Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro

The relative rates of the synthetic, interconversion and catabolic reactions of purine metabolism in chopped mouse cerebrum were studied. The rates of incorporation of [(14)C]adenine and [(14)C]hypoxanthine into purine ribonucleotides were much less than the potential activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, and the rates of incorporation were stimulated by the addition of guanosine to the incubation mixture. The availability of ribose phosphates may be a limiting factor for the formation of ribonucleotides from purine bases. The rate of incorporation of [(14)C]adenosine into purine ribonucleotides was at least seven- to eight-fold higher than that of adenine. The radioactivity in adenine ribonucleotides synthesized from adenine and hypoxanthine was about 100- and ten-fold respectively higher than that in the radioactive guanine ribonucleotides. The conversion of inosinate into guanine ribonucleotides was probably limited by the amount of inosinate available, and the conversion of adenine ribonucleotides into guanine ribonucleotides was probably limited by the activity of adenylate deaminase. The rate of catabolism of [(14)C]adenosine was low in comparison with its rate of utilization for ribonucleotide synthesis. A fraction of the [(14)C]hypoxanthine was catabolized to xanthine and urate. [(14)C]Guanine was completely converted into xanthine, mostly by the guanine deaminase that was released during incubation of chopped mouse cerebrum.     

What determines the intracellular ATP concentration

Analysis is made of the mechanisms that control the intracellular ATP level. The balance between energy production and expenditure determines the energy charge of the cell and the ratio of [ATP] to the adenylate pool. The absolute ATP concentration is determined by the adenylate pool, which, in its turn, depends on the balance between the rates of AMP synthesis and degradation. Experimental data are discussed that demonstrate an increase in the adenylate pool in response to activation of energy-consuming processes. A hypothesis is proposed according to which variation in the adenylate pool and absolute ATP concentration affords a cell the possibility of additional control over processes fulfilling useful work. A mechanism involved in this regulation is described using human erythrocytes as an example. The hypothesis explains why different metabolic pathways (protein and DNA syntheses, polysaccharide synthesis, and lipid synthesis) use different trinucleotides (GTP, UTP, and CTP, respectively) as an energy source. This allows the cell to independently control these metabolic processes by varying the individual nucleotide pools.     

Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.     

Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN^?-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN^?) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min^?1 · 10^?11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min^?1 · 10^?11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.     

Factors affecting inosinate synthesis and inosine triphosphate accumulation in human erythrocytes.

Measurements of rates of inosinate synthesis from radioactive hypoxanthine by human erythrocytes show a large degree of individual variation. Rates of inosinate synthesis also vary with the pH and phosphate concentration of the incubation medium. This may be due to changes in the rate of phosphoribosyl pyrophosphate synthesis, and the stimulatory effect of phosphate on this process seems to be more important than the inhibitory effect of 2,3-diphodphoglycerate. The rate of inosinate synthesis, and especially the extent of accumulation of inosine triphosphate, increase disproportionately with time of incubation up to at least 24 h. Storage of erythrocytes also tends to increase inosinate synthesis and inosine triphosphate accumulation.     

Enzymes involved in adenine nucleotide metabolism of developing chick embryo liver.

1. This paper describes the changes in the activity of adenylate deaminase, adenylate and inosinate phosphatase, and adenosine deaminase in the developing chick embryo liver. 2. The adenylate and inosinate phosphatase and adenosine deaminase activity appears considerably higher in chick embryo liver with respect to other chick embryo tissues previously examined. 3. During development the control exerted by ATP on AMP breakdown undergoes variations. Consequently, in the first period of incubation AMP is degraded by the direct pathway (AMP-IMP) and in the last period of incubation by the indirect pathway (AMP-adenosine). In the intermediate period (from the 12th to the 15th day of incubation) both pathways may be followed. 4. The ability to synthesize purine nucleotides through "salvage pathway" seems to be acquired by embryonic liver at least at the 15th day.     

