Cloning, DNA sequence, and expression of flagellins from high and low virulence strains of Edwardsiella tarda and their macrophage-stimulating activities.

Edwardsiella tarda is a causative pathogen of edwardsiellosis in fish. Our previous studies on high (NUF251) and low (NUF194) virulent strains of E. tarda demonstrated that NUF251 strain induced significantly higher levels of NO and TNF-α from fish and mouse macrophages than NUF194 strain. Subsequent studies suggested that a flagellin-like protein secreted from E. tarda might be a responsible factor for the macrophage-stimulating activities. To evaluate the activities of flagellins of E. tarda, in this study, the flagellin genes of NUF251 and NUF194 strains were isolated by PCR and cloned into pQE-30 and pCold I expression vectors, and then the recombinant flagellins of two strains were overexpressed in E. coli JM109 and pG-Tf/BL21, respectively. The molecular weight of the purified recombinant flagellins of NUF251 and NUF194 strains were estimated to be 45 kDa and 37 kDa, respectively on SDS-PAGE analysis. Referring the three-dimensional structure of Salmonella flagellin, which has been reported to have 4 domains (D0, D1, D2, and D3), high sequence homology between two flagellins of E. tarda was observed at conservative domain (D0 and D1) regions, whereas the sequences equivalent to D2 and D3 domains were different, and even equivalent to 57 amino acids were deleted in NUF194. Both recombinant flagellins induced NO production, mRNA expression level of inducible NO synthase (iNOS), and intercellular ROS production in mouse macrophage cell line RAW264.7 cells. Also, the secretion of TNF-α and its mRNA expression level were increased by treatment of both recombinant flagellins. These results indicate that the recombinant flagellins from different virulent E. tarda strains can stimulate macrophages with nearly equal levels as judged by the parameters tested, even though they are differences in the structure and molecular weight, suggesting that conservative D0 and D1 domains are sufficient structural elements for the recombinant flagellins to induce a certain level of macrophage-stimulation in vitro. Further studies are necessary focusing on the role of D2 and D3 domain regions of the recombinant flagellins as macrophage-stimulating agent as well as their influence on host immune system in vivo.

Identification and Characterization of the Larval Settlement Pheromone Protein Components in Adult Shells of Crassostrea gigas: A Novel Function of Shell Matrix Proteins.

The global decline of natural oyster populations emphasizes the need to improve our understanding of their biology. Understanding the role of chemical cues from conspecifics on how oysters occupy appropriate substrata is crucial to learning about their evolution, population dynamics, and chemical communication. Here, a novel role of a macromolecular assembly of shell matrix proteins which act as Crassostrea gigas Settlement Pheromone Protein Components in adult shells is demonstrated as the biological cue responsible for gregarious settlement on conspecifics. A bioassay-guided fractionation approach aided by biochemical and molecular analyses reveals that Gigasin-6 isoform X1 and/or X2 isolated from adult shells is the major inducing cue for larval settlement and may also play a role in postlarva-larva settlement interactions. Other isolated Stains-all-stainable acidic proteins may function as a co-factor and a scaffold/structural framework for other matrix proteins to anchor within this assembly and provide protection. Notably, conspecific cue-mediated larval settlement induction in C. gigas presents a complex system that requires an interplay of different glycans, disulfide bonds, amino acid groups, and phosphorylation crosstalk for recognition. These results may find application in the development of oyster aquacultures which could help recover declining marine species and as targets of anti-fouling agents.

A bacterial polysaccharide biosynthesis-related gene inversely regulates larval settlement and metamorphosis of Mytilus coruscus.

Larval settlement and metamorphosis is essential for the development of marine invertebrates. Although polysaccharides are involved in larval settlement and metamorphosis of Mytilus coruscus, the molecular basis of polysaccharides underlying this progression remains largely unknown. Here, the roles of the polysaccharide biosynthesis-related gene 01912 of Pseudoalteromonas marina ECSMB14103 in the regulation of larval settlement and metamorphosis were examined by gene-knockout technique. Compared with biofilms (BFs) of the wild-type P. marina, Δ01912 BFs with a higher colanic acid (CA) content showed a higher inducing activity on larval settlement and metamorphosis. Deletion of the 01912 gene caused an increase in c-di-GMP levels, accompanied by a decrease in the motility, an increase in cell aggregation, and overproduction of CA. Thus, the bacterial polysaccharide biosynthesis-related gene 01912 may regulate mussel settlement by producing CA via the coordination of c-di-GMP. This work provides a deeper insight into the molecular mechanism of polysaccharides in modulating mussel settlement.

