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Chiral ITP of the weak base methadone using inverse cationic configurations with H + as leading component and multiple isomer sulfated β-CD (S-β-CD) as leading electrolyte (LE) additive, has been studied utilizing dynamic computer simulation, a calculation model based on steady-state values of the ITP zones, and capillary ITP. By varying the amount of acidic S-β-CD in the LE composed of 3-morpholino-2-hydroxypropanesulfonic acid and the chiral selector, and employing glycylglycine as terminating electrolyte (TE), inverse cationic ITP provides systems in which either both enantiomers, only the enantiomer with weaker complexation, or none of the two enantiomers form cationic ITP zones. For the configuration studied, the data reveal that only S-methadone migrates isotachophoretically when the S-β-CD concentration in the LE is between about 0.484 and 1.113 mM. Under these conditions, R-methadone migrates zone electrophoretically in the TE. An S-β-CD concentration between about 0.070 and 0.484 mM results in both S- and R-methadone forming ITP zones. With >1.113 mM and < about 0.050 mM of S-β-CD in the LE both enantiomers are migrating within the TE and LE, respectively. Chiral inverse cationic ITP with acidic S-β-CD in the LE is demonstrated to permit selective ITP trapping and concentration of the less interacting enantiomer of a weak base., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The structure of three forms of a dimeric enzyme, human inosine triphosphate pyrophosphatase, is considered to identify the enzyme conformation changes causing the inactivation effect of a P32T mutation. Analysis of a nanosecond molecular dynamics is performed; the mean square deviations of the atoms between the wild-type and mutant homodimers, and also the heterodimer are calculated. A 3 ns modeling shows a greater displacement of atoms in mutant protomers. During molecular dynamics simulation, the strongest changes are observed in the loop between α2 and β2 (amino acid residues 28-33, an area of the P32T mutation), the loop between β5 and β6, and the C-terminal amino acid residues. The loop between (α2 and β2 has two conformations characterized by different positions of the Phe31 aromatic group. The distance between Cys33 (Cα) and Phe31 (C(z)) for wild-type and mutant protomers was -9 and 5.5 Å, respectively. These conformations were kept constant.

A catalytic fragment of soluble NTPase has been isolated from bovine kidneys.The 236-fold purification was carried out to obtain the preparation with a specific activity of 37.7 U/mg of protein. The purification scheme included the enzyme extraction followed by four column chromatography steps. The catalytic fragment was activated with divalent metal ions, had a pH optimum of 7.0, and possessed specificity for ITP, GTP, UTP and XTP. The apparent K(m) for Mg-ITP, Mg-GTP and Mg-UTP complexes were calculated from Hanes plots to be 1.70 mM, 0.93 mM and 0.48 mM, respectively. As estimated by gel filtration and SDS-PAAGE, the catalytic fragment has Mw 54.7 kDa being composed of two identical polypeptide chains. Our results suppose soluble NTPase from bovine kidney to consist of regulatory and catalytic structural units.

Background: Thiopurine drugs, widely used in cancer chemotherapy, inflammatory bowel disease, and autoimmune hepatitis, are responsible for common adverse events. Only some of these may be explained by genetic polymorphism of thiopurine S-methyltransferase. Recent articles have reported that inosine triphosphate pyrophosphatase (ITPase) deficiency was associated with adverse drug reactions toward thiopurine drug therapy. Here, we report a weak anion exchange high-performance liquid chromatography method to determine ITPase activity in red blood cells and to investigate the relationship with the occurrence of adverse events during azathioprine therapy., Methods: ITPase activity was assessed by the enzymatic conversion of inosine triphosphate (ITP) to inosine monophosphate (IMP). The reaction was stopped by heating for 3 minutes at 120°C. IMP, inosine diphosphate, and ITP were analyzed on a Hypersil APS-2 column, a weak anion exchange phase that exhibits both ionic and hydrophobic properties., Results: The chromatographic method reported allows the analysis of IMP, inosine diphosphate, and ITP in a single run in <12.5 minutes. The method was linear in the range 5-1500 μmole/L of IMP. Intraassay and interassay precisions were <5% for red blood cell lysates supplemented with 50, 500, and 1000 μmole/L IMP. Km and Vmax evaluated by Lineweaver-Burk plot were 677.4 μmole/L and 19.6 μmole·L·min, respectively. The frequency distribution of ITPase from 73 patients was investigated., Conclusions: The method described is useful to determine the ITPase phenotype from patients on thiopurine therapy and to investigate the potential relation between ITPase deficiency and the occurrence of adverse events.

