MOLECULAR-CLONING AND QUANTIFICATION OF SARCOPLASMIC-RETICULUM CA-2+-ATPASE ISOFORMS IN RAT MUSCLES

A cDNA encoding the full-length adult rat fast-twitch muscle Ca2+-adenosinetriphosphatase (ATPase) was cloned. The deduced amino acid sequence of this molecule has 97 and 90% identity with those of rabbit fast-twitch muscle and chicken skeletal muscle Ca2+-ATPases, respectively. Specific probes from the 3'-untranslated region of each sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) gene product and full-length mRNA transcript standards were used to determine the quantity of mRNA encoding each isoform in various rat muscles. Quantitative immunoblotting was also used to determine the protein content of each SERCA isoform. Fast-twitch fibers expressed both SERCA1 mRNA and protein at a level two- to fivefold higher than SERCA2 was expressed in slow-twitch fibers. We observed a protein-to-mRNA ratio that varied from approximately 500,000 molecules per molecule in the fast-twitch muscles to approximately 200,000 in cardiac and smooth muscles. There was no difference, however, between the ratio for different isoforms in the same muscle. The content of Ca2+ pump in a given muscle therefore depends on at least three factors: 1) the efficiency of gene transcription and message stability (fiber type dependent), 2) the efficiency of translation and protein stability (muscle identity dependent), and 3) fiber composition of the muscle.