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M Porcelli

Publications and source records attributed to M Porcelli.

At least 37 records · Page 2Linked to original sources
PubMed1989

Biosynthesis and metabolism of 9-[5'-deoxy-5'-(methylthio)-beta-D-xylofuranosyl]adenine, a novel natural analogue of methylthioadenosine.

The biosynthesis of 9-[5'-deoxy-5'-(methylthio)-beta-D-xylofuranosyl]adenine (xylosyl-MTA), a naturally occurring analogue of 5'-deoxy-5'-methylthioadenosine (MTA) recently characterized, was studied in the nudibranch mollusc Doris verrucosa. Experiments performed in vivo with putative labelled precursors such as [8-14C]adenine, [Me-14C]methionine and [Me-14C]MTA indicate that xylosyl-MTA originates from MTA. Experiments with MTA double-labelled at critical positions are consistent with a 3'-isomerization of the nucleoside through the formation of a 3'-oxo intermediate. In addition, experiments with the newly synthesized [3'-3H]xylosyl-MTA are indicative for a very low turnover rate of this molecule, which therefore accumulates in the mollusc.

Adenosine↗
PubMed1988

S-adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus. Purification and characterization of two isoforms.

Two isoforms of methionine adenosyltransferase (S-adenosylmethionine synthetase), A and B, have been partially purified from Sulfolobus solfataricus, a thermophilic archaebacterium optimally growing at 87 degrees C. The chromatographic procedure, involving hydrophobic chromatography on a phenyl-Sepharose column as a major step, results in 330-fold and 150-fold purification of adenosylmethionine synthetase A and B respectively. The apparent molecular masses, estimated by gel filtration, are 180 kDa for A and 75 kDa for B. The A and B isoforms follow Michaelis-Menten kinetics with apparent Km values of 10 microM and 20 microM for L-methionine and of 50 microM and 150 microM for ATP respectively. Adenosylmethionine, a product of the reaction, acts as a powerful non-competitive inhibitor (Ki = 50 microM) of the A isoform while it inhibits only slightly the B isoform. Both isozymes exhibit tripolyphosphatase activity but only that associated with the form A is stimulated by 5 microM adenosylmethionine concentration. The two enzymes absolutely require a divalent cation for the activity, but are not affected by monovalent ions and reducing agents. The optimum temperature is 90 degrees C and no significant loss of activity is observable after incubation of the two isoforms at 100 degrees C in the presence of ATP. The Arrhenius plots observed for both isozymes are biphasic, indicating different activation energies below and above 75 degrees C. The cytoplasmic levels of ATP, methionine and adenosylmethionine are evaluated.

Adenosine Triphosphate↗
PubMed1988

High-performance liquid chromatographic determination of a naturally occurring xylosyl thioether, 9-[5'-deoxy-5'(methylthio)-beta-D-xylofuranosyl]adenine.

A high-performance liquid chromatographic (HPLC) method for the separation and purification of 9-[5'-deoxy-5'(methylthio)-beta-D-xylofuranosyl]adenine (xylosyl-MTA), a naturally occurring analogue of 5'-deoxy-5'-methylthioadenosine (MTA), has been developed. The compound was purified from perchloric extracts of biological samples by reversed-phase HPLC on an Ultrasil ODS RP-18 column. Isocratic elution resulted in a complete separation between the xylosyl thioether and MTA. The determination of xylosyl-MTA in the nudibranch mollusc Doris verrucosa was also performed. Its concentration ranged from 330 to 380 nmol/g, considerably exceeding the cellular levels of MTA.

Adenosine Deaminase↗
PubMed1986

Purification and characterization of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium.