2',3'-O-(N-Methylanthraniloyl)-ITP (MANT-ITP) is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, K(i), 1 nM) yet discovered and surpasses the potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329:1156-1165, 2009). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP. MANT-ITP was a considerably more potent inhibitor of the purified catalytic domains VC1 and IIC2 of mAC than MANT-GTP (K(i), 0.7 versus 18 nM). Moreover, there was considerably more efficient fluorescence resonance energy transfer between Trp1020 of IIC2 and the MANT group of MANT-ITP compared with MANT-GTP, indicating optimal interaction of the MANT group of MANT-ITP with the hydrophobic pocket. The crystal structure of MANT-ITP in complex with the G(s)α- and forskolin-activated catalytic domains VC1:IIC2 compared with the existing MANT-GTP crystal structure revealed only subtle differences in binding mode. The higher affinity of MANT-ITP to mAC compared with MANT-GTP is probably due to fewer stereochemical constraints upon the nucleotide base in the purine binding pocket, allowing a stronger interaction with the hydrophobic regions of IIC2 domain, as assessed by fluorescence spectroscopy. Stronger interaction is also achieved in the phosphate-binding site. The triphosphate group of MANT-ITP exhibits better metal coordination than the triphosphate group of MANT-GTP, as confirmed by molecular dynamics simulations. Collectively, the subtle differences in ligand structure have profound effects on affinity for mAC.

The genome of the mesophilic Gram-negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth&#95;I1158 gene) that has been annotated as a putative ribokinase and PFK-B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK-B protein, and the specificity extended not only to purine and purine-analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest k(cat)/K(m) value (80 s(-1).mm(-1)) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK-B family.

DNA complementarity is expressed by way of three hydrogen bonds for a G:C base pair and two for A:T. As a result, careful control of the denaturation temperature of PCR allows selective amplification of AT-rich alleles. Yet for the same reason, the converse is not possible, selective amplification of GC-rich alleles. Inosine (I) hydrogen bonds to cytosine by two hydrogen bonds while diaminopurine (D) forms three hydrogen bonds with thymine. By substituting dATP by dDTP and dGTP by dITP in a PCR reaction, DNA is obtained in which the natural hydrogen bonding rule is inversed. When PCR is performed at limiting denaturation temperatures, it is possible to recover GC-rich viral genomes and inverted Alu elements embedded in cellular mRNAs resulting from editing by dsRNA dependent host cell adenosine deaminases. The editing of Alu elements in cellular mRNAs was strongly enhanced by type I interferon induction indicating a novel link mRNA metabolism and innate immunity.

Inosine triphosphate pyrophosphatases, which are ubiquitous house-cleaning enzymes, hydrolyze noncanonical nucleoside triphosphates (inosine triphosphate (ITP) and xanthosine triphosphate (XTP)) and prevent the incorporation of hypoxanthine or xanthine into nascent DNA or RNA. Here we present the 1.5-A-resolution crystal structure of the inosine triphosphate pyrophosphatase RdgB from Escherichia coli in a free state and in complex with a substrate (ITP+Ca(2+)) or a product (inosine monophosphate (IMP)). ITP binding to RdgB induced a large displacement of the alpha1 helix, closing the enzyme active site. This positions the conserved Lys13 close to the bridging oxygen between the alpha- and beta-phosphates of the substrate, weakening the P(alpha)-O bond. On the other side of the substrate, the conserved Asp69 is proposed to act as a base coordinating the catalytic water molecule. Our data provide insight into the molecular mechanisms of the substrate selectivity and catalysis of RdgB and other ITPases.

Inosine triphosphate (ITP) and xanthosine triphosphate (XTP) are formed upon deamination of ATP and GTP as a result of exposure to chemical mutagens and oxidative damage. Nucleic acid synthesis requires safeguard mechanisms to minimize undesired lethal incorporation of ITP and XTP. Here, we present the crystal structure of YjjX, a protein of hitherto unknown function. The three-dimensional fold of YjjX is similar to those of Mj0226 from Methanococcus janschii, which possesses nucleotidase activity, and of Maf from Bacillus subtilis, which can bind nucleotides. Biochemical analyses of YjjX revealed it to exhibit specific phosphatase activity for inosine and xanthosine triphosphates and have a possible interaction with elongation factor Tu. The enzymatic activity of YjjX as an inosine/xanthosine triphosphatase provides evidence for a plausible protection mechanism by clearing the noncanonical nucleotides from the cell during oxidative stress in E. coli.

