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PubMed1990

Interrelation between nucleotide degradation and aldehyde formation in red blood cells. Influence of xanthine oxidase on metabolism: an application of nucleotide and aldehyde analyses by high-performance liquid chromatography.

The mechanism by which hypoxia leads to irreversible cellular damage is poorly understood. A decrease in purine nucleotides is common to all ischaemic tissues, yielding hypoxanthine as the substrate of the xanthine oxidase reaction. Excessive production of radicals via xanthine oxidase induces peroxidation of unsaturated fatty acids, accompanied with the formation of aldehydes. The nucleotides and aldehydes were determined by high-performance liquid chromatography (HPLC) of red blood cell extracts. Nucleotides and their derivatives were determined by HPLC on an ODS column and elution with 10 mM phosphate buffer containing 2 mM tert.-butylammonium phosphate. The aldehyde production in glucose deprived red blood cells was stimulated by addition of xanthine oxidase and by inhibition of different haemotype enzymes with sodium azide. Aldehydes were analysed by derivatization to dinitrophenylhydrazones, followed by thin-layer chromatographic and HPLC separation with aqueous methanol on an ODS column. The HPLC methods presented are appropriate for the determination of nucleotides, nucleosides and nucleobases, in addition to alkenals and hydroxyalkenals in extracts of oxidatively stressed red blood cells.

Aldehydes↗
PubMed1991

On the frequencies of nucleotides and nucleotide substitutions in conservative regulatory DNA sequences.

We present data on the frequencies of nucleotides and nucleotide substitutions in conservative DNA regions involved in the regulation of gene expression. Data on prokaryotes and eukaryotes are considered separately. In both cases DNA strands complementary to those which serve as templates for RNA-polymerase have low frequencies of cytosine. The most conservative positions also have an increased frequency of adenine. Various substitutions in the series of homologous regulatory DNA sequences, as compared to their consensuses, have different frequencies. In prokaryotes guanine in a consensus sequence is substituted for at the lowest and adenine at the highest frequency, whereas in eukaryotes cytosine is substituted for at the lowest and guanine at the highest frequency. In both cases the nucleotides substituted for are most frequently replaced with cytosine. Deviations from consensus sequences tend to cluster in adjacent positions. The more pronounced the consequences of a nucleotide substitution are the higher is the frequency of substitutions in adjacent positions. Possible explanations for these phenomena are discussed.

Animals↗
PubMed2003

Optimization of cellular nucleotide extraction and sample preparation for nucleotide pool analyses using capillary electrophoresis.

Cell extraction and further sample preparation for nucleotide pool analysis using capillary electrophoresis was faster and simpler using volatile extraction solvents (e.g. organic solvents and de-ionized water) compared to the commonly applied acids dissolved in water (e.g. perchloric acid and trichloracetic acid). Temperature had to be controlled during the whole sample preparation process to prevent degradation, and extracts had to be cleaned from proteins and other large molecules prior to capillary electrophoretic analysis to improve reproducibility. Capillary electrophoresis using borate and cyclodextrins in the background electrolyte was used for determining 11 cellular nucleotides simultaneously. In order to optimize the assay, 0-100% acetonitrile, 0-100% ethanol, and 0-100% methanol in de-ionized water were applied to extract nucleotides from mouse lymphoma cells, and nucleotide yields, recovery, and reproducibility were compared. The assay met the commonly accepted validation limits for biological fluids, if 20-80% acetonitrile in water and 40-60% ethanol in water were used as extraction solvents.

Acyclovir↗
PubMed1988

Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates.

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed2006

Molecular basis for differential nucleotide binding of the nucleotide-binding domain of ABC-transporter CvaB.