The Flagellar Gene Regulates Biofilm Formation and Mussel Larval Settlement and Metamorphosis.

Biofilms are critical components of most marine systems and provide biochemical cues that can significantly impact overall community composition. Although progress has been made in the bacteria-animal interaction, the molecular basis of modulation of settlement and metamorphosis in most marine animals by bacteria is poorly understood. Here, Pseudoalteromonas marina showing inducing activity on mussel settlement and metamorphosis was chosen as a model to clarify the mechanism that regulates the bacteria-mussel interaction. We constructed a flagellin synthetic protein gene fliP deletion mutant of P. marina and checked whether deficiency of fliP gene will impact inducing activity, motility, and extracellular polymeric substances of biofilms. Furthermore, we examined the effect of flagellar proteins extracted from bacteria on larval settlement and metamorphosis. The deletion of the fliP gene caused the loss of the flagella structure and motility of the ∆fliP strain. Deficiency of the fliP gene promoted the biofilm formation and changed biofilm matrix by reducing ß-polysaccharides and increasing extracellular proteins and finally reduced biofilm-inducing activities. Flagellar protein extract promoted mussel metamorphosis, and ∆fliP biofilms combined with additional flagellar proteins induced similar settlement and metamorphosis rate compared to that of the wild-type strain. These findings provide novel insight on the molecular interactions between bacteria and mussels.

An ɑ2-adrenergic receptor is involved in larval metamorphosis in the mussel, Mytilus coruscus.

Metamorphosis is crucial in the life-cycle transition between the larval and juvenile stages of marine invertebrates. Although a number of agonists and antagonists of the adrenergic receptor (AR) are known to regulate larval metamorphosis in Mytilus coruscus (Mc), the molecular basis of the modulation of larval metamorphosis by the AR gene in this species remains elusive. Herein, the role of the AR gene in M. coruscus larval metamorphosis using the RNA interference technique was examined. The Mcα2AR transcript was observed to be present during the entire process of larval development and its level in the post-larvae was significantly increased compared to that in the pediveligers. Mcα2AR-knockdown resulted in a substantial reduction in the abundance of the Mcα2AR transcript and significantly inhibited the metamorphosis of M. coruscus larvae. These findings provide new insights into the molecular basis of modulation of larval metamorphosis in M. coruscus by the AR gene.

Purification and Characterization of Cathepsin B from the Muscle of Horse Mackerel Trachurus japonicus.

An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s), such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.

Transcriptional regulation of the Japanese flounder Cu,Zn-SOD (Jfsod1) gene in RAW264.7 cells during oxidative stress caused by causative bacteria of edwardsiellosis.

Edwardsiellosis is one of the most important bacterial diseases in fish, sometimes causing extensive economic losses in the aquaculture industry. Our previous studies demonstrated that the Cu,Zn-SOD (sod1) activity has significantly increased in Japanese flounder, Paralichthys olivaceus, hepatopancreas infected by causative bacteria of edwardsiellosis Edwardsiella tarda NUF251. In this study, NUF251 stimulated intracellular superoxide radical production in mouse macrophage RAW264.7 cells, which was reduced by N-acetylcysteine. This result suggests that NUF251 infection causes oxidative stress. To evaluate the regulatory mechanism of Jfsod1 at transcriptional levels under oxidative stress induced by NUF251 infection, we cloned and determined the nucleotide sequence (1124 bp) of the 5'-flanking region of the Jfsod1 gene. The sequence analysis demonstrated that the binding sites for the transcription factors C/EBPα and NF-IL6 involved in the transcriptional regulation of the mammalian sod1 gene existed. We constructed a luciferase reporter system with the 5'-flanking region (-1124/-1) of the Jfsod1 gene, and a highly increased transcriptional activity of the region was observed in NUF251-infected RAW264.7 cells. Further studies using several mutants indicated that deletion of the recognition region of NF-IL6 (-272/-132) resulted in a significant decrease in the transcriptional activity of the Jfsod1 gene in NUF251-infected RAW264.7 cells. In particular, the binding site (-202/-194) for NF-IL6 might play a major role in upregulating the transcriptional activity of the 5'-flanking region of the Jfsod1 gene in response to oxidative stress induced by NUF251 infection. These results could be provided a new insight to understand the pathogenic mechanism of causative bacteria of edwardsiellosis.

