Inactivation and morphological changes of avian influenza virus by copper ions.

The infectivity of the H9N2 virus to MDCK cells was time-dependently inhibited by Cu(2+) at concentrations of 2.5-250 microM. In 25 microM Cu(2+) solution, the virus titer decreased by approximately 3 and 4 log within 3 and 6 h, respectively. Compared to Cu(2+), Zn(2+) was much less effective in virus inactivation. The H9N2 virus hemagglutinin activity was not affected by 2.5-250 microM Cu(2+). The H9N2 virus neuraminidase (NA) activity was drastically reduced by 25 mM Cu(2+), marginally reduced by 250 microM Cu(2+), and not affected by 25 microM Cu(2+). Thus, we found that copper ions suppress the infectivity of influenza virus at lower concentrations at which neither NA nor hemagglutination inhibition occurs. Electron microscopic analysis revealed morphological abnormalities of the Cu(2+)-treated H9N2 virus. Additional studies should be undertaken to clarify the mechanism underlying the antiviral effect of copper ions on influenza virus.

Purification, characterization and inhibition of D-3-aminoisobutyrate aminotransferase from the rat liver.

D-3-Aminoisobutyrate-pyruvate aminotransferase was purified 2000-fold from rat liver extract using heat treatment, ammonium sulfate fractionation, carboxylmethyl-Sepharose CL-6B, DEAE-Sepharose CL-6B, hydroxyapatite, Sephacryl S-200 and electrofocusing chromatographies. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 220 kDa and the subunit molecular mass was 52 kDa. The enzyme exhibited absorption maxima at 280 nm and 412 nm with a shoulder at 330 nm at neutral pH. The pH optimum for enzyme activity was 9.5 and the Km values for beta-alanine and pyruvic acid were calculated to be 0.81 mM and 0.45 mM, respectively. The purified enzyme catalyzed the transamination of omega-amino acids; beta-alanine and D-3-aminoisobutyric acid served as good amino donors, and pyruvic acid, glyoxylic acid and oxaloacetic acid were favorable amino acceptors. 6-Azauracil and 6-azathymine were found to be potent inhibitors of purified rat liver D-3-aminoisobutyrate-pyruvate aminotransferase. 6-Azauracil acted as a competitive inhibitor with respect to beta-alanine, and was an uncompetitive inhibitor with respect to pyruvic acid with a Ki of approximately 8.9 mM.

Inhibitory effect of 6-azauracil on beta-alanine metabolism in rat.

The effect of 6-azauracil on beta-alanine metabolism was investigated in vivo in the rat. Both of the enzymes beta-alanine-oxoglutarate aminotransferase (aminobutyrate aminotransferase) and D-3-aminoisobutyrate-pyruvate aminotransferase [R)-3-amino-2-methylpropionate-pyruvate aminotransferase), which are beta-alanine catabolizing enzymes from rat liver and kidney, were inactivated by 6-azauracil injection, while dihydrouracil dehydrogenase, dihydropyrimidinase, and beta-ureidopropionase, which are pyrimidine metabolizing enzymes, were not affected. The content of beta-alanine was increased, but the level of uridine and uracil in rat liver was not affected, by 6-azauracil. When a crude enzyme preparation was passed through a Sephacryl S-200 column, both enzymes could be separated from each other. beta-Alanine-oxoglutarate aminotransferase and beta-alanine-pyruvate aminotransferase activities in rat liver decreased to 27.4% and 63.9%, respectively, upon 6-azauracil injection, and those in kidney were 11.7% and 38.3%, respectively. From these findings, it is suggested that the accumulation of beta-alanine in 6-azauracil-treated rat liver might be caused by the inhibition of beta-alanine catabolizing enzymes, but not by an increase in the uridine pool nor by the activation of pyrimidine metabolism.

Effect of pyridoxine deficiency and prednisolone on beta-alanine-oxoglutarate aminotransferase and D-3-aminoisobutyrate aminotransferase in rat liver and kidney.

beta-Alanine-oxoglutarate aminotransferase (beta-Ala-TI) was found to be distributed mainly in liver, brain, kidney, and testis (decreasing order of enzyme activity in the rat). D-3-Aminoisobutyrate aminotransferase (beta-Ala-T II) was distributed in kidney and liver. Both beta-Ala-T I and beta-Ala-T II were localized in the mitochondrial fraction in rat kidney. beta-Ala-T I in the liver of rats fed on pyridoxine-deficient or control diets was induced by injecting with prednisolone, while beta-Ala-T II in the liver of these rats was unaffected by prednisolone injection. The activities of beta-Ala-T I and beta-Ala-T II in the liver of rats fed on pyridoxine deficient diet did not change. However, in kidney, pyridoxine deficiency suppressed both enzyme activities, while treatment with prednisolone did not induce either enzyme. The ratios of the apo- to holo-enzyme for beta-Ala-T I and beta-Ala-T II in control rat kidney were 1.04 and 0.11, respectively. The values increased to 2.69 and 1.53, respectively, in pyridoxine-deficient rat kidney. These experiments indicate that pyridoxine deficiency and prednisolone affect the activities of beta-alanine degrading enzymes, but that the degree is different between liver and kidney.