The cytoplasmic membrane protein CvaB, involved in colicin V secretion in Escherichia coli, belongs to the ABC-transporter family in which ATP hydrolysis is typically the driving force for substrate transport. However, our previous studies indicated that the nucleotide-binding domain of CvaB could also bind and hydrolyze GTP and, indeed, highly preferred GTP over ATP at low temperatures. In this study, we have examined the molecular basis of this preference. Sequence alignment and homology modeling of the CvaB nucleotide-binding domain predicted that the aromatic stacking region of CvaB (Y501DSQ loop) had a role in the differential binding of nucleotides, and Ser503 and Gln504 provided potential hydrogen bonds to GTP but not to ATP. Site-directed mutagenesis of the Y501DSQ loop, mutations S503A, Q504L, and double mutation S503A/Q504L, was made to test the predicted hydrogen bonds with GTP. The double mutation S503A/Q504L increased the affinity for ATP by 6-fold, whereas the affinity for GTP was reduced slightly: the ATP/GTP-binding ratio increased about 10-fold. The temperature effect assays on nucleotide binding and hydrolysis further indicated that the double mutant protein had largely eliminated the difference for substrates ATP and GTP, and behaved more similarly to the NBD of typical ABC-transporter HlyB. Therefore, we conclude that Ser503 and Gln504 in aromatic stacking region of CvaB block the ATP binding and are important for the GTP-binding preference.

ATP-Binding Cassette Transporters↗
PubMed1997

Guanine nucleotide binding properties of Rac2 mutant proteins and analysis of the responsiveness to guanine nucleotide dissociation stimulator.

The Rac GTPases are currently being subjected to intensive study due to their involvement in a wide array of cellular phenomena. Many studies of Rac function have relied upon the use of relatively uncharacterized Rac dominant active, dominant negative, and effector domain mutants on the basis of the analogy to Ras structure. We have generated and purified such Rac2 mutants and characterized their guanine nucleotide binding properties in vitro. The Rac2 G12V and Q61L activating mutations were shown to hydrolyze bound GTP very slowly and were unresponsive to p190 Rac GTPase-activating protein. Distinct differences in the kinetics of nucleotide binding to individual mutant proteins were observed, accounting for the behavior of these proteins in biological assays. The structural features required for the responsiveness of Rac2 to the guanine nucleotide exchange protein smgGDS were examined. We show that guanine nucleotide exchange by smgGDS is dependent upon intact switch 1 and switch 2 regions in Rac2. Functional interactions between the switch 1 and switch 2 regions and the G12V mutation of Rac2 are described. These data form the basis for rational use of Rac mutants in biological studies.

GTP Phosphohydrolases↗
PubMed1997

Regulation of the mitochondrial Ca2+ uniporter by external adenine nucleotides: the uniporter behaves like a gated channel which is regulated by nucleotides and divalent cations.

We have previously used measurements of uncoupler-enforced reverse activity to demonstrate that the mitochondrial Ca2+ uniporter is strongly inhibited by external EGTA plus free Mg2+, following a brief period of rapid activity. Using the same approach, we now show that in addition to divalent cations, the uniporter is regulated by external adenine nucleotides and by other components of the cytosol. Inhibition produced by EGTA plus free Mg2+ is reversed by spermine (EC0.5 approximately 40 microM) and reduced when mitochondria are purified by an isoosmotic density-gradient method. Under either condition, inhibition is restored by external adenine nucleotides in a concentration-dependent manner. The order of effectiveness is ATP > ADP > AMP, with the nucleoside adenosine being ineffective. Among nucleotide triphosphates, the order is ATP > CTP approximately UTP > GTP. The effectiveness of ATP (EC50 approximately 0.6 mM) is the same in mitochondria and mitoplasts, the same as that of AMPPNP, and is not altered by the presence of oligomycin, carboxyatractyloside, or AP5A, used alone or in combinations. These findings indicate that ATP acts at a site located on the outer surface of the inner membrane through a mechanism which does not require its hydrolysis. Phosphate also inhibits reverse uniport under some conditions (EC50 approximately 20 microM). The sites at which free ATP and free Mg2+ inhibit the uniporter can be distinguished by chymotrypsin treatment of mitoplasts, which eliminates the action of Mg2+ but does not affect the action of ATP. Data are interpreted within the context of a model in which the uniporter is considered to be a gated channel that is controlled, in part, by specific external effector sites that accept divalent cations or nucleotides. The possible consequences of the model for cell Ca2+ regulation by mitochondria and regulation of TCA cycle activity by the matrix free Ca2+ concentration are considered.

