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PubMed1996

An intracellular adenine nucleotide binding site inhibits guanyly cyclase C by a guanine nucleotide-dependent mechanism.

Guanylyl cyclase C (GCC), the receptor for the Escherichia coli heat-stable enterotoxin (ST), is inhibited by 2-substituted adenine nucleotides in an allosteric fashion. In confluent cultures of Caco-2 intestinal epithelial cells, extracellular 2-methylthioadenosine triphosphate (2MeSATP) had no effect on basal or ST-stimulated cyclic GMP (cGMP) accumulation. However, this nucleotide inhibited cGMP accumulation in digitonin-permeabilized Caco-2 human colon carcinoma cells, demonstrating that allosteric inhibition of GCC by adenine nucleotides is mediated by an intracellular adenine nucleotide binding site rather than purinergic receptors. The role of guanine nucleotides in the regulation of GCC by adenine nucleotides was examined. Increasing GTP concentrations from 5 to 100 microM increased the potency of 2MeSATP inhibition of GCC 20-fold, with a shift in the Ki from 447 to 22 microM, respectively. Also, the hydrolysis-resistant analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), supported 2MeSATP inhibition of GCC with a potency which was 10-fold greater than GTP. In addition, GTP alone, in the absence of adenine nucleotides and at concentrations greater than 1 mM, inhibited GCC through a mechanism convergent with 2MeSATP. Guanine nucleotides supported adenine nucleotide inhibition of GCC at low concentrations and directly inhibited this enzyme at high concentrations when these studies were conducted with receptors expressed in Caco-2 cells, native rat intestine, or cloned rat GCC heterologously expressed in 293 monkey kidney cells. These observations demonstrate that adenine nucleotide inhibition of GCC is mediated through an intracellular mechanism which is guanine nucleotide-dependent.

Adenine Nucleotides↗
PubMedDate not supplied

[Concentration of free nucleotides and turnover of adenine nucleotides in the liver of birds of different ages].

The content of free nucleotides and exchangeability of adenine nucleotides were studied in the liver of 1- and 18-month cocks. It is established that adenine nucleotides in the young cocks liver account for 40%, uridine nucleotides for 29%, cytidine nucleotides for 13% of the total amount of nucleotides. These ratios are maintained with age but the amount of adenine nucleotides decreases by 35% and that of uridine nucleotides by 22%. Against a background of a relatively constant level of adenine and uridine nucleotides the content of cytidine and guanine nucleotides varies synchronously within wide ranges, which can evidence for the presence of a common mechanism of regulating synthesis of these metabolites. A direct correlation is detected between the amount of adenine nucleotides and their specific radioactivity. This is due to a higher intensity of synthetic processes in young cocks. The inverse correlation is observed in adult cocks, that results from the effect of "label dilution".

Adenine Nucleotides↗
PubMed1993

Negative cooperativity in the binding of nucleotides to Escherichia coli replicative helicase DnaB protein. Interactions with fluorescent nucleotide analogs.

The interactions of nucleotides with Escherichia coli replicative helicase DnaB protein have been systematically studied using fluorescent nucleotide analogs, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP), 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP), 3'-O-(N-methylantraniloyl) 5'-diphosphate (MANT-ADP), and 1,N6-ethenoadenosine diphosphate (epsilon ADP). The binding of the analogs is accompanied by strong quenching of the protein fluorescence; 0.76 +/- 0.05, 0.76 +/- 0.05, 0.58 +/- 0.05, and 0.53 +/- 0.5 for TNP-ATP, TNP-ADP, MANT-ADP, and epsilon ADP, respectively. A thermodynamically rigorous method has been applied to obtain all binding parameters from fluorescence titration curves independent of the assumption of strict proportionality between the observed quenching of the protein fluorescence and the degree of nucleotide binding. An exact representation of the observed fluorescence quenching, as a function of the nucleotide binding, is introduced through an empirical function which enables analysis of single binding isotherms without the necessity of determining all quenching constants for different binding sites. Using this method, we determined that, at saturation, the DnaB hexamer binds six molecules of TNP-ATP, TNP-ADP, MANT-ADP, and epsilon ADP, and that there is strong heterogeneity among nucleotide binding sites. The binding isotherms are biphasic. Three molecules of nucleotide are bound in the first high-affinity binding phase, and the subsequent three molecules are bound in the second low-affinity binding phase. The separation of the two binding steps is even more pronounced at higher temperatures. The change of the monitored fluorescence is sequential. The binding of the first nucleotide causes the largest quenching of the protein fluorescence with subsequent nucleotide binding inducing progressively less quenching. The simplest explanation of this behavior is that there is a negative cooperativity among nucleotide binding sites on a DnaB hexamer. The negative cooperativity is an intrinsic property of the DnaB helicase, since it is observed in the binding of nucleotide analogs which are different in type and location of the modifying group. A statistical thermodynamic model is proposed, the hexagon, which provides an excellent description of the binding process using only two interaction parameters, intrinsic binding constant K and cooperativity parameter sigma. The data suggest an important role of the phosphate groups in binding and in recognition of nucleotides by the DnaB helicase.

