| Budget Amount *help |
¥15,600,000 (Direct Cost: ¥15,600,000)
Fiscal Year 1998: ¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 1997: ¥10,400,000 (Direct Cost: ¥10,400,000)
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| Research Abstract |
It has been reported that carp myosin is composed of two heavy chains of approximately 200 kDa and two pairs each of alkali (essential) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) (regulatory) light chains of approximately 20 kDa. In this study at first, light meromyosin, C-terminal one third of myosin heavy chain, was isolated from fast skeletal muscle of thermally acclimated carp and determined for N-terminal amino acid sequences. While cDNA library was constructed from carp fast muscle, PCR primers were regenerated from these sequences and used for amplification of cDNA fragments encoding myosin heavy chain subunits. Three types of cDNA clones were isolated, where the 10 and 30ﾟC types were predominating in carp acclimated to 10 and 30ﾟC, respectively. In addition, the intermediate type was found as a minor component in the 10ﾟC-acclimated carp with an intermediate feature in both DNA nucleotide and deduced amino acid sequences between those of the 10 and 300C types. The three myosin heavy chain isoforms consisted of 1,849-1,845 amino acid residues.
The cDNA library of thermally acclimated carp was screened with antibodies against skipjack myosin light chains and clones encoding carp myosin alkali light chains were isolated. The amino acid sequences of A1 and A2 light chains were deduced from cDNA nucleotide sequences, predicting 193 and 151 amino acid residues, respectively. A1 light chain contained the so-called difference peptide in the N-terminal regions, where alanine, proline and lysine are abundant. Southern blot analyses probed with cDNA clones specific to A1 and A2 light chains showed different hybridization patterns from each other, demonstrating that they are encoded by different genes. On the other hand, two types of cDNA clone encoding carp DTNB-light chain were found with identical deduced amino acid sequence consisting of 166 amino acid residues.
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