Adenine Nucleotides↗
PubMed2002

Single-nucleotide polymorphism characterization in species with limited available sequence information: high nucleotide diversity revealed in the avian genome.

As a case study for single-nucleotide polymorphism (SNP) identification in species for which little or no sequence information is available, we investigated several approaches to identifying SNPs in two passerine bird species: pied and collared flycatchers (Ficedula hypoleuca and F. albicollis). All approaches were successful in identifying sequence polymorphism and over 50 candidate SNPs per species were identified from approximately 9.1 kb of sequence. In addition, 17 sites were identified in which the frequency of alternative bases differed by > 50% between species (termed interspecific SNPs). Interestingly, polymorphism of microsatellite/intron loci in the source species appeared to be a positive predictor of nucleotide diversity in homologous flycatcher sequences. The overall nucleotide diversity of flycatchers was 2.3-2.7 x 10(-3), which is approximately 3-6 times higher than observed in recent studies of human SNPs. Higher nucleotide diversity in the avian genome could be due to the relatively older age of flycatcher populations, compared with humans, and/or a higher long-term effective population size.

Animals↗
PubMed1992

Hydrolytic elimination of a mutagenic nucleotide, 8-oxodGTP, by human 18-kilodalton protein: sanitization of nucleotide pool.

8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies. Once 8-oxodGTP is formed in the cellular nucleotide pool, this mutagenic nucleotide is incorporated into DNA and would cause transversion mutations. The MutT protein of Escherichia coli possesses enzyme activity to hydrolyze 8-oxodGTP to the corresponding nucleoside monophosphate and thus may be responsible for preventing the occurrence of such mutations. Here we show that the human cell has an enzyme specifically hydrolyzing 8-oxodGTP in a fashion similar to that seen with MutT protein. The human 8-oxodGTPase has been found in cell-free extracts from Jurkat cells and purified > 400-fold. Analyses by gel filtration and gel electrophoresis revealed that the molecular mass of the native form of human 8-oxodGTPase is 18 kDa. Mg2+ ion is required for the enzyme action and the optimum pH for the reaction is pH 8.0. The enzyme hydrolyzes 8-oxodGTP to 8-oxodGMP with a Km value of 12.5 microM. dGTP and dATP are also degraded to dGMP and dAMP, respectively, with Km values 70 times greater than that for 8-oxodGTP. dTTP and dCTP are not hydrolyzed. These properties of the human 8-oxodGTPase are similar to those observed with the E. coli MutT protein, suggesting that the function of protecting the genetic information from the threat of endogenous oxygen radicals is widely distributed in organisms.

Chromatography, Affinity↗
PubMed1997

Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase.

Stimulation of the mitochondrial permeability transition (MPT) in de-energized mitochondria by phenylarsine oxide (PheArs) is greater than that by diamide and t-butylhydroperoxide (TBH), yet the increase in CyP binding to the inner mitochondrial membrane (Connern, C. P. and Halestrap, A. P. (1994) Biochem. J. 302, 321-324) is less. From a range of nucleotides tested only ADP, deoxy-ADP, and ATP inhibited the MPT. ADP inhibition involved two sites with Ki values of about 1 and 25 microM which were independent of [Ca2+] and CyP binding. Carboxyatractyloside (CAT) abolished the high affinity site. Following pretreatment of mitochondria with TBH or diamide, the Ki for ADP increased to 50-100 microM, whereas pretreatment with PheArs or eosin maleimide increased the Ki to >500 microM; only one inhibitory site was observed in both cases. Eosin maleimide is known to attack Cys159 of the adenine nucleotide translocase (ANT) in a CAT-sensitive manner (Majima, E., Shinohara, Y., Yamaguchi, N., Hong, Y. M., and Terada, H. (1994) Biochemistry 33, 9530-9536), and here we demonstrate CAT-sensitive binding of the ANT to a PheArs affinity column. In adenine nucleotide-depleted mitochondria, no stimulation of the MPT by uncoupler was observed in the presence or absence of thiol reagents, suggesting that membrane potential may inhibit the MPT by increasing adenine nucleotide binding through an effect on the ANT conformation. We conclude that CsA and ADP inhibit pore opening in distinct ways, CsA by displacing bound CyP and ADP by binding to the ANT. Both mechanisms act to decrease the Ca2+ sensitivity of the pore. Thiol reagents and oxidative stress may modify two thiol groups on the ANT and thus stimulate pore opening by both means.

