Mg(2+)-ATPase from rabbit skeletal muscle transverse tubules is 67-kilodalton glycoprotein

Abstract

There exists a Mg(2+)-ATPase in transverse tubules which has properties that are very different from other ATPases located in skeletal muscle cells. Several groups have suggested that a 100-kDa glycoprotein is the molecular entity responsible for this Mg(2+)-ATPase activity. In this study we have extended the methods utilized in the purification of this integral membrane glycoprotein. Although the apparent pI (isoelectric point) of this protein is pH 5, most of the net charge is due to the presence of sialic acid on the associated glycan chains. After these residues are removed, the behavior of this protein on an anion exchange column changes dramatically, allowing it to be further purified. Using a combination of the earlier procedures (Kirley, T.L. (1988) J. Biol. Chem. 263, 12682-12689 and Kirley, T. L. (1991) Biochem. J. 278, 375-400.) and the one reported here, purification to specific activities of approximately 400,000 mumol of ATP hydrolyzed/mg of protein/h were obtained and all traces of the 100-kDa protein were removed. The digitonin-solubilized Mg(2+)-ATPase appears to be a dimer of two identical 67-kDa subunits as assessed by high performance size exclusion chromatography. A single diffuse protein band is observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at approximately 67 kDa, which reduced to a single tight protein band at 52 kDa after deglycosylation with the following unique N-terminal amino acid sequence: Ala-Lys-Lys-Val-Leu-Pro-Leu-Leu-Leu-Pro- Pro-Leu-Val-X-Ala-Ala-Leu-Gly-Leu-Ala-X-Phe. Therefore, the Mg(2+)-ATPase appears to be an enzyme of very high specific activity, present in small quantities in transverse tubule membranes and unrelated to the known classes of ATPases present in skeletal muscle. The data reported here on the orientation of the transverse-tubule membrane preparations are consistent with the very recent report (Saborido, A., Moro, G., and Megias A. (1991) J. Biol. Chem. 266, 23490-23498) that the Mg(2+)-ATPase is an ecto-enzyme.