Purification and characterization of transglutaminase from squid gill

ABSTRACT: The present study used squid gill as a source of transglutaminase (TGase) because it has extremely high TGase activity compared with other tissues. The enzyme was purified using successive chromatographies of Sephacryl S-300 and hydroxyapatite columns. The yield and purification-fold of the enzymatic activity was 12.6% and 14.1-fold, respectively. The molecular mass of the purified enzyme was estimated to be 94 kDa by using sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis. Enzyme activity was enhanced 15-fold with an increase in NaCl concentration. Although the activity was dependent on Ca²⁺ concentration, it was not sufficiently activated even by 50 mM CaCl₂ in the absence of NaCl, but could be fully activated with 10 mM CaCl₂ in 0.7 M NaCl. However, in the absence of substrates, the enzyme was rapidly inactivated. The pH and temperature optima of the enzyme were approximately pH 8.0 and 20°C, respectively. It was stable in the absence of Ca²⁺ at pH 7.5–9.0 and had a rate constant (K _D ) of 1.6 × 10^–5 s^–1 for thermal inactivation at 50°C. These results in which squid gill TGase could be activated at higher concentrations of Ca²⁺ and NaCl than at a physiological concentration, suggest that contact with seawater or body fluid seems to activate the enzyme if the tissue is disrupted.     

Purification and characterization of the amylase from a small abalone <Emphasis Type="Italic">Haliotis sieboldii</Emphasis>

ABSTRACT:   Amylase, with MW of 59 kDa, was purified from small abalone Haliotis sieboldii by ammonium sulfate fractionation, CM Sepharose Fast Flow and Sephacryl S-100 HR chromatographies. The optimal pH and temperature of purified amylase were 6.0 and 37°C, respectively. The purified enzyme was stable at pH 6.0–8.0 and low temperatures. It was activated by Ba²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, K⁺, Ag⁺, Na⁺ and Li⁺, but completely or partially inhibited by Al³⁺, Cu²⁺, Cd²⁺, Hg²⁺ and Zn²⁺. EDTA could completely inhibit, while iodoacetamide, N-ethylmaleimide and urea partially inhibit the purified amylase. According to the digestion mode of various polysaccharides, the purified enzyme was considered to be an α-amylase.     

Effects of environmental Ca2+ levels on branchial chloride cell morphology in freshwater-adapted killifish Fundulus heteroclitus

ABSTRACT: To examine the involvement of chloride cells in the uptake of Ca²⁺ in freshwater (FW) killifish, chloride cell morphology was compared in fish acclimated to different defined FW environments with Ca²⁺ concentrations of either 0.1 mM, 0.5 mM, or 2.5 mM. Numerous chloride cells were detected in whole-mount preparations of the gill filaments, which were stained with an antiserum specific for Na⁺, K⁺-ATPase. Chloride cells, located mostly on the afferent–vascular edge of the filaments, were larger at lower Ca²⁺ concentrations. Electron microscopic observations showed that in the 0.1 mM and 0.5 mM Ca²⁺ experimental groups, the apical membrane of chloride cells were flat or slightly projecting and equipped with numerous microvilli. In the 2.5 mM Ca²⁺ group, some chloride cells formed an apical pit, whereas other cells were similar to those observed in the 0.1 mM and 0.5 mM Ca²⁺ groups. Plasma osmolality decreased with decreasing ambient Ca²⁺ concentration, suggesting that environmental Ca²⁺ affects the permeability of the body surfaces. Gill Na⁺, K⁺-ATPase activity in the 0.1 mM and 0.5 mM Ca²⁺ groups were significantly higher than that in the 2.5 mM Ca²⁺ group, implying the involvement of the Na⁺–Ca²⁺ exchanger in Ca²⁺ uptake in the gills. These findings suggest that chloride cells function as the site for Ca²⁺ uptake in killifish acclimated to low Ca²⁺ environments.     

Thermal denaturation and autolysis profiles of myofibrillar proteins of mantle muscle of jumbo squid Docidicus gigas

ABSTRACT:    Jumbo squid was very similar to Japanese common squid in terms of myofibrillar Ca²⁺-, Mg²⁺- and K⁺(EDTA)-ATPase activities. Myofibrils of jumbo squid were significantly stabilized upon addition of Ca²⁺ and destabilized by increasing KCl concentration for heating. Incubation of muscle homogenate of jumbo squid induced a selective cleavage of myosin into two major fragments and the cleavage was inhibited by EDTA. Autolysis was prominent at and above 0.3 M NaCl where myosin filaments dissolve. The enzyme involved in the autolysis was proved to be unstable showing maximal autolysis rate at 25°C. Washing the homogenate partially reduced the autolysis activity.     

