Specific Alkylation of the Sarcoplasmic Reticulum ATPase by <i>N</i>‐Ethyl‐[1‐¹⁴C]maleimide and Identification of the Labeled Protein in Acrylamide Gel‐Electrophoresis

Abstract

The total sulfhydryl content of sarcoplasmic reticulum determined in the presence of sodium dodecylsulfate is 14 sulfhydryl equivalents per 10 5 g of membrane protein, but in the absence of sodium dodecylsulfate only 10 sulfhydryl groups are titrateable with 5,5′‐dithiobis (2‐nitrobenzoic acid). The rate of ATPase inactivation by N ‐ethylmaleimide in the presence of ATP yields an ATP‐ATPase dissociation constant of 45μM. When the sulfhydryl group essential for ATPase activity was reacted with N ‐ethyl‐[1‐ 14 C]maleimide after all the sulfhydryl groups unrelated to ATPase activity had been reacted previously with unlabeled reagent, only one protein band in electrophorograms was strongly labeled. The presence of ATP during trypsin proteolysis preserves ATPase activity and the band that can be specifically labeled with N ‐ethylmaleimide is demonstrated in gel electrophorograms. When trypsin proteolysis was carried out in the absence of ATP this band can no longer be seen. The N ‐ethyl‐[1‐ 14 C]maleimide labeledband is tentatively identified with the ATPase.