The occurrence of two types of fast skeletal myosin heavy chains from abdominal muscle of kuruma shrimp Marsupenaeus japonicus and their different tissue distribution.

Shrimps belong to the class Crustacea, which forms a large, diverse group in the invertebrates. However, the physiology and biochemistry of their skeletal muscles have been poorly understood compared with those from vertebrates including mammals and fish. The present study focused on myosin, the major protein in skeletal muscle, from adult specimens of kuruma shrimp Marsupenaeus japonicus. Two types of the gene encoding myosin heavy chain (MHC), a large subunit of the myosin molecule, were cloned from abdominal fast skeletal muscle and defined as MHCa and MHCb. Protein analysis revealed that the MHCa isoform was expressed at a higher level than the MHCb isoform. The full-length cDNA clones of MHCa and MHCb consisted of 5929 bp and 5955 bp, respectively, which encoded 1912 and 1910 amino acids, respectively. Both were classified into fast muscle type by comparison with the partially deduced amino acid sequences of fast-type and slow-type (S(1), slow twitch) MHCs reported previously for the American lobster Homarus americanus. The amino acid identities between MHCa and MHCb of kuruma shrimp were 78%, 60% and 72% in the regions of subfragment-1, subfragment-2 and light meromyosin, respectively, and 71% in total. In situ hybridisation using anti-sense RNA-specific probes, along with northern blot analysis using different tissues from abdominal muscle, revealed the different localisation of MHCa and MHCb transcripts in abdominal fast skeletal muscle, suggesting their distinct physiological functions.

Rheological properties of fast skeletal myosin rod and light meromyosin from walleye pollack and white croaker: contribution of myosin fragments to thermal gel formation.

Myosin rod and light meromyosin (LMM) of walleye pollack and white croaker were examined for their rheological properties by measuring dynamic viscoelastic parameters. Rods from walleye pollack and white croaker increased their storage moduli (G') in the ranges of 29-43 degrees C and 31-38 degrees C, respectively, in temperature sweep analysis. Walleye pollack LMM showed no peak of G' upon heating, whereas the white croaker counterpart exhibited a single sharp peak of G' at 35 degrees C. Loss modulus (G") showed similar temperature-dependent changes for the two fish species as the case of G', irrespective of rod and LMM, although G" values were lower than those of G'. Thus, rheological properties of rod and LMM were different between walleye pollack and white croaker. Taken together with data previously reported for myosin, it was considered that both myosin rods from walleye pollack and white croaker are attributed to thermal gel formation of myosin in a low-temperature range, though in a species-specific manner.

Characterization of the pufferfish Takifugu rubripes apolipoprotein multigene family.

We have characterized the apolipoprotein multigene family of the pufferfish Takifugu rubripes. The pufferfish mainly contains 28-kDa, 27-kDa, and 14-kDa apolipoproteins in its plasma and was designated apo-28 kDa, apo-27 kDa, and apo-14 kDa, respectively. N-terminal amino acid sequencing revealed that pufferfish apo-28 kDa and apo-27 kDa have an identical amino acid sequence except an additional propeptide in the former; and both are homologues of apoA-I from other animals. The sequence of pufferfish apo-14 kDa is homologous to that of eel apo-14 kDa previously reported, both being apparently specific to fish. In silico screening, using the publicly available Fugu genome database confirmed the pufferfish apoA-I and apo-14 kDa genes. The database further contained the genes encoding four types of apoA-IV, one apoC-II and two types of apoE. Thus, pufferfish contains nine genes encoding apolipoprotein multigene family. Two apoA-IV and one apoE genes were tandemly arrayed and located on one scaffold. Thus two sets of these genes formed two gene clusters. The apoC-II and apo-14 kDa genes are also located on a single scaffold. apoA-I and apo-14 kDa gene transcripts were mainly expressed in liver and less abundantly in brain. The transcripts of the former gene were also observed in intestine. In contrast, the transcripts encoding four apoA-IVs, one apoC-II, and two apoEs were mainly expressed in intestine. These structural details of pufferfish apolipoproteins and tissue distribution of their gene transcripts provide a novel evidence for better understanding of evolutionary relationships of apolipoprotein multigene family.

Characterization of goldfish heat shock protein-30 induced upon severe heat shock in cultured cells.

Temperature-dependent changes of growth rate and protein components were investigated for primary cultured cells derived from goldfish caudal fin. When the culture temperature was shifted from 20 degrees C to 35 degrees C and 40 degrees C, the growth rate was increased at 35 degrees C as compared with that at 20 degrees C, but no cell growth was observed at 40 degrees C. The differential scanning calorimetry demonstrated the onset of the endothermic reaction for goldfish cellular components at 40 degrees C. Therefore, the temperature shift to 40 degrees C was found to be of severe heat shock for goldfish cultured cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that, although expression of 70-kDa components was slightly induced at 35 degrees C, the temperature shift to 40 degrees C markedly induced the expression of the 30-kDa component in addition to that of 70-kDa component. The N-terminal amino acid sequencing identified the 30- and 70-kDa components to be heat shock protein (Hsp)-30 and Hsp70, respectively. Northern blot analysis revealed that the enhanced Hsp30 messenger ribonucleic acid (mRNA) levels were only observed at 40 degrees C, whereas Hsp70 mRNA was slightly accumulated at 35 degrees C. These results indicated that Hsp30 might have important functions under severe heat stress condition.

Differential scanning calorimetry and circular dichroism spectrometry of walleye pollack myosin and light meromyosin.

The thermodynamic properties of myosin and its C-terminal fragment, light meromyosin (LMM), from walleye pollack, a typical cold-water fish efficiently utilized on an industrial scale, were analyzed by using differential scanning calorimetry (DSC) and circular dichroism (CD) spectrometry. Recombinant walleye pollack LMM expressed in Escherichia coli was also subjected to DSC and CD measurements for reference. The two proteins prepared from frozen surimi showed three endothermic peaks, the transition temperatures (T(m)) of which were quite similar, although overall DSC patterns differed considerably from one another. Their alpha-helical contents determined by CD were low compared to values reported before for other species. On the other hand, recombinant LMM gave four endothermic peaks at 27.4, 30.8, 36.5, and 43.4 degrees C in DSC and showed an alpha-helical content of approximately 80%. The peak at 27.4 degrees C could not be observed in walleye pollack LMM prepared from frozen surimi and thus was possibly attributed to its C terminus, because this extreme C-terminal region is supposedly truncated during preparation of LMM by tryptic digestion.