Quality improvement of threadfin bream (Nemipterus virgatus) surimi-gel using soy protein as a natural food additive.

Previously, we identified sarcoplasmic serine proteinase (SSP) as a modori-inducing proteinase from threadfin bream belly muscle. In this study, we investigated the autolytic activity of commercial threadfin bream surimi under modori-inducing conditions. High autolytic activity was detected in commercial surimi and was inhibited by a soybean trypsin inhibitor, indicating that SSP still remained in the commercial surimi. The effects of soy protein, defatted soy protein (DSP) and isolated soy protein (ISP), on SSP activity and surimi-gel properties were evaluated. The results showed that the modori phenomenon was induced at 70 °C, and that both DSP and ISP suppressed SSP activity and strengthened the breaking strength and breaking distance of the modori-induced gel. Surimi-gel with DSP performed better on gel whiteness than that of ISP, and 1 g/kg DSP had optimal gel properties. In conclusion, soy protein proved to be a good natural food additive for surimi-gel production of threadfin bream.

Identification of a Tissue Inhibitor of Metalloproteinase-2: An Endogenous Inhibitor of Modori-Inducing Insoluble Metalloproteinase from Yellowtail Seriola quinqueradiata Muscle.

The existence of an endogenous protease inhibitor (EPI) was expected from the comparison of the gel properties between washed and nonwashed yellowtail surimi gels. A possible candidate, tissue inhibitor of metalloproteinase-2 (TIMP-2), was partially purified from the soluble fraction of yellowtail muscle, and an 18 kDa protein band was detected by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions and western blot analysis. Its N-terminal amino acid sequence was determined as XSXSPAHPQQAF, with high homology to TIMP-2 from other fish species, suggesting that it was identified as yellowtail TIMP-2. Subsequently, full-length cDNA of two isoforms (TIMP-2a and TIMP-2b) was successfully cloned from yellowtail muscle. The N-terminal sequence of purified TIMP-2 completely corresponded to TIMP-2b. When the surimi gel quality decreased after spawning, the mRNA expression of TIMP-2b also decreased. Human TIMP-2 could inhibit autolysis of myofibrillar proteins from yellowtail muscle. Thus, TIMP-2b was considered the major EPI of the modori-inducing insoluble metalloproteinase in yellowtail muscle.

Impact of different enzymes on biofilm formation and mussel settlement.

Enzymes have been known to impact the biofilm forming capacity. However, how the enzymes mediate the biofilm formation and macrofouling remains little known. Here, we investigated the effects of the three kinds of proteases, four kinds of glycosidases and one kind of lipase on the detachment of biofilms of Shewanella marisflavi ECSMB14101, identified biofilm total proteins response to enzyme treatments, and then tested the effects of biofilms treated with enzymes on the settlement of the mussel Mytilus coruscus plantigrades. The results showed that the cell density of bacteria in biofilms formed at different initial bacterial density were noticeably reduced after treating with all tested enzymes, and Neutrase and α-Amylase exhibited best removing efficiency of > 90%. Bacterial total proteins in S. marisflavi biofilm noticeably reduced or disappeared after treated by Alcalase. For the settlements of the mussel M. coruscus plantigrades, inducing capacities of S. marisflavi biofilm were noticeably suppressed and downregulation was > 75% at the initial density of 5 × 106 cells/cm2. Thus, the tested enzymes could effectively remove the adhered bacterial cell, inhibit the biofilm formation and finally suppress the mussel settlement. Our findings extend novel knowledge to developing eco-friendly approach to control micro- and macro-fouling.

Purification and characterization of pepsinogens and pepsins from the stomach of rice field eel (Monopterus albus Zuiew).

