Purification and partial characterization of a thermostable trithionate hydrolase from the acidophilic sulphur oxidizer Thiobacillus acidophilus.

Cell-free extracts of Thiobacillus acidophilus catalysed the quantitative conversion of trithionate (S3O6(2-) to thiosulphate and sulphate. A continuous assay for quantification of experimental results was based on the difference in absorbance between trithionate and thiosulphate at 220 nm. Trithionate hydrolase was purified to near homogeneity from cell-free extracts of T. acidophilus. The molecular masses of the native enzyme and the subunit were 99 kDa (gel filtration) and 34 kDa (SDS/PAGE). The purified enzyme has a pH optimum of 3.5-4.5 and a temperature optimum of 70 degrees C. Enzyme activity was stimulated by sulphate. The stimulation of the enzyme activity by sulphate was half maximal at a concentration of 0.23 M. The Km for trithionate is 70 microM at 30 degrees C and 270 microM at 70 degrees C. Enzyme activity was lost after 36 days at 0 degrees C, 27 days at 70 degrees C; but after 97 days at 30 degrees C, 40% of the initial activity was still present: The enzyme activity was inhibited by mercury chloride, N-ethylmaleimide, thiosulphate and tetrathionate. Tetrathionate S4O6(2-) was not hydrolysed by trithionate hydrolase.     

Enzyme inactivation by a cellular neutral protease: enzyme specificity, effects of ligands on inactivation, and implications for the regulation of enzyme degradation.

A protease from Tetrahymena pyriformis inactivated eight of nine commercially available enzymes tested, including lactate deyhdrogenase, isocitrate dehydrogenase (TPN-specific), glucose-6 phosphate dehydrogenase, D-amino acid oxidase, fumarase, pyruvate kinase, hexokinase, and citrate synthase. Urate oxidase was not inactivated. Inactivation occurred at neutral pH, was prevented by inhibitors of the protease, and followed first order kinetics. In those cases tested, inactivation was enhanced by mercaptoethanol. Most of the enzyme-inactivating activity was due to a protease of molecular weight 25,000 that eluted from DEAE-Sephadex at 0.3 M KCl. A second protease of this molecular weight, which was not retained by the gel, inactivated only isocitrate dehydrogenase and D-amino acid oxidase. These two proteases could also be distinguished by temperature and inhibitor sensitivity. Two other protease peaks obtained by DEAE-Sephadex chromatography had little or no no enzyme inactivating activity, while another attacked only D-amino acid oxidase. At least six of the enzymes could be protected from proteolytic inactivation by various ligands. Isocitrates dehydrogenase was protected by isocitrate, TPN, or TPNH, glucose-6-dehydrogenase by glucose-6-P or TPN, pyruvate kinase by phosphoenolypyruvate or ADP, hexokinase by glucose, and fumarase by a mixture of fumarate and malate. Lactate dehdrogenase was not protected by either of its substrates of coenzymes. Citrate synthase was probably protected by oxalacetate. Our data suggest that the protease or proteases discussed here may participate in the inactivation or degradation of a least some enzymes in Tetrahymena. Since the inactivation occurs at neutral pH, this process could be regulated by variations in the cellular levels of substrates, coenzymes, or allosteric regulators resulting form changes in growth conditions or growth state. Such a mechanism would permit the selective retention of enzymes of metabolically active pathways.     

Aurinetricarboxylic acid: a potent inhibitor of glucose-6-phosphate dehydrogenase.

Inhibition by aurinetricarboxylic acid (ATA) of glucose-6-phosphate (G6P) dehydrogenase was "competitive" with respect to G6P and "mixed type" with respect to NADP+. Inhibited enzyme bound two molecules of ATA. Kinetic constants, Km, Ki at varying pH suggested possible binding of the inhibitor by the sulfhydryl of the enzyme; of the several enzymes tested only milk xanthine oxidase and G6P dehydrogenase from bovine adrenal was inhibited by ATA.     

Contribution of aldehyde oxidizing enzymes on the metabolism of 3,4-dimethoxy-2-phenylethylamine to 3,4-dimethoxyphenylacetic acid by guinea pig liver slices.

