1. (Na+ + K+)-ATPase from rectal glands of Squalus acanthias contains 34 SH groups per mol (Mr 265000). 15 are located on the alpha subunit (Mr 106000) and two on the beta subunit (Mr 40000). The beta subunit also contains one disulphide bridge. 2. The reaction of (Na+ + K+)-ATPase with N-ethylmaleimide shows the existence of at least three classes of SH groups. Class I contains two SH groups on each alpha subunit and one on each beta subunit. Reaction of these groups with N-ethylmaleimide in the presence of 40% glycerol or sucrose does not alter the enzyme activity. Class II contains four SH groups on each alpha subunit, and the reaction of these groups with 0.1 mM N-ethylmaleimide in the presence of 150 mM K+ leads to an enzyme species with about 16% activity. The remaining enzyme activity can be completely abolished by reaction with 5-10 mM N-ethylmaleimide, indicating a third class of SH groups (Class III). This pattern of inactivation is different from that of the kidney enzyme, where only one class of SH groups essential to activity is observed. 3. It is also shown that N-ethylmaleimide and DTNB inactivate by reacting with the same Class II SH groups. 4. Spin-labelling of the (Na+ + K+)-ATPase with a maleimide derivative shows that Class II groups are mostly buried in the membrane, whereas Class I groups are more exposed. It is also shown that spin label bound to the Class I groups can monitor the difference between the Na+- and K+-forms of the enzyme.