Selective modification of functionally distinct sulfhydryl groups of sarcoplasmic reticulum Ca2+,Mg2+-adenosine triphosphatase with N-ethylmaleimide.

Rabbit sarcoplasmic reticulum vesicles were treated with N-ethylmaleimide (NEM) at pH 7.0. At 1.5 mM NEM, only 4 SH groups per mol of ATPase peptide were modified in 25 min at 30 degrees C. Two of these are essential for Ca2+ transport, one being involved in E-P formation (SHF), and the other in its decomposition (SHD), whereas the other two are apparently non-essential (SHN and SHN'). SHN was modified first, followed by SHD, SHN', and SHF, in this order. Modification of SHD was accompanied by the loss of Ca2+-transport activity, while E-P forming activity survived until the least reactive one (SHF) was modified. At a lower NEM concentration (4 x 10(-5) M) SHN could be selectively modified without loss of the enzyme activity. SHF could be protected by adenyl-5'-yl-imidodiphosphate (AMP-P(NH)P) in the presence of Ca2+ ions, whereas SHD was not. SHD was distinctly less reactive in the absence of Ca2+ (less than 10(-7) M) than in its presence. Changes in the reactivity of these SH groups may be related to conformational changes of the ATPase molecule induced by the binding of Ca2+ and ATP.