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Biophysical characterization of rat cardiac Ca2+/Mg2+ ecto-ATPase (myoglein)
S Kannan1, V Elimban, K Dakshinamurti
1St. Boniface General Hospital Research Centre, Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Abstract:
Sarcolemmal Ca2+/Mg2+ ecto-ATPase (Myoglein; MW 180 kD) is a membrane bound enzyme which requires a millimolar concentration of either Ca2+ or Mg2+ for maximal hydrolysis of ATP. The isoelectric point (pI) of the cardiac ecto-ATPase was 5.7. The purified Ca2+/Mg2+ ecto-ATPase from the rat heart sarcolemmal appeared as a single band with MW approximately 90 kD in the SDS-PAGE. In order to understand the nature of this enzyme, the 90 kD band in the SDS-PAGE was electroeluted; the analysis of the eluate showed 2 prominent bands with MW approximately 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 pI 3.0-10.0) also showed two distinct bands. Mass spectroscopic analysis of the enzyme using different matrix combinations revealed the presence of multi-components indicating microheterogeneity in the protein structure. Treatment of the ecto-ATPase with DL-dithiothreitol did not alter the pattern of mass spectroscopic analysis and this indicated that the microheterogeneity may be due to some posttranslational modifications. It is concluded that rat cardiac Ca2+/Mg2+ ecto-ATPase is an acidic protein having two subunits. Furthermore, the enzyme shows microheterogeneity in its molecular structure.
Insights
Rat cardiac Ca2+/Mg2+ ecto-ATPase is an acidic protein with two subunits, exhibiting microheterogeneity in its molecular structure due to posttranslational modifications.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Sarcolemmal Ca2+/Mg2+ ecto-ATPase (Myoglein) is a membrane-bound enzyme crucial for ATP hydrolysis.
- This enzyme requires millimolar concentrations of Ca2+ or Mg2+ for maximal activity.
- The cardiac ecto-ATPase has an isoelectric point (pI) of 5.7.
Purpose of the Study:
- To elucidate the molecular nature and structure of rat cardiac Ca2+/Mg2+ ecto-ATPase.
- To investigate the potential microheterogeneity within the purified enzyme.
- To determine if posttranslational modifications contribute to the enzyme's structure.
Main Methods:
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess molecular weight.
- Electroelution of protein bands from SDS-PAGE for further analysis.
- Gradient gel electrophoresis and non-equilibrium linear two-dimensional electrophoresis to analyze protein components.
- Mass spectroscopic analysis to identify protein components and assess structural heterogeneity.
- Treatment with DL-dithiothreitol to investigate the role of disulfide bonds.
Main Results:
- Purified rat heart sarcolemmal Ca2+/Mg2+ ecto-ATPase initially appeared as a single ~90 kD band on SDS-PAGE.
- Electroelution and subsequent electrophoresis revealed two distinct protein bands (~90 kD and ~85 kD).
- Mass spectrometry indicated the presence of multiple components, suggesting microheterogeneity.
- DL-dithiothreitol treatment did not affect the mass spectroscopic profile, pointing towards non-disulfide bond related modifications.
Conclusions:
- Rat cardiac Ca2+/Mg2+ ecto-ATPase is an acidic protein composed of two subunits.
- The enzyme exhibits significant microheterogeneity in its molecular structure.
- Posttranslational modifications are likely responsible for the observed microheterogeneity.