In vitro adenine nucleotide catabolism in African catfish spermatozoa

It has been shown recently that African catfish (Clarias gariepinus) spermatozoa possess relatively low ATP content and low adenylate energy charge (AEC). One of the possible explanations for this phenomenon is that the spermatozoa actively catabolize adenine nucleotides. A relatively high rate of such catabolism could then contribute to the low ATP concentration and low adenylate energy charge observed in the spermatozoa in vitro. To check this hypothesis, we investigated ATP content and adenine nucleotide catabolism in African catfish spermatozoa stored at 4 °C in the presence of glycine as an energetic substrate. Our results indicate that the storage of African catfish sperm at 4 °C in the presence of glycine causes time-dependent ATP depletion. In contrast to ATP, the AMP content increases significantly during the same period of sperm storage, while the ADP increases only slightly. Moreover, a significant increase of inosine and hypoxanthine content was also found. Hypoxanthine was accumulated in the storage medium, but xanthine was found neither in spermatozoa nor in the storage medium. It indicates that hypoxanthine is not converted to xanthine, probably due to lack of xanthine oxidase activity in catfish spermatozoa. Present results suggest that adenine nucleotides may be converted to hypoxanthine according to the following pathway: ATP→ADP→AMP (adenosine/IMP)→inosine→hypoxanthine. Moreover, hypoxanthine seems to be the end product of adenine nucleotide catabolism in African catfish spermatozoa. In conclusion, our results suggest that a relatively high rate of adenine nucleotide catabolism contributes to the low ATP concentration and low adenylate energy charge observed in African catfish spermatozoa in vitro.     

Purine metabolism in Saccharomyces cerevisiae.

The synthesis, interconversion, and catabolism of purine bases, ribonucleosides, and ribonucleotides in wild-type Saccharomyces cerevisiae were studied by measuring the conversion of radioactive adenine, hypoxanthine, guanine, and glycine into acid-soluble purine bases, ribonucleosides, and ribonucleotides, and into nucleic acid adenine and guanine. The pathway(s) by which adenine is converted to inosinate is (are) uncertain. Guanine is extensively deaminated to xanthine. In addition, some guanine is converted to inosinate and adenine nucleotides. Inosinate formed either from hypoxanthine or de novo is readily converted to adenine and guanine nucleotides.     

Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli.

Methods for measurements of catabolic reduction charge (defined as NADH/(NADH+NAD+)) and anabolic reduction charge (defined as NADPH/(NADPH + NADP+)) are described using [14C]nicotinamide labeling of Escherichia coli cultures. Together with these parameters the adenylate energy charge (ATP + 1/2ADP)/(ATP + ADP + AMP) was measured using labeling with [2-3H]adenine. These three charges were found under different exponential growth conditions to have values independent of the growth conditions: catabolic reduction charge, 0.05; anabolic reduction charge, 0.45; and adenylate energy charge, 0.9. The charges were examined during interruption of growth primarily affecting catabolism, respiration, or anabolism, leading to changes of the charges. The changes of charges are evaluated as a possible regulation of the metabolic rates utilizing or producing the nucleotides by their respective charges.     

Role of adenylate kinase, AMP deaminase and 5'-nucleotidase in the metabolism of adenylic nucleotides

The activities of adenylate kinase, AMP-deaminase and 5'-nucleotidase in various tissues of the rat were studied. The activity of the forward adenylate kinase reaction (ATP + AMP----2 ADP) against the back one (2 ADP----ATP + AMP) was predominant. The liver was shown to contain two, while the blood serum--three adenylate kinase isoenzymes. In the skeletal muscles, the catabolism of adenylic acid involving AMP-deaminase and 5'-nucleotidase predominantly occurred via deamination, in the liver--via dephosphorylation, while in the leucocytes, erythrocytes and blood serum the activity of these processes was essentially the same. In vitro, ATP enhanced the activity of AMP-deaminase in the liver, leucocytes and erythrocytes and decreased it in the blood serum. Under effects of ATP, the activity of 5'-nucleotidase in the leucocytes and blood serum was markedly elevated, that in the liver and erythrocytes was unaffected.     