The enzyme propylamine transferase, catalyzing the transfer of the propylamine moiety from S-adenosyl(5')-3-methylthiopropylamine to several amine acceptors, has been purified 643-fold in 20% yield from Sulfolobus solfataricus, an extreme thermophilic archaebacterium optimally growing at 87 degrees C. The purified enzyme (specific activity 2.05 units/mg protein), is homogeneous by criteria of gel electrophoresis, gel filtration, isoelectric focusing and ultracentrifugation analysis. The molecular mass of the native enzyme was estimated to be about 110 kDa by gel permeation and ultracentrifugation analysis. The protein migrates on SDS/polyacrylamide gel electrophoresis as a single band of 35 kDa, suggesting that the enzyme is a trimer composed by identical subunits. An optimum pH of 7.5 and an acidic isoelectric point of 5.3 have been calculated. The optimum temperature was 90 degrees C and no loss of activity is observable even after exposure of the purified enzyme to 100 degrees C for 1 h. No reducing agents are required for enzymatic activity. Substrate specificity towards the amine acceptors is rather broad in that 1,3-diaminopropane (Km = 1675 microM), putrescine (Km = 3850 microM), sym-norspermidine (Km = 954 microM) and spermidine (Km = 1539 microM) are recognized as substrates. Conversely S-adenosyl(5')-3-methylthiopropylamine is the only propylamine donor (Km = 7.9 microM) and the deamination of the sulfonium compound prevents the recognition by the enzyme. The reaction is irreversible and initial-rate kinetic studies indicate that the propylamine transfer is operated through a sequential mechanism. 5'-Methylthioadenosine, a product of the reaction, acts as a powerful competitive inhibitor with a Ki of 3.7 microM. Enzyme-substrate binding sites have been investigated with the aid of several substrate analogs and products. Among the compounds assayed, 5'-methylthiotubercidin, S-adenosyl(5')-3-thiopropylamine and S-adenosyl-3-thio-1,8-diaminooctane are the most active inhibitors.

Archaea↗
PubMed1985

Escherichia coli S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action.

S-Adenosylhomocysteine/5'-methylthioadenosine nucleosidase (EC 3.2.2.9) was purified to homogeneity from Escherichia coli to a final specific activity of 373 mumol of 5'-methylthioadenosine cleaved/min per mg of protein. Affinity chromatography on S-formycinylhomocysteine-Sepharose is the key step of the purification procedure. The enzyme, responsible for the cleavage of the glycosidic bond of both S-adenosylhomocysteine and 5'-methylthioadenosine, was partially characterized. The apparent Km for 5'-methylthioadenosine is 0.4 microM, and that for S-adenosylhomocysteine is 4.3 microM. The maximal rate of cleavage of S-adenosylhomocysteine is approx. 40% of that of 5'-methylthioadenosine. Some 25 analogues of the two naturally occurring thioethers were studied as potential substrates or inhibitors of the enzyme. Except for the analogues modified in the 5'-position of the ribose moiety or the 2-position of the purine ring, none of the compounds tested was effective as a substrate. Moreover, 5'-methylthioformycin, 5'-chloroformycin, S-formycinylhomocysteine, 5'-methylthiotubercidin and S-tubercidinylhomocysteine were powerful inhibitors of the enzyme activity. The results obtained allow the hypothesis of a mechanism of enzymic catalysis requiring as a key step the protonation of N-7 of the purine ring.

Chromatography, Affinity↗
PubMed1984

Studies on the metabolic effects of methylthioformycin.

5'-Methylthioformycin, a structural analog of 5'-methylthioadenosine in which the N-C glycosidic bond is substituted by a C-C bond, has been synthesized by a newly developed procedure. Membrane permeability of the molecule has been compared to that of methylthioadenosine in intact human erythrocytes and Friend erythroleukemia cells. The formycinyl compound is taken up with a rate significantly lower than that of 5'-methylthioadenosine and is not metabolized by the cells. 5'-Methylthioformycin inhibits Friend erythroleukemia cells' growth: the effect is dose-dependent, fully reversible and not caused by cytotoxicity. Several enzymes related to methylthioadenosine metabolism are inhibited by methylthioformycin. Rat liver methylthioadenosine phosphorylase is competitively inhibited with a Ki value of 2 microM. Among the propylamine transferases tested only rat brain spermine synthase is significantly inhibited, while rat brain spermidine synthase is less sensitive. Rat liver S-adenosylhomocysteine hydrolase is irreversibly inactivated with 50% inhibition at 400 microM methylthioformycin. 5'-Methylthioformycin does not exert any significant effect on protein carboxyl-O-methyltransferase. Inferences about the mechanism of the antiproliferative effect of the drug have been drawn from the above results.

Adenosine↗
PubMed1984

Inhibition of dimethyl sulfoxide-induced differentiation of Friend erythroleukemic cells by 5'-methylthioadenosine.

