Molecular Cloning of Rat Cardiac Sarcolemmal Ca2+/Mg2+ectoATPase (Myoglein)

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 2.0 kb transcript in canine, rabbit and porcine heart, whereas transcripts of a 4.1 kb, 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 inE. coliproduced 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.