Purine catabolism in rat brain (author's transl)

In soluble rat brain fraction, the specific activities of purine nucleoside phosphorylase, guanine deaminase, 5'Nucleotidase and adenosine deaminase, decrease in their mentioned order. A kinetic parameter comparison between these enzymes shows that 5'Nucleotidase with AMP has the lowest KM and the greatest Vmax values, while purine nucleoside phosphorylase has its lowest KM and its greatest Vmax values with guanosine and with inosine, respectively. The enzymes activity is not modified by the metabolic intermediates differently from their own reaction products which behave as competitive inhibitors.     

Pentose phosphates in nucleoside interconversion and catabolism

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.     

Cytokinin biosynthesis and interconversion

To maintain hormone homeostasis, the rate of cytokinin biosynthesis, interconversion, and degradation is regulated by enzymes in plant cells. Cytokinins can be synthesized via direct (de novo) or indirect (tRNA) pathways. In the de novo pathway, a cytokinin nucleotide is synthesized from 5'-AMP and isopentenyl pyrophosphate; a key enzyme which catalyzes this synthesis has been isolated from plant tissues, slime mold, and some microorganisms. Studies on the in vitro synthesis of the isopentenyl side chain of cytokinin in tRNA demonstrated that the isopentenyl group was derived from mevalonate, and turnover of the cytokinin-containing tRNA may serve as a minor source of free cytokinins in plant cells. The interconversion of cytokinin bases, nucleosides and nucleotides is a major feature of cytokinin metabolism; and enzymes that regulate the interconversion have been identified. The N⁶-side chain and purine moiety of cytokinins are often modified and some of the enzymes involved in the modifications have been isolated. Most of the cytokinin metabolites have been characterized but very few enzymes regulating their metabolism have been purified to homogeneity. It remains a significant challenge to isolate plant genes involved in the regulation of cytokinin biosynthesis, interconversion and degradation.     

Purine catabolism in advanced carotid artery plaque

This study was carried out on carotid artery plaque and plasma of 50 patients. We analyzed uric acid, hypoxanthine, xanthine, and allantoin levels to verify if enzymatic purine degradation occurs in advanced carotid plaque; we also determined free radicals and sulphydryl groups to check if there is a correlation between oxidant status and purine catabolism. Comparing plaque and plasma we found higher levels of free radicals, hypoxanthine, xanthine, and a decrease of some oxidant protectors, such as sulphydryl groups and uric acid, in plaque. We also observed a very important phenomenon in plaque, the presence of allantoin due to chemical oxidation of uric acid, since humans do not have the enzyme uricase. The hypothetical elevated activity of xanthine oxidase in atherosclerosis could be reduced by specific therapies using its inhibitors, such as oxypurinol or allopurinol.     

Purine biosynthesis in Staphylococcus aureus

Summary The auxanographic analysis of 67 purine-dependent mutants and chromatographic analysis of their culture fluids were used to study purine biosynthesis in Staphylococcus aureus. The de novo biosynthesis of IMP from SAICAR, and the conversion of IMP to AMP and GMP were shown to occur via the conventional pathways reported for other organisms. Mutants blocked prior to the formation of SAICAR could not be differentiated by the tests used, and no substantial information on this portion of the pathway was obtained. The auxanographic characteristics of double mutants requiring both histidine and purines provided evidence that the sole route whereby S. aureus can convert AMP to IMP (and hence to GMP) is via those reactions of the histidine biosynthetic pathway leading to the formation of IGP and AICAR. In addition, we were able to mutationally separate AICAR transformylase and inosinocase; this separation has not been accomplished in other microorganisms.     

Purine and pyrimidine biosynthesis in methanogenic bacteria

Following long-term labeling with [1-¹³C]acetate, [2-¹³C]acetate, ¹³CO₂, H¹³COOH, or ¹³CH₃OH, NMR spectroscopy was used to determine the labeling patterns of the purified ribonucleosides of Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii. Major differences were observed among the methanogens studied, specifically at carbon positions 2 and 8 of the purines, positions at which one-carbon carriers are involved during synthesis. In Methanospirillum hungatei and Methanosarcina barkeri, the labcl at both positions came from carbon atom C-2 of acetate, as predicted from known eubacterial pathways, whereas in Methanococcus voltae and Methanobacterium bryantii both originated from CO₂. In Methanosphaera stadtmanae grown in the presence of formate, the C-2 of purines originated exclusively from formate and the C-8 was labeled by the C-2 of acetate. When grown in media devoid of formate, the C-2 of the purine ring originated mainly from the C-2 of acetate and in part from CH₃OH. In Methanobrevibacter smithii grown in the presence of formate, C-2 and C-8 of purines were derived from CO₂ and/or formate. The labeling patterns obtained for pyrimidines are consistent with the biosynthetic pathways common to eubacteria and eucaryotes.Abbreviations CODH Carbon monoxide dehydrogenase - FH₄ tetrahydrofolate - H₄MPT tetrahydromethanopterin Issued as NRCC Publication No. 37383     

Purine biosynthesis and catabolism in soybean root nodules: incorporation of 14C from 14CO2 into xanthine

Nodulated root systems of soybean plants were exposed to ¹⁴CO₂ in the presence and absence of allopurinol. After 5 h about one-fifth of the label in the perchloric acid-soluble fraction of the nodules was found to be in xanthine in the allopurinol-treated plants. Control plants contained much lower levels of xanthine, but with similar specific activity. Hypoxanthine was not detected in either control or allopurinol-treated plants, even though it would be expected to accumulate in the latter. Degradation of labeled xanthine from allopurinol-treated plants using xanthine oxidase and uricase resulted in the loss of most of the label. The preferential incorporation and accumulation of ¹⁴C from ¹⁴CO₂ into C₆ of xanthine in allopurinol-treated plants is consistent with the involvement of phosphoribosylaminoimidazole carboxylase in the de novo synthesis of purines. The accumulation of xanthine and absence of hypoxanthine in nodules of allopurinol-treated plants confirms earlier observations. In addition, the similar specific activities of ¹⁴C in xanthine in allopurinol-treated and control plants indicate that the xanthine which accumulates in allopurinol-treated plants is the product of de novo purine biosynthesis.     

