A cellulase [endo-b-1,4-D-glucanase (EC 3.2.1.4)] was isolated from the hepatopancreas of abalone Haliotis discus hannai by successive chromatographies on TOYOPEARL CM-650M, hydroxyapatite and Sephacryl S-200 HR. The molecular mass of the cellulase was estimated to be 66 000 Da by SDS/PAGE, thus the enzyme was named HdEG66. The hydrolytic activity of HdEG66 toward carboxymethylcellulose showed optimal temperature and pH at 38°C and 6.3, respectively. cDNAs encoding HdEG66 were amplified by the polymerase chain reaction from an abalone hepatopancreas cDNA library with primers synthesized on the basis of partial amino-acid sequences of HdEG66. By overlapping the nucleotide sequences of the cDNAs, a sequence of 1898 bp in total was determined. The coding region of 1785 bp located at nucleotide position 56-1840 gave an amino-acid sequence of 594 residues including the initiation methionine. The N-terminal region of 14 residues in the deduced sequence was regarded as the signal peptide as it was absent in HdEG66 protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Thus, matured HdEG66 was thought to consist of 579 residues. The C-terminal region of 453 residues in HdEG66, i.e. approximately the C-terminal three quarters of the protein, showed 42-44% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)-cellulases from arthropods and Thermomonospora fusca. While the N-terminal first quarter of HdEG66 showed 27% identity to the carbohydrate-binding module (CBM) of a Cellulomonas fimi cellulase, CenA. Thus, the HdEG66 was regarded as the GHF9-cellulase possessing a family II CBM in the N-terminal region. By genomic PCR using specific primers to the 3¢-terminal coding sequences of HdEG66-cDNA, a DNA of 2186 bp including three introns was amplified. This strongly suggests that the origin of HdEG66 is not from symbiotic bacteria but abalone itself.

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SUMMARY: The amino acid sequences of myosin rod containing subfragment‐2 (S2) and light meromyosin (LMM) were determined by cDNA cloning for walleye pollack fast skeletal myosin heavy chain. While S2 and LMM were composed of 442 and 656 amino acid residues, a total of 1937 amino acid residues accounted for the whole myosin heavy chain molecule with previously determined sequence for the subfragment‐1 heavy chain region of this fish. Both regions for S2 and LMM showed a seven‐residue repeat pattern characteristic to fibrous proteins with a coiled‐coil structure of two α‐helices, displaying a, b, c, d, e, f, and g where positions a and d were frequently occupied by hydrophobic amino acids and c and g often contained charged residues. The occurrence of a 28‐residue unit with repetitive sequence was also strongly suggested, when one and three skip residues were adopted into S2 and LMM, respectively. Thus, walleye pollack S2 and LMM consisted of 17 and 24 zones with a 28‐residue repeat rearrangement. There were several amino acid substitutions which might account for a low thermal stability of walleye pollack myosin heavy chain in comparison with the sequences of higher vertebrate counterparts. However, it seemed difficult to interpret such low stability only from the comparison in the 28‐residue repeat arrangement at the primary structure.

FTIR spectroscopy has been applied to study the coordination structures of Mg2+ and Ca2+ ions bound in Akazara scallop troponin C (TnC), which contains only a single Ca2+ binding site. The region of the COO– antisymmetric stretch provides information about the coordination modes of COO– groups to the metal ions: bidentate, unidentate, or pseudo‐bridging. Two bands were observed at 1584 and 1567 cm−1 in the apo state, whereas additional bands were observed at 1543 and 1601 cm−1 in the Ca2+‐bound and Mg2+‐bound states, respectively. The intensity of the band at 1567 cm−1 in the Mg2+‐bound state was identical to that in the apo state. Therefore, the side‐chain COO– group of Glu142 at the 12th position in the Ca2+‐binding site coordinates to Ca2+ in the bidentate mode but does not interact with Mg2+ directly. A slight upshift of COO– antisymmetric stretch due to Asp side‐chains was also observed upon Mg2+ and Ca2+ binding. This indicates that the COO– groups of Asp131 and Asp133 interact with both Ca2+ and Mg2+ in the pseudo‐bridging mode. Therefore, the present study directly demonstrated that the coordination structure of Mg2+ was different from that of Ca2+ in the Ca2+‐binding site. In contrast to vertebrate TnC, most of the secondary structures remained unchanged among apo, Mg2+‐bound and Ca2+‐bound states of Akazara scallop TnC, as spectral changes upon either Ca2+ or Mg2+ binding were very small in the infrared amide‐I′ region as well as in the CD spectra. Fluorescence spectroscopy indicated that the spectral changes upon Ca2+ binding were larger than that upon Mg2+ binding. Moreover, gel‐filtration experiments indicated that the molecular sizes of TnC had the order apo TnC > Mg2+‐bound TnC > Ca2+‐bound TnC. These results suggest that the tertiary structures are different in the Ca2+‐ and Mg2+‐bound states. The present study may provide direct evidence that the side‐chain COO– groups in the Ca2+‐binding site are directly involved in the functional on/off mechanism of the activation of Akazara scallop TnC.

Vertebrate troponin regulates muscle contraction through alternative binding of the C‐terminal region of the inhibitory subunit, troponin‐I (TnI), to actin or troponin‐C (TnC) in a Ca2+‐dependent manner. To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI. The C‐terminal fragment of Akazara scallop TnI (ATnI232−292), which contains the inhibitory region (residues 104–115 of rabbit TnI) and the regulatory TnC‐binding site (residues 116–131), bound actin‐tropomyosin and inhibited actomyosin‐tropomyosin Mg‐ATPase. However, it did not interact with TnC, even in the presence of Ca2+. These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On the other hand, ATnI130−252, which contains the structural TnC‐binding site (residues 1–30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC. Moreover, the ternary complex consisting of this fragment, troponin‐T, and TnC activated the ATPase in a Ca2+‐dependent manner almost as effectively as intact Akazara scallop troponin. Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC‐binding site to the inhibitory region of TnI. Together with the observation that corresponding rabbit TnI‐fragment (RTnI1−116) shows similar activating effects, these findings suggest the importance of the TnI N‐terminal region not only for maintaining the structural integrity of troponin complex but also for Ca2+‐dependent activation.

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Troponins which confer Ca-sensitivity to skeletal actomyosin ATPase were successfully isolated from striated and smooth adductor muscles of "Akazara" scallop (Chlamys nipponensis akazara). SDS-gel electrophoresis showed that striated and smooth adductor troponins were composed of three components having molecular weights of about 52K (52,000), 40K, and 20K, and about 40K, 21K, and 20K, respectively. The Mg-ATPase activity of actomyosin reconstituted from rabbit actin and either Akazara striated adductor myosin or smooth adductor myosin, along with the respective tropomyosin and troponin, indicated that the Ca2+ concentration required for the activation of actomyosin ATPase appeared to be favorable to myosin-linked regulation.