Multicatalytic proteinase in fish muscle

Proteinase II, a high-molecular-mass proteinase previously identified in white croaker skeletal muscle, was purified to apparent homogeneity by DEAE-Sephacel, phenyl-Sepharose CL 4B, and Sephacryl S-300 chromatographies. Under denaturing conditions, the enzyme dissociated into a cluster of subunits with Mr ranging from 18,000 to 26,000 and a large subunit with a Mr 60,000. The proteinase was able to hydrolyze N-terminal-blocked 4-methyl-7-coumarylamide substrates having either an aromatic amino acid (chymotrypsin-like activity) or an arginine residue (trypsin-like activity) adjacent to the fluorogenic group. The trypsin-like activity of the enzyme was inhibited by fatty acids and sodium dodecyl sulfate, whereas the chymotrypsin-like activity was stimulated by those compounds but inhibited by nonionic and cationic detergents. Several thiol reagents inhibited both proteinase II activities. However, leupeptin and Cu2+ strongly inhibited its trypsin-like activity but only slightly affected its chymotrypsin-like activity. Dithiothreitol stimulated both activities, but at different extents and in different concentration ranges. These results suggest that the enzyme is multicatalytic, having at least two different active sites.     

Multicatalytic proteinase in fish muscle

A partially active and a latent form of multicatalytic protease (MCP) were isolated from fish skeletal muscle. Both forms were inactive against protein substrates, but their activity against peptide substrates differed in one order of magnitude. The chymotrypsin-like activity of the partially active form was moderately stimulated by fatty acids and SDS, whereas its trypsin-like activity was inhibited by the same reagents. In contrast, both activities of the latent form were strongly stimulated by SDS. The chymotrypsin-like activity of the latent form was also stimulated by heating or high urea concentrations, whereas its trypsin-like activity did not change or was inhibited respectively by these treatments. These activation effects were irreversible. Pre-treatment of the latent form with SDS or urea in the absence of substrate led to its irreversible inactivation, whereas activation by pre-heating occurred in the presence or absence of substrate. These results suggest that MCP can exist in several active states with distinct properties. Studies on the distribution of MCP in fish tissues showed a much higher level of the enzyme in gonads than in any other tissue, suggesting a role of MCP in development.Abbreviations MCP multicatalytic proteinase - Suc succinyl - Bz benzoyl - Z carbobenzoxy - NMec 4-methyl-7-coumarylamide - CTAB cetyl trimethylammonium bromide     

Intracellular distribution of fish muscle alkaline proteinase

1. Intracellular distribution of a muscle alkaline proteinase was investigated on four kinds of fish. 2. The total activity of the muscle proteinase of carp, Cyprinus carpio, was larger in both myofibrillar (Mf) and microsomal (Mic) fractions than the activity in mitochondrial, lysosomal, and supernatant fractions. The activity found in Mf fraction seemed to due to the Mic enzyme which was not separated from Mf fraction. 3. The relative specific activity was mostly found in Mic fraction in the species tested. 4. The results indicate that the distribution pattern of fish muscle alkaline proteinase is different from those of cathepsin D and acid phosphatase. 5. The Mic fraction hydrolyzed Mf and sarcoplasmic proteins. The rates were 40 and 55%, respectively, of the rate when casein was used as a substrate.     

五种淡水鱼类幼鱼游泳能力的比较

为了探讨栖息于不同生境中鱼类的游泳能力和偏好游泳速度及其生理机制,本研究以中华倒刺鲃(Spinibarbus sinensis)、异育银鲫(Carassius auratus gibelio)、岩原鲤(Procypris rabaudi)、青鱼(Mylopharyngodon piceus)和胭脂鱼(Myxocryprinus asiaticus) 5种鱼的幼鱼为对象,在(25±1)℃条件下测定了5种鱼类的标准代谢率(SMR)、最大代谢率(MMR)、有氧代谢范围(MS)、临界游泳速度(U_crit)、最大匀加速游泳速度(U_cat)和偏好游泳速度(U_pref)。结果发现:5种实验鱼中,中华倒刺鲃的游泳能力最强,游泳能力较差的为青鱼和胭脂鱼; 5种鱼之间的代谢和游泳能力差异显著,其偏好游泳速度主要集中在(10～24.5cm·s^-1)区域。研究表明,鱼类游泳能力的种间差异可能主要由心鳃系统相关的呼吸能力和体型相关的游泳效率所决定。本研究提供的有关鱼类游泳能力、偏好游泳速度等资料对于鱼道设计等有一定的参考价值...     