Purification of transglutaminase from scallop striated adductor muscle and NaCl-induced inactivation

SUMMARY: Tissue type transglutaminase (TGase) was purified from scallop striated adductor muscle with successive chromatographies of DE-52 cellulose, Sephacryl S-300, and Mono Q columns. The yield and purification of the enzymatic activity was 16.6% and 101.9-fold, respectively. The molecular mass of purified enzyme was estimated to be 95 kDa by sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis. Scallop TGase was Ca²⁺-dependent and strongly inactivated by ρ-chloromercuribenzoic acid, N -ethylmaleimide, Cu²⁺, and Zn²⁺, meaning it belongs to the thiol group of enzymes as well as being a mammalian enzyme. When scallop TGase was incubated in 0.5 M NaCl without substrate for 2 h at 20°C and pH 7.5, enzymatic activity decreased to 14.4% of its original. However, a conformational change in the TGase molecule was not detected by either fluorescent, ultraviolet, and circular dichroism spectra analyses compared to the enzyme incubated without NaCl. In addition, the enzyme inactivated by NaCl was partially recovered by the dilution of salt concentration, which means that the NaCl-induced inactivation process is reversible to some extent. These results suggest that NaCl-induced modulation of the TGase molecule occurs via a small conformational change.     

Short-term dietary supplementation with the microalga Parietochloris incisa enhances stress resistance in guppies Poecilia reticulata

Two trials were conducted to determine the effects of dietary enrichments with the microalga Parietochloris incisa , rich in arachidonic acid (ARA), on stress resistance in guppies Poecilia reticulata . The microalga was added to commercial diets as a neutral lipid (NL) extract and its fractions or as broken cells. Experimental diets were applied for a period of 14 days. In trial 1, commercial diets were supplemented with NL (containing 25 mg ARA and 0.11 mg β-carotene g⁻¹ feed), its triacylglycerol (TAG) fraction (containing 25 mg ARA g⁻¹ feed and no β-carotene) and the β-carotene fraction (containing 0.11 mg carotenoid g⁻¹ feed and minute amounts of ARA). Neutral lipid-fed fish demonstrated the highest resistance ( P <0.05) to osmotic stress (32-ppt NaCl), followed by fish fed with diets supplemented with TAG and β-carotene alone, which were more resistant than control ( P <0.05). In trial 2, fish fed diets supplemented with higher levels of broken alga (26.1 mg ARA g⁻¹ feed) were more resistant ( P <0.05) to stress as compared with fish fed lower ARA (16.3 mg g g⁻¹) or an unsupplemented control diet. We suggest a dietary supplementation with broken P. incisa cells to enhance stress resistance in guppies before a stressful event.     

Insight into nucleotide and Ca<Superscript>2+</Superscript> binding to carp α-actin

ABSTRACT:   Nucleotides and Ca²⁺ binding to α-actin prepared from ordinary skeletal muscle of carp Cyprinus carpio was studied. When bound Ca²⁺ was removed with ethylenediaminetetraacetic acid, carp α-actin denatured more rapidly than chicken α-actin. Kinetic studies of the denaturation process showed that in the absence of divalent cations, the binding constants of ATP to carp and chicken actin were 5.0 × 10⁴/M and 1.2 × 10⁵/M, respectively. Competitive binding of Ca²⁺ between actin and 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N',N'-tetraacetic acid (Quin 2) showed that affinity of Ca²⁺ for carp actin was also lower than that for chicken actin by a factor of 1.6. These results indicated that carp actin could relatively easily denature due to the low affinities of these ligands. Enthalpy changes upon ATP binding to carp and chicken actin were −65 kJ/mol and −110 kJ/mol, respectively. Thermodynamic analyses of our results revealed that the entropy change associated with ATP binding to carp actin was significantly smaller than that to chicken actin, suggesting that structural stabilization upon ATP binding was less effective in carp actin.     

