Selective Modification of Functionally Distinct Sulfhydryl Groups of Sarcoplasmic Reticulum Ca2+, Mg2+-Adenosine Triphosphatase with N-Ethylmaleimide

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Abstract
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°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 × 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+ (2+ and ATP.

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