Maintenance requirements: energy supply from simultaneous endogenous respiration and substrate consumption

Abstract A model is presented that describes energy for maintenance purposes (ATP) as being obtained simultaneously from biomass degradation as well as from substrate degradation in excess of growth requirements. The ratio between both catabolic processes was taken to be growth rate dependent. As such, this approach is intermediate between established models; its significant features are negative growth and the absence of substrate consumption at zero substrate concentration, and the attainability of the maximum specific growth rate (the model parameter μ _max) at elevated substrate concentrations. As a simple case, the amounts of ATP obtained from direct substrate catabolism or from the degradation of an equivalent amount of biomass were taken as identical. Also, the maintenance demand in terms of ATP per unit time and biomass was taken to be constant. True growth rate dependency of maintenance can be implemented by relaxing either of these assumptions.     

Activation by GTP of liver adenylate cyclase in the presence of high concentrations of ATP

Effect of GTP on adenylate cyclase of liver plasma membrane was examined using ATP which was extensively purified by DEAE-cellulose column chromatography. In the incubation containing 2mM purified ATP as substrate, GTP enhanced basal and glucagon- or fluoride-stimulated activities. When the unpurified ATP at 2mM was used, all the activities were high and the stimulatory effect of GTP was not detected. The substance(s) which was recovered from a small but significant peak on DEAE-cellulose column was equivalent to 10–100μM GTP in stimulating adenylate cyclase. These results indicate that, if highly purified ATP is used as substrate, GTP can enhance adenylate cyclase activity in the presence of millimolar concentration of ATP and that GTP enhances not only the glucagon-stimulated adenylate cyclase but also the basal as well as fluoride-stimulated adenylate cyclase activities.     

ATP requirement for acidic resistance in Escherichia coli

ATP participates in many cellular metabolic processes as a major substrate to supply energy. Many systems for acidic resistance (AR) under extremely acidic conditions have been reported, but the role of ATP has not been examined. To clarify whether or not ATP is necessary for the AR in Escherichia coli, the AR of mutants deficient in genes for ATP biosynthesis was investigated in this study. The deletion of purA or purB, each of which encodes enzymes to produce AMP from inosinate (IMP), markedly decreased the AR. The content of ATP in these mutants decreased rapidly at pH 2.5 compared to that of the wild type. The AR was again decreased significantly by the mutation of adk, which encoded an enzyme to produce ADP from AMP. The DNA damage in the purA and purB mutants was higher than that in the wild type. These results demonstrated that metabolic processes that require ATP participate in survival under extremely acidic conditions, and that one such system is the ATP-dependent DNA repair system.     

Close correlation between platelet responses and adenylate energy charge during transient substrate depletion

The relation between availability of metabolic energy and thrombin-induced platelet aggregation and secretion was investigated in a system of transient substrate depletion followed by restoration of ATP resynthesis. Substrate depletion induced a fall in the concentration of metabolic ATP and in the adenylate energy charge and a concurrent decline in aggregation and secretion of dense and α-granule contents. Restoration of energy generation completely restored the adenylate energy charge and restored aggregation and secretion, but led to incomplete recovery of the ATP concentration. A close correlation between the adenylate energy charge and aggregation and between the adenylate energy charge and the secretion of dense and α-granule contents could be demonstrated. No such correlation existed between these responses and the concentration of ATP. These results show that the adenylate energy charge monitors an energetic condition which is crucial for preservation of platelet aggregation and secretion of dense and α-granule contents.     

