The NH2 terminus of the (Ca2+ + Mg2+)-adenosine triphosphatase is located on the cytoplasmic surface of the sarcoplasmic reticulum membrane

• Published in: The Journal of biological chemistry Vol. 256; no. 12; pp. 5957 - 5960
• Main Authors: Reithmeier, R A, MacLennan, D H
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25-06-1981
• Subjects: Amino Acid Sequence; Animals; Ca(2+) Mg(2+)-ATPase; Calcium-Transporting ATPases; Chemical Phenomena; Chemistry; Cysteine; Ethylmaleimide - metabolism; Intracellular Membranes - enzymology; Peptide Fragments; Sarcoplasmic Reticulum - drug effects; Sarcoplasmic Reticulum - enzymology; Sarcoplasmic Reticulum - metabolism; Sulfhydryl Reagents - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)69108-8

The (Ca2+ + Mg2+)-adenosine triphosphatase (ATPase) of sarcoplasmic reticulum contains a cysteine residue at position 12 of
its sequence. This sulfhydryl group was 1 out of a total of 10-11 that were labeled by treatment of sarcoplasmic reticulum
vesicles with N-[3H]ethylmaleimide under saturating conditions. This was shown by isolating a 31-residue NH2-terminal peptide
from a tryptic digest of the succinylated ATPase, prepared from N-[3H]ethylmaleimide-labeled vesicles. Reaction of the vesicles
with glutathione maleimide, parachloromercuribenzoic acid, or parachloromercuriphenyl sulfonic acid, membrane-impermeant reagents,
prevented further reaction of sulfhydryl groups with N-ethylmaleimide. This result indicates that all sulfhydryl groups that
are reactive with N-ethylmaleimide are on the outside of the vesicles. Since Cys12 is located in a hydrophilic NH2-terminal
portion of the ATPase, the labeling results suggest that the NH2 terminus of the ATPase is on the cytoplasmic side of the
membrane. These results are consistent with earlier observations (Reithmeier, R. A. F., de Leon, S., and MacLennan, D. H.
(1980) J. Biol. Chem. 255, 11839-11846) that the (Ca2+ + Mg2+)-ATPase is synthesized without an NH2-terminal signal sequence.