Meiko Kimura

Aspolin, a Novel Extremely Aspartic Acid-rich Protein in Fish Muscle, Promotes Iron-mediated Demethylation of Trimethylamine-N-oxide

Trimethylamine-N-oxide (TMAO) is abundant in marine fish. Formaldehyde synthesis by TMAO demethylation during storage markedly deteriorates fish meat. In the present work, we cloned the extremely aspartic acid-rich proteins from skeletal muscle of a commercially important species, walleye pollack, in the course of molecular identification of trimethylamine-N-oxide demethylase (TMAOase). One of the cDNAs, designated as aspolin1, encodes an extremely aspartic acid-rich protein of 228 amino acids which is converted to the TMAOase after processing between Ala42 and Asp43. Mature aspolin1/TMAOase protein contains 179 Asp in 186 total amino acids. The other cDNA, designated as aspolin2, has a common nucleotide sequence with aspolin1 in the 5′ part and encodes a protein which has an additional Asp polymer and a C-terminal cysteine-rich region. The amino acid sequence of the C-terminal cysteine-rich region of aspolin2 is highly homologous to the mammalian histidine-rich Ca2+-binding protein. Aspolin1/TMAOase and aspolin2 mRNA was most abundant in the skeletal muscle. A lower level of the mRNA was also detected in kidney, heart, spleen, and brain. Synthetic Asp polymer showed marked TMAOase activity in the presence of Fe2+, whereas a monomer and oligomers did not. Purified TMAOase protein bound to Fe2+ with low affinity, which may be responsible for the catalytic activity. Poly aspartic acid-Fe2+ complex generated after death would be involved in formaldehyde synthesis by the demethylation of TMAO during the storage of fish meat.

Effects of microbial transglutaminase and starch on the thermal gelation of salted squid muscle paste

The addition of microbial transglutaminase (MTGase) to salted squid muscle paste greatly strengthened the elasticity of the thermal gel, which was produced by the preferential crosslinking of myosin heavy chains through a two-step heating process that consisted of setting at 40°C and subsequent heating to 80°C or 90°C. Starch increased the breaking strength of thermal squid gels, but decreased the deformation. Thus, the starch-added gels became harder and less elastic. Although, when both MTGase and starch were added to squid muscle paste, the changes in viscoelasticity and myosin cross-linking reaction were similar to those with MTGase alone during setting, the storage and loss moduli of the paste sharply increased with an increase in starch content above 70°C. Subsequently, the thermal gel texture became more brittle or breakable than gels prepared by setting with MTGase only.

The existence of aspolin and its trimethylamine‐N‐oxide demethylating activity in the muscle of freshwater fish

Aspolin is a polyaspartic acid-like protein, which is originally isolated from walleye pollack Theragra chalcogramma muscle as trimethylamine-N-oxide (TMAO) demethylase. Although carp Cyprinus carpio muscle contains a trace amount of the enzyme substrate, TMAO, aspolin can be extracted and purified by acid treatment, successive chromatographies and polyacrylamide gel electrophoresis, and has twice the amount of that in walleye pollack muscle. Carp aspolin showed a low enzymatic activity in the presence of Fe2+ and reductants, and its Km value (100 mM) to TMAO was extremely high. It was a thermostable protein and had an unfolded conformation. The amino acid sequence of carp aspolin 1 deduced from cDNA revealed that it contained a long Asp polymer, an uninterrupted stretch of 138 Asp residues, followed by four amino acid residues, His-Glu-Glu-Leu, in C-terminus. The chain length was shorter by 42 Asp residues than that of its walleye pollack counterpart.

Thermally induced gelation of paramyosin from scallop adductor muscle

Thermally induced gelation of paramyosin from scallop smooth adductor muscle was investigated by dynamic rheological measurements under various conditions. The paramyosin thermal gel was produced at pH 6.5 and 7.2 at temperatures above 30°C through a two-step increase in storage (G′) and loss (G″) moduli; these values were higher than in gels produced from actomyosin at a high temperature. The thermal gel properties were very firm and brittle. In contrast, one main peak of G′ was observed during gelation at pH 8.0. The gel produced at pH 8.0 was more transparent and less soluble in a 6 M urea-0.5 M NaCl solution than those formed either at pH 6.5 or 7.2. These differences in the thermal gel properties are presumed to derive from the pH dependence of the gel matrix-forming process, such as oxidative cross-linking between cysteine residues, rather than from the thermal unfolding of the paramyosin molecules. The thermal gelation profile of chymotrypsin-digested paramyosin showed marked depression of G′ at high temperature.

Effects of microbial growth on the preservation of scallop muscle in a vital state

Adductor muscles dissected from live scallop Patinopecten yessoensis were stored in oxygenated artificial sea water. The initial muscle adenosine triphosphate (ATP) level, approximately 7.5 μmol/g, remained longer at 5°C than at either 0 or 10°C. The pH of sea water, decreased continuously and the consumption of dissolved oxygen increased even after muscle ATP was almost exhausted. The number of viable microbes, measured as colony-forming units (c.f.u.) in the muscle, increased to reach a plateau at approximately 107–108 c.f.u./g, while muscle ATP remained at high levels. After this time, muscle ATP sharply decreased. Antibiotics or sorbate added into the oxygenated sea water effectively inhibited both the growth of microbes and the decrease in the pH of sea water. Under these conditions, the retention period of muscle ATP was greatly extended. Thus, it seems most likely that scallop adductor muscle cells are suffocated by the limitation of oxygen supply caused by aerobic microbes grown on the surface of muscle tissue.