Role of sodium bicarbonate on the initiation of sperm motility in the Japanese eel

ABSTRACT:   In order to find out the role of sodium bicarbonate (NaHCO₃) on the initiation of sperm motility in the Japanese eel Anguilla japonica , interactions were investigated between NaHCO₃ and various reagents (K⁺ channel blocker 4-aminopyridine [4-AP], ammonium chloride [NH₄Cl], sodium acetate and calcium chloride [CaCl₂]) that could regulate internal factors (intracellular K⁺, intracellular pH [[pH]_i] and intracellular Ca²⁺) in sperm motility. Contradictory effects of NaHCO₃ were observed (i.e. an inhibitory effect when 4-AP was absent and a promoting effect when 4-AP was present). Sodium bicarbonate inhibited the initiation of sperm motility in the Japanese eel. However, NaHCO₃ restored the motility of immotile sperm that 4-AP inhibited. The inhibitory effect of NaHCO₃ disappeared with the addition of NH₄Cl, which raised [pH]_i, but the promoting effect was not affected by [pH]_i. Although NaHCO₃ recovered motility in the presence of 4-AP, this recovery was also observed with the addition of CaCl₂ instead of NaHCO₃. In the initiation of sperm motility in the Japanese eel, two roles for NaHCO₃ are suggested: an inhibitory role relating to the regulation of [pH]_i and a promoting role relating to the uptake of another initiation factor, which could be Ca²⁺.     

Preparation of heavy meromyosin from the autolyzed squid mantle muscle homogenate

ABSTRACT:   Incubation of squid mantle muscle homogenate caused a selective cleavage of myosin into heavy meromyosin (HMM) and light meromyosin (LMM). HMM was isolated from the incubated homogenate by using ammonium sulfate fractionation. The purified HMM retained two types of light chain components. Its Mg²⁺-ATPase activity with or without F-actin showed a Ca-sensitivity. HMM was cleaved into subfragment-1 and subfragment-2 upon chymotryptic digestion with or without Ca²⁺, possessing different light chain composition. Two types of light chain component were kept intact when digested in the presence of Ca²⁺. Ca²⁺ stabilized HMM especially in a bound form to F-actin.     

Purification and properties of β-galactosidase from Tilapia intestine: Digestive enzyme of Tilapia-X

ABSTRACT:   β-galactosidase of the intestine of Tilapia nilotica was purified by ammonium sulfate precipitation, followed by PAPTG-Sepharose 4B affinity chromatography, ethylenediamineetetraacetic acid ion-exchange chromatography, polyexchanger PBE 94 chromatofocusing, and Sephadex G-100 gel filtration. β-galactosidase was found to be a single band when examined by poly-acrylamide gel electrophoresis. The purifications of β-galactosidase were 27-fold from the crude extract. β-galactosidase showed optimum activity at pH 5.0 at 40°C, and was specifically found to be able to hydrolyze p -nitrophenyl β-galactopyranoside. It degrades galactan and agarose, and produces galactose. β-galactosidase was strongly inhibited by Hg²⁺ and PCMB. β-galactosidase is considered to be secreted by the upper and middle parts of the intestine and most of the activity was detected in the intestinal juice.     

Thermal stability of carp G-actin monitored by loss of polymerization activity using an extrinsic fluorescent probe

ABSTRACT:   The thermal stability of carp G-actin was investigated by monitoring loss of actin polymerization ability. To determine the amount of native actin remaining after heat treatment, actin was labeled with a fluorescence reagent, N-(1-pyrene)iodoacetamide. The loss of polymerization ability of carp actin during heat treatment, at between 45 and 55°C, occurred faster than that of chicken actin. The inactivation rate was influenced by concentrations of ATP and Ca²⁺ in solution. With the increase of Ca²⁺ concentration, the inactivation of carp actin was markedly suppressed. Furthermore, the activation energy of the inactivation of carp actin obtained from an Arrhenius plot was similar to that of chicken actin. These results indicated that the thermal instability of carp G-actin was due to the low affinites of ATP and Ca²⁺ for carp actin described in a previous report.     

Amoxycillin resistance in Scottish isolates of Aeromonas salmonicida

Abstract. Eighty isolates of Aeromonas salmonicida , recovered from separate outbreaks of furunculosis in farmed and wild salmon in Scotland during 1988 and 1989, were examined for susceptibility to the β-lactam antibiotic amoxycillin. Susceptibility was determined in terms of minimum inhibitory concentration (MIC). All of the A. salmonicida subsp. salmonicida isolates investigated were susceptible to amoxycillin, with MICs of 0.30–1.50mg1^-1. All of the A. salmonicida subsp. achromogenes isolates tested were resistant to amoxycillin, with MICs in excess of 500mgl^-1. The A. salmonicida subsp. achromogenes produced a β-lactamase enzyme with a pI of approximately 8.0. The enzyme was inducible and its production was unaffected by plasmid curing with ethidium bromide, suggesting that resistance was chromosomal rather than plasmid mediated.     

