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

1. (Na⁺ +K⁺)-ATPase from rectal gland of Squlus acanthias contains 34 SH groups per mol (M_r 265000). 15 are located on the α subunit (M_r 106 000) and two on the β subunit (M_r 40 000). The β 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 α subunit and one on each β subunit. Reaction of these groups with N-methylmaleimide in the presence of 40% glycerol or sucrose does not alter the enzyme activity. Class II contains four SH groups on each α 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 nM 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.