Adenosine↗
PubMed2003

Comparative analysis of the ATP-binding sites of Hsp90 by nucleotide affinity cleavage: a distinct nucleotide specificity of the C-terminal ATP-binding site.

The 90-kDa heat shock protein (Hsp90) is a molecular chaperone that assists both in ATP-independent sequestration of damaged proteins, and in ATP-dependent folding of numerous targets, such as nuclear hormone receptors and protein kinases. Recent work from our lab and others has established the existence of a second, C-terminal nucleotide binding site besides the well characterized N-terminal, geldanamycin-sensitive ATP-binding site. The cryptic C-terminal site becomes open only after the occupancy of the N-terminal site. Our present work demonstrates the applicability of the oxidative nucleotide affinity cleavage in the site-specific characterization of nucleotide binding proteins. We performed a systematic analysis of the nucleotide binding specificity of the Hsp90 nucleotide binding sites. N-terminal binding is specific to adenosine nucleotides with an intact adenine ring. Nicotinamide adenine dinucleotides and diadenosine polyphosphate alarmones are specific N-terminal nucleotides. The C-terminal binding site is much more unspecific-it interacts with both purine and pirimidine nucleotides. Efficient binding to the C-terminal site requires both charged residues and a larger hydrophobic moiety. GTP and UTP are specific C-terminal nucleotides. 2',3'-O-(2,4,6-trinitrophenyl)-nucleotides (TNP-ATP, TNP-GTP) and pyrophosphate access the C-terminal binding site without the need for an occupied N-terminal site. Our data provide additional evidence for the dynamic domain-domain interactions of Hsp90, give hints for the design of novel types of specific Hsp90 inhibitors, and raise the possibility that besides ATP, other small molecules might also interact with the C-terminal nucleotide binding site in vivo.

Adenosine Triphosphate↗
PubMed1984

Effect of nucleotides on translocation of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi apparatus vesicles.

Recent studies from this laboratory have suggested that rat-liver Golgi apparatus derived membranes contain different proteins which can translocate in vitro CMP-N-acetylneuraminic acid, GDP-fucose and adenosine 3'-phosphate 5'-phosphosulfate from an external compartment into a lumenal one. The aim of this study was to define the role of the nucleotide, sugar and sulfate moieties of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate in translocation of these latter compounds across Golgi vesicle membranes. Indirect evidence was obtained suggesting that the nucleotide (but not sugar or sulfate) is a necessary recognition feature for binding to the Golgi membrane (measured as inhibition of translocation) but is not sufficient for overall translocation; this latter event also depends on the type of sugar. Important recognition features for inhibition of translocation of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate were found to be the type of nucleotide base (purine or pyrimidine) and the position of the phosphate group in the ribose. Thus, UMP and CMP were found to be competitive inhibitors of translocation of CMP-N-acetylneuraminic acid, while AMP did not inhibit. Structural features of the nucleotides which were less important in inhibition of translocation (and thus presumably in binding) of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate were the number of phosphate groups in the nucleotide (CDP and CMP inhibited to a similar extent), the presence of ribose or deoxyribose in the nucleotide, a replacement of hydrogen in positions 5 of pyrimidines or 8 in purines by halogens or an azido group. The sugar or sulfate did not inhibit translocation of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi vesicles and therefore appear not to be involved in their binding to the Golgi membrane.

Adenine Nucleotides↗
PubMed1986

Neuromodulation by adenine nucleotides, as indicated by experiments with inhibitors of nucleotide inactivation.