Adenosine Diphosphate↗
PubMed2001

O-helix mutant T664P of Thermus aquaticus DNA polymerase I: altered catalytic properties for incorporation of incorrect nucleotides but not correct nucleotides.

Previous studies indicate that the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) plays an important role in the replication fidelity of the enzyme. This study examines the role of Thr-664, which lies in the middle of the O-helix of Taq pol I. A mutant of Taq Pol I with a proline substitution of Thr-664 (T664P) exhibits much lower replication fidelity than the wild type enzyme in a forward mutation assay. T664P produces base substitution, single-base deletion, and single-base insertion errors at 20-, 5, and 50-fold higher rates than wild type, respectively. In specific activity and steady-state kinetic experiments, T664P was catalytically robust for insertion of correct nucleotides. In contrast, it incorporated incorrect nucleotides 6.1- to 10-fold more efficiently than wild type at a template dC. Mismatched primer termini were extended by T664P 4.2- to 9.5-fold more efficiently than wild type. These data imply that the O-helix with a proline at position 664 functions like wild type Taq pol I for correct nucleotide incorporations, but bends and enlarges the catalytic pocket of the enzyme and increases the rate of nucleotide misincorporation.

Base Sequence↗
PubMed2001

Thermodynamic stability of base pairs between 2-hydroxyadenine and incoming nucleotides as a determinant of nucleotide incorporation specificity during replication.

We investigated the thermodynamic stability of double-stranded DNAs with an oxidative DNA lesion, 2-hydroxyadenine (2-OH-Ade), in two different sequence contexts (5'-GA*C-3' and 5'-TA*A-3', A* represents 2-OH-Ade). When an A*-N pair (N, any nucleotide base) was located in the center of a duplex, the thermodynamic stabilities of the duplexes were similar for all the natural bases except A (N = T, C and G). On the other hand, for the duplexes with the A*-N pair at the end, which mimic the nucleotide incorporation step, the stabilities of the duplexes were dependent on their sequence. The order of stability is T > G > C >> A in the 5'-GA*C-3' sequences and T > A > C > G in the 5'-TA*A-3' sequences. Because T/G/C and T/A are nucleotides incorporated opposite to 2-OH-Ade in the 5'-GA*C-3' and 5'-TA*A-3' sequences, respectively, these results agree with the tendency of mutagenic misincorporation of the nucleotides opposite to 2-OH-Ade in vitro. Thus, the thermodynamic stability of the A*-N base pair may be an important factor for the mutation spectra of 2-OH-Ade.

Base Pairing↗
PubMed1986

Normal levels of ATP, total nucleotides and activities of three enzymes related to nucleotide metabolism in fetal erythrocytes.

Pure fetal blood was obtained by direct-vision fetoscopy from 30 fetuses at 17-23 weeks gestation. The erythrocyte concentrations of ATP and total nucleotides and the activities of the enzymes pyrimidine-5'-nucleotide nucleosidase (Pyr5N), phosphoribosylpyrophosphate (PRPP) synthetase and adenylate kinase (AK) were analysed by established techniques to find the normal ranges for this gestational age. The ranges were relatively narrow and could serve as reference values for the prenatal diagnosis of defects in nucleotide metabolism. The results from the fetal erythrocytes were compared with the corresponding values from the maternal blood collected and analysed concurrently. The ATP and total nucleotide concentrations and the activity of Pyr5N in the fetal cells were substantially higher than those of the maternal blood. The activities of PRPP synthetase and AK were much lower. The significance of these findings is discussed.

