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Two specific methods for the determination of 5'-deoxy-5'-methylthioadenosine (MTA) in biological samples have been developed. The chromatographic procedure requires a preliminary step on a phenylboronate column to remove non-cis-diol compounds. The sample is then analysed using a high-performance liquid chromatography system equipped with a reversed-phase column. 5'-Deoxy-5'-methyl-thio[2-3H]adenosine with high specific activity was synthesized and employed as an internal standard. An alternative radioimmunoassay (RIA) procedure has also been developed. The RIA method is based on competition between the unlabelled thio-ether and 3H-labelled MTA for the binding to a specific antiserum. Anti-MTA antibodies were obtained from rabbits immunized with the nucleoside covalently linked to carrier proteins. Both the chromatographic and RIA procedures gave identical results when employed to determine MTA in human urine.

5'-Methylthioadenosine phosphorylase has been purified to homogeneity (30,000-fold) from human full-term placenta by a procedure involving covalent chromatography on organomercurial-agarose as the major step. The specific activity of the homogeneous enzyme is 10.2 mumol of 5'-methylthioadenosine cleaved per min per mg of protein, and the overall yield is about 20%. The enzyme has a molecular weight of 98,000, as determined by gel filtration on Sephacryl S-200 and Superose 6B, and is composed by three apparently identical subunits with a molecular weight of 32,500. The isoelectric point is 5.5, and the optimal pH ranges from 7.2 to 7.6. The resistance of the enzyme to thermal inactivation is increased remarkably by the addition of 5'-methylthioadenosine or phosphate. The homogeneous enzyme shows an absolute requirement for -SH-reducing agents and is specifically and rapidly inactivated by thiol-blocking compounds. The reaction catalyzed by the enzyme is fully reversible with a Keq of 1.39 X 10(-2) (in the direction of phosphorolysis) at 37 degrees C and pH 7.4. The Km values for 5'-methylthioadenosine, phosphate, adenine, and 5-methylthioribose 1-phosphate are 5, 320, 23, and 8 microM, respectively.

The effects of a number of nucleosides related to 5'-methylthioadenosine on the activities of S-adenosylhomocysteine hydrolase, 5'-methylthioadenosine phosphorylase, spermidine synthase and spermine synthase were investigated. Both 5'-methylthioadenosine and 5'-isobutylthioadenosine gave rise to an enzyme-activated irreversible inhibition of S-adenosylhomocysteine hydrolase, but 5'-methylthiotubercidin (5'-methylthio-7-deaza-adenosine), 5'-deoxy-5'-chloroformycin, 5'-ethylthio-2-fluoro-adenosine and 1,N6-etheno-5'-methylthioadenosine were totally ineffective in producing this inactivation. Of the nucleosides tested, only 5'-methylthioadenosine, 5'-methylthiotubercidin and 5'-isobutylthioadenosine were inhibitory towards the aminopropyltransferases responsible for the synthesis of spermine and spermidine. 5'-Methylthiotubercidin, 5'-deoxy-5'-chloroformycin and 5'-isobutylthioadenosine were inhibitors of the degradation of 5'-methylthioadenosine by 5'-methylthioadenosine phosphorylase, but only 5'-isobutylthioadenosine was also a substrate for this enzyme. These results suggest that the effects of 5'-isobutylthioadenosine of the cell may result from the combination of inhibitory actions on polyamine synthesis, 5'-methylthioadenosine degradation and S-adenosylhomocysteine degradation. The resulting increased concentrations of S-adenosylhomocysteine could bring about inhibition of methyltransferase reactions. A new convenient method for the assay of S-adenosylhomocysteine hydrolase in the direction of synthesis is described.

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.

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.

In the present study the effect of 5'methylthioadenosine (MTA), a natural metabolite of S-adenosylmethionine (AdoMet), on the enzymatic methyl esterification of intact erythrocyte membrane proteins has been investigated. The thioether significantly affects the methylation process :50% inhibition being observable at 100 microM MTA. This inhibition is due to the action of MTA on the enzyme protein methylase II. Since MTA is present in micromolar amounts in the cells, the reported effect could be of physiological interest and suggests a new regulatory role of this AdoMet metabolite.

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.

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.