In this paper, we describe the synthesis and thermal stabilities of the triplexes containing either 2'-deoxyinosine (1) or 2'-deoxyxanthosine (3) in their second strands. It was found that the triplexes with the 2'-deoxy-5-methylcytidine(dM)*1:dC and dM*1:dA base triplets are thermally stable, but those containing the dM*1:T and dM*1:dG base triplets are unstable under both neutral and slightly acidic conditions. On the other hand, it was found that the oligonucleotide containing 3 could form thermally stable triplexes with the oligonucleotides that involve four natural bases opposite the sites of 3. The rank of the thermal stabilities of the triplexes was as follows: the triplex containing the dM*3:dC base triplet > that containing the dM*3:dA base triplet > that containing the dM*3:T base triplet > that containing the dM*3:dG base triplet.

The RecA protein from Escherichia coli promotes an ATP-dependent three-strand exchange reaction between a circular single-stranded DNA (ssDNA) and a homologous linear double-stranded (dsDNA). We have now found that under certain conditions, the RecA protein is also able to promote the three-strand exchange reaction using the structurally related nucleoside triphosphate, ITP, as the nucleotide cofactor. However, although both reactions are stimulated by single-stranded DNA-binding (SSB) protein, the ITP-dependent reaction differs from the ATP-dependent reaction in that it is observed only at low SSB protein concentrations, whereas the ATP-dependent reaction proceeds efficiently even at high SSB protein concentrations. Moreover, the circular ssDNA-dependent ITP hydrolysis activity of the RecA protein is strongly inhibited by SSB protein (suggesting that SSB protein displaces RecA protein from ssDNA when ITP is present), whereas the ATP hydrolysis activity is uninhibited even at high SSB protein concentrations (because RecA protein is resistant to displacement by SSB protein when ATP is present). These results suggest that SSB protein does not stimulate the ITP-dependent strand exchange reaction presynaptically (by facilitating the binding of RecA protein to the circular ssDNA substrate) but may act postsynaptically (by binding to the displaced strand that is generated when the circular ssDNA invades the linear dsDNA substrate). Interestingly, the mechanistic characteristics of the ITP-dependent strand exchange reaction of the E. coli RecA protein are similar to those of the ATP-dependent strand exchange reaction of the RecA protein from Streptococcus pneumoniae. These findings are discussed in terms of the relationship between the dynamic state of the RecA-ssDNA filament and the mechanism of the SSB protein-stimulated three-strand exchange reaction.

Under experimental conditions in which the self-association of the purine-nucleoside 5'-triphosphates (PuNTPs) GTP and ITP is negligible, potentiometric pH titrations were carried out to determine the stabilities of the M(H;PuNTP) and M(PuNTP)2-complexes where M2+ = Mg2+, Ca2+, Sr2+. Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, or Cd2+ (I = 0.1 M, 25 degrees C). The stabilities of all M(GTP)2- and M(ITP)2- complexes are significantly larger than those of the corresponding complexes formed with pyrimidine-nucleoside 5'-triphosphates (PyNTPs), which had been determined previously under the same conditions. This increased complex stability is attributed, in agreement with previous 1H MNR shift studies, to the formation of macrochelates of the phosphate-coordinated metal ions with N7 of the purine residues. A similar enhanced stability (despite relatively large error limits) was observed for the M(H;PuNTP) complexes, in which H+ is bound to the terminal y-phosphate group, relative to the stability of the M(H;PyNTP)- species. The percentage of the macrochelated isomers in the M(GTP)2- and M(ITP)2- systems was quantified by employing the difference log KMM(PuNTP)-log KMM(PyNTP); the lowest and highest formation degrees of the macrochelates were observed for Mg(ITP)2- and Cu(GTP)2- with 17 +/- 11% and 97 +/- 1%, respectively. From previous studies of M(ATP)2- complexes, it is known that innersphere and outersphere macrochelates may form; that is, in the latter case a water molecule is between N7 and the phosphate-coordinated M2+. Similar conclusions are reached now by comparisons with earlier 1H MNR shift measurements, that is, that Mg(GTP)2- (21 +/- 11%), for example, exists largely in the form of an outersphere macrochelate and Zn(GTP)2- (68 +/- 4%) as an innersphere one. Generally, the overall percentage of macrochelate falls off for a given metal ion in the order M(GTP)2- > M(ITP)2- > M(ATP)2-; this is in accord with the decreasing basicity of N7 and the steric inhibition of the (C6)NH2 group in the adenine residue. Furthermore, although the absolute stability constants of the previously studied M(GMP), M(IMP), and M(AMP) complexes differ by about two to three log units from the present M(PuNTP)2- results, the formation degrees of the macrochelates are astonishingly similar for the two series of nucleotides for a given metal ion and purine-nucleobase residue. The conclusion that N7 of the guanine residue is an especially favored binding site for metal ions is also in accord with observations made for nucleic acids.