Reduction in the IgE reactivity of Pacific mackerel parvalbumin by mutations at Ca2+-binding sites

ABSTRACT:   Parvalbumin is a sarcoplasmic Ca²⁺-binding protein of 12 kDa and represents the major fish allergen. Several peptide segments are identified as immunoglobulin E (IgE)-binding epitopes of cod parvalbumin. However, carp parvalbumin (Cyp c 1) shows a markedly reduced IgE-binding ability upon depletion of Ca²⁺, suggesting the importance of conformational epitopes associated with Ca²⁺-chelating. In this study, the IgE reactivity of Pacific mackerel Scomber japonicus parvalbumin (Sco j 1) was demonstrated to be markedly reduced (60–100% reduction) by Ca²⁺-depletion, similar to Cyp c 1. Three Sco j 1 mutants (D51A, D90A, D51/90A), with modifications in either one or both of the two Ca²⁺-binding sites, were then constructed by site-directed mutagenesis, followed by expression in Escherichia coli , and evaluated for their IgE reactivity. Interestingly, the double mutant (D51/90A), probably devoid of Ca²⁺-binding capacity, exhibited a significantly reduced IgE reactivity (equivalent to 0.0–7.5% of the IgE reactivity of natural Sco j 1). The results suggest that the IgE-binding ability of Sco j 1 largely depends on the solid conformation mediated by Ca²⁺-chelating, and that the hypoallergenic D51/90A will be a useful tool for the specific immunotherapy of fish allergy.     

Comparative study on general properties of alginate lyases from some marine gastropod mollusks

Alginate lyase (EC 4.2.2.3) is an enzyme that splits glycosyl linkages of alginate chain via β-elimination, producing unsaturated oligoalginates. This enzyme is widely distributed in herbivorous marine mollusks, brown algae, and marine and soil bacteria. In the present study, we determined the general properties and partial amino acid sequences of alginate lyases from three Archeogastropoda, i.e., Haliotis discus hannai, H. iris, and Omphalius rusticus, and one Mesogastropoda, i.e., Littorina brevicula, in order to enrich the information about functional and structural diversity in gastropod alginate lyases. The alginate lyases were extracted from hepatopancreas of these animals and purified by ammonium sulfate fractionation followed by conventional column chromatography. Single alginate lyases with molecular masses of approximately 28, 34, and 34 kDa were isolated from H. discus, H. iris, and O. rusticus, respectively. While three alginate lyases with molecular masses of 35, 32, and 28 kDa were isolated from L. brevicula. These enzymes were identified as poly(M) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate. Western blot analysis using an antiserum raised against H. discus enzyme suggested that H. iris, and O. rusticus enzymes shared similar primary/higher-order structure with H. discus enzyme, but the L. brevicula enzymes did not. H. discus, H. iris, and O. rusticus enzymes were classified to polysaccharide-lyase family-14 by the analysis of partial amino acid sequences, while the L. brevicula enzymes were not.     

Natural abundance of 15N and 13C in fish tissues and the use of stable isotopes as dietary protein tracers in rainbow trout and gilthead sea bream

For developing efficient diets, two sets of experiments examined whether the use and allocation of dietary protein can be traced by labelling with stable isotopes (¹⁵N and ¹³C) in two culture fish ( Oncorhynchus mykiss and Sparus aurata) . In the first experiment, natural abundance and tissue distribution of these isotopes were determined, by measuring the δ¹³C and δ¹⁵N values by isotopic ratio mass spectrometry, in fingerlings (14–17 g) adapted to diets differing in the percentage of fish meal replacement by plant protein sources. For both species, δ¹⁵N and δ¹³C were greater in tissues with higher protein and lower lipid content. Delta ¹⁵N of diets and tissues decreased as replacement increased, suggesting δ¹⁵N can be used as a marker for dietary protein origin. The ¹⁵N fractionation (δ¹⁵N fish − δ¹⁵N diet) differed between groups, and could thus be used to indicate protein catabolism. In the second experiment, fish (75–90 g) of each species ingested a diet enriched with ¹⁵N-protein (10 g kg⁻¹ diet) and ¹³C-protein (30 g kg⁻¹ diet). These proportions were suitable for determining that the delta values of tissue components were high enough above natural levels to allow protein allocation to be traced at 11 and 24 h after feeding, and revealed clear metabolic differences between species.     