Alterations of inosinate branchpoint enzymes in cultured human lymphoblasts

The specific activities of the three enzymes of the inosinate branchpoint are independently regulated when lymphoblasts are grown under various tissue culture conditions. In comparison to rapidly dividing cells, lymphoblasts at high cell density with no cellular division have decreased activity of the enzymes which commit inosinate to adenylate or guanylate, while cytoplasmic 5'-nucleotidase is relatively preserved. A linear relationship between inosinate dehydrogenase activity and growth rate (r = 0.92) exists in lymphoblasts with slowed growth rates. In contrast, in dividing cells adenylosuccinate synthetase and 5'-nucleotidase do not vary with growth rate. Adenylosuccinate synthetase and inosinate dehydrogenase activities appear to be related to the presence or rate of cellular division, as opposed to the presence or degree of neoplastic transformation. Lymphoblast lines with alterations of specific purine metabolic enzymes have characteristic alteration of the inosinate utilizing enzymes. Deficiencies of purine nucleoside phosphorylase or hypoxanthine phosphoribosyltransferase, abnormalities which render the cell unable to salvage purine effectively, are associated with depressed inosinate dehydrogenase activity. Insertion of the hypoxanthine phosphoribosyltransferase gene into hypoxanthine phosphoribosyltransferase-deficient cells normalizes inosinate dehydrogenase activity, while a hypoxanthine phosphoribosyltransferase-deficient mutant selected from a hypoxanthine phosphoribosyltransferase-containing line has depressed inosinate dehydrogenase activity. In contrast, overactivity of phosphoribosylpyrophosphate synthetase, with enhanced excretion of purines due to excessive production, is associated with elevated inosinate dehydrogenase activity. Inosinate dehydrogenase appears to be regulated according to the availability of purine nucleotides. Patients who overproduce uric acid and potentially have undescribed purine metabolic defects are now being screened for abnormalities in the inosinate branchpoint enzymes.     

Role of magnesium in the (Ca2+ + Mg2+)-stimulated membrane ATPase of human red blood cells.

(Ca2+ + Mg2+)-stimulated ATPase of human red cell membranes as a function of ATP concentration was measured at fixed Ca2+ concentration and at two different but constant Mg2+ concentrations. Under the assumption that free ATP rather than Mg-ATP is the substrate, a value for Km (for ATP) of 1-2 micron is found which is in good agreement with the value obtained in the phosphorylation reaction by A.F. Rega and P.J. Garrahan (1975. J. Membrane Biol. 22:313). Mg2+ increases both the maximal rate and the affinity for ATP, whereas Ca2+ increases the maximal rate without affecting Km for ATP. As a by-product of these experiments, it was shown that after thorough removal of intracellular proteins the adenylate kinase reaction at approximately 1 mM substrate concentration is several times faster than maximal rate of (Ca2+ + Mg2+)ATPase in red cell membranes.     

Tryptophanyl adenylate formation by tryptophanyl-tRNA synthetase from Escherichia coli

By gel filtration and titration on DEAE-cellulose filters we show that Escherichia coli tryptophanyl-tRNA synthetase forms tryptophanyl adenylate as an initial reaction product when the enzyme is mixed with ATP-Mg and tryptophan. This reaction precedes the synthesis of the tryptophanyl-ATP ester known to be formed by this enzyme. The stoichiometry of tryptophanyl adenylate synthesis is 2 mol per mole of dimeric enzyme. When this reaction is studied either by the stopped-flow method, by the fluorescence changes of the enzyme, or by radioactive ATP depletion, three successive chemical processes are identified. The first two processes correspond to the synthesis of the two adenylates, at very different rates. The rate constants of tryptophanyl adenylate synthesis are respectively 146 +/- 17 s-1 and 3.3 +/- 0.9 s-1. The third process is the synthesis of tryptophanyl-ATP, the rate constant of which is 0.025 s-1. The Michaelis constants for ATP and for tryptophan in the activation reaction are respectively 179 +/- 35 microM and 23.9 +/- 7.9 microM, for the fast site, and 116 +/- 45 microM and 3.7 +/- 2.2 microM, for the slow site. No synergy between ATP and tryptophan can be evidenced. The data are interpreted as showing positive cooperativity between the subunits associated with conformational changes evidenced by fluorometric methods. The pyrophosphorolysis of tryptophanyl adenylate presents a Michaelian behavior for both sites, and the rate constant of the reverse reaction is 360 +/- 10 s-1 with a binding constant of 196 +/- 12 microM for inorganic pyrophosphate (PPi).(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of L-isoleucyl-tRNA synthetase with L-isoleucyl-bromomethyl ketone. The effect of the catalytic steps.

