Gastric proteases of the Greenland cod Gadus ogac. I. Isolation and kinetic properties

The zymogens of three gastric proteases of the Greenland cod (Gadus ogac) were isolated by exclusion chromatography and chromatofocusing. The cod zymogens were activated more rapidly at lower temperatures than porcine pepsinogen and, after activation, were further purified by exclusion chromatography. The cod proteases had more alkaline pH optima and were active over a wider range of pH than porcine pepsin. The specific activity of porcine pepsin on protein substrates was greater than that of the individual cod proteases. However, the cod proteases had cumulative activity on protein substrates that was greater than the sum of their individual activities. Cod protease 1 was active on pepsin-specific substrates, and cod proteases 2 and 3 were active as gastricsin-specific substrates. All three cod proteases had greater milk-clotting activity and hydrolysed hemoglobin to a greater extent than porcine pepsin. The Vmax and Km,app of the cod proteases were dependent upon the substrate, and Vmax/Km,app values of the cod proteases were generally lower than porcine pepsin. It is suggested that the cod proteases together exhibit broad substrate specificity and maintain activity over a wide range of conditions to enhance protein digestion in the cod stomach.     

Gastric proteases of the Greenland cod Gadus ogac. II. Structural properties

Three gastric proteases were isolated from the stomach mucosa of the Greenland cod (Gadus ogac). The cod proteases were all less stable to heating and protease 1 retained less activity at 5 degrees C when the pH was greater than 5 in comparison with porcine pepsin. The activities of cod proteases 1 and 2, with hemoglobin as the substrate, were doubled in the presence of 25 mM NaCl, while cod protease 3 and porcine pepsin were not stimulated by the salt. The cod proteases did not cross-react with antibodies raised against porcine pepsin. However, some cross-reactivity was noted with antibodies raised against proteases from psychotrophic pseudomonads. The molecular weights of all the cod proteases were in the range of 36,000-38,000. The amino acid compositions of the cod proteases as compared by the Metzger difference index differed from the mammalian gastric proteases by about the same extent that pepsin, gastricsin, and chymosin differ from each other. Of the cod enzymes, protease 1 differed from mammalian gastric proteases, while cod proteases 3 was more like chymosin with respect to amino acid composition. Cod protease 1 had the lowest hydrophobicity index and chymosin had the highest. The hydrophobicity indices of cod proteases 2 and 3 were intermediate between that of porcine pepsin and bovine chymosin. It is suggested that the Greenland cod proteases represent less differentiated forms of gastric proteases than the mammalian pepsins, gastricsins, and chymosins.     

Biology of Greenland cod,Gadus ogac,at Saqvaqjuac,northwest coast of Hudson Bay

Synopsis The distribution and relative abundance, life history parameters, food habits, and metabolic rate were determined forGadus ogac in Sagvagjuac Inlet, northwest coast of Hudson Bay (63° N). Fish were demersal, non-schooling, and distributed evenly down to 35 m depth. Growth was slow (maximum age 12 y) and mortality relatively low (0.5 y^–1).G. ogac first spawned at 2–3 y and spawned annually thereafter, in late March – early April. They tended to remain in the inlet and were not taken on the open coast. They are top carnivores, taking primarily capelin when available, benthic crustacea (crabs, amphipods) when not. The metabolic rate ofG. ogac is intermediate between the elevated rate of Arctic cod,Boreogadus saida, and eurythermal temperate species. Available data indicate they are not important in marine mammal and bird food webs. Their biology is contrasted with that of Arctic cod, which are short-lived, cryopelagic, feed on pelagic crustacea, and are an extremely important component of Arctic marine food webs.Direct reprint requests     

Cathepsin D from Atlantic cod (Gadus morhua L.) liver. Isolation and comparative studies

