Molecular characterization of rat cardiac sarcolemmal Ca2S+/Mg2S+ ecto-ATPase

Abstract

Sarcolemmal Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase (Myoglein; MW 180 kD) is a membrane bound enzyme which requires millimolar concentrations of either Ca$\sp{2+}$ or Mg$\sp{2+}$ for maximal hydrolysis of ATP. In order to elucidate the structural and functional properties of the rat cardiac sarcolemmal Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase, this protein was purified to homogeneity and utilized for the biophysical, molecular and immunochemical characterization. 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. For determining the molecular structural properties, the purified Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase was subjected to tryptic digestion and amino acid sequencing. The amino acid sequence was utilized to design an oligonucleotide probe. Screening of a rat heart cDNA library produced a partial cDNA clone (pND2.1) that was 100% homologous to human platelet CD36. A high degree of homology ($>$70%) with other cell adhesion molecules was also noted. The oligomer probe detected a 4.4 kB transcript in heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. An additional 3.1 kB transcript was detectable in heart, lung, liver and skeletal muscle while transcripts of $\sim$2.0 kB and $\sim$1.0 kB were also evident in the heart whereas ND2.1 detected a $\sim$3.1 kB transcript in cardiac tissue. To determine the immunochemical properties of the rat cardiac Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase, a polyclonal antiserum was raised against purified $\rm Ca\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase. As assessed by Western blot analysis, the antiserum as well as the purified immunoglobulin were specific for Ca$\sp{2+}$/Mg$\sp{2+}$ ATPase; no crossreaction was observed towards other membrane bound enzymes such as rat sarcoplasmic reticulum Ca$\sp{2+}$-pump ATPase or plasma membrane Ca$\sp{2+}$-pump ATPase On the other hand, the cardiac Ca$\sp{2+}$/Mg$\sp{2+}$ ATPase was not recognized by either rat SR Ca$\sp{2+}$ ATPase or liver ecto-ATPase (Ca$\sp{2+}$/Mg$\sp{2+}$ ATPase) antibodies. In this study, we have produced an antiserum which is specific for plasma membrane Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase in cardiocytes. This antiserum can localize the plasma membrane of cardiomyocytes as well as brain, skeletal muscle, liver and kidney. Western immunoblots revealed that a polyclonal antiserum raised against the purified cardiac Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase in rabbit recognized the CD36 molecule, whereas a monoclonal antibody directed against human CD36 cross-reacted with cardiac Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase, in cardiac plasma membrane preparation. On the basis of these observations, the role of the Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase in cell adhesion phenomenon was tested by carrying out a cell-cell adhesion bioassay in neonatal cardiomyocyte culture. The purified IgG fraction of the anti-cardiac Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase serum was found to depress the spreading and attachment of cardiomyocytes to their substratum. These results suggest that the rat cardiac Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase is an acidic protein having two subunits. Furthermore, Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase shows microheterogeneity in its molecular structure. It is concluded that rat cardiac Ca$\sp{2+}$/Mg$\sp{2+}$ ecto-ATPase may either contain a fragment with cell adhesion properties or is tightly associated with a protein which is homologous to the adhesion molecule CD36. (Abstract shortened by UMI.)