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.     

Trypsin from Greenland cod, Gadus ogac. Isolation and comparative properties

Trypsin(ogen) was isolated from the pyloric ceca of Greenland cod. Greenland cod trypsin catalyzed hydrolysis of N alpha-benzoyl-DL-arginine p-nitroanilide, tosyl arginine methyl ester and protein and was inhibited by the serine protease inhibitor PMSF and other well-known trypsin inhibitors. Greenland cod trypsin was more stable at alkaline pH than at acid pH; and was inactivated by relatively low thermal treatment. Like other trypsins, the enzyme was rich in potential acidic amino acid residues but poor in basic amino acid residues and had a molecular weight of 23,500; but it had less potential disulfide pairs, less alpha-helix and a lower H phi ave than other trypsins previously characterized. Reactions catalyzed by Greenland cod trypsin were not very responsive to temperature change, such that specific activity was relatively high at low reaction temperature.     

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     

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.     

Gastric emptying in cod, Gadus morhua L.: emptying and retention of indigestible solids

Particles 2 mm or smaller may be passed relatively easily through the pylorus of the cod, Gadus morhua L., but, under some conditions, 5 mm particles are held in the stomach for prolonged periods. The emptying of indigestible particles appears to be impaired by the consumption of additional meals, and prolonged retention of these particles may occur in fish fed multiple meals. The findings agree in general with those reported for mammals and the results are discussed in relation to mechanisms thought to control gastric motor activity patterns.     

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.     

Visual contrast thresholds in the cod Gadus morhua L.

The contrast discrimination of Gadus morhua L. was studied by means of a cardiac conditioning technique. Fish were trained to respond to a projected pattern of spots and the contrast of pattern and background then reduced until the minimum contrast required to elicit a response was established. This was repeated at seven different background light levels to cover the range of light conditions naturally encountered by the cod. The minimum detectable contrast decreased with increasing light level to a minimum of around 2.0%. The contrast threshold curve showed a discontinuity at a light level of approximately 8.0 × 10^-6 W sr^-1 m^-2. This was thought to be linked to the change from photopic to scotopic vision. Significant differences were found in cod taken from two different locations. Overall, cod contrast detection compared very favourably with figures available for other species including man. Using the obtained contrast discrimination figures some estimates were made of the cod sighting distance of hypothetical targets under natural conditions.     

Gastric emptying in cod, Gadus morhua L.: effects of food particle size and dietary energy content

Whole herring, Clupea harengus , were digested and emptied from cod stomachs much more slowly than meals of minced herring, showing that prey integrity is an important factor controlling emptying. Increasing the energy content of the minced herring by addition of fish meal and oil led to a reduction in the percentage of the meal emptied in the 24 h subsequent to feeding, but rates of energy throughput on the enriched herring diet were high. The physical form of the diet appears to be more important than energy content in the control of gastric emptying in cod. The results are discussed with respect to possible effects of dietary type on absorptive and metabolic processes.     

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.     

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.     

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.     

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.     

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.     

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.     

Glycogen phosphorylase from human leukocytes. Isolation and kinetic properties

Homogeneous glycogen phosphorylase from human leukocytes has been obtained. A one-step bioluminescent procedure for the enzyme activity assay has been developed. This method is based on a continuous recording of the product of the glycogen phosphorylase-catalyzed reaction using a coimmobilized multienzyme system (phosphoglucomutase, glucose-6-phosphate dehydrogenase, NADH:FMN oxidoreductase and bacterial luciferase). The method sensitivity is 10 times as high compared to earlier described methods. The Km values for glycogen (0.2 mg/ml) and phosphate (3.9 mM) at pH 7.9 were determined. AMP was shown to be the enzyme effector.     

Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11.5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.     

Effects of cod bradykinin and its analogs on vascular and intestinal smooth muscle of the Atlantic cod, Gadus morhua.

The effects of [Arg(0),Trp(5),Leu(8)]-BK (cod [Arg(0)]BK) on vascular preparations from branches of the cod celiac artery and on longitudinal smooth muscle preparations from the cod intestine were investigated. Cod [Arg(0)]BK (3 x 10(-8) M) caused a relaxation of the celiac artery precontracted with adrenaline. The relaxation was abolished by the cyclooxygenase inhibitor indomethacin, suggesting that the effect is mediated through the release of prostaglandins, but there was no evidence for the involvement of leukotrienes or nitric oxide in the response. In the intestinal preparations, cod [Arg(0)]BK produced concentration-dependent contractions (pD(2) = 8.28 +/- 0.16). Experiments with N-terminally and C-terminally truncated analogs and with alanine-substituted analogs of cod [Arg(0)]BK demonstrate that the central amino acid Gly(4) and the C-terminal amino acids Leu(8) and Arg(9) are the most important in determining the conformation of the peptide that interacts with the receptor. The results indicate that the ligand binding properties of the cod BK receptor are considerably different from the receptor present in trout tissues and may resemble those of the mammalian B(2) receptor more closely.