Three pepsinogens (PG1, PG2, and PG3) were highly purified from the stomach of freshwater fish rice field eel (Monopterus albus Zuiew) by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephacryl S-200 HR. The molecular masses of the three purified PGs were all estimated as 36 kDa using SDS-PAGE. Two-dimensional gel electrophoresis (2D-PAGE) showed that pI values of the three PGs were 5.1, 4.8, and 4.6, respectively. All the PGs converted into corresponding pepsins quickly at pH 2.0, and their activities could be specifically inhibited by aspartic proteinase inhibitor pepstatin A. Optimum pH and temperature of the enzymes for hydrolyzing hemoglobin were 3.0-3.5 and 40-45 °C. The K (m) values of them were 1.2 × 10⁻4 M, 8.7 × 10⁻5 M, and 6.9 × 10⁻5 M, respectively. The turnover numbers (k(cat)) of them were 23.2, 24.0, and 42.6 s⁻¹. Purified pepsins were effective in the degradation of fish muscular proteins, suggesting their digestive functions physiologically.

Extracellular products from virulent strain of Edwardsiella tarda stimulate mouse macrophages (RAW264.7) to produce nitric oxide (NO) and tumor necrosis factor (TNF)-alpha.

We have previously found that high virulent strain (NUF251) of Edwardsiella tarda, but not low virulent strain (NUF194), was able to survive and multiply within Japanese flounder (Paralichthys olivaceus) peritoneal macrophages. Further studies demonstrated that NUF251 induced much higher levels of NO and TNF-alpha productions than NUF194 in both Japanese flounder peritoneal macrophages and mouse macrophage cell line RAW264.7. In this study, we examined the effects of extracellular products (ECP) from two strains of E. tarda on RAW264.7 cells in terms of the induction of NO and TNF-alpha. ECP from NUF251 stimulated RAW264.7 cells to induce NO production in a concentration-dependent manner. The activity of NUF251-ECP completely disappeared by heat-treatment (at 100 degrees C for 5 min), but it could not be removed by dialysis. Polymyxin B, an endotoxin inhibitor, had no effect on NUF251-ECP-induced NO production. These results suggest that active agents in NUF251-ECP responsible for NO induction may be heat-labile high molecular weight substances rather than the cell wall derived endotoxin like substances. Since NO synthase (NOS) inhibitor, l-NAME, suppressed NUF251-ECP-induced NO production, inducible NO synthase (iNOS) in RAW264.7 cells may be a main source of NO. Furthermore, NUF251-ECP-induced high level of TNF-alpha secretion from RAW264.7 cells. Both NO and TNF-alpha productions induced by NUF251-ECP were significantly blocked by a JNK inhibitor. In contrast to NUF251-ECP, no significant activities of NUF194-ECP to induce NO and TNF-alpha productions were detected. SDS-PAGE and subsequent proteomic analysis of ECP from both strains suggested that NUF251-specific protein, which has sequence homology with flagellin, is present in NUF251-ECP as a main component. Our results suggest that the high virulent strain (NUF251) of E. tarda may specifically produce an exotoxin capable of inducing high levels of NO and TNF-alpha from macrophages through the activation of JNK system, and most probable candidate for such exotoxin might be a flagellin-like protein.

Identification of a modori-inducing proteinase in the threadfin bream: Molecular cloning, tissue distribution and proteinase leakage from viscera during ice storage.

Previously we purified and characterized a sarcoplasmic serine proteinase (SSP) from the belly muscle of the threadfin bream as a modori-inducing proteinase. In our attempt to clarify the structure and physiological functions of SSP, we successfully cloned the full-length cDNA of SSP (ORF 726 bp). The deduced amino acid sequence of SSP (241 residues) was highly homologous to fish trypsinogen. The distribution of SSP mRNA and the proteinase activity in the tissue indicated that SSP was mainly synthesized and existed in the digestive system under physiological conditions. After ice storage of the threadfin bream without gutting, a high SSP activity was detected only in the belly muscle because of SSP leaked from the viscera. Therefore, it is desirable to use edible proteinase inhibitor to inactivate the leaked SSP during production of surimi-based products or to take effective measures to prevent the proteinase leakage during post-harvest storage.

Comparative analysis of the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) from macrophages exposed to high virulent and low virulent strains of Edwardsiella tarda.

