Abstract
We determined the full-length primary structure of the tropomyosin (TM)-1 and -2 proteins from the adductor muscle of the Japanese pearl oyster Pinctada fucata (Pifuc-TM-1 and Pifuc-TM-2), and found that they are each composed of 284 amino acid residues. We predicted the gene structure of P. fucata TM (Pifuc-TM) using Splign alignment of our cDNA with genomic sequences and elucidated that Pifuc-TM consists of 10 exons. Exons 1 - 3 and 5 - 10 are used to transcribe Pifuc-TM-1 mRNA, and exons 1 - 4 and 6 - 10 are used to transcribe Pifuc-TM-2 mRNA. Both genes share the same start and stop codons located in exon 1 and exon 10, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TM-1 gene was mainly expressed in adductor phasic muscle, and at a relatively weaker level in adductor catch muscle, whereas the Pifuc-TM-2 gene was expressed equally in both phasic and catch muscles. They were weakly expressed in gill and mantle. Immunoblot analysis using anti-Pifuc-TM-1 and anti-Pifuc-TM-2 antibodies revealed that adductor phasic muscle contained Pifuc-TM-1, while adductor catch muscle contained both Pifuc-TM-1 and Pifuc-TM-2. Differential scanning calorimetry (DSC) analysis was carried out for Pifuc-TM-1 and Pifuc-TM-2 expressed in bacteria, as well as TM purified from P. fucata phasic and catch muscle tissues (phasic-TM and catch-TM). The DSC data indicated that phasic-TM was mainly composed of Pifuc-TM-1, whereas catch-TM contained Pifuc-TM-1 and Pifuc-TM-2. These findings suggest that the distribution of Pifuc-TM-1 and Pifuc-TM-2 in adductor muscle is specific to the muscle fiber type, and reflects the properties of each.
Highlights
Bivalve adductor muscles are composed of two muscle fiber types: phasic and catch
Differential scanning calorimetry (DSC) analysis was carried out for Pifuc-TM-1 and Pifuc-TM-2 expressed in bacteria, as well as TM purified from P. fucata phasic and catch muscle tissues
These findings suggest that the distribution of Pifuc-TM-1 and Pifuc-TM-2 in adductor muscle is specific to the muscle fiber type, and reflects the properties of each
Summary
Bivalve adductor muscles are composed of two muscle fiber types: phasic and catch. The larger phasic muscle is used for quick closure of shells, whereas the smaller catch muscle is involved in the sustained closure of shells. Catch muscles can develop a long-lasting high-tension state with little energy expenditure [1]. They begin to contract following an increase in intracellular Ca2+ concentrations, which activates myosin and develops the tension. They subsequently enter the catch state once Ca2+ concentrations decrease to resting levels. We recently analyzed troponin C, a subunit of troponin, of the Japanese pearl oyster Pinctada fucata (Pifuc-TnC), and revealed that it is distributed only in phasic muscle, suggesting that troponin does not participate in the regulation of catch contraction [5]. We determined the full sequences of Pifuc-TM-1 and Pifuc-TM-2, predicted the gene structure of Pifuc-TM, and investigated the tissue distribution of the two isoforms
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