BACKGROUND/AIMS: 3,4-Dimethoxy-2-phenylethylamine is catalyzed to its aldehyde derivative by monoamine oxidase B, but the subsequent oxidation into the corresponding acid has not yet been studied. Oxidation of aromatic aldehydes is catalyzed mainly by aldehyde dehydrogenase and aldehyde oxidase. METHODS: The present study examines the metabolism of 3,4-dimethoxy-2-phenylethylamine in vitro and in freshly prepared and cryopreserved guinea pig liver slices and the relative contribution of different aldehyde-oxidizing enzymes was estimated by pharmacological means. RESULTS: 3,4-Dimethoxy-2- phenylethylamine was converted into the corresponding aldehyde when incubated with monoamine oxidase and further oxidized into the acid when incubated with both, monoamine oxidase and aldehyde oxidase. In freshly prepared and cryopreserved liver slices, 3,4-dimethoxyphenylacetic acid was the main metabolite of 3,4-dimethoxy-2- phenylethylamine. 3,4-Dimethoxyphenylacetic acid formation was inhibited by 85% from disulfiram (aldehyde dehydrogenase inhibitor) and by 75-80% from isovanillin (aldehyde oxidase inhibitor), whereas allopurinol (xanthine oxidase inhibitor) inhibited acid formation by only 25-30%. CONCLUSIONS: 3,4- Dimethoxy-2-phenylethylamine is oxidized mainly to its acid, via 3,4-dimethoxyphenylacetaldehyde, by aldehyde dehydrogenase and aldehyde oxidase with a lower contribution from xanthine oxidase.     

Thiosulfate Oxidation and Electron Transport in Thiobacillus novellus.

Aleem, M. I. H. (Research Institute for Advanced Studies, Baltimore, Md.). Thiosulfate oxidation and electron transport in Thiobacillus novellus. J. Bacteriol. 90:95-101. 1965.-A cell-free soluble enzyme system capable of oxidizing thiosulfate was obtained from Thiobacillus novellus adapted to grow autotrophically. The enzyme systems of autotrophically grown cells brought about the transfer of electrons from thiosulfate to molecular oxygen via cytochromes of the c and a types; the reactions were catalyzed jointly by thiosulfate oxidase and thiosulfate cytochrome c reductase. The levels of both of these enzymes were markedly reduced in the heterotrophically grown organism. Cell-free extracts from the autotrophically grown T. novellus catalyzed formate oxidation and enzymatically reduced cytochrome c with formate. Both formate oxidation and cytochrome c reduction activities were abolished under heterotrophic conditions. The thiosulfate-activating enzyme S(2)O(3) (-2)-cytochrome c reductase, as well as thiosulfate oxidase, was localized chiefly in the soluble cell-free fractions, and the former enzyme was purified more than 200-fold by ammonium sulfate fractionation and calcium phosphate gel adsorption procedures. Optimal activity of the purified enzyme occurred at pH 8.0 in the presence of 1.67 x 10(-1)m S(2)O(3) (-2) and 2.5 x 10(-4)m cytochrome c. The thiosulfate oxidase operated optimally at pH 7.5 and thiosulfate concentrations of 1.33 x 10(-3) to 3.33 x 10(-2)m in the presence of added cytochrome c at a concentration of 5 x 10(-4)m. Both enzymes were markedly sensitive to cyanide and to a lesser extent to some metal-binding agents. Although a 10(-3)m concentration of p-hydroxymercuribenzoate had no effect on S(2)O(3) (-2)-cytochrome c reductase, it caused a 50% inhibition of S(2)O(3) (-2) oxidase, which was completely reversed in the presence of 10(-3)m reduced glutathione. Carbon monoxide also inhibited S(2)O(3) (-2) oxidase; the inhibition was completely reversed by light.     

Immobilized enzyme system for determination of sialic acid in serum or urine.

An immobilized enzyme system has been developed and employed to determine the concentration of sialic acid (N-acetylneuraminic acid) in human serum and urine. Two enzyme pairs, neuramindiase-Neu-5-Ac lyase and pyruvate oxidase-peroxidase, have been respectively co-immobilized onto 1,12-aminododecane-agarose with glutaraldehyde. The relative specific activity of the co-immobilized neuraminidase and Neu-5-Ac lyase were 60% and 78%, and those of pyruvate oxidase and peroxidase were 50% and 95% of the corresponding soluble enzymes, respectively. The optimal reaction pH at 37 degrees C for each of the co-immobilized enzymes was about one pH unit higher than that of the corresponding soluble enzyme. The optimal reaction temperature of each enzyme was increased as a result of immobilization. The thermal stability at 45 degrees C of the immobilized neuraminidase, Neu-5-Ac lyase, pyruvate oxidase, and peroxidase were increased 80-, 83-, 115-, and 147-fold, respectively. Km and Vm of each immobilized and co-immobilized enzyme have also been determined. The system provided a convenient and rapid method to determine the concentration of total sialic acid without pretreatment of the sample. The results correlated satisfactorily with those obtained by using a soluble enzyme system. The co-immobilized enzymes were stable for at least 1 year of 500 tests when used repeatedly. The system is thus a reproducible and reliable novel assay method for sialic acid in the serum or urine sample.     

Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures of Aspergillus niger

Batch cultures of Aspergillus niger grown from conidia on a medium with high C/N ratio accumulated gluconate from glucose with a yield of 57%. During almost the whole time of accumulation there was no net synthesis of total protein in the mycelium but the activity per flask and the specific activity of glucose oxidase (EC 1.1.3.4) in mycelial extracts increased whereas both values decreased for glucose dehydrogenase (EC 1.1.99.10) gluconate 6-phosphatase (cf. EC 3.1.3.1, 3.1.3.2), gluconokinase (EC 2.7.1.12), glucose 6-phosphate and phosphogluconate dehydrogenases (EC 1.1.1.49, EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), and most enzymes of the Embden-Meyerhof pathway and the tricarboxylic acid cycle. Gluconate dehydratase (EC 4.2.1.39), gluconate dehydrogenase (EC 1.1.99.3) and enzymes of the Entner-Doudoroff pathway could not be detected. By cycloheximide the increase of glucose oxidase activity was inhibited. It is concluded that the high yield of gluconate was due mainly to the net (de novo) synthesis of glucose oxidase which occurred during protein turnover after the exhaustion of the nitrogen source, and which was not accompanied by a net synthesis of the other enzymes investigated. Some gluconate may also have been formed by hydrolytic cleavage of gluconate 6-phosphate.Abbreviations GOD glucose oxidase - GD glucose dehydrogenase - PP pentose phosphate - EM Embden-Meyerhof - TCA tricarboxylic acid     

Peroxisomal straight-chain Acyl-CoA oxidase and D-bifunctional protein are essential for the retroconversion step in docosahexaenoic acid synthesis

Docosahexaenoic acid (DHA, C22:6n-3) is essential for normal brain and retinal development. The nature and subcellular location of the terminal steps in DHA biosynthesis have been controversial. Rather than direct Delta4-desaturation of C22:5n-3, it has been proposed that this intermediate is elongated to C24:5n-3, desaturated to C24:6n-3, and "retroconverted" to DHA via peroxisomal beta-oxidation. However, this hypothesis has recently been challenged. The goal of this study was to determine the mechanism and specific enzymes required for the retroconversion step in human skin fibroblasts. Cells from patients with deficiencies of either acyl-CoA oxidase or D-bifunctional protein, the first two enzymes of the peroxisomal straight-chain fatty acid beta-oxidation pathway, exhibited impaired (5-20% of control) conversion of either [1-14C]18:3n-3 or [1-14C]22:5n-3 to DHA as did cells from peroxisome biogenesis disorder patients comprising eight distinct genotypes. In contrast, normal DHA synthesis was observed in cells from patients with rhizomelic chondrodysplasia punctata, Refsum disease, X-linked adrenoleukodystrophy, and deficiency of mitochondrial medium- or very long-chain acyl-CoA dehydrogenase. Acyl-CoA oxidase-deficient cells accumulated 2-5 times more radiolabeled C24:6n-3 than did controls. Our data are consistent with the retroconversion hypothesis and demonstrate that peroxisomal beta-oxidation enzymes acyl-CoA oxidase and D-bifunctional protein are essential for this process in human skin fibroblasts.     

Effect of copper on liver key enzymes of anaerobic glucose metabolism from freshwater tropical fish Prochilodus lineatus

We investigated the effect of copper on liver key enzymes of the anaerobic glucose metabolism (hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK; lactate dehydrogenase, LDH) as well as of the pentose pathway (glycose-6-phosphate dehydrogenase, G6PDH) from the fish Prochilodus lineatus. The fish were acclimated at either 20 degrees C or 30 degrees C at pH 7.0, transferred to water at pH 4.5 or 8.0, and exposed to 96 h-CL(50) copper concentrations. Copper accumulation in liver was higher in fish acclimated at 20 degrees C and maintained in water pH 8.0. Three-way analysis of variance revealed a significant effect of temperature on all enzymes, a significant effect of pH on all enzymes except for PK, and a significant effect of copper on only PFK, and LDH in pH 4.5 at 20 degrees C and, at 30 degrees C, on PFK and PK at pH 4.5 and 8.0, HK at pH 4.5 and G6PDH at pH 8.0. There were significant interactions between treatments for many enzymes. These changes suggest that the activity of enzymes in question is modified by a change in ambient water. At least at 30 degrees C, the overall reduction in the glycolytic enzyme activities of copper-exposed fish seems to reduce energy availability via glucose metabolism, thereby contributing to enhance copper toxic effects.     