The adenine nucleotides AMP, ADP and ATP (3 X 10(-7) M and above) inhibited contractile responses to transmural nerve stimulation in guinea-pig ileum longitudinal muscle via a prejunctional action. Nucleotides assumed to inhibit the degradation of adenine nucleotides were employed to determine whether inhibition of contractile responses was elicited by adenine nucleotides per se, or required breakdown to adenosine. The IMP or 2'-deoxy AMP enhanced the prejunctional inhibitory effect elicited by AMP. A similar enhancement of the inhibitory effect of ADP and ATP was seen after administration of IDP and ITP, respectively. The inhibitory effect of adenosine was not enhanced by inosine, IMP or IDP. The 5'-nucleotidase inhibitor, TDP enhanced inhibition elicited by ADP. In contrast, alpha, beta-meADP did not influence the prejunctional inhibitory effect elicited by the adenine nucleotides. However, the combination of alpha, beta-meADP and IMP enhanced the inhibitory effect of ATP. The postjunctional contractile effect elicited by ADP and ATP was enhanced by pretreatment with inosine nucleotides, alpha, beta-meADP or TDP, indicating decreased inactivation of ADP and ATP during concurrent nucleotide administration. The fact that the prejunctional effect of adenine nucleotides can be enhanced by forms of pretreatment known to antagonize the breakdown of adenine nucleotides, constitutes strong evidence for prejunctional action per se by adenine nucleotides.

Adenine Nucleotides↗
PubMed1982

Residual nucleotide and tubulin's ability to polymerize with nucleotide analogs.

The nucleotide requirements for microtubule assembly following two different methods of nucleotide removal were defined. Microtubular protein extracted with charcoal in a buffer containing 1 mM EDTA showed an absolute dependence on GTP for assembly, whereas, microtubular protein extracted with charcoal in a buffer containing 1 mM MgCl2 could assemble with the nucleotide analogs, guanosine-5'-(beta,gamma-imido)triphosphate (GMP-PNP) and guanosine-5'-(beta-gamma-methylene)triphosphate. Assembly promoted by the nucleotide analogs proceeded at slower rate and to a lesser extent than the assembly induced by GTP. Furthermore, after the analog-induced assembly had reached a steady state level, the addition of GTP immediately induced further tubulin assembly to the level produced by GTP alone. Microtubules assembled with GMP-PNP or GTP contained approximately 2.8 mol of bound nucleotide per mol of dimer. The two different methods of charcoal extraction produced microtubular protein preparations that differed in their residual nucleotide and Mg2+ content. The EDTA-charcoal-extracted microtubular protein retained 1 mol of tightly bound nucleotide (GDP) and 1 mol of Mg2+ per mol of dimer, whereas, the Mg2+ charcoal-extracted microtubular protein retained 1.4 mol of bound nucleotide (1.0 mol of GDP + 0.4 mol of GTP) and 1.5 mol of Mg2+ per mol of dimer. The results suggest that only that fraction of tubulin subunits containing bound GTP can assemble with the nucleotide analogs and that microtubules may possess a third or polymer binding site for nucleotide.

Animals↗
PubMed1982

Estimation of average number of nucleotide substitutions when the rate of substitution varies with nucleotide.

A formal mathematical analysis of Kimura's (1981) six-parameter model of nucleotide substitution for the case of unequal substitution rates among different pairs of nucleotides is conducted, a new formulae for estimating the number of nucleotide substitutions and its standard error are obtained. By using computer simulation, the validities and utilities of Jukes and Cantor's (1969) one-parameter formula, Takahata and Kimura's (1981) four-parameter formula, and our six-parameter formula for estimating the number of nucleotide substitutions are examined under three different schemes of nucleotide substitution. It is shown that the one-parameter and four-parameter formulae often give underestimates when the number of nucleotide substitutions is large, whereas the six-parameter formula generally gives a good estimate for all the three substitution schemes examined. However, when the number of nucleotide substitutions is large, the six-parameter and four-parameter formulae are often inapplicable unless the number of nucleotides compared is extremely large. It is also shown that as long as the mean number of nucleotide substitutions is smaller than one per nucleotide site the three formulae give more or less the same estimate regardless of the substitution scheme used.

Base Sequence↗
PubMed1995

Nucleotide-free actin: stabilization by sucrose and nucleotide binding kinetics.