5'-Methylthioadenosine is a sulfur-containing nucleoside derived from the metabolism of polyamines which is known to exert an antiproliferative effect on several cell systems in vitro, including the Friend leukemia cell system. We have investigated the role of 5'-methylthioadenosine on the dimethyl sulfoxide-induced differentiation of this system. At a concentration of 400 microM, the drug strongly inhibited (80%) the induced differentiation of Friend cells, and this effect was already observable at a concentration as low as 10 microM (36% inhibition), as evidenced by the benzidine staining procedure and by the dot-blot hybridization of globin mRNA with a human beta-globin probe. Similar results have been obtained by using 5'-S-isobutylthioadenosine, which is a synthetic structural analogue of 5'-methylthioadenosine. The block of differentiation produced by these nucleosides was not mediated by adenine (a catabolite of both molecules) and was not reverted by spermine or spermidine, the two polyamines whose synthesis is inhibited by 5'-methylthioadenosine. We report a decrease of the aminopropyltransferases activities (the enzymes responsible for 5'-methylthioadenosine biosynthesis) in dimethyl sulfoxide-treated Friend cells, which could lead to a decrease of the intracellular content of 5'-methylthioadenosine during the erythroid maturation of Friend cells. The results obtained are consistent with the hypothesis that 5'-methylthioadenosine may act as an endogenous regulator of Friend cell differentiation.

Adenosine↗
PubMed1983

Transport and metabolism of 5'-methylthioadenosine in human erythrocytes.

The transport and metabolism of 5'-deoxy-5'-S-methylthioadenosine have been studied in intact human erythrocytes. The sulfur nucleoside is rapidly accumulated into red cells and the extent of uptake largely exceeds the theoretical equilibrium between inner and outer compartment owing to its conversion into a non-permeable compound, namely 5-methylthioribose 1-phosphate. To characterize the nucleoside transport, phosphate-depleted erythrocytes, in which the methylthioadenosine metabolism is negligible, have been employed. The results indicate that: (i) the transport occurs via a facilitated-diffusion mechanism; (ii) the process is not energy-dependent and (iii) no specific cation is required. The kinetic analyses of both the transport and the metabolism show that the uptake of methylthioadenosine is a result of the tandem action of a transport step of high capacity (Vmax = 604 +/- 51 pmol/10(6) cells per min) and low affinity (Km = 3270 +/- 321 microM) followed by a metabolic step of low capacity (Vmax = 6.6 pmol/10(6) cells per min) and high affinity (Km = 30 microM). Furthermore, a substrate inhibition exerted by methylthioadenosine at high concentration (over 200 microM) on its specific phosphorylase is reported for the first time. Experiments performed with several analogs of the thioether indicate that the adenine amino group and the hydrophobic substituent at the 5'-position are critical for the transport carrier recognition. Adenine is the most powerful inhibitor of methylthioadenosine transport.

Adenosine↗
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

[A new method of measuring 5'-methylthioadenosine with high pressure liquid chromatography].

A rapid, sensitive and specific high performance liquid chromatographic method for the estimation of 5'-methylthioadenosine in biological samples has been developed. A double-step chromatographic on Dowex-50, H+ form, and Affi-Gel 601 were required before the chromatographic separation of sample on Partisil 10 SCX. The yield is quantitative and the procedure is simple and highly reproducible.

Adenosine↗
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↗
PubMed1980

[New 5'-sulfurated nucleosides as inhibitors of the biosynthesis of polyamines].

The inhibitory effect of the deaminated analogs of S-adenosyl(5')-3-methylthiopropylamine on spermidine synthase (EC 2.5.1.16.) from E. coli, AdoMet decarboxylase (EC 4.1.1.50.) from human placenta and E. coli and AdoMet lyase (EC 3.3.1.-) from rat liver have been reported. 5'-isobutylthioadenosine, a new powerful antiproliferative drug has been assayed as substrate of MTA phosphorylase (EC 2.4.2.1). The apparent Km is 1,8x10(-5) M. The inhibitory effect of 5'-isobutylthiadenosine on AdoMet lyase and spermidine synthase was also demonstrated.

Animals↗
PubMed1980

[S-adenosyl(5')-3-methylthiopropylamine as substrate and inhibitor of S-adenosylmethionine lyase from rat liver].

The present paper reports the study on a lyase in rat liver which decomposes S-adenosylmethionine into 5'-methylthioadenosine and alpha-amino-gamma-butyrolactone. The partial purification of this enzyme and some of its properties are discussed. S-adenosyl(5')-3-methylthiopropylamine, the decarboxylated analog of AdoMet, exerts a non-competitive inhibition of the enzyme whereas is inactive as substrate. The reported results demonstrate the relevance of the carboxyl group of AdoMet for the mechanism of the enzymatic hydrolysis of the sulfonium compound.

Animals↗