Characterization of an alkaline proteinase of fish muscle

1. The alkaline proteinase showing pH optimum 8.0 from white croaker (Sciaena schlegeli) skeletal muscle was purified electrophoretically homogeneously (2000-fold) using a combination of DEAE-cellulose chromatography, hydroxylapatite chromatography and Ultrogel AcA 34 gel filtration. 2. It was stable for 1 hr at 50 degrees C. The molecular weight of the enzyme was estimated to be 430,000 by gel filtration, with the enzyme composed of four kinds of subunits, the chain molecular weights of which were 45,000, 48,000, 51,000 and 57,000. 3. From the effects of inhibitors, the enzyme was identified as cysteine proteinase. ATP and Cu2+ inhibited the activity 50% at 10 mM and 70% at 0.1 mM, respectively. 4. Thus the enzyme was characterized as a high molecular weight, heat-stable, alkaline cysteine proteinase (HAP). 5. The enzyme showed hardly any activity below 50 degrees C but considerable activity at around 60 degrees C against myofibrils, digesting myosin heavy chain, actin and tropomyosin. With the addition of 5 M urea the enzyme hydrolyzed myofibrils well at around 30 degrees C.     

Comparison of carbonic anhydrase activity among various species of plantlets

During plant tissue culture, the culture container is small and sealed; the concentration of CO₂ in the microenvironment is relatively low. The plantlet growth is restrained for the shortage of CO₂ in the culture container. Carbonic anhydrase is a zinc-containing metalloenzyme that catalyzes the reversible conversion of bicarbonate to CO₂. The determination of carbonic anhydrase of leaves from Atractylodes lancea (thunb.) DC, Orychophragmus violaceus (L.) O.E. Schulz, Brassica juncea (L.) Czern.et Coss. cv. Luzhousileng, Brassica campestris L. cv. Chuanyou No.8, Brassica napus L cv. Oro, Brassica carinata Braun, Raphanus sativa L. var. raphanistroides Makino and their plantlets indicates that the carbonic anhydrase activity of leaves from both plantlets and fields varies from plant species to plant species, the carbonic anhydrase activity of leaves of Atractylodes lancea (thunb.) DC is the lowest among those plants, and the leaves of all plantlets are lower in carbonic anhydrase activity than the same species of plants from fields. The comparison of the growth rates of those plantlets shows that their relative growth rates are significantly different, plantlets of Atractylodes lancea have the slowest relative growth rate among those plants, and plantlets of Brassica juncea have the greatest relative growth rate. The relationship between RGR of plantlets and their CA activities is a significant linear function. It seems that there was certain correlation between carbonic anhydrase activities of plants and their growth rates. It suggests that in vitro, the greater the carbonic anhydrase activity of plantlet is, the higher its net photosynthetic rate, and the faster its growth rate. Those results offer a foundation to a rational medium choice in plant tissue culture.     

Action of a serine proteinase from fish skeletal muscle on myofibrils

The action of a serine proteinase from fish skeletal muscle on myofibrils was studied. The enzyme was able to destroy the structural integrity of myofibrils, and to degrade both their major contractile and cytoskeletal constituent proteins. Proteolysis could be completely prevented by the addition of a trypsin inhibitor isolated from the same muscle.     

Cysteine proteinase content of rat muscle lysosomes. Evidence for an unusual proteinase activity

Lysosomal cysteine proteinases were fractionated from partially purified rat muscle lysosomes. By gel filtration on Sephadex G75, cathepsin D was separated from two thiol-requiring proteolytic fractions of Mr 25 000 and 55 000, respectively. By chromatofocusing, the first fraction (Mr = 25 000) was resolved into three isoenzymic forms of cathepsin H, eluted at pH 5.8, 6.0 and 7.2, respectively, and two isoenzymic forms of cathepsin B, eluted at pH 5.5 and 5.25. Cathepsin H isoenzymes hydrolyzed Arg-NNap and BANA, were totally inhibited by 1 mM p-CMB and only to 60% by 5.10(-5) M leupeptin. The two forms of cathepsin B which degraded Z-Phe-Arg-NMec, Z-Arg-Arg-NNap and BANA were very sensitive to p-CMB and leupeptin. In addition to cathepsins B and H, a typical cathepsin-L- like activity was found in this fraction but only as a very minor component. The high Mr fraction (Mr = 55 000) contained a cysteine proteinase hydrolyzing, at pH 6.0, Z-Phe-Arg-NMec, and to a lesser extent Z-Arg-Arg-NNap and BANA. Unlike cathepsins B and H, it was very sensitive to p-CMB and HgCl2 and was fully activated only in the presence of 10 mM DTT, and inhibited to 93% by 2.10(-8) M leupeptin. By chromatofocusing, it was resolved into several isoenzymatic forms, eluted between pH 5.8 and 4.0.     