We prepared nucleotide-free actin in buffer containing 48% (w/v) sucrose. Sucrose inhibits the irreversible denaturation of actin that follows nucleotide dissociation [Kasai et al. (1965) Biochim. Biophys. Acta 94, 494-503]. Our conditions removed nucleotide from approximately 80% of the actin. Stabilization of nucleotide-free actin depends on the sucrose concentration. The CD ellipticity (x 10(3) deg cm2 dmol-1) at 222 nm of nucleotide-free actin in 48% sucrose is -3.54. The ellipticity of denatured nucleotide-free actin in dilute buffer is -2.01 and that of native actin is -4.19. In 48% sucrose nucleotide-free actin has 1.12 and native actin has 0.5 solvent-exposed thiol residues. The conformation of native actin is recovered when ATP and Mg2+ are added. Our ability to generate stable nucleotide-free actin permitted us to study the kinetics of nucleotide binding to actin. The observed rate constant of the reaction is linearly dependent on the concentration of epsilon ATP, a fluorescent analog of ATP. The inverse of the association rate constant is proportional to the viscosity of the solvent with an intercept near the origin as expected for a diffusion-limited reaction. The second-order association rate constant for Mg(2+)-ATP and Ca(2+)-ATP binding to nucleotide-free actin in water at 22 degrees C is 5 x 10(6) M-1 s-1. The Smoluchowski collision rate constant for actin and ATP is calculated to be 6.5 x 10(9) M-1 s-1, which makes the "orientation factor" 7.7 x 10(-4). From the ratio of the dissociation and association rate constants, we calculate dissociation equilibrium constants of 1.2 x 10(-9) M for Mg(2+)-ATP-actin, 4.4 x 10(-9) M for Mg(2+)-epsilon ATP-actin, and 1.2 x 10(-10) M for Ca(2+)-ATP-actin.

Actins↗
PubMed1999

Pseudomonas aeruginosa exoenzyme S disrupts Ras-mediated signal transduction by inhibiting guanine nucleotide exchange factor-catalyzed nucleotide exchange.

Pseudomonas aeruginosa exoenzyme S double ADP-ribosylates Ras at Arg(41) and Arg(128). Since Arg(41) is adjacent to the switch 1 region of Ras, ADP-ribosylation could interfere with Ras-mediated signal transduction via several mechanisms, including interaction with Raf, or guanine nucleotide exchange factor-stimulated or intrinsic nucleotide exchange. Initial experiments showed that ADP-ribosylated Ras (ADP-r-Ras) and unmodified Ras (Ras) interacted with Raf with equal efficiencies, indicating that ADP-ribosylation did not interfere with Ras-Raf interactions. While ADP-r-Ras and Ras possessed equivalent intrinsic nucleotide exchange rates, guanine nucleotide exchange factor (Cdc25) stimulated the nucleotide exchange of ADP-r-Ras at a 3-fold slower rate than Ras. ADP-r-Ras did not affect the nucleotide exchange of Ras, indicating that the ADP-ribosylation of Ras was not a dominant negative phenotype. Ras-R41K and ADP-r-Ras R41K possessed similar exchange rates as Ras, indicating that ADP-ribosylation at Arg(128) did not inhibit Cdc25-stimulated nucleotide exchange. Consistent with the slower nucleotide exchange rate of ADP-r-Ras as compared with Ras, ADP-r-Ras bound its guanine nucleotide exchange factor (Cdc25) less efficiently than Ras in direct binding experiments. Together, these data indicate that ADP-ribosylation of Ras at Arg(41) disrupts Ras-Cdc25 interactions, which inhibits the rate-limiting step in Ras signal transduction, the activation of Ras by its guanine nucleotide exchange factor.

ADP Ribose Transferases↗
PubMed2003

Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating.

CFTR, the protein defective in cystic fibrosis, functions as a Cl- channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [alpha32P]8-N3ATP or [gamma32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0 degrees C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30 degrees C, with extensive washing, also at 30 degrees C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30 degrees C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore.

Adenosine Diphosphate↗
PubMed1994

The interaction between the first and last intron nucleotides in the second step of pre-mRNA splicing is independent of other conserved intron nucleotides.

Virtually all pre-mRNA introns begin with the sequence /GU and end with AG/ (where / indicates a border between an exon and an intron). We have previously shown that the G residues at the first and last positions of the yeast actin intron interact during the second step of splicing. In this work, we ask if other highly conserved intron nucleotides also take part in this /G-G/ interaction. Of special interest is the penultimate intron nucleotide (AG/), which is important for the second step of splicing and is in proximity to other conserved intron nucleotides. Therefore, we tested interactions of the penultimate intron nucleotide with the second intron nucleotide (/GU) and with the branch site nucleotide. We also tested two models that predict interactions between sets of three conserved intron nucleotides. In addition, we used random mutagenesis and genetic selection to search for interactions between nucleotides in the pre-mRNA. We find no evidence for other interactions between intron nucleotides besides the interaction between the first and last intron nucleotides.

Base Sequence↗
PubMedDate not supplied

[Effectiveness of complex-formation of nucleotides with human DNA polymerase alpha from data of enzyme modification by reactive nucleotide analogs].

The modification of the human placenta DNA polymerase alpha by the imidazolides of dNMP was investigated. The modification was shown to occur only in the simultaneous presence of the