We previously reported that high virulent strain (NUF251) of Edwardsiella tarda has an ability to prevent the production of reactive oxygen species by macrophages, and is even capable of surviving and multiplying within Japanese flounder (Paralichthys olivaceus) peritoneal macrophages, whereas the low virulent strain (NUF194) has no such ability. In this study, we found that NUF251 and NUF194 induced NO and TNF-alpha production from Japanese flounder peritoneal macrophages, and NUF251 caused faster induction of NO release and much higher level of TNF-alpha production than NUF194. In addition, similar differences between two strains in terms of the induction of NO and TNF-alpha production were also observed in mouse macrophage cell line RAW264.7 cells. Our results suggest that the potent ability to induce the production of NO and TNF-alpha from macrophages may be one of the factors responsible for the virulence of E. tarda.

Purification and characterization of a sarcoplasmic serine proteinase from threadfin bream Nemipterus virgatus muscle.

A sarcoplasmic serine proteinase (SSP) was purified from threadfin bream (Nemipterus virgatus) belly muscle by ammonium sulfate precipitation and a series of chromatographies including Q-Sepharose, Phenyl Sepharose and Superdex 200. The SSP was purified 1967 folds with a yield of 4.8%. The molecular weight of the SSP was estimated to be 43.5 kDa and 22.5 kDa on SDS-PAGE under non-reducing and reducing conditions, respectively. The N-terminal amino acid sequence of the two protein bands were determined as IVGGYEXQPYSQAHQVSLNSGY and corresponded. It is suggested that the SSP exists as a homodimer. Optimum pH and temperature were 9.5 and 50 °C, using Boc-Val-Pro-Arg-MCA as a substrate. Substrate specificity and effects of inhibitors indicated that the SSP was a trypsin-like serine proteinase. The SSP was responsible for hydrolyzing myosin heavy chain (MHC) and inducing modori phenomenon in the threadfin bream surimi gel. Thus, the SSP was considered as a modori-inducing proteinase.

Polyurethane, epoxy resin and polydimethylsiloxane altered biofilm formation and mussel settlement.

In many environments, biofilms are a major mode and an emergent form of microbial life. Biofilms play crucial roles in biogeochemical cycling and invertebrate recruitment in marine environments. However, relatively little is known about how marine biofilms form on different substrata and about how these biofilms impact invertebrate recruitment. Here, we performed a comparative analysis of a 28-day-old biofilm community on non-coated (a control glass) and coated substrata (polyurethane (PU), epoxy resin (EP) and polydimethylsiloxane (PDMS)) and examined the settlement of Mytilus coruscus plantigrades on these biofilms. PU, EP and PDMS deterred the development of marine biofilms by reducing the biofilm biomass including the biofilm dry weight, cell density of the bacteria and diatoms and chlorophyll a concentrations. Further analysis of bacterial community revealed that EP altered the bacterial community composition compared with that on the glass substrata by reducing the relative abundance of Ruegeria (Alphaproteobacteria) and by increasing the relative abundance of Methylotenera (Betaproteobacteria) and Cyanobacteria in the biofilms. However, bacterial communities developed on PU and PDMS, as well as glass and PU, EP and PDMS did not exhibit differences from each other. The M. coruscus settlement rates on biofilms on PU, EP and PDMS were reduced by 20-41% compared with those on the glass after 28 days. Thus, the tested coatings impacted the development of marine biofilms by altering the biofilm biomass and/or the bacterial community composition. The mussel settlements decreased in the biofilms that formed on the coatings compared with those on non-coated glass.

Elevated Seawater Temperatures Decrease Microbial Diversity in the Gut of Mytilus coruscus.

The gut microbial community is critical for the host immune system, and in recent years, it has been extensively studied in vertebrates using 'omic' technologies. In contrast, knowledge about how the interactions between water temperature and diet affect the gut microbiota of marine invertebrates that do not thermoregulate is much less studied. In the present study, the effect of elevated seawater temperature and diet (Isochrysis zhanjiangensis and Platymonas helgolandica var. tsingtaoensis) on the gut microbial community of the commercial mussel, Mytilus coruscus, was investigated. The 16S rRNA gene sequencing was used to characterize the microbial community in M. coruscus gut. The mortality of M. coruscus exposed to a high water temperature (31°C) increased after 3 days and the diversity of the bacterial community in the gut of live M. coruscus was significantly reduced. For example, the abundance of Bacteroides (Bacteroidetes) and norank_Marinilabiaceae (Bacteroidetes) increased in the gut of M. coruscus fed I. zhanjiangensis. In M. coruscus fed P. helgolandica, the abundance of Arcobacter (Proteobacteria) and norank_Marinilabiaceae increased and the abundance of unclassified_Flavobacteriaceae (Bacteroidetes) decreased. The results obtained in the present study suggest that high temperatures favored the proliferation of opportunistic bacteria, including Bacteroides and Arcobacter, which may increase host susceptibility to disease. Microbial community composition of the gut in live M. coruscus was not impacted by the microalgal diet but it was modified in the group of mussels that died. The present study provides insight into the potential effects on the gut microbiome and mussel-bacteria interactions of rising seawater temperatures.

