Purification and characterization of the ecto-Mg-ATPase of chicken gizzard smooth muscle.

The ecto-Mg-ATPase isolated from chicken gizzard smooth muscle was solubilized, purified and characterized. The purification did not require the use of expensive or specialized apparatus. The chromatographic and electrophoretic characteristics of the ecto-Mg-ATPase from chicken are similar to those reported earlier for the ecto-Mg-ATPase isolated from rabbit skeletal muscle transverse tubule membranes [1992, J. Biol. Chem. 267, 11777-11782]. One obvious difference found was that the solubilized chicken ecto-Mg-ATPase can be stimulated approximately 1900% by the lectin Concanavalin A (Con A) under the same conditions that the rabbit enzyme is inhibited by approximately 50%. This stimulatory effect of Con A is useful for following the purification, and also increases the specific activity of the chicken enzyme to a very high level similar to that observed for the rabbit enzyme. After purification of the solubilized chicken ecto-Mg-ATPase by three steps of anion exchange chromatography, as well as Con A and erythroagglutinating Phaseolus vulgaris (PHA-E) lectin affinity chromatographies, a single diffuse glycoprotein band at approximately 66 kDa is observed after SDS-PAGE. This protein could be deglycosylated to a core protein of 53 kDa. Thus, the chicken gizzard protein is very similar in molecular size to the rabbit skeletal muscle ecto-Mg-ATPase both before and after deglycosylation [1992, J. Biol. Chem. 267, 11777-11782]. The N-terminal sequence of the 66 kDa chicken gizzard protein was found to be: Ala-Arg-Arg-Ala-Ala-Ala-Val-Leu-Leu-Leu-Leu-Ala. This is a unique sequence which, while very different from the rabbit ecto-Mg-ATPase N-terminus, exhibits some of the same characteristics, since it contains basic residues as the second and third amino acids, with the remainder of the N-terminus being very hydrophobic in nature. Furthermore, the chicken gizzard ecto-Mg-ATPase can be separated from the adhesion molecule, truncated cadherin (T-cadherin) by anion exchange chromatography, and is therefore not identical to that protein, as had been recently proposed [1993, Arch. Biochem. Biophys. 303, 32-43].