The isolated cathepsin D-like enzyme from Atlantic cod (Gadus morhua L.) liver was shown to be a monomer with a molecular mass of approximately 40 kDa. It was inhibited by Pepstatin A and had an optimum for degradation of haemoglobin at pH 3.0. The purified enzyme had lower temperature stability than bovine cathepsin D. Antibodies raised against the purified enzyme and against two C-terminal peptides of cod cathepsin D recognized a 40 kDa protein in immunoblotting of the samples from the purification process. Both antisera showed cross reactivity with a similar sized protein in liver from cod, saithe (Pollachius virens L.), Atlantic herring (Clupea harengus L.) and Atlantic salmon (Salmo salar L.). A protein of same size was detected in wolffish (Anarhichas lupus L.) liver with the antibody directed against the purified enzyme. This antibody also recognized the native enzyme and detected the presence of cathepsin D in muscle of cod, saithe, herring and salmon. These antibodies may be useful in understanding the mechanisms of post mortem muscle degradation in fish by comparing immunohistochemical localization and enzyme activity, in particular in cod with different rate of muscle degradation. They may also be used for comparing muscle degradation in different fish species.     

Pancreatic bile salt dependent lipase from cod (Gadus morhua): purification and properties.

The enzymatic basis for cod digestive lipolysis has been investigated. Lipase activity was found in aqueous extracts from pyloric caeca as well as in pancreatic tissue surrounding the caeca and the bile duct. A bile salt-dependent lipase (BSDL) was purified from either defatted powder of cod pyloric caeca or aqueous pancreatic extracts by combined affinity chromatography on cholate-Sepharose and gel filtration on Sephacryl S-200 HR. By SDS-PAGE analysis the molecular weight of purified cod BSDL was estimated to 60 kDa. The enzyme was totally dependent on bile salts for hydrolysis of insoluble fatty acid esters. Antiserum raised against purified cod BSDL reacted specifically with selected mammalian pancreatic BSDLs by Western blot analysis. Results presented in this paper strongly suggest that the bile salt-dependent lipase is the only pancreatic enzyme involved in lipid digestion in cod. The enzyme has been characterized and compared to human pancreatic BSDL with respect to substrate specificity, temperature- and pH-dependence and inhibitors. Both soluble and insoluble fatty acid esters were hydrolysed and the enzyme was 1,3-specific in hydrolysis of triolein. The enzyme was inhibited by di-isopropyl fluorophosphate and phenyl boronic acid, but not significantly by phenyl methyl sulfonyl fluoride. The cod BSDL is probably homologous to mammalian pancreatic BSDLs.     

Functional,bioactive and antioxidative properties of hydrolysates obtained from cod (Gadus morhua) backbones

Fish protein hydrolysates (FPH) have good and well documented functional properties. Peptides obtained from various fish protein hydrolysates have also shown bioactive and antioxidative activities.The aim of this study was to evaluate how storage and preparation of cod (Gadus morhua) backbones influence the yield, functionality, bioactivity (CGRP and gastrin/CCK related molecules) and antioxidative properties of fish protein hydrolysates. A series of hydrolysis trials have been carried out using backbones from cod that were initially fresh or frozen and further hydrolysed for different times (10, 25, 45 and 60 min). Use of fresh raw material significantly increased yield of dry FPH, gave lighter and less yellow powders with better emulsification properties. Longer time of hydrolysis gave higher FPH yield, increased degree of hydrolysis and decreased water holding capacity of the powders. Among the hydrolysis times tested, 25 and 45 min hydrolysis demonstrated the best emulsification properties.FPH have potential to enhance product stability by preventing oxidative deterioration. The DPPH scavenging activity showed that antioxidative activity of hydrolysates could be due to the ability to scavenge lipid radicals. The ability of hydrolysates to inhibit iron induced lipid oxidation was not influenced by time of hydrolysis.This work also shows that it is possible to obtain bioactive molecules from cod backbones by protein hydrolysis. The content of bioactive peptides (gastrin/CCK- and CGRP-like peptides) could make the cod hydrolysates useful for incorporation in functional foods.     

