神澤 信行

Myosin heavy chain (MHC) from fish muscle is less resistant for heal treatment than that from mammalian. It has been reported that the degree of the thermal stability of fish MHC is different from species to species. To elucidate the causes of the difference, mRNAs are extracted from the skeletal muscle of sardine, greenling and yellowfin, and cDNA fragments (about 600 bp) encoding a C-terminal region of open reading frame were amplified by RT-PCR. The fragments obtained were cloned and sequenced. The amino acid sequences deduced from these cDNA fragments were compared with those of carp and rabbit MHC. The homology of each sequence was very high except 10 residues at the tip of C-terminal region. These data show the difference of the sequences in this region plays one of an important role for the thermal stability of the myosin molecules. To determine the full nucleotide sequence corresponding to MHC and clarify all of the causes for the difference of thermal stability of fish myosin, we constructed cDNA library from the skelatal muscle of amberjack. Relationships between amino acid sequence of MHC deduced from cDNA and thermal stability of amberjack myosin will be discussed.

Myosin was partially purified from ciliated protozoan Tetrahymena pyriformis. Tetrahymena myosin has a fibrous tail with two globular heads at one end and contains 220-kDa heavy chains. The tail length of the molecule (200 nm) is longer than that of myosins from other animals (approximately 160 nm). A sample after HPLC column chromatography containing 220-kDa peptide showed a myosin-specific K+-/NH4+-EDTA-ATPase activity. Polyclonal anti-crayfish myosin heavy chain antibody reacted with Tetrahymena 220-kDa myosin heavy chain, and monoclonal anti-pan myosin antibody reacted with Tetrahymena 180-kDa peptide. The isolated 180-kDa peptide was identified as a clathrin heavy chain. Copyright (C) 1996 Elsevier Science Inc.

Myosin and paramyosin were purified to homogeneity from deep abdominal flexor and extensor, and claw closer and opener muscles of crayfish. Myosins from fast muscle (deep abdominal flexor and extensor) consisted of one species of heavy chain and two species of light chains, Lf21 and Lf18. Myosin from slow muscle (opener) consisted of two species of heavy chains and three species of light chains, Ls31, Ls21 and Ls18. Closer myosin containing both fast and slow types of muscles contained Lf21, Lf18, Ls31, Ls21 and Ls18. The actin-Mg2+-activated, Ca2+-activated, and K+-EDTA-activated ATPase activities of the fast muscle type of myosins were much higher than those of the slow muscle type of myosin. The ATPase activities of closer myosin were in-between. The optimal KCl concentration of the K+-EDTA-activated ATPase activity of crayfish myosins was around 0.4 M as compared to 1 M in rabbit skeletal muscle. Opener myosin was contaminated with a 130 kDa protein. The latter turned out to be a paramyosin isoform. Slow type of muscle including closer muscle contained both 130 kDa and 105 kDa paramyosin isoforms. On the other hand, fast muscle expressed 110 kDa paramyosin isoform.

Myosin was purified to a homogeneity from sea sponge, Halichondria okadai. The myosin consisted of 220 kDa heavy chain, 18 kDa calcium binding light chain and 21 kDa phosphorylatable light chain. Rotary shadowed images showed the two headed myosin ( myosin II) with a 160 nm tail. The myosin was less soluble in a KCl solution as compared to rabbit skeletal myosin. The K+-stimulated and Ca2+-stimulated ATPase activities of sea sponge myosin were 0.46 and 0.07 mu mol Pi min(-1) mg(-1), respectively. The Mg2+ activated myosin ATPase activity showed no significant enhancement by the addition of rabbit skeletal muscle actin despite that the light chain was phosphorylated by myosin light chain kinase from chicken gizzard. Sea sponge myosin 18 kDa light chain bound to Ca2+ ion but was not phosphorylated like Physarum plasmodia myosin light chains.

A partial cDNA encoding 811 amino acids of connectin (titin), a giant elastic protein of muscle (3,000 kDa), was cloned from a chicken embryonic skeletal muscle cDNA library using antibodies to muscle connectin. The encoded product was the C terminal segment of connectin. The predicted sequences consisted of 5 type II motifs (immunoglobulin C2 type) separated by 5 interdomain insertions. One interdomain insertion had significant homology (RSP) to KSP repeats found in human cardiac C-terminal connectin and another had a high sequence homology to porin (67.7%; 31 amino acids).

1. Myosin was purified to homogeneity from both tentacle and body of the sea anemone, Actinia equina. 2. Sea anemone myosin was two-headed myosin (Type II), approximately 160 nm long. 3. The tail tended to bend at about 60 nm from the tip of the tail. 4. Sea anemone myosin consisted of heavy chain (about 230 kDa) and two species of light chains: 18 and 19 kDa for tentacle myosin; 17 and 22 kDa for body myosin. 5. The K+-EDTA-activated and Ca2+-activated ATPase activities were approximately 0.40 and 0.07 (tentacle), 0.39 and 0.06 (body) mumol mg-1 min-1, respectively. 6. In both myosins, the Mg2+-actin activated ATPase activities were as low as 0.02 mumol mg-1 min-1. 7. However, it was elevated to 0.055 mumol mg-1 min-1, when myosin light chain was phosphorylated by myosin light chain kinase from chicken gizzard. 8. It appears that sea anemone myosin-actin interaction was regulated by myosin light chain phosphorylation.

Electron microscopy revealed that bodywall muscle of the annelid Urechis unicinctus consisted of B type smooth muscle cells containing parallel thick (40 nm in diameter) and thin (7 nm) filaments. Myosin was extracted from Urechis bodywall muscle and purified to homogeneity. Urechis myosin, two-headed myosin, consisted of 200 kDa heavy chain and two species of light chains, 24 and 19 kDa. The ATPase activity was approximately 0.1-mu-mole/mg/min in the presence of Ca2+ or EDTA. The Mg2+-ATPase activity was remarkably enhanced from 0.005 to 0.2-mu-mole/mg/min by rabbit skeletal muscle actin. EGTA inhibited this actin-activated ATPase activity suggesting that the regulation by calcium ion is of the scallop type.