The rapidly reacting cysteine-sulfhydryl group of L-isoleucyl-tRNA synthetase has been specifically alkylated with L-isoleucyl-bromomethyl ketone [Rainey, P., Holler, E. & Kula, M.-R. (1976) Eur. J. Biochem. 63, 419-426]. We have now investigated the catalytic and substrate binding properties of the modified protein by radioactive and fluorescence techniques. The rate constants for the transfer of AMP and isoleucine from the protein - adenylate complex to form ATP or Ile-tRNAIle were only 3% of those for native enzyme, whereas the rate constant for the formation of adenylate was essentially unchanged. The tendency to form synthetase - substrate complexes remained almost unchanged with the exception of L-isoleucine which exhibited a 20-fold reduction. Similarly, complex formation of L-isoleucinol together with its synergistic coupling to complex formation of ATP was partially inhibited. The results rule out the essential participation of the rapidly alkylatable cysteine-sulfhydryl group during catalysis.     

Manganese and manganese-ATP interactions with bovine sperm adenylate cyclase

The adenylate cyclase of mammalian spermatozoa shares some of the properties of the isolated catalytic component from somatic cell adenylate cyclases. One of these properties is the large apparent stimulation by Mn2+. We have used the direct linear plot according to Eisenthal and Cornish-Bowden to explore whether this apparent stimulation is due to direct stimulation by Mn2+ or due to complexation of free ATP, a postulated inhibitor of cyclase activity. We have observed the activity of the particulate adenylate cyclase from bovine caudal epididymal spermatozoa as a function of calculated equilibrium values for the concentrations of Mn2+, free ATP, and the enzyme's substrate, the manganese-ATP complex. Direct linear plots for activity and substrate concentration over the apparent inhibitory concentration range of free ATP give the pattern expected for a hyperbolic substrate response. By contrast, direct linear plots in which Mn2+ concentration varies over its apparent stimulatory range show that as Mn2+ concentration increases, activities are higher than would be predicted for a hyperbolic substrate response. We conclude that for particulate bovine sperm adenylate cyclase, free ATP is not strongly inhibitory, and Mn2+ is a positive effector, reaching half-maximal stimulation at 0.2 mM. The unique nature of the sperm adenylate cyclase and its possible regulation by Mn2+ under physiological conditions is discussed.     

Stability of the Adenosine 5′-Triphosphate Pool in Coxiella burnetii: Influence of pH and Substrate

The ability of Coxiella burnetii to couple oxidation of metabolic substrates to adenosine 5'-triphosphate (ATP) synthesis in axenic reaction buffers was examined. Pyruvate, succinate, and glutamate were catabolized and incorporated at the highest rates of 11 substrates tested. Glutamate oxidation, however, resulted in the greatest stability of the ATP pool and highest intracellular ATP levels over a 48-h period. At pH 4.5, the optimum for metabolism by C. burnetii, glutamate oxidation resulted in maintenance of the ATP pool at a concentration of approximately 0.7 nmol of ATP per mg of dry weight over a 96-h period. In the absence of substrate, ATP declined by 96 h to less than 0.01 nmol/mg of dry weight. When cells were maintained at pH 7.0 in the presence or absence of glutamate, ATP pools were considerably more stable, presumably due to the minimal metabolic activity displayed by C. burnetii at pH 7. The stability of the ATP pool reflected viability as there was greater than an 8-log decrease in viable C. burnetii after incubation for 7 days at pH 4.5 in the absence of glutamate. Viability was retained in the presence of glutamate at pH 4.5 or 7.0 in the absence of any added substrate. The stability of the ATP pool was due to endogenous synthesis of ATP coupled to substrate oxidation as shown by depression of ATP levels in the presence of inhibitors of electron transport or oxidative phosphorylation. In addition, the adenylate energy charge increased from an initial value of 0.57 to 0.73 during glutamate oxidation with a concomitant rise in the total adenylate pool size. C. burnetii therefore appears able to regulate endogenous ATP levels in response to substrate availability and pH, thus effecting a conservation of metabolic energy in neutral or alkaline environments. Such a mechanism has been proposed to play a role in the resistance of C. burnetii to environmental conditions and subsequent activation upon entry into the phagolysosome in which this organism replicates.