Isolation of two C-reactive protein homologues from cod (Gadus morhua L.) serum

Pentraxins are important molecules in innate defence and play a role in the acute phase response of both mammals and fish. Isolation of cod pentraxins by affinity chromatography using phosphorylcholine agarose revealed two pentraxin-like proteins, referred to as PI and PII proteins. These varied in their overall charge, pentameric and subunit molecular size, glycosylation and N-terminal amino acid sequences. The PI protein was homologous with the CRP-like pentraxin previously described in cod whereas the PII protein was a new CRP homologue, which was characterized by substantial individual heterogeneity with regard to subunit size and relative density. The results indicate considerable genetic variations in the cod pentraxins.     

Conformation and stability of elastase from Atlantic cod, Gadus morhua

Metal binding and conformational stability characteristics of psychrophilic elastase (ACE) from Atlantic cod (Gadus morhua) has been investigated. Chelation to Ca(2+) was found to be important for maintaining the biologically active conformation and for the thermal stability of the enzyme. However, presence of metal ions such as Zn(2+), Fe(3+) and Cu(2+) was found to inhibit its hydrolytic activity and so did the chelating agent EDTA. Both pH and guanidinium chloride induced denaturation of the enzyme was followed by monitoring the changes in the tryptophan fluorescence. ACE exhibited a simple two-state unfolding pattern in both acidic and basic conditions with the midpoint of transition at pH values 4.08 and 10.29, respectively. Guanidinium chloride and heat induced denaturation of the enzyme was investigated at two pH values, 5.50 and 8.00, wherein the enzyme possesses similar tertiary structure but differ in its hydrolytic activity. Guanidinium chloride induced denaturation indicated that the enzyme unfolds with a C(m) of 1.53 M at pH 8.0 and a DeltaG(H2O) of 6.91 kJ mol(-1) (28.65 J mol(-1) residue(-1)) which is the lowest reported for psychrophilic enzymes investigated till-date. However, at pH 5.50, DeltaG(H2O) value is slightly lowered by 0.65 kJ mol(-1) consistent with the observed increase in the apparent quenching constant obtained with acrylamide. On the other hand, increase in T(m) by 38.45 degrees C was observed for the enzyme at acid pH (5.50) in comparison to the heat induced unfolding at pH 8.0. The increase in the apparent T(m) has been attributed to the possible weak intermolecular association of the enzyme molecules at moderately high temperatures that is favoured by the increase in the accessible surface area / dynamics under acidic conditions. The stability characteristics of ACE have been compared with the available data for mesophilic porcine pancreatic elastase and possible mechanism for the low temperature adaptation of ACE has been proposed.     

Catalytic properties and chemical composition of pepsins from Atlantic cod (Gadus morhua)

1. Three pepsins were purified from the gastric mucosa of Atlantic cod (Gadus morhua). 2. The enzymes, called Pepsin I and Pepsin IIa and b, had isoelectric points 6.9, 4.0 and 4.1, respectively, and digested hemoglobin at a maximal rate at a pH of approximately 3. 3. They resembled bovine cathepsin D in being unable to digest the mammalian pepsin substrate N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine. 4. Specificity constants (kcat/Km) for the cod pepsins were lower than for porcine pepsin, and they expressed higher substrate affinity and physiological efficiency at pH 3.5 than at pH 2. 5. The cod pepsins are glycoproteins, and their amino acid composition resembles that of porcine cathepsin D more than that of porcine pepsin. 6. The N-terminal sequence of Atlantic cod pepsins is substantially different from that of porcine pepsin. This indicates a significant evolutionary gap between fish and mammalian pepsins.     

The spawning activity of cod, Gadus morhua L.

Aspects of the reproduction of reared cod, Gadus morhua L., with special emphasis on the females, were studied under laboratory conditions. The fecundity and condition factor were 2–5 and 1–5 times, respectively, that of wild cod. A total of 18 spawning females were kept in separate tanks/ chambers, each with one or two males. Seven of the 18 females were classified as stressed, based upon behaviour, irregular spawning intervals and low fertilization rates of the eggs. The reared cod were found to spawn 17–19 batches. The number of eggs liberated in each batch normally followed a smooth, dome-shaped curve with time. The fertilization rate was normally 100%. Egg size decreased from first to last batch and the egg dry weight decreased by about 20–30%. The reared cod showed the same egg diameter to dry weight relation as wild cod. Egg diameter of first batch and maternal fish length were significantly positively correlated. The mean spawning interval for the female and the mean water temperature during its spawning were negatively correlated. The reared cod spawned in both the night and the day for about 50–60 days.     

