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 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.
Kannan
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S
St. Boniface General Hospital Research Centre, Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Elimban
V
V
Dakshinamurti
K
K
Dhalla
N S
NS
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010446:Peptide Fragments; D000251:Adenosine Triphosphatases; C027201:ectoATPase