Acid proteinase activity in fish II. Purification and characterization of cathepsins B and D from Mujil auratus muscle

Two cathepsins were detected in Mujil auratus muscle extracts. They were classified as a thiol- and aspartyl-proteinase (cathepsins B and D, respectively) on the basis of their catalytic behaviour in presence of specific inhibitors. Following extraction in 1% KCl, the proteinases were purified by autolysis, acetone fractionation, affinity chromatography, and gel permeation chromatography. The haemoglobin-agarose column chromatography allowed us to separate the two activities. Sephadex G-75 column chromatography resulted in apparent molecular weights of 25,000 (cathepsin B) and 35,000 (cathepsin D). The molecular size, together with pH-activity profiles and kinetic parameters are similar to those reported for mammalian cathepsins B and D. This was not the case with the temperature-activity profiles, the optimum temperature as well as the heat stability being higher for fish cathepsins than for those obtained from other sources. Cathepsin B was characterized by its ability to inactivate aldolase. Fluorescence quenching experiments showed that tryptophyl residues of cathepsin B were less occluded and located in a more electronegative microenvironment that those pertaining to cathepsin D.     

Purification and characterization of a latent form of multicatalytic proteinase from fish muscle.

1. A latent form of multicatalytic proteinase (MCP) was purified to apparent homogeneity from white croaker muscle by DEAE-Sephacel, Mono-Q, Sephacryl S-300 and second Mono-Q chromatographies. 2. The enzyme preparation was electrophoretically and immunologically similar to MCP purified from the same source by a different method (Folco et al., 1988b, Archs Biochem. Biophys. 267, 599-605) but showed much lower chymotrypsin- and trypsin-like activities. 3. These activities responded to sodium dodecyl sulphate (SDS), urea and heat treatments in different ways: SDS stimulated both activities, urea stimulated the former and inhibited the latter and heating stimulated the former and did not affect the latter. 4. The stimulation of chymotrypsin-like activity by the three treatments was irreversible. 5. Exposure of MCP to SDS or urea in the absence of substrate rapidly inactivated it, whereas heat activation took place irrespective of the presence of substrate. 6. The stimulating effect of SDS on chymotrypsin-like activity was lost in the presence of urea. 7. These results suggest that the enzyme may be activated by different mechanisms.     

Arginase activity in different fish species and tissues

• 1.1. Arginase activity was measured in different tissues from eight species of fish.
• 2.2. Spur dogfish showed a very high arginase activity compared with the other species analysed.
• 3.3. The activity in teleosts was mainly found in tissues of high metabolic activity (liver, kidney and red muscle).

     

Interspecific trophic relationships among pelagic fish species underneath FADs

Fortnightly experimental purse-seine hauls at fish aggregation devices (FADs) and open water control sites, over a 2-year period in oceanic waters o. the eastern coast of Majorca revealed that carangid, coryphaenid, serranid, balistid and centrolophid fishes caught there were mostly planktivores. Most of the species had a high food intake. The dominance of neustonic and holoplanktonic epipelagic prey could indicate a direct link between FADs, invertebrates (biofouling) and fish. Polyprion and Schedophilus were more generalist predators than the more specialized Naucrates and Trachurus spp. There was low variation in feeding intake and the types of prey categories important for each species. Naucrates , Coryphaena and Schedophilus characterized the autumn community under FADs, while Trachurus , Seriola and Balistes were present throughout the summer. There was little diet overlap among the species suggesting only limited competition for the food resources among Trachurus spp, Naucrates and Seriola , and among Seriola and Coryphaena.     

Activation of an alkaline proteinase from fish skeletal muscle by fatty acids and sodium dodecyl sulphate

1. Fish skeletal muscle contains an alkaline thiol proteinase with a temperature optimum of 60 degrees C and undetectable activity below 50 degrees C. 2. The present study shows that fatty acids and sodium dodecyl sulphate (SDS) shifted the temperature-activity curve of the enzyme toward the lower temperature side. 3. All unsaturated fatty acids tested strongly stimulated proteolytic activity at 37 degrees C, whereas myristic acid was the only saturated fatty acid that produced an important degree of activation. 4. These effects could be observed at millimolar concentrations of the reagents.     