Halothane depletes cardiac microvasculature enzymes in the rat

Left ventricular arterioles from Sprague-Dawley rats were analyzed histochemically to determine the effects of halothane administration on key enzymes of aerobic and anaerobic metabolism, as well as on key enzymes of the hexose monophosphate shunt. Significant decreases occurred in cytochrome oxidase (-42%) and beta-hydroxybutyrate dehydrogenase (-57%). No significant changes were observed in isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, or lactate dehydrogenase. These data suggest that anesthetic levels of halothane can cause impaired metabolism in the coronary microvasculature.     

Activities and subcellular localization of enzymes responsible for lipolysis and gluconeogenesis during the germination of Brassica campestris cv. esculenta seeds

During the growth of turnip seedlings, two new lipases have been demonstrated, one with a maximum activity at pH 4.5 (acid lipase) and the other with a maxima at pH 8.6 (alkaline lipase). Many different enzymes are involved in gluconeogenesis: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase, aconitase, citrate synthetase, fumarase, glycolate oxidase, phosphoenol-pyruvate carboxykinase. All of these show maximum activity coinciding with the stage in which lipid hydrolysis is maximal and when the accumulation of soluble carbohydrates has also reached its peak. The alkaline lipase as found to be located mainly in the spherosomes, whereas the glyoxysomes contained the following main activities: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase and citrate synthetase. Aconitase, together with cytochrome oxidase and fumarase showed their highest activity in the mitochondria, and the presence of malate dehydrogenase, citrate synthetase and glycolate oxidase was also observed in these organelles. In the membrane-bound fraction, the activities of cytochrome reductase, glycolate oxidase and phosphoenol-pyruvate kinase were marked, although the latter enzyme was even more active in the soluble fraction.     

Oxidation--reduction potentials of turkey liver xanthine dehydrogenase and the origins of oxidase and dehydrogenase behaviour in molybdenum-containing hydroxylases.

Redox potentials for the various centres in the enzyme xanthine dehydrogenase (EC 1.2.1.37) from turkey liver determined by potentiometric titration in the presence of mediator dyes, with low-temperature electron-paramagnetic-resonance spectroscopy. Values at 25 degrees C in pyrophosphate buffer, pH 8.2, are: Mo(VI)/Mo(V)(Rapid),-350 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -362 +/- 20mV; Fe-S Iox./Fe-S Ired., -295 +/- 15mV; Fe-S IIox./Fe-S IIred., -292 +/- 15mV; FAD/FADH,-359+-20mV; FADH/FADH2, -366 +/- 20mV. This value of the FADH/FADH2 potential, which is 130mV lower than the corresponding one for milk xanthine oxidase [Cammack, Barber & Bray (1976) Biochem. J. 157, 469-478], accounts for many of the differences between the two enzymes. When allowance is made for some interference by desulpho enzyme, then differences in the enzymes' behaviour in titration with xanthine [Barber, Bray, Lowe & Coughlan (1976) Biochem. J. 153, 297-307] are accounted for by the potentials. Increases in the molybdenum potentials of the enzymes caused by the binding of uric acid are discussed. Though the potential of uric acid/xanthine (-440mV) is favourable for full reduction of the dehydrogenase, nevertheless, during turnover, for kinetic reasons, only FADH and very little FADH2 is produced from it. Since only FADH2 is expected to react with O2, lack of oxidase activity by the dehydrogenase is explained. Reactivity of the two enzymes with NAD+ as electron acceptor is discussed in relation to the potentials.     