Bisfunction of propionic acid on purified rat liver beta-ureidopropionase.

Propionic acid and isobutyric acid, which are structural analogues of N-carbamoyl-beta-alanine and N-carbamoyl-beta-aminoisobutyric acid, respectively, acted as an allosteric activator as well as a competitive inhibitor of purified rat liver beta-ureidopropionase. Propionic acid and isobutyric acid had a Ki value of approx. 0.3 mM at pH 7.0. The Hill coefficient for N-carbamoyl-beta-alanine was 2.0, but the cooperatively decreased to 1.0 in the presence of 1 mM propionic acid. The K1/2 value towards N-carbamoyl-beta-alanine was calculated to be 0.17 mM from Hill plots and the Km value was determined to be 0.06 mM from replots of the apparent Km vs propionic acid.

Submitochondrial localization of rat liver beta-alanine-oxoglutarate aminotransferase.

beta-Alanine-oxoglutarate aminotransferase from rat liver was mainly distributed in the mitochondrial fraction, while beta-ureidopropionase, the last one of uracil-metabolizing enzymes to beta-alanine, was predominantly distributed in the cytosolic fraction. When rat liver mitochondria were separated into submitochondrial fractions, beta-alanine-oxoglutarate aminotransferase was localized in the mitochondrial matrix. beta-Alanine was transported into mitochondria with time and the influx of beta-alanine into the matrix was 0.47 nmol/mg of mitochondrial protein at 2 min after incubation.

Purification and properties of beta-ureidopropionase from the rat liver.

beta-Ureidopropionase was purified 1000-fold over the initial rat liver extract, using heat treatment, ammonium sulfate fractionation, CM-Sepharose CL-6B, DEAE-Sepharose CL-6B, hydroxyapatite and Sephacryl S-300 chromatographies. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. Its molecular mass, determined by gel filtration and sucrose density gradient centrifugation, was 327,000 +/- 9000 and 323,000 +/- 13,000 respectively, and the subunit molecular mass was 54,000 +/- 600. The pH optimum for enzyme activity was 7.0 and pI was 6.4. The enzyme catalyzed the amidohydrolysis of N-carbamoyl-beta-alanine and N-carbamoyl-DL-beta-aminoisobutyrate but did not catalyze that of other ureido compounds including N-carbamoyl-DL-aspartate. With N-carbamoyl-beta-alanine and N-carbamoyl-DL-beta-aminoisobutyrate as substrate, the enzyme exhibited positive cooperativity with a Hill coefficient h = 2. The enzyme activity was proportional to the enzyme concentration between 0.2 nM and 0.5 microM. Arrhenius plots of the influence of temperature on the catalytic activity of the enzyme showed a sharp break at 19 degrees C.

Identity of beta-alanine-oxo-glutarate aminotransferase and L-beta-aminoisobutyrate aminotransferase in rat liver.

L-beta-Aminoisobutyrate served as an amino donor for purified beta-alanine-oxo-glutarate aminotransferase from rat liver when 2-oxoglutarate was employed as an amino acceptor, but the D-isomer did not. L-beta-Aminoisobutyrate acted as a competitive inhibitor with respect to beta-alanine and had a Ki of approximately 2.6 mM, which is the same value as the Km of 2.7 mM. When the crude extract was applied to a DEAE-Sepharose CL-6B column, L-beta-aminoisobutyrate aminotransferase and beta-alanine-oxo-glutarate aminotransferase activities were found in the same fractions with a single peak. Antiserum to rat liver beta-alanine-oxo-glutarate aminotransferase inhibited L-beta-aminoisobutyrate aminotransferase activity in rat liver in the same way as beta-alanine-oxo-glutarate aminotransferase activity.

The level of beta-alanine aminotransferase activity in regenerating and differentiating rat liver.

beta-Alanine aminotransferase from rat liver was purified to electrophoretic homogeneity. The immunological and kinetic properties of this enzyme were similar to those of the enzyme from rat brain. However, the liver enzyme transaminates from beta-alanine to 2-oxoglutaric acid, while the brain enzyme transaminates from gamma-aminobutyric acid. beta-Alanine aminotransferase activity in regenerating rat liver was lower than that in control rat liver. Activity of this enzyme, as well as of other uracil-catabolizing enzymes (Weber, G., Queener, S.F. and Ferdinandus, A. (1970) in Advances in Enzyme Regulation (Weber, G., ed.), Vol. 9, pp. 63-95, Pergamon Press, Oxford), was low in newborn rat liver and increased about 5-fold, reaching the level observed in adult rat liver. beta-Alanine and prednisolone induced beta-alanine aminotransferase in rat liver.

Increased beta-aminoisobutyric acid in rat liver with 6-azauracil and its enantiomer.

When 6-azauracil was subcutaneously injected, beta-aminoisobutyric acid and beta-alanine contents were increased 22 and 61-fold, respectively, in rat liver. Incorporation of [methyl-14C]thymine into beta-aminoisobutyric acid was increased to 42-fold by 6-azauracil treatment. The absolute configuration of this amino acid was proved to be the (R)-form by means of a gas-chromatographic technique. 6-Azauracil inhibited beta-alanine-pyruvate aminotransferase activity with an I50 of approx. 2.5 mM.