5'-Nucleotidase↗
PubMed1980

Interactions of elongation factor 2 (EF-2) with guanine nucleotides and ribosomes. Binding of periodate-oxidized guanine nucleotides to EF-2.

Interactions of rat liver elongation factor 2 (EF-2) with guanine nucleotides and ribosomes were studied by equilibrium dialysis and sedimentation methods. GDP (Kd = 0.5 microM) or GDP-Mg2+ (Kd = 1.57 microM) displayed a higher affinity in the formation of a binary complex with EF-2 than GTP (Kd = 2.68 microM), GTP-Mg2+ (Kd = 2.77 microM), or guanosine 5'-[beta, gamma-methylene]triphosphate (GuoPP[CH2]P) (Kd = 24.0 microM). NaIO4-oxidized guanine nucleotides (oGDP) (Kd = 38 microM) and oxidized/reduced guanine nucleotides (orGDP) (Kd = 27 microM) had lower affinites to the binding site on EF-2 than those of GDP or GTP. However, the binding of oGDP, oGTP or oGuoPP[CH2]P to EF-2 resulted in the formation of a stable product which could be recovered by the nitrocellulose filter technique or by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In the presence of ribosomes and EF-2 the formation of a new binding site (or a different conformation of the binding site) with a higher affinity for GuoPP[CH2]P-Mg2+ (Kd = 0.26 microM) than fof GDP-Mg2+ (kd = 9.3 microM) became apparent. The presence of ribosomes thus appeared to favor the formation of a complex involving guanosine triphosphates. Adenosine diphosphate ribosylated EF-2 (ADP-Rib-EF-2) in its turn could bind to the ribosome with high affinity even without guanosine nucleotides (Kd = 0.18 microM). GuoPP[CH2]P increased to some extent the affinity of ADP-Rib-EF-2 for its ribosomal binding site (Kd = 0.05 microM).

Animals↗
PubMed1992

Dissociation of guanosine 5'-[gamma-thio]triphosphate from guanine-nucleotide-binding regulatory proteins in native cardiac membranes. Regulation by nucleotides and muscarinic acetylcholine receptors.

Binding of the poorly hydrolyzable GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to purified guanine-nucleotide-binding regulatory proteins (G proteins) has been shown to be nonreversible in the presence of millimolar concentrations of Mg2+. In porcine atrial membranes, binding of [35S]GTP[S] to G proteins was stable in the presence of 1 mM Mg2+. However, either large dilution or, even more strongly, addition of unlabelled guanine nucleotides, in the potency order, GTP[S] greater than GTP greater than or equal to guanosine 5'-[beta,gamma-imino]triphosphate greater than GDP greater than or equal to guanosine 5'-[beta-thio]diphosphate greater than GMP, markedly enhanced the observed dissociation, with 20-30% of bound [35S]GTP[S] being released by unlabelled guanine nucleotide within 20 min at 25 degrees C. Most interestingly, dissociation of [35S]GTP[S] was rapidly and markedly stimulated by agonist (carbachol) activation of cardiac muscarinic acetylcholine receptors. Carbachol-stimulated release of [35S]GTP[S] was strictly dependent on the presence of Mg2+ and an unlabelled guanine nucleotide. Although having different potency and efficiency in releasing [35S]GTP[S] from the membranes by themselves, the guanine nucleoside triphosphates and diphosphates studied, at maximally effective concentrations, promoted the carbachol-induced dissociation to the same extent, while GMP and ATP were ineffective. GTP[S]-binding-saturation experiments indicated that one agonist-activated muscarinic acetylcholine receptor can cause release of bound GTP[S] from three to four G proteins. The data presented indicate that binding of GTP[S] to G proteins in intact membranes, in contrast to purified G proteins, is reversible, and that agonist-activated receptors can even, either directly or indirectly, interact with GTP[S]-bound G proteins, resulting in release of bound guanine nucleoside triphosphate.