NADH dehydrogenase of Corynebacterium glutamicum. Purification of an NADH dehydrogenase II homolog able to oxidize NADPH

NADPH oxidase activity, in addition to NADH oxidase activity, has been shown to be present in the respiratory chain of Corynebacterium glutamicum. In this study, we tried to purify NADPH oxidase and NADH dehydrogenase activities from the membranes of C. glutamicum. Both the enzyme activities were simultaneously purified in the same fraction, and the purified enzyme was shown to be a single polypeptide of 55 kDa. The N-terminal sequence of the enzyme was consistent with the sequence deduced from the NADH dehydrogenase gene of C. glutamicum, which has been sequenced and shown to be a homolog of NADH dehydrogenase II. In addition to high NADH-ubiquinone-1 oxidoreductase activity at neutral pH, the purified enzyme showed relatively high NADPH oxidase and NADPH-ubiquinone-1 oxidoreductase activities at acidic pH. Thus, NADH dehydrogenase of C. glutamicum was shown to be rather unique in having a relatively high reactivity toward NADPH.     

Genetics and physiology of proline utilization in Saccharomyces cerevisiae: enzyme induction by proline.

Proline is converted to glutamate in the yeast Saccharomyces cerevisiae by the sequential action of two enzymes, proline oxidase and delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase. The levels of these enzymes appear to be controlled by the amount of proline in the cell. The capacity to transport proline is greatest when the cell is grown on poor nitrogen sources, such as proline or urea. Mutants have been isolated which can no longer utilize proline as the sole source of nitrogen. Mutants in put1 are deficient in proline oxidase, and those in put2 lack P5C dehydrogenase. The put1 and put2 mutations are recessive, segregate 2:2 in tetrads, and appear to be unlinked to one another. Proline induces both proline oxidase and P5C dehydrogenase. The arginine-degradative pathway intersects the proline-degradative pathway at P5C. The P5C formed from the breakdown of arginine or ornithine can induce both proline-degradative enzymes by virtue of its conversion to proline.     

The effects of storage on the retention of enzyme activity in cryostat sections. A quantitative histochemical study on rat liver

Summary The effect of storage of unfixed cryostat sections from rat liver for 4 h, 24 h, 3 days and 7 days at -25°C was studied on the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine oxidoreductase, glutamate dehydrogenase, succinate dehydrogenase (all demonstrated with tetrazolium salt procedures), glucose-6-phosphatase (cerium-diaminobenzidine method), 5-nucleotidase (lead salt method), dipeptidyl peptidase II, acid phosphatase (both simultaneous azo coupling methods), d-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure) and catalase (diaminobenzidine method). The effect of drying of the cryostat sections at room temperature for 5 and 60 min was investigated as well. The enzyme activities were quantified by cytophotometric measurements of test and control reactions. The test minus control reaction was taken as a measure for specific enzyme activity. It was found that the activities of all the enzymes investigated, with one exception, were affected neither by storage of the cryostat sections at -25°C for up to 7 days, nor by drying of the sections at room temperature for up to 60 min. The exception was xanthine oxidoreductase, whose activity was reduced by 20% after 5 min drying of sections or after 4 h storage. Therefore, only incubations for xanthine oxidoreductase activity have to be performed immediately after cutting cryostat sections, whereas for the other enzymes a considerable margin appears to exist.     

Repression of oxalic acid-mediated mineral phosphate solubilization in rhizospheric isolates of Klebsiella pneumoniae by succinate

Two strains of Klebsiella (SM6 and SM11) were isolated from rhizospheric soil that solubilized mineral phosphate by secretion of oxalic acid from glucose. Activities of enzymes for periplasmic glucose oxidation (glucose dehydrogenase) and glyoxylate shunt (isocitrate lyase and glyoxylate oxidase) responsible for oxalic acid production were estimated. In presence of succinate, phosphate solubilization was completely inhibited, and the enzymes glucose dehydrogenase and glyoxylate oxidase were repressed. Significant activity of isocitrate lyase, the key enzyme for carbon flux through glyoxylate shunt and oxalic acid production during growth on glucose suggested that it could be inducible in nature, and its inhibition by succinate appeared to be similar to catabolite repression.     