We have determined the binding affinity for binding of the four purine nucleoside triphosphates GTP, ITP, XTP, and ATP to E-site nucleotide- and nucleoside diphosphate kinase-depleted tubulin. The relative binding affinities are 3000 for GTP, 10 for ITP, 2 for XTP, and 1 for ATP. Thus, the 2-exocyclic amino group in GTP is important in determining the nucleotide specificity of tubulin and may interact with a hydrogen bond acceptor group in the protein. The 6-oxo group also makes a contribution to the high affinity for GTP. NMR ROESY experiments indicate that the four nucleotides have different average conformations in solution. ATP and XTP are characterized by a high anti conformation, ITP by a medium anti conformation, and GTP by a low anti conformation. Possibly, the preferred solution conformation contributes to the differences in affinities. When the tubulin E-site is saturated with nucleotide, there appears to be little difference in the ability of the four nucleotides to stimulate assembly. The critical protein concentration is essentially identical in reactions using the four nucleotides. All four of the nucleotides were hydrolyzed during the assembly reaction, and the NDPs were incorporated into the microtubule. We also examined the binding of two gamma-phosphoryl-modified GTP photoaffinity analogues, p(3)-1, 4-azidoanilido-GTP and p(3)-1,3-acetylanilido-GTP. These analogues are inhibitors of the assembly reaction and bind to tubulin with affinities that are 15- and 50-fold lower, respectively, than the affinty for GTP. The affinity of GTP is less sensitive to substitutions at the gamma-phosphoryl position that to changes in the purine ring.

Protein kinases are essential for the regulation of cellular growth and metabolism. Since their dysfunction leads to debilitating diseases, they represent key targets for pharmaceutical research. The rational design of kinase inhibitors requires an understanding of the determinants of ligand binding to these proteins. In the present study, a theoretical model based on continuum electrostatics and a surface-area-dependent nonpolar term is used to calculate binding affinities of balanol derivatives, H-series inhibitors, and ATP analogues toward the catalytic subunit of cAMP-dependent protein kinase (cAPK or protein kinase A). The calculations reproduce most of the experimental trends and provide insight into the driving forces responsible for binding. Nonpolar interactions are found to govern protein-ligand affinity. Hydrogen bonds represent a negligible contribution, because hydrogen bond formation in the complex requires the desolvation of the interacting partners. However, the binding affinity is decreased if hydrogen-bonding groups of the ligand remain unsatisfied in the complex. The disposition of hydrogen-bonding groups in the ligand is therefore crucial for binding specificity. These observations should be valuable guides in the design of potent and specific kinase inhibitors.

The noncovalent fluorescent probe 6-propionyl-2-(dimethylamino)naphthalene (prodan) binds stoichiometrically to myosin subfragment-1 (S-1) without affecting the ATPase and actin-binding properties of S-1. Neither ATP nor actin interferes with the prodan binding. Free prodan exhibits a green emission peak at 520 nm. However, the prodan bound to S-1 and the S-1.ADP complex shows blue emission peaks at 460 and 450 nm, respectively, which allow easy separation of the fluorescence contributions from the free and bound probes. In the S-1.ADP.Pi state, the blue emission peak is further shifted to 445 nm with a large (4.5-fold) fluorescence enhancement. Thus, prodan in the presence of S-1 exhibits predominantly blue fluorescence only during ATP hydrolysis, and so visualizes the ATPase reaction continuously. The initial velocities of the steady state of the Mg2+-, Ca2+-, and actin-activated ATPases can be conveniently calculated from the blue fluorescence changes. The ability of different nucleoside triphosphates (NTP) to enhance the blue fluorescence of prodan follows the order ATP > CTP > UTP > ITP > GTP. This order agrees with those of the extent of hydrophobicity near the ribose of the corresponding nucleoside diphosphates (NDP) trapped to S-1 with orthovanadate (Vi) [Hiratsuka, T. (1984) J. Biochem. (Tokyo) 96, 155-162] and the ability of different NTPs to support force production in muscle fibers [Regnier, M., et al. (1993) Biophys. J. 64, A250]. The rate of formation of the corresponding S-1.NDP.Vi complex also follows this order, whereas the NTPase rate follows the reverse order. These results indicate that nucleotide-induced changes in prodan fluorescence correspond to the nucleotide-induced conformational states of S-1. Thus, the use of prodan in studies of the myosin ATPase offers a new and promising approach not only to monitoring the ATPase reaction but also to investigating the structural changes during ATP hydrolysis.

