The inosinic branch point and its hormonal regulation: evaluation through a mathematical model.
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Biomedical subjects
Publications and source records attributed to E Marinello.
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Reverse phase high performance liquid chromatography was used for the determination of urinary hypoxanthine and xanthine in normal subjects. The values observed in normal subjects agree with those reported by other authors. Excretion of hypoxanthine and xanthine was not related to sex. Urinary oxypurines were related to age, and, when corrected for body surface area (1.73 m2), they were significantly higher in normal children under 15 years of age than in adults.
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Rat liver L-serine dehydratase (E.C.4.2.1.13) catalyzes the deamination both of L-serine and of L-threonine. These reactions show different rates and, at this moment, the "preferential" substrate of the enzyme is not clear. We have analysed, in various experimental conditions, the behaviour of the deaminase reaction toward the two substrates. From the obtained data, it is evident that at lower pH values L-serine and at higher pH values L-threonine, are the preferred substrates, respectively. A peculiar behaviour is shown by Km values, because they are different by changing the pH in the assay mixtures, and changes are related to the presence of pyridoxal-5'-phosphate in the assay mixtures.
Phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase is the "key anabolic enzyme" of purine nucleotide synthesis; PRPP synthetase connects the pentose cycle with the same pathway. We have studied their behavior in 5 control subjects and in 8 affected by CLL. Determination of PRPP amidotransferase was carried out through the evaluation of 14C-glutamic acid (released by 14C-glutamine) in the incubation mixture. PRPP synthetase was followed by adding ATP and ribose 5-phosphate to the incubation mixtures, and by evaluating the PRPP formed through the release of CO2 in a coupled reaction. In the case of PRPP-amidotransferase, our values are in the range reported in the literature: in patients affected by CLL, the enzyme activity is much higher and the increase is more evident when values referred to the patients, than when to the cells. Our values of PRPP synthetase are consistent with those of Peters and Veerkamp, but no definite conclusion is possible in the case of leukemic patients.
The behavior of plasma and urine oxypurines (hypoxanthine and xanthine) and of uric acid has been studied in normal subjects and in gout patients. Oxypurines and uric acid were increased in the plasma of gout patients but only the urinary excretion of hypoxanthine was higher in this group. The interpretation of the observed variations is discussed.
The clearance of uric acid, hypoxanthine and xanthine has been examined in gout patients and in normal subjects compared to creatinine, after a purine-free diet. The treatment decreased the clearance in normal subjects, but showed an opposite effect in gout patients. The clearances both of uric acid, hypoxanthine and xanthine were enhanced by allopurinol. The interpretation of the observed variations is discussed.
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We have elaborated a kinetic method which allows us to evaluate whether a Michaelis-Menten-type enzyme acting on two different substrates has one or two active sites. This method has been used with the rat liver L-threonine dehydratase, which catalyzes the dehydrative deamination of both serine and threonine. The experimental data can be fitted to the theoretical plot obtained for the case of a single active site.
We examined the effects of a two-thirds hepatectomy in the adult rat on the activities of the three L-threonine-degrading enzymes, L-threonine dehydratase, L-threonine aldolase and L-threonine dehydrogenase. Noticeable variations were observed which did not occur in either sham-operated or turpentine-treated rats and were not linked to food intake. They were considered specific to the regenerating liver. When the reactions were followed in vitro, L-threonine deaminase and L-threonine aldolase were significantly lower for the first 12-24 h: L-threonine dehydrogenase decreased only after 48 h. These results are linked to a decrease in the enzyme concentration in the tissue. L-Serine and L-threonine liver concentrations increased 2-3-fold during the same periods. When the activities were evaluated in vivo, the levels of the first two enzymes remained constant for 24 h, but increased after 48 h; L-threonine dehydrogenase increased between 12 and 48 h. The in vivo activity of the enzymes was reflected by total L-threonine degradation, which had a single sharp peak at 48 h. The asynchronous variations in enzyme activity are related to the differences in protein metabolism which occur in the regenerating liver, and are the consequence of a new transient differential control. The changes observed are significant in liver regeneration; they regulate the consumption and the serum and liver levels of L-serine and L-threonine, setting them aside for protein synthesis. They minutely control the flux of amino acids toward gluconeogenesis, since, during the first 48 h after partial hepatectomy, the production of glucose is ensured principally by lactate; the contribution of L-threonine seems to be more significant only at 48 h. These findings are useful in the study of the regulation of the enzymes involved in amino acid metabolism during liver regeneration.
We examined in vivo the influence of testosterone on purine synthesis de novo, in the levator ani and gastrocnemius muscles of the rat. The hypoxanthine, adenine and guanine contents and the rate of incorporation of [14C]formate into these purine bases were determined in castrated adult and prepubertal rats (groups 1 and 2) both before and after orchiectomy and, in the second case, at different times after testosterone treatment. Substantially similar behavior was found in both groups, with some specific differences. The results showed an increase in the basal levels after castration (except for a dramatic decrease in adenine and a rise in the Gua/Ade molar ratio in prepubertal rats) and a return to basal levels after hormone administration, which was also accompanied by variations in the Gua/Ade molar ratio. The kinetics of purine nucleotide synthesis de novo in vivo and, specifically, of the overall reactions: IMP formation from PRib-PP, IMP----AMP and IMP----GMP, were followed by evaluating the incorporation curves of [14C]formate into hypoxanthine, adenine and guanine. Our results show that testosterone administration enhanced the incorporation rate and gave characteristic patterns: a diphasic cyclic oscillation of the Ade values in adult castrated rats, and single peaks having a specific shape in the other cases. The Gua/Ade labeling ratio was unchanged in castrated rats and increased in both groups during the first 5 days after testosterone treatment, after which values even fell below normal; in most cases, values overlapped the pattern of the Gua/Ade molar ratio. The specific profile of the curves indicated that testosterone initially accelerated the turnover of guanylic acid and in the second phase re-established the normal behavior and ratio of AMP and GMP formation. These results indicate that the 'inosinic branch point' was subject to regulation by testosterone. The profiles of the incorporation curves and of the Gua/Ade ratio were indicative of a primary and secondary response to hormone action.
Plasma levels and urinary excretion of oxypurines--hypoxanthine and xanthine--were evaluated by reverse-phase high-pressure liquid chromatography in 13 patients affected by gastric tumors and in 19 colorectal tumor-bearing patients. Preliminary results indicate higher values of urinary xanthine and an increase in the xanthine/hypoxanthine ratio in cancer patients. The increase was not generalized to all subjects, and did not appear related either to the stage of the disease or to CEA values. The limits within which the determination of urinary oxypurines can be employed as a tumor marker are discussed.
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