The synthesis and turnover of rat liver peroxisomes. I. Fractionation of peroxisome proteins

Rat liver peroxisomes isolated by density gradient centrifugation were disrupted at pH 9, and subdivided into a soluble fraction containing 90% of their total proteins and virtually all of their catalase, D-amino acid oxidase, L-α-hydroxy acid oxidase and isocitrate dehydrogenase activities, and a core fraction containing urate oxidase and 10% of the total proteins. The soluble proteins were chromatographed on Sephadex G-200, diethylaminoethyl (DEAE)-cellulose, hydroxylapatite, and sulfoethyl (SE)-Sephadex. None of these methods provided complete separation of the protein components, but these could be distributed into peaks in which the specific activities of different enzymes were substantially increased. Catalase, D-amino acid oxidase, and L-α-hydroxy acid oxidase contribute a maximum of 16, 2, and 4%, respectively, of the protein of the peroxisome. The contribution of isocitrate dehydrogenase could be as much as 25%, but is probably much less. After dissolution of the cores at pH 11 , no separation between their urate oxidase activity and their protein was achieved by Sephadex G-200 chromatography.     

Reduced pyridine nucleotide oxidases of Eugena gracilis var. bacillaris.

Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (Km) determined were as follows: NADH diaphorase, 350 muM; NADPH oxidase 150 muM ; NADH lipoyl dehydrogenase, 0.35 muM. Enzyme activities after storage at -5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles.     

Effect of the proline analogue baikiain on proline metabolism in Salmonella typhimurium

A proline analogue, 4,5-dehydro-l-pipecolic acid (baikiain) induces the formation in Salmonella typhimurium of the two enzymes catalyzing the degradation of proline, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid (P5C) dehydrogenase. The level of induction by 20 mm baikiain is about 10% of the maximum level induced by proline. Since the analogue is a substrate of proline oxidase the first enzyme of the proline catabolic pathway, the oxidation derivative rather than baikiain itself might be the actual effector. Baikiain is also an inducer of proline oxidase in Escherichia coli K-12 and E. coli W. An additional effect of this analogue on proline degradation in S. typhimurium is inhibition of P5C dehydrogenase. At a concentration of 5 x 10(-4)m, baikiain inhibits completely the growth of strains constitutive for proline oxidase. This inhibition, which can be overcome by proline, occurs in the presence or absence of P5C dehydrogenase activity. Three spontaneously occurring mutants resistant to baikiain were isolated from constitutive strains. All are pleiotropic-negative for the proline-degrading enzymes. The sites of these mutations are linked to the put region. Although the mechanism of toxicity has not been determined, baikiain provides a simple and direct selection for obtaining mutants unable to degrade proline. In addition, it allows selection for strains with an inducible rather than constitutive phenotype.     

Relation between growth rate and metabolic organization of white muscle,liver and digestive tract in cod,Gadus morhua

To determine whether the aerobic capacity of tissues required for growth specifically reflects growth rates, we monitored the activities of key enzymes of oxidative, glycolytic and amino acid metabolism in muscle, liver and intestine of Atlantic cod (Gadus morhua) growing at different rates. Fish were maintained individually in small tanks at 10°C and fed on rations that allowed growth rates ranging from-0.6 to 1.6% per day. The correlation between growth rate and muscle enzyme activity was pronounced for the glycolytic enzymes (LDH, PFK and PK). The activities of glycolytic enzymes were more than four times higher for fish having higher growth rates compared to those that did not grow. Mitochondrial enzyme (cytochrome c oxidase, citrate synthase and -hydroxyacyl-CoA dehydrogenase) activities remained unchanged in fish with positive growth. The liver seems to respond to requirements of growth by an increase in size. In the liver, the activities of the enzymes of amino acid metabolism expressed as units · g DNA^-1 specifically increases with growth rate. In contrast to the two other tissues, the specific activities of mitochondrial enzymes in the intestine increased with growth rate while the relative mass of the intestine remained constant. Intestinal cytochrome c oxidase activity increased from a minimum of about 2 to more than 8 units · g intestine^-1. Cytochrome c oxidase activity increased in parallel with the food conversion efficiency. This suggests that the aerobic capacity of the intestine may initially limit the rates of digestion and growth in this species.Abbreviations AA amino acid(s) - BM body mass - CCO cytochrome c oxydase - CS citrate synthase - DTNB 5,5 dithiobis-2-nitrobenzoic acid - GDH glutamate dehydrogenase - GOT glutamate oxalacetate transaminase - GPT glutamate pyruvate transaminase - GR growth rate(s) - HOAD -hydroxyacyl-CoA dehydrogenase - HSI hepatosomatic index - LDH lactate dehydrogenase - MR metabolic rate(s) - PCA perchloric acid - PFK phosphofructokinase - PK pyruvate kinase - PMSF phenylmethylsulphonyl fluoride; TRIS