The molecular structures of 3'-azido-2',3'-dideoxyribosylthymine 5'-triphosphate (AZTTP), 2',3'-dideoxyribosylinosine 5'-triphosphate (ddlTP), 3'-azido-2',3'-dideoxyribosylthymine 5'-monophosphate (AZTMP) and 2',3'-dideoxyribosyladenine 5'-monophosphate (ddAMP) have been studied by NMR to understand their anti-HIV activity. For ddAMP and ddITP, conformations are almost identical with their nucleoside analogues with sugar ring pucker equilibriating between C3'-endo (approximately 75%) and C2'-endo (approximately 25%). AZTMP and AZTTP on the other hand show significant variations in the conformational behaviour compared with 3'-azido-2',3'-dideoxyribosylthymine (AZT). The sugar rings for these nucleotides have a much larger population of C2'-endo (approximately 75%) conformers, like those observed for natural 2'-deoxynucleosides and nucleotides. The major conformers around C5'-O5', C4'-C5' and the glycosidic bonds are the beta 1, gamma + and anti, respectively.

The contractile protein actin contains one mole of firmly bound nucleotide and a number of divalent cations bound with different affinities. During recent years evidence for a second nucleotide interacting site on actin has been reported. Therefore, a specific search for the presence of a second nucleotide-interacting site on actin was undertaken. For this purpose G- and F-actin or actin in complex with deoxyribonuclease I (DNase I) was passed over ADP-agarose which was found to retain all three forms of actin. Nucleotide bound to the high affinity site of actin did not exchange during passage and retention to agarose-immobilized ADP, thus indicating the presence of a second nucleotide interacting site. This site was found to be equally accessible in G- and F-actin and in the actin-DNase I complex, whereas DNase I alone passed unretained through this column. A number of nucleotides and phosphorylated compounds were tested for their ability to compete with immobilized ADP for actin interaction. It was found that all forms of actin are liberated only by high concentrations (5mM) of ADP, ATP and NADH, by 1mM CTP and ITP, and by high salt concentrations (150mM NaCl). Since it was found that EDTA- and heat-treated actin were also retained on ADP-agarose, we conclude that this second nucleotide interacting site is of limited specificity, low affinity, and not dependent on the native configuration of actin. It exhibits characteristics of an unspecific, polyanionic site, but may represent the low affinity phosphate binding site.

The structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), are all hydrolyzed by the recA protein with the same turnover number (17.5 min-1). The S0.5 values for these nucleotides increase progressively in the order ATP (45 microM), PTP (100 microM), ITP (300 microM), and GTP (750 microM). PTP, ITP, and GTP are each competitive inhibitors of recA protein-catalyzed ssDNA-dependent ATP hydrolysis, indicating that these nucleotides all compete for the same catalytic site on the recA protein. Despite these similarities, ATP and PTP function as cofactors for the recA protein-promoted three-strand exchange reaction, whereas ITP and GTP are inactive as cofactors. The strand exchange activity of the various nucleotides correlates directly with their ability to support the isomerization of the recA protein to a strand exchange-active conformational state. The mechanistic deficiency of ITP and GTP appears to arise as a consequence of the hydrolysis of these nucleotides to the corresponding nucleoside diphosphates, IDP and GDP. We speculate the nucleoside triphosphates with S0.5 values greater than 100 microM will be intrinsically unable to sustain the strand exchange-active conformational state of the recA protein during ongoing NTP hydrolysis and will therefore be inactive as cofactors for the strand exchange reaction.