Spectral sensitivity of the cod,Gadus morhua L.

The spectral sensitivity of the cod was determined under both dark adapted and light adapted conditions in the laboratory. Cod were trained by cardiac conditioning to detect a difference in radiance between an image of spots and the background radiance of a screen. Thresholds for this response were measured for a range of different wavelengths, and expressed as quantum adjusted values. Electroretino‐graphic studies were also performed on the eyes of cod, and spectral sensitivity curves prepared. Under dark adapted conditions both the behavioural and e.r.g. derived curves showed greatest sensitivity in the blue/green at 490 nm, matching the absorption curve for rhodopsin. A secondary peak in the behaviourally derived curve in the green/yellow at 550 nm indicated that a population of yellow cones may be implicated with the rods in scotopic vision. Under light adapted conditions the behavioural curves showed a shift to the blue, perhaps indicating an adaption to the high red content of the illuminating source. The e.r.g. curve showed greatest sensitivity to blue/green, as in the scotopic experiments but with an enhanced response at 550 nm, indicating greater cone activity. It is suggested that there is complex interaction between rods and cones in the cod retina, both types of receptor being active over a wide range of light intensities.     

Isolation and biochemical characterisation of lipid rafts from Atlantic cod (Gadus morhua) intestinal enterocytes

Lipid rafts are glycosphingolipid/cholesterol-enriched membrane microdomains that have been extensively studied during the past two decades. Our aim was to isolate and perform biochemical characterization of lipid rafts from the intestinal brush border membrane (BBM) of Atlantic cod (Gadus morhua) to confirm their existence in a cold-water species and compare their characteristics with lipid rafts from other species in terms of lipid and protein content. To validate the isolation process, we assayed marker enzymes for subcellular organelles, including alkaline phosphatase (AP) and leucine aminopeptidase (LAP), both well-known marker enzymes for BBM and lipid rafts. All biochemical methods showed enrichment of AP in both the BBM and lipid raft fractions. Proteomic studies were performed by MALDI-TOF mass spectrometry using trypsin digested SDS-PAGE samples. Various proteins were associated with the cod intestinal lipid raft preparation such as aminopeptidase-N, prohibitin, and beta-actin. Lipid analysis with ³¹P NMR and thin layer chromatography on BBMs and lipid rafts samples gave higher content of sphingomyelin than previously reported in the BBM and lower content of phosphatidylcholine. Furthermore, sphingomyelin was highly dominant in the lipid rafts together with cholesterol. The existence of lipid rafts containing previously reported lipid raft characteristics from the cod intestine has, therefore, been confirmed in a ray-finned fish for the first time to the best of our knowledge.     

Isolation by distance in the Atlantic cod, Gadus morhua, at large and small geographic scales

Genetic isolation by distance (IBD) has rarely been described in marine species with high potential for dispersal at both the larval and adult life-history stages. Here, we report significant relationships between inferred levels of gene flow and geographic distance in the Atlantic cod, Gadus morhua, at 10 nuclear restriction-fragment-length-polymorphism (RFLP) loci at small regional scales in the western north Atlantic region (< 1,600 km) that mirror those previously detected over its entire geographic range (up to 7,300 km). Highly significant allele frequency differences were observed among eight northwestern Atlantic populations, although the mean FST for all 10 loci was only 0.014. Despite this weak population structuring, the distance separating populations explained between 54% and 62% of the variation in gene flow depending on whether nine or 10 loci were used to estimate Nm. Across the species' entire geographic range, highly significant differences were observed among six regional populations at nine of the 10 loci (mean FST = 0.068) and seven loci exhibited significant negative relationships between gene flow and distance. At this large geographic scale, natural selection acting in the vicinity of one RFLP locus (GM798) had a significant effect on the correlation between gene flow and distance, and eliminating it from the analysis caused the coefficient of determination to increase from 17% to 62%. The role of vicariance was assessed by sequentially removing populations from the analysis and was found to play a minor role in contributing to the relationship between gene flow and distance at either geographic scale. The correlation between gene flow and distance detected in G. morhua at small and large spatial scales suggests that dispersal distances and effective population sizes are much smaller than predicted for the species and that the recent age of populations, rather than extensive gene flow, may be responsible for its weak population structure. Our results suggest that interpreting limited genetic differences among populations as reflecting high levels of ongoing gene flow should be made with caution.     