A pharmacokinetic study of flumequine in sea bass, Dicentrarchus labrax (L.), after a single intravascular injection

The pharmacokinetic properties of flumequine following a single intravascular injection (10 mg kg^–1 fish) were studied in sea bass, Dicentrarchus labrax (L.), 120 g held at 18 °C. The absorption half life ( t _1/2α) and the elimination half life ( t _1/2β) of the drug were calculated to be 1.05 and 10.71 h, respectively. Tissue penetration of flumequine seemed to be moderate because both the apparent volume of distribution of the drug at steady-state ( V _d(ss)) and the extensive apparent volume of the central compartment ( V _c) were found to be small (1.51 and 0.626 L kg^–1). The mean residence time ( MRT ) was short (09.73 h) and the total clearance (CL_T) of the drug was rapid (0.156 L kg^–1 h^–1).     

Binding characteristics of the Zn-binding membrane protein from common carp

ABSTRACT:    This study incorporated the 43 kDa Zn-binding membrane protein isolated from common carp into liposome. The specificity and strength of the binding of ⁶⁵Zn to the 43 kDa protein-liposomes, and the binding of the ⁶⁵Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that ⁶⁵Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of ⁶⁵Zn to the protein-liposomes was detected. The binding parameter of Zn to the protein was found to be: maximum binding site (N_max), 76.7 pmole/µg protein (approx. 3 mole of Zn²⁺/mole); and equilibrium dissociation constant (Kd), 0.19 µM. Of the cations introduced (Ca²⁺, Cd²⁺,Co²⁺, Cr²⁺, Cu²⁺, Fe²⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺), only Co²⁺ competed significantly with Zn. The protein-liposomes were also found to bind specifically to laminin with a N_max of 1.1 pmole/µg laminin, and Kd of 4.79 µM. No significant protein-liposome binding occurred to other extracellular matrix proteins (fibronectin, fibrinogen or vitronectin). Furthermore, the binding was specifically inhibited by the Arg-Gly-Asp (RGD) peptide or GRGDSPG, while two other analogs (GRGESPG and GRADSPG) were without effect.     

Effects of Rhizopus extract administration on somatic growth and sexual maturation in lacustrine sockeye salmon Oncorhynchus nerka

ABSTRACT: The filamentous fungi Rhizopus , like many fungal species, possesses physiologically active substances. Rhizopus extract (RU) is reported to be effective for various aspects of growth and reproduction in many vertebrates. The effects of RU administration on body growth and plasma levels of steroid hormones were investigated in lacustrine sockeye salmon Oncorhynchus nerka . One-year-old fish were fed daily with RU (20 mg/kg feed) from July 1999 to October 2000 for 15 months. Fish were sampled every month and plasma levels of testosterone (T), 11-ketotestosterone (11-KT), estradiol-17β (E₂) and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) were measured by time-resolved fluoroimmunoassay. Body growth of RU-fed fish of both sexes increased significantly in 1⁺ and 2⁺ October, and 2⁺ January–March and July. All RU-fed males and one female matured in 2⁺ October. RU-fed 1⁺ precociously mature males showed increased plasma levels of T, 11-KT and DHP in 1⁺ October. In 2⁺ males, RU significantly elevated plasma levels of T from May to June, 11-KT from June to July, and DHP in October. In sockeye salmon, administration of RU accelerated body growth of both sexes and sexual maturation in males, suggesting physiologically active substances present in RU enhance somatic growth and sexual maturation by sex-specific mechanisms.     

Effects of dietary supplementation of alga Haematococcus pluvialis (Flotow), synthetic astaxanthin and β-carotene on survival, growth, and pigment distribution of red devil, Cichlasoma citrinellum (Günther)

Dietary carotenoids of various types and concentration can affect the pigmentation efficiency in an ornamental fish red devil, Cichlasoma citrinellum . Astaxanthin (AX) containing alga Haematococcus pluvialis , a synthetic AX, and a synthetic β-carotene (BC) were supplemented in formulated diets at two concentrations, 80 and 160 mg kg⁻¹, resulting in six pigmented diets. Formulated diet without carotenoids supplementation served as a control. These diets were fed to the fish, for 8 weeks. Astaxanthin dominated in body carotenoids deposition. Dietary BC hardly had contribution to body AX. Control fish had much lower AX content in skin, fin and muscle than fish fed pigmented diets, but had equal AX content in liver, intestine and gonad as those fish. Dietary synthetic AX had equal efficiency in depositing AX in skin and fin as natural AX but higher efficiency in gonad than natural AX. Fish fed AX supplemented at 160 mg kg⁻¹, either natural or synthetic AX, had higher AX content in skin than fish at 80 mg kg⁻¹ but had equal AX content in fin as fish at 80 mg kg⁻¹. Disregarding the treatment effects, the overall average AX content in tissue in descending order was gonad>fin≧(intestine=skin)>liver>muscle.