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Biomedical subjects

V Zappia

Publications and source records attributed to V Zappia.

At least 91 records · Page 5Linked to original sources
PubMed1981

Studies on 5'-methylthioadenosine uptake by human erythrocytes.

The transport of 5'-methylthioadenosine across plasma membrane of human erythrocytes occurs by a carrier-mediated mechanism displaying a Km of congruent to 3 mM and a Vmax of congruent to 600 pmol/10(6) cells/min. Phosphate-depleted erythrocytes were employed to distinguish between 5'-methylthioadenosine transport and metabolic trapping. In phosphate medium, where 5'-methylthioadenosine phosphorylase is operative, the uptake of radioactivity increases over the theoretical calculated equilibrium owing principally to conversion of the molecular into 5-methylthioribose-1-phosphate. Newly developed HPLC techniques have been applied to analyze intracellular 5'-methylthioadenosine and its metabolites after exposure of the erythrocytes to [14C]-labelled 5'-methylthioadenosine.

Adenosine↗
PubMed1980

Mechanism of propylamine-transfer reactions. Kinetic and inhibition studies on spermidine synthase from Escherichia coli.

Spermidine synthase (EC 2.5.1.16) purified from Escherichia coli has been subjected to a kinetic analysis including initial velocity and substrate analogs inhibition studies. Evidence is reported for a ping-pong mechanism, indicating that a propylaminated form of the enzyme is an obligatory intermediate in the reaction mechanism. S-Adenosyl(5')-3-methylthiopropylamine exerts a competitive substrate inhibition by combining with the improper stable enzyme form, while putrescine does not show any inhibitory effect. In order to investigate the substrate binding sites, new sulfonium-deaminated analogs of S-adenosyl(5')-3-methylthiopropylamine have been synthesized and assayed as substrates and as inhibitors of the reaction. The replacement of the amino group of adenine, or propylamine moiety of the sulfonium compound by the hydroxyl group, or both, resulted in a complete loss of activity as substrate. On the other hand, the deaminated analogs exert a competitive inhibition with respect to putrescine. On the basis of these results and in analogy with methyltransfer reactions, three recognition sites for S-adenosyl(5')-3-methylthiopropylamine on propylamine transfer enzymes are proposed.

Binding Sites↗
PubMed1980

High-performance liquid chromatographic separation of natural adenosyl-sulphur compounds.

A rapid, sensitive and specific high-performance liquid chromatographic method for the simultaneous separation of natural adenosyl-sulphur compounds has been developed. The compounds were separated by using the strong cation-exchange resin Partisil 10 SCX with isocratic elution. The adenosyl-compounds were monitored by an ultraviolet detector operating at 254 nm. Sensitivity was greater than 50 pmoles for all compounds tested and standard curves were found to be linear for concentrations of up to 50 nmoles. The method can be applied to biological samples for the estimation of S-adenosyl-L-methionine,S-adenosyl-L-homocysteine and S-adenosyl-(5')-3-methyl-thiopropylamine and for measurement of enzyme activities involving the above-mentioned compounds.

Animals↗
PubMed1979

5'-Methylthioadenosine phosphorylase from Caldariella acidophila. Purification and properties.

The occurrence of 5'-methylthioadenosine phosphorylase in Caldariella acidophila, a thermophilic bacterium growing optimally at 87 degrees C, is reported. It represents the first example in prokaryotes of a phosphoryolytic cleavage of the thioether. The reaction products, purified by ion-exchange chromatography, have been identified as 5-methylthioribose-1-phosphate and adenine by several analytical procedures. The enzyme has been purified to homogeneity in 32% yield by using DEAE-cellulose and hydroxyapatite chromatography, gel filtration and isoelectric focusing. The enzyme shows a high degree of thermophilicity, its temperature optimum being at 93 degrees C; furthermore no loss of activity is observable after exposure for 1 h at 100 degrees C. The kinetic data indicate a sequential mechanism of the reaction. The apparent Km values are 0.095 mM for 5'-methylthioadenosine and 6.1 mM for phosphate. The specificity of the reaction is rather strict. Experiments performed with analogues of the substrate, i.e. 5'-methylthioinosine, 5'-dimethylthioadenosine sulfonium salt, 5'-n-butylthioadenosine, 5'-isobutylthioadenosine, 5'-isobutylthioinosine, adenosylhomocysteine, 5'-thioethanoladenosine, adenosine, indicate the relevance of the adenine amino group and the sulfur in thioether form in the binding to the enzyme protein.

Adenosine↗
PubMed1978

[5'-methylthioadenosine phosphorylase from Caldariella acidophila. 1. Purification and partial characterization].