Animals↗
PubMed1962

Occurrence of nucleotides in culture fluids of microorganisms. III. Nucleotide excretion by Brevibacterium liquefaciens sp. n. with special reference to uridine diphospho-N-acetylglucosamine compounds.

Okabayashi, T. (Shionogi Research Laboratory, Fukushimaku, Osaka, Japan). Occurrence of nucleotides in culture fluids of microorganisms. III. Nucleotide excretion by Brevibacterium liquefaciens sp. n. with special reference to uridine diphospho-N-acetylglucosamine compounds. J. Bacteriol. 84:1-8. 1962.-Among various nucleotides excreted in the culture broth of Brevibacterium liquefaciens sp. n., four uridine nucleotides containing N-acetylglucosamine were isolated. These were found to be uridine diphospho-N-acetylglucosamine and Park's compounds 1, 2, and 3. The significance of the appearance of these cell-wall precursors in the culture fluid of B. liquefaciens is discussed.

Acetylglucosamine↗
PubMed1990

Differentiation of the nucleotide-binding sites on nucleotide-depleted mitochondrial F1-ATPase by means of a fluorescent ADP analogue.

The interaction of the fluorescent ADP analogue lin-benzo-ADP (containing a linearly extended version of adenine, in which a benzene ring is inserted between pyrimidine and imidazole ring) with nucleotide-depleted mitochondrial F1 was investigated. It was found that lin-benzo-ADP is able to occupy all six nucleotide-binding sites present on the enzyme. Two sites exhibit a very high affinity for the analogue (dissociation constant, Kd, less than 10 nM) and bind it rapidly (association rate constant, k+1, about 1.10(6) M-1 S-1). A third site shows a lower affinity for the analogue (Kd = 1-2 microM) and is occupied relatively fast (k+1 approximately 10(4) M-1 S-1). Binding of lin-benzo-ADP to these three sites is prevented not only in the presence of excess ADP and ATP, but also by IDP and ITP, thus indicating that these sites are the catalytic ones. As it will be discussed, this conclusion is further corroborated by the finding that release of the analogue from the two high affinity sites can be promoted by binding of nucleoside di- and triphosphates to the third site. The remaining three sites were found to bind lin-benzo-ADP with identical affinity (Kd = 1-2 microM) and with a rather low association rate (k+1 = 300-600 M-1 S-1). Binding of the analogue to them is only prevented by ADP and ATP, but not by IDP and ITP, which confirms that these sites are the noncatalytic ones. The analogue could be displaced by excess ADP also from these sites; however, in contrast to the catalytic sites, no promotive effect was observed here. The obvious changes in the nucleotide binding behavior of the non-catalytic sites after depletion of endogenous nucleotides will be discussed.

Adenosine Diphosphate↗
PubMed1981

[Mathematical modelling of glycolysis and of adenine nucleotide metabolism of human erythrocytes. II. Simulation of adenine nucleotide breakdown following glucose depletion].

The breakdown of adenine nucleotides in human erythrocytes in physiological and alkaline medium at 37 degrees C after glucose depletion is simulated by a mathematical model of energy metabolism. The simulation consists of time dependent solutions of a system of 16 differential equations derived from the stoichiometry of chemical pathways and kinetic properties of enzymes. Starting with the stationary characteristics of the model (M. Schauer et al.: Acta biol. med. germ. 40, 1659, 1981) the results of the simulation were analysed as a function of 1) the share of adenylate deaminase in the degradation of adenine nucleotides and 2) the interaction between adenylate kinase equilibrium and magnesium ions. The experimental data and the simulated concentration changes are in good accord, provided that the assumed activity of adenylate deaminase is very low so that the degradation of adenine nucleotides proceeds mainly via AMP-hydrolysis. The extensive activation of adenylate deaminase during incubation is explained by its sigmoid kinetics with respect to AMP. To improve the results of simulation changes in the adenylate kinase equilibrium have to taken into consideration. These have been measured during incubation of erythrocytes; they can be attributed only partly to the increasing concentration of magnesium ions and to differences among the constants of magnesium association to adenine nucleotides.

Adenine Nucleotides↗