Structural and kinetic properties of chymotrypsin from Atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin.

1. Two chymotrypsins with isoelectric points pI 6.2 and 5.8 were purified from the pyloric caeca of Atlantic cod using a phenyl-Sepharose column and chromatofocusing chromatography. The apparent molecular weight was 26,000 as judged by SDS-polyacrylamide gel electrophoresis and gel filtration. 2. The cod enzymes differed from bovine chymotrypsin in having a slightly higher molecular weight and more acidic pI points. N-terminal amino acid sequence analysis of cod chymotrypsin B showed considerable similarity with bovine chymotrypsin. 3. Heat stability and stability towards acidic pH were reduced in the cod enzymes. Generally, the cod and bovine chymotrypsins responded similarly to various protease inhibitors. However, the cod chymotrypsins were less sensitive to aprotinin inhibition but more sensitive towards soybean trypsin inhibitor and cysteine. 4. Kinetic properties were examined and the cod enzymes found to be more active towards both ester (N-benzoyl-tyrosine ethyl ester) and amide (N-benzoyl-tyrosine-p-nitroanilide) substrates. The observed differences in kinetic properties are indicative of an adaptive response towards the low temperature environment in which the cod lives.     

Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua)

The epidermal mucus of fish species has been found to contain antimicrobial proteins and peptides, which is of interest in regard to fish immunity. An acidic extract from the epidermal mucus of the Atlantic cod (Gadus morhua) was found to exhibit antimicrobial activity against Bacillus megaterium, Escherichia coli and Candida albicans. This activity varied significantly when salt was added to the antimicrobial assay, and was eliminated by pepsin digestion. No lysozyme activity was detected in the extract. By using weak cationic exchange chromatography together with reversed-phase chromatography, and monitoring the antimicrobial activity, we have isolated four cationic proteins from the mucus extract. Using N-terminal and C-terminal amino acid sequence analysis, together with MS, the antimicrobial proteins were identified as histone H2B (13 565 Da), ribosomal protein L40 (6397 Da), ribosomal protein L36A (12 340 Da) and ribosomal protein L35 (14 215 Da). The broad spectra of antimicrobial activities in the cod mucus and the characterization of four antimicrobial polypeptides suggest that mucus compounds contribute to the innate host defence of cod.     

Organization of the mitochondrial genome of Atlantic cod, Gadus morhua.

The mitochondrial DNA (mtDNA) from the Atlantic cod, Gadus morhua, was mapped using 11 different restriction enzymes and cloned into plasmid vectors. Sequence data obtained from more than 10 kilobases of cod mtDNA show that the genome organization, genetic code, and the overall codon usage have been conserved throughout the evolution of vertebrates. Comparison of the derived amino acid sequences of proteins encoded by cod mtDNA to the ones encoded by Xenopus laevis mtDNA revealed that the amino acid identity range from 46% to 93% for the different proteins. ND4L is most divergent while COI is most conserved. GUG was found as the translation initiation codon of the COI gene, indicating a dual coding function for this codon. The sequences of the 997 base pair displacement-loop (D-loop)-containing region and the origin of L-strand replication (oriL), are presented. Only few of the primary and secondary structure features found to be conserved among mammalian mitochondrial D-loops, can be identified in cod. Presence of CSB-2 in the D-loop-containing region and the conserved hairpin structure at oriL, indicates that replication of bony fish mtDNA may follow the same general scheme as described for higher vertebrates.