Concentration-dependent suppressive effect of shrimp head protein hydrolysate on dehydration-induced denaturation of lizardfish myofibrils.

To utilize fishery waste products as functional food material, the shrimp head protein hydrolysate (SHPH) was produced from three species of shrimp wastes, Northern pink shrimp, Endeavour shrimp and black tiger shrimp, by enzymatic hydrolysis. The SHPH was used as a natural food preservative by adding to lizardfish myofibrils at concentrations ranging from 2.5% to 10%. Their effects on the state of water and the denaturation of myofibrils during dehydration were evaluated. The amount of monolayer and multilayer water in myofibrils containing SHPH were higher than those without SHPH (control). DSC analyses revealed that the amount of unfrozen water increased significantly after addition of SHPH. The Ca-ATPase inactivation rate of myofibrils containing SHPH decreased during dehydration while 5-7.5% concentrations of SHPH exhibited optimum effect regardless of the species. The results implicated that SHPH can be used as an alternative food preservative for suppressive the dehydration-induced denaturation of myofibrils.

Identification of Translin/Trax complex as a glucose response element binding protein in liver.

Previously, we found a novel protein factor in the livers of rats fed a high-carbohydrate diet, which binds to the major late transcription factor (MLTF)-like site within the glucose response element (GRE) of the liver-type pyruvate kinase (L-PK) gene [J. Biol. Chem. 274 (1999) 1100]. This factor, termed glucose response element binding protein (GRBP), exists in both liver cytosol and nucleus. In order to identify GRBP, we purified to homogeneity cytosolic GRBP from rat liver extract and identified it as a Translin/Trax heteromeric complex. Based on partial amino acid sequences, we have cloned full-length rat cDNAs of both Translin and Trax. The nuclear and the cytosolic Translin/Trax complex were both large polymers of 240 and 420 kDa, respectively. The molar ratio of Translin/Trax in the polymers was 2:1 in the liver cytosols. The nuclear and cytosolic Translin/Trax complexes as well as expressed His-tagged Translin bound to double- and single-stranded MLTF sites of the GRE of L-PK gene more avidly than to single-stranded Bcl-CL1, which was initially thought to be specific for Translin. Our findings indicate that the Translin/Trax complex constitutes the previously described GRBP, and that this complex binds the GRE of the L-PK gene with high affinity. The precise physiologic role of GRBP, however, remains unclear.

A novel type of myofibril-bound serine protease from white croaker (Argyrosomus argentatus).

Myofibril-bound serine protease (MBSP) was purified from the myofibril fraction of white croaker (Argyrosomus argentatus) muscle and its enzymatic properties were compared with other fish MBSPs. White croaker MBSP was extracted by the heat treatment of myofibrils and then purified by a series of column chromatographies on Q-Sepharose, Sephacryl S-300, hydroxyapatite and Benzamidine Sepharose. The purified MBSP migrated as a single protein band at 67 kDa in SDS-PAGE under both reducing and non-reducing conditions. It was inhibited by Pefabloc SC, soybean trypsin inhibitor (STI), aprotinin and benzamidine, and was not affected by E-64, pepstatin A and EDTA. The enzyme was most active against Boc-Phe-Ser-Arg-MCA at pH 7.0 and 50 degrees C, and preferentially hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Asp-Pro-Arg-MCA. Unlike other marine fish MBSPs, white croaker MBSP considerably hydrolyzed Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA. Some enzymatic characteristics including the molecular structure and the substrate specificity for a lysine residue at the P(1) position are quite different not only from other fish MBSPs but also from soluble serine protease obtained from white croaker muscle (MSSP). White croaker MBSP could be therefore classified into a novel type of fish muscle MBSP.