BIOPHYSICAL CHARACTERIZATION OF RAT CARDIAC CA2+ MG2+ ECTO-ATPASE (MYOGLEIN)/

Sarcolemmal Ca2+/Mg2+ ecto-ATPase (Myoglein; MW 180 kD) is a membrane bound enzyme which requires a millimolar concentration of either Ca2or Mg2+ for maximal hydrolysis of ATP. The isoelectric point (pI) of t he cardiac ecto-ATPase was 5.7. The purified Ca2+/Mg2+ ecto-ATPase fro m the rat heart sarcolemmal appeared as a single band with MW similar to 90 kD in the SDS-PAGE. In order to understand the nature of this en zyme, the 90 kD band in the SDS-PAGE was electroeluted; the analysis o f the eluate showed 2 prominent bands with MW similar to 90 and 85 kD. The presence of 2 bands was further confirmed by gradient gel (10-20% ) electrophoresis in 0.375 M Tris-HCl buffer, pH 8.8. Analysis of the purified Ca2+/Mg2+ ecto-ATPase as well as the electroeluted protein in a non-equilibrium linear two dimensional electrophoresis (Ampholyte p I 3.0-10.0) also showed two distinct bands. Mass spectroscopic analysi s of the enzyme using different matrix combinations revealed the prese nce of multi-components indicating microheterogeneity in the protein s tructure. Treatment of the ecto-ATPase with DL-dithiothreitol did not alter the pattern of mass spectroscopic analysis and this indicated th at the microheterogeneity may be due to some posttranslational modific ations. It is concluded that rat cardiac Ca2+/Mg2+ ecto-ATPase is an a cidic protein having two subunits. Furthermore, the enzyme shows micro heterogeneity in its molecular structure.