Delta-8 desaturation activity varies among fatty acyl desaturases of teleost fish: high activity in delta-6 desaturases of marine species

The benefits of dietary fish and fish oil are derived from n-3 long-chain polyunsaturated fatty acids (LC-PUFA) that have beneficial effects in a range of human diseases and pathologies such as cardiovascular and other inflammatory disorders, neural development and neurological pathologies. The precursor of n-3 LC-PUFA, 18:3n-3 does not have the same beneficial effects prompting interest in the pathways of endogenous synthesis of LC-PUFA in vertebrates. The LC-PUFA biosynthesis pathway classically involves Δ6 and Δ5 fatty acyl desaturases (Fad), but it was recently shown that Δ6 Fad in mammals also displayed Δ8 activity demonstrating a possible alternative "Δ8-pathway" for the synthesis of LC-PUFA. Our primary hypothesis was that Δ8 desaturase activity would be a common feature of vertebrate Δ6 Fads, and so the aim of the present study was to determine the ability of teleostei Fads for Δ8 desaturation activity. To this end, cDNAs for Fads from a range of freshwater, diadromous and marine teleost fish species were assayed for Δ8 activity in the heterologous yeast expression system. In summary, the present study has demonstrated that Δ8 desaturation activity was also a characteristic of fish orthologs, although the activity varied notably between freshwater/diadromous and marine fish species, with the latter possessing Fads2-like proteins with Δ8 activity far higher than mammalian FADS2. The data showed that, generally, the fish Fad are technically υ-3 desaturases, with new double bonds introduced 3C beyond a pre-existing double bond. However, the ability of zebrafish and rabbitfish Fads, previously characterised as Δ6/Δ5 bifunctional desaturases, to introduce non-methylene interrupted double bonds in 20:3n-3 and 20:2n-6 suggested that a novel combination of regioselectivity modes operates within these enzymes.     

Antibacterial activity of marine macroalgae against fish pathogenic Vibrio species

In mariculture, diseases of microbial origin can cause significant economic losses worldwide; the evolution of microorganism resistance to antibiotics has resulted in a growing need for new antibacterial compounds that are effective in veterinary medicine and characterized by limited undesirable side effects. Increased attention has recently been turned to seaweeds as a promising source for metabolites with antimicrobial activity. Vibriosis is a common disease, caused by bacteria of the genus Vibrio, that can result in high mortality in aquaculture. The aim of this study was to identify seaweeds with antibacterial activity against some pathogenic Vibrio species, in order to identify a possible alternative to the commonly used antibiotics in aquaculture. Chloroform/methanol lipidic extracts of six seaweed species (Chaetomorpha linum, Cladophora rupestris, Gracilaria dura, Gracilaria gracilis, Gracilariopsis longissima, Ulva prolifera) were tested for their antibacterial activities against six fish pathogenic Vibrio species using the disc diffusion method. Different susceptibilities to lipidic algal extracts were observed. All six of the seaweed extracts tested demonstrated inhibition of Vibrio ordalii. The best was that from Gracilariopsis longissima, showing activity against Vibrio ordalii, Vibrio salmonicida, Vibrio alginolyticus and Vibrio vulnificus. The results confirmed the potential use of seaweed extracts as a source of antibacterial compounds or as a health-promoting feed for aquaculture.     

Variation in posing behaviour among fish species visiting cleaning stations

The adaptive significance of posing behaviour by fish visiting cleaning stations on a Barbadian fringing reef was investigated. The probability of a visitor being cleaned by cleaning gobies ( Elacatinus spp.) was significantly higher after posing than after failing to pose upon arrival at a cleaning station. Despite this, not all visitors posed, and there was much variation among species in tendency to pose. This interspecific variation was not related to the probability of being cleaned, either after posing or after failing to pose, nor was it related to trophic level or fish total length. The latter was true both for cross-species analyses and phylogenetically independent contrasts. A cost-benefit model is proposed to understand interspecific variation in posing behaviour, which considers both decisions by clients and by cleaners. As well as explaining the results, this may reconcile differences among anecdotal and experimental observations from previous studies.     