We have examined the effects of the structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), on the recA protein-promoted DNA renaturation reaction (phi X DNA). In the absence of nucleotide cofactor, the recA protein first converts the complementary single strands into unit-length duplex DNA and other relatively small paired DNA species; these initial products are then slowly converted into more complex multipaired network DNA products. ATP and PTP stimulate the conversion of initial product DNA into network DNA, whereas ITP and GTP completely suppress network DNA formation. The formation of network DNA is also inhibited by all four of the corresponding nucleoside diphosphates, ADP, PDP, IDP, and GDP. Those nucleotides which stimulate the formation of network DNA are found to enhance the formation of large recA-ssDNA aggregates, whereas those which inhibit network DNA formation cause the dissociation of these nucleoprotein aggregates. These results not only implicate the nucleoprotein aggregates as intermediates in the formation of network DNA, but also establish the functional equivalency of ITP and GTP with the nucleoside diphosphates. Additional experiments indicate that the net effect of ITP and GTP on the DNA renaturation reaction is dominated by the corresponding nucleoside diphosphates, IDP and GDP, that are generated by the NTP hydrolysis activity of the recA protein.

Mammalian phosphoenolpyruvate carboxykinase (PEPCK) specifically requires a guanosine or inosine nucleotide as a substrate; however, the structural basis for this nucleotide specificity is not yet known. Because affinity labels derived from guanosine have not yielded a stable, modified peptide in quantities sufficient for sequence analysis, we have investigated the utility of direct photochemical cross-linking of GTP to PEPCK in order to identify the nucleotide binding site. UV irradiation at a distance of 2 cm by a Mineralight lamp (330 microW/cm2) results in the attachment of [alpha-32P]GTP to PEPCK via a stable, covalent linkage in a reaction that is dependent upon GTP concentration and duration of irradiation. After 10 min of irradiation, more than 0.2 mol of [alpha-32P] GTP is incorporated per mole of PEPCK; under these conditions the GTP concentration required for half-maximal labeling is 69 microM. The substrates phosphoenolpyruvate, ITP, and GDP provide protection against photolabeling, as do Mn2+ and Mg2+. One major and one minor radioactive peptide derived from proteolytic digests of photolabeled PEPCK have been isolated and identified. The major modified peptide has been provisionally assigned to an acidic region near the C-terminus, and the minor peptide has been identified as Ser462-Lys471.

The acid-base properties of the nucleic base residues of ITP, GTP, and ATP, and for comparison also as far as possible of the corresponding nucleosides, were studied in dependence on their concentration, i.e. on the effect of self-association. From the dependence between the 1H-NMR chemical shifts of H-2 (where applicable), H-8, and H-1', and the pD of the solutions, the acidity constants for the deprotonation of the D+(N-7) site in D2(ITP)2-, D2(GTP)2-, D(Ino)+, and D(Guo)+, and of the D+(N-1) site in D2(ATP)2- and D(Ado)+ were calculated. Chemical shift/pD profiles for a whole series of varying concentrations of the nucleic base derivatives (= N) were constructed, including those for infinite dilution (delta o), which give the acidity constant for the monomeric N, and for infinitely concentrated solutions (delta infinity), which give the acidity constant of an N in an infinitely long stack. The acidity constants determined from the delta o/pD plots are in excellent agreement with the pKa values measured by potentiometric pH titrations of highly diluted solutions of N. The effects of self-association are striking; e.g. the pKa value of the D+(N-7) site in D2(GTP)2- is lowered by about 1 (as calculated from the delta o/pD and delta infinity/pD profiles), while the pKa value of the D+(N-1) site in D2(ATP)2- is increased by approximately 0.3; i.e. in the first case deprotonation is facilitated and in the second it is inhibited. The increasing inhibition of the H+(N-1) deprotonation with an increasing ATP concentration is due to the high stability of the dimeric [H2(ATP)]2(4-) stack for which the intermolecular H+(N-1)/gamma-P(OH)(O)2- ion pairs between the two ATP molecules are crucial. In those cases where no other significant interaction but aromatic-ring stacking in the self-association process occurs, the release of protons from protonated nitrogen-ring sites is facilitated with increasing stacking; this holds not only for D2(GTP)2- as indicated above, but also for D2(ITP)2-, D(Ino)+, and D(Ado)+. The latter example especially suggests that the situation for the D2(ATP)2- system is exceptional. Some consequences of the considered acid-base properties for biological systems are indicated.