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Updated: Aug 10, 2026
Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging
Published on: September 17, 2009
Molecular cloning of rat cardiac sarcolemmal Ca2+/Mg2+ ectoATPase (Myoglein)
S Kannan1, C Lalonde, P Zahradka
1Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, University of Manitoba, Winnipeg, Canada.
Abstract:
Rat cardiac sarcolemmal Ca2+/Mg2+ ectoATPase (Myoglein), a membrane-bound enzyme requiring millimolar concentrations of Ca2+ or Mg2+ for maximal hydrolysis of ATP, has been purified to apparent homogeneity. Tryptic digestion and amino acid sequencing was used to design an oligonucleotide probe for screening a rat heart cDNA library; this produced a partial cDNA clone (pND2.1), and sequencing of a 400 base pair portion revealed a 100% homology to human platelet CD36. Northern blotting with pND2.1 detected a 3.1 kb transcript in rat heart but not in other tissues. Interspecies expression analysis (cardiac tissue total RNA blot probed with pND2.1) detected a approximately 2.0 kb transcript in canine, rabbit and porcine heart, whereas transcripts of a 4.1 kb, approximately 3.0 kb and 2.1 kb were observed in human cardiac tissue. A rat genomic DNA Southern blot, probed with pND2.1, indicated that there was a single copy of the gene in the rat genome. Expression of the pND2.1 cDNA in E. coli produced an 89 kDa polypeptide recognized by anti-human CD36 antibody but not by anti-rat Ca2+/Mg2+ ectoATPase antibody. It is concluded that rat cardiac Ca2+/Mg2+ ectoATPase is tightly associated with a protein highly homologous to the adhesion molecule CD36.
Insights
Rat cardiac Ca2+/Mg2+ ectoATPase (Myoglein) was purified and found to be highly homologous to the adhesion molecule CD36. This enzyme plays a role in cardiac function and is associated with CD36 across species.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiology
Background:
- Rat cardiac sarcolemmal Ca2+/Mg2+ ectoATPase (Myoglein) is a membrane-bound enzyme crucial for ATP hydrolysis.
- This enzyme requires millimolar concentrations of calcium (Ca2+) or magnesium (Mg2+) for optimal activity.
Purpose of the Study:
- To purify and characterize the rat cardiac Ca2+/Mg2+ ectoATPase.
- To investigate the molecular identity and genetic basis of this enzyme.
Main Methods:
- Purification of the enzyme to homogeneity.
- Oligonucleotide probe design based on amino acid sequencing for cDNA library screening.
- Northern and Southern blot analyses for gene expression and copy number determination.
- Expression of cDNA in E. coli for protein characterization.
Main Results:
- A partial cDNA clone (pND2.1) showed 100% homology to human platelet CD36.
- A 3.1 kb transcript was detected in rat heart, with varying transcript sizes in other species' cardiac tissues.
- Southern blot indicated a single gene copy in the rat genome.
- Expressed protein was recognized by anti-human CD36 antibody.
Conclusions:
- Rat cardiac Ca2+/Mg2+ ectoATPase is tightly associated with a protein highly homologous to the adhesion molecule CD36.
- This association suggests a potential functional link between ectoATPase activity and CD36-mediated cellular processes in the heart.