Serine proteinase inhibitors of seminal plasma of teleost fish: distribution of activity, electrophoretic profiles and relation to proteinase inhibitors of blood

Anti-proteinase activity has been found in seminal plasma of eight teleost fish species: brown trout, rainbow trout, brook trout, lake whitefish, bream, northern pike, Danube salmon and burbot. This activity correlated with seminal plasma protein and sperm concentrations. Using a mammalian (bovine) trypsin for detecting proteinase inhibitors it was found for the first time that there are species-specific electrophoretic profiles of anti-proteinase activity. One to three bands could be identified by this method. However, additional proteinase inhibitors could be identified by using fish (cod) trypsin. These inhibitors were detected in seminal plasma of salmonids and coregonids and have a slow migration rate. Fast-migrating proteinase inhibitors were present in rainbow, brown and brook trout, northern pike, whitefish and burbot. These inhibitors could be detected in brook and brown trout by using either trypsins. However, they were detected only with bovine trypsin in rainbow trout, northern pike, whitefish and burbot. These results suggest that multiple forms of serine proteinase inhibitors exist in seminal plasma of teleost fish and they differ in their affinity toward serine proteinases. Seminal plasma serine proteinase inhibitors of rainbow trout migrated during electrophoresis similarly to blood plasma proteinase inhibitors, and suggests that the two inhibitors may be similar or the same. Anti-proteinase specific activity was similar in blood and seminal plasma. Proteinase inhibitors of fish seminal plasma seem to be an important part of sperm physiology, possibly related to protection of spermatozoa. Staining for detection of serine proteinase inhibitors also allowed detection of presence of nonspecific esterase in seminal plasma of most species.     

Patterns in the species composition of fish assemblages among Wisconsin streams

Synopsis To better understand patterns of fish assemblage composition in Wisconsin streams in relation to major environmental gradients, I carried out multivariate direct gradient analysis (canonical correspondence analysis) of two large independent datasets on fish species abundance in Wisconsin streams. Analysis of the two datasets yielded similar results, suggesting that observed patterns and relationships were real. Stream sites were distributed along fish species-environment gradients, but segregation into distinct stream temperature and geographic groups was also evident. The strongest gradient in both datasets was related to summer water temperature patterns, and encompassed a transition from small, coldwater streams dominated by salmonids, cottids, certain cyprinids, and few other species, to both small and large, warmwater streams dominated by a high diversity of different cyprinids, catostomids, ictalurids, centrarchids, and percids. A second gradient in both datasets was complex but largely geographic. Within it, sites from each of the four ecoregions that occupy Wisconsin formed fairly discrete groups. The strongest differences were between sites in the two southern Wisconsin ecoregions, the Driftless Area and the Southeastern Wisconsin Till Plains, that were dominated by certain cyprinids, ictalurids, and centrarchids, and sites in the two northern Wisconsin ecoregions, the North Central Hardwood Forests and the Northern Lakes and Forests, that were dominated by a different set of cyprinids and ictalurids, plus some petromyzontids, salmonids, catostomids, and percids. Sites from the Driftless Area that were mostly higher-gradient (steep stream slope) and had many riffle-dwelling species could also be distinguished from sites in the Southeastern Wisconsin Till Plains that were mostly lower-gradient and had many pool-dwelling species. The patterns of fish assemblage composition among sites and the associated fish species-environment relationships that were revealed by the analyses provided a framework for developing an ecologically meaningful hierarchical classification of Wisconsin stream sites based on stream thermal regime, ecoregion, stream size, and stream gradient.     

Arsenolipids show different profiles in muscle tissues of four commercial fish species

Identification of arsenolipids in biological samples is today a challenge and in particular the need for speciation data for toxicological assessment. Fish is one of the major contributors of arsenic in diet. However, the majority of work in this area has only focused on the water soluble compounds. The aim of this study is to provide some data on total arsenic and in particular to gain insights into the types of arsenolipids in the muscle tissues of four commercial and commonly consumed fish species. Determination of total arsenic was carried out by ICP-MS following microwave-assisted acid digestion of the samples and the concentrations found for total arsenic in the muscles ranged from 4.8 to 6.0 μg/g d.w. Sequential extraction was carried out using hexane and MeOH/DCM followed by reversed phase HPLC-ICP-MS/ESI-MS analysis of the MeOH/DCM fraction. Eight arsenolipids including three arsenic fatty acids (AsFAs) and five arsenic hydrocarbons (AsHCs) were identified. The result showed that fish with higher arsenolipid (AsLp) content (brill and sardine) are dominated by AsHC, while those with the smaller proportion of AsLp (mackerel and red mullet) have predominately arsenic in the form of AsFA.