5'-Methylthioadenosine phosphorylase has been isolated from C.acidophila, a thermophilic bacterium living in acid hot springs at temperatures ranging from 63 to 89 degrees C. The enzyme has been purified to homogeneity in 32% yield. The enzyme shows a high degree of thermophilicity, its temperature optimum being 93 degrees C in the in vitro assay. The enzyme is exceptionally stable; no loss of activity was observable after exposure for 1 h at 100 degrees C. The optimum pH is about 7,2, with one-half of the maximal activity occurring at pH 6 and 9. The apparent Km for the substrates are: 8,3 x 10(-5) M for MTA and 4,3 x 10(-4) M for phosphate ions.

Adenosine↗
PubMed1978

Substrate specificity of 5'-methylthioadenosine phosphorylase from human prostate.

5'-Methylthioadenosine phosphorylase was purified approx. 340-fold from human prostate by using affinity chromatography by Hg-coupled Sepharose. The enzyme, responsible for the breakdown of 5'-methylthioadenosine into adenine and methylthioribose 1-phosphate, was partially characterized. The apparent Km for 5'-methylthioadenosine is 25 microM. It is activated by thiols and shows an absolute requirement for phosphate ions. New analogues of 5'-methylthioadenosine were prepared and their activity as substrates or inhibitors of the reaction was investigated. The replacement of the 6-amino group of the adenine moiety by a hydroxy group, as well as the replacement of N-7 by a methinic radical, resulted in an almost complete loss of activity. Otherwise the replacement of sulphur by selenium, as well as that of the methyl group by an ethyl one, is compatible with the activity as substrate. The positively charged sulphonium group also prevents catalytic interaction with the enzyme. The inhibitory effect of 5'-methylthiotubercidin (competitive) and 5'-dimethylthioadenosine sulphonium salt (non-competitive) was also demonstrated. The reported results suggest three binding sites between the substrate and the enzyme.

Adenosine↗
PubMed1978

[5'-methylthioadenosine phosphorylase from the human prostate. 1. Purification and partial characterization].

5'-Methylthioadenosine phosphorylase has been purified approximately 340-fold in 20% yield from human prostate: the use of affinity chromatography by Sepharose-Hg has been found particularly advantageous. The enzyme has been partially characterized and an apparent Km of 2.5 x 10(-5) M has been calculated for 5'-methylthioadenosine. The reaction is activated by thiols and shows an absolute requirement for phosphate ions.

Adenosine↗
PubMed1978

[Incorporation of S-adenosylmethionine in the isolated and perfused rat liver. Preliminary study].

The uptake of labeled S-adenosylmethionine (SAM) by isolated and perfused rat liver has been compared to that of methionine: the rate of incorporation of the amino acid exceeds by about three times that of the sulfonium compound. S-Adenosylmethionine transport system shows saturation kinetics with an apparent Km of 59,5 microM and is energy-dependent, as demonstrated by the inhibition by 2,4-dinitrophenol.

Animals↗
PubMed1978

The biosynthetic pathway of new polyamines in Caldariella acidophila.

1. Spermidine and sym-nor-spermine (1,11-diamino-4,8-diazaundecane) were identified as the major components of the polyamine pool in Caldariella acidophila, an extreme thermoacidophilic bacterium. A minor component, a new polyamine, sym-nor-spermidine (1,7-diamino-4-azaheptane) was isolated and characterized. 2. To elucidate the biosynthetic pathway, labelled methionine, putrescine, spermidine and spermine were fed to Caldariella acidophila. Incubation of the bacterium in the presence of putrescine or spermidine labelled in the tetramethylene moiety gave unlabelled sym-nor-spermidine and sym-nor-spermine, whereas the radioactivity of propylamine-labelled methionine or spermidine was incorporated into these molecules. No radioactivity was recovered in the polyamines pool when spermine was fed to Caldariella acidophila. 3. S-Adenosylmethionine and S-(5'-adenosyl)-3-methylthiopropylamine were identified as intermediates of the biosynthetic pathway; the cellular contents of the two sulphonium compounds, measured with a new isotope-dilution technique, are 60 and 15nmol/g wet wt. of cells respectively. 4. The above results are indicative of a new pathway characterized by three propylamine-transfer reactions, decarboxylated S-adenosylmethionine being the common donor of the propylamine moiety. The reactions yielding sym-nor-spermidine and sym-nor-spermine are reported for the first time. 5. The probable intermediates related to the catabolism of the tetramethylene moiety of spermidine, gamma-aminobutyraldehyde, gamma-aminobutyric acid or Delta(1)-pyrroline were not detectable. Experiments with [3-aminopropyl-3(n)-(3)H]spermidine trihydrochloride plus [tetramethylene-1,4-(14)C]spermidine trihydrochloride gave rise to an amount of labelled CO(2) equivalent to the spermidine catabolized.

Bacteria↗