Characterization of yellowfin tuna (Thunnus albacares, Scombroidei) tryptophan hydroxylase.

Tryptophan hydroxylase (EC 1.14, 16.4) was purified from yellowfin tuna liver and properties of this enzyme were compared with those of tryptophan hydroxylase from some other species (mouse mastocytoma and rat brain-stem). The molecular weight of the yellowfin tuna enzyme was estimated to be about 280,000 Da. This value is similar to that for the enzymes from mouse mastocytoma and rat brain-stem. On SDS-polyacrylamide gel electrophoresis analysis, yellowfin tuna enzyme was estimated to be about 96,000 Da. This value is different from that for the enzymes from mouse mastocytoma (53,000 Da) and rat brain-stem (59,000 Da) and suggests that yellowfin tuna enzyme may be a dimer of identical subunits of Mr 96,000 Da.

Two kinds of neutral serine proteinases in salted muscle of anchovy, Engraulis japonica.

Two kinds of proteinases, type-I and type-II, were purified or partially purified from salted muscle of anchovy, Engraulis japonica. Mol. wts. of type-I and type-II proteinases were estimated to 25,000 and 37,000, respectively, on electrophoretic analysis. Both proteinases strongly hydrolyzed synthetic tri or tetrapeptide substrates specific to trypsin, alpha-thrombin, and an activated protein C, while they hardly hydrolyzed Arg-MCA and benzoyl Arg-MCA derivatives. The proteinases were inhibited by common trypsin inhibitors. Optimal pH for the proteinase activities were pH 6.8 (type-I) and pH 7.0 to 7.5 (type-II), and the proteinases showed the highest activities at 45 degrees C (type-I) and 50 degrees C (type-II). The N-terminal amino acid sequence of type-I proteinase, 1I-2V-3G-4G ... (29 residues were identified), was significantly similar to sequences of trypsins and tryptases. Based on these findings, both proteinases were presumed to be kinds of tryptases in E. japonica muscle.

Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli.

An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose.

Liver 3-hydroxyanthranilic acid oxygenase activity in rainbow trout (Oncorhynchus mykiss).

1. In rainbow trout, 3HAA activity was comparable with those of terrestrial animals; 3HAA:PC activity ratio suggests ineffective conversion of tryptophan to niacin. 2. Inactivation as well as reactivation under different conditions was investigated. 3. Some characteristics of the enzyme extract were studied with the aim of optimizing assay in fish.

Purification and characterization of liver arylformamidase in rainbow trout and cattle.

1. Arylformamidases were purified from the liver of rainbow trout and cattle. 2. Optimal pH's were 7.7 and 8.0 for the fish and cattle enzyme, respectively. Both enzymes showed optimum temperature at 40 degrees C. Thermal and pH stability ranges and Arrhenius plots of the enzymes differed. 3. Km and molecular weight of the fish enzyme were determined to be 0.28 mM and 32,700, respectively, while those for the cattle enzyme were 0.78 mM and 42,5000, respectively.

Inhibition studies on liver arylformamidases of rainbow trout and cattle.

The effects of L-tryptophan, L-kynurenine, 3-hydroxy-L-kynurenine, ascorbate, some amino acids, 5-hydroxy-L-tryptophan, anthranilate, sodium bisulfite, EDTA, divalent ions and ionic strength on purified liver arylformamidases of rainbow trout and cattle were investigated.

Enzymatic properties of glyceraldehyde-3-phosphate dehydrogenase from fish muscle.

1. Glyceraldehyde-3-phosphate dehydrogenase was isolated from the ordinary muscle of red sea bream Pagrus major, Pacific mackerel Scomber japonicus and carp Cyprinus carpio by ammonium sulfate fractionation, followed by DEAE-Sepharose CL-6B and DEAE-cellulose column chromatography and Sephadex G-150 gel filtration, and examined for enzymatic properties. 2. Their optimum pH values in the backward reaction ranged from 7.8 to 8.2, and Km values from 1.56 to 1.90 mM. 3. Irrespective of the species of fish, the enzymatic activity was non-competitively inhibited by inorganic phosphate in the backward reaction. Divalent metal ions were not necessary to activate these glyceraldehyde-3-phosphate dehydrogenases. In the presence of 1 mM Zn(2+), these enzymes showed relative activities of 42-64% the activities measured in the absence of those ions. 5. Thermal stability of carp enzyme was higher than those of red sea bream and Pacific mackerel; the enzyme activity of the latter two species was almost lost on incubation at 45 degrees C for 10-20 min, whereas carp enzyme retained half the activity even when incubated at 60 degrees C for 30 min.

Enzymatic properties of fish muscle aldolase.

1. Aldolases were isolated from the ordinary muscle of red sea bream Pagrus major, Pacific mackerel Scomber japonicus, and carp Cyprinus carpio by ammonium sulfate fractionation, followed by ion-exchange chromatography on DEAE-cellulose and CM-Sepharose CL-6B columns, and examined for enzymatic properties. 2. The aldolases showed the highest activity in a pH range from 6.8-7.8 Km values for fructose-1,6-bisphosphate ranged from 0.025-0.10 mM. 3. Irrespective of fish species, aldolase activity was inhibited by ATP, ADP, and AMP. ATP showed the strongest inhibition and was competitive with fructose-1,6-bisphosphate. 4. The aldolases did not require divalent metal ions for activation and were completely inhibited at 0.1 mM Cu2+. 5. Thermal inactivation of the enzymes was of the first-order reaction. Red sea bream, Pacific mackerel and carp enzymes lost the activity by 50% when incubated at 50 degrees C for 8, 14 and 23 min, respectively.

A hexokinase from fish liver with wide specificity for nucleotides as phosphoryl donor.

The liver of rainbow trout contains two hexokinases (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) designated C and D from the elution pattern in DEAE-cellulose column chromatography. Hexokinase D has been purified about 50-fold from the liver of rainbow trout by chromatography with DEAE-cellulose and Sephadex G-200, and by isoelectric focusing. The properties of hexokinase D were similar to those of mammalian hexokinase III with respect to the Km values for ATP and glucose and the substrate inhibition by glucose at high concentration. However, the enzyme showed a wide specificity for nucleotides as the phosphoryl donor. Although it has been reported that the only effective nucleotide as the phosphoryl donor for hexokinase from various origin in ATP, and that ADP, a reaction product, inhibits the enzyme, hexokinase D from the rainbow-trout liver was found to be able to form glucose 6-phosphate (Glc-6-P) from glucose and various nucleotides such as ATP, ADP, CTP, GTP, UTP and UDP. The reaction products from ADP and glucose, Glc-6-P and AMP, were identified by chromatography on ion-exchange resin column and paper. The enzyme D was not inhibited by ADP but was strongly inhibited by AMP, which is a reaction product from ADP.