Purine and pyrimidine biosynthesis in higher plants

Purine and pyrimidine nucleotides have important functions in a multitude of biochemical and developmental processes during the life cycle of a plant. In higher plants the processes of nucleotide metabolism are poorly understood, but it is in principle accepted that nucleotides are essential constituents of fundamental biological functions. Despite of its significance, higher plant nucleotide metabolism has been poorly explored during the last 10–20 years (Suzuki and Takahashi 1977, Schubert 1986, Wagner and Backer 1992). But considerable progress was made on purine biosynthesis in nodules of ureide producing tropical legumes, where IMP-synthesis plays a dominant role in primary nitrogen metabolism (Atkins and Smith 2000, Smith and Atkins 2002). Besides these studies on tropical legumes, this review emphasises on progress made in analysing the function in planta of genes involved in purine and pyrimidine biosynthesis and their impact on metabolism and development.     

A multifunctional protein possessing glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase activities in de novo purine biosynthesis

Three activities on the pathway of purine biosynthesis de novo in chicken liver, namely, glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase, have been found to reside on the same polypeptide chain. Three diverse purification schemes, utilizing three different affinity resins, give rise to the same protein since the final material has identical specific activities for all three enzymatic reactions and a molecular weight on sodium dodecyl sulfate gels of about 110 000. A single antibody preparation precipitates all three activities and binds to the multifunctional protein obtained by two methods in Western blots. Partial chymotryptic digestion of the purified protein gives rise to two fragments, one possessing glycinamide ribonucleotide synthetase activity and the other containing glycinamide ribonucleotide transformylase activity.     

In vitro biosynthesis of sialosylgalactosylceramide (G7) by mouse brain microsomes.

A sialytransferase activity which catalyzes the synthesis of sialosylgalactosylceramide (G7) from added galactocerebroside and CMP-N-acetylneuraminic acid has been demonstrated in mouse brain microsomes. The enzyme reaction shows a pH optimum of 6.3 and requires detergents. Both Mn2+ and Ca2+ inhibited the reaction, whereas Mg2+ had no effect. The apparent Km for galactocerebroside leading to G7 was estimated to be 8.7 X 10(-4) M. The same microsomal preparation also synthesized hematoside when ceramide lactoside was the glycolipid acceptor. The apparent Km for ceramide lactoside was about one-tenth that for galactocerebroside. When the preparations were partially inactivated by heat the synthesis of G7 and of hematoside was reduced at approximately the same rate. Liver appeared to have the highest activity for G7 synthesis (as well as of hematoside), followed by brain. The synthesis of B7 by mouse brain microsomes in vitro demonstrates a new pathway for brain ganglioside synthesis.     

Purine catabolism in isolated rat hepatocytes. Influence of coformycin

1. The catabolism of purine nucleotides was investigated by both chemical and radiochemical methods in isolated rat hepatocytes, previously incubated with [¹⁴C]adenine. The production of allantoin reached 32±5nmol/min per g of cells (mean±s.e.m.) and as much as 30% of the radioactivity incorporated in the adenine nucleotides was lost after 1h. This rate of degradation is severalfold in excess over values previously reported to occur in the liver in vivo. An explanation for this enhancement of catabolism may be the decrease in the concentration of GTP. 2. In a high-speed supernatant of rat liver, adenosine deaminase was maximally inhibited by 0.1μm-coformycin. The activity of AMP deaminase, measured in the presence of its stimulator ATP in the same preparation, as well as the activity of the partially purified enzyme, measured after addition of its physiological inhibitors GTP and Pi, required 50μm-coformycin for maximal inhibition. 3. The production of allantoin by isolated hepatocytes was not influenced by the addition of 0.1μm-coformycin, but was decreased by concentrations of coformycin that were inhibitory for AMP deaminase. With 50μm-coformycin the production of allantoin was decreased by 85% and the formation of radioactive allantoin from [¹⁴C]adenine nucleotides was completely suppressed. 4. In the presence of 0.1μm-coformycin or in its absence, the addition of fructose (1mg/ml) to the incubation medium caused a rapid degradation of ATP, without equivalent increase in ADP and AMP, followed by transient increases in IMP and in the rate of production of allantoin; adenosine was not detectable. In the presence of 50μm-coformycin, the fructose-induced breakdown of ATP was not modified, but the depletion of the adenine nucleotide pool proceeded much more slowly and the rate of production of allantoin increased only slightly. No rise in IMP concentration could be detected, but AMP increased manyfold and reached values at which a participation of soluble 5′-nucleotidase in the catabolism of adenine nucleotides is most likely. 5. These results are in agreement with the hypothesis that the formation of allantoin is controlled by AMP deaminase. They constitute further evidence that 5′-nucleotidase is inactive on AMP, unless the concentration of this nucleotide rises to unphysiological values.