Molecular characterization and expression of the equine M(1) and M(2)-pyruvate kinase gene

To elucidate the molecular properties of the equine glycolytic enzymes equine M(1) (eM(1)) and M(2) (eM(2))-pyruvate kinase (PK), mRNAs were isolated from thoroughbred horse skeletal muscle and hair roots, respectively. The full-length eM(1) and eM(2)-PK cDNAs consist of 2,320 and 2,376 bp, respectively, containing a 1596 bp open reading frame. The cDNAs were mapped to equine chromosome 1, and the equine pyruvate kinase M (PKM) gene consists of twelve exons. Exon 9 of eM(1)-PK and exon 10 of eM(2)-PK were further investigated in five equine species. Out of 55 amino acids encoded by exon 9 in equines, Glu 418 and Lys 422, which are conserved in all PK isozymes among other vertebrates, were substituted by Gln 418 and His 422. Also, the transcriptional regulatory element(s), which have potential for involvement in alternative splicing between these exons, were completely conserved among the equines. In semi-quantitative RT-PCR analysis, strong expression of both eM(1) and eM(2)-PK mRNAs was found in skeletal muscle, heart, and brain of thoroughbred horses. In addition, the authors made the novel finding that eM(2)-PK derived from hair roots has a transcriptional start site different from that of other tissues and is more specific in its expression. These results suggest that eM(1) and eM(2)-PKs may have kinetic properties and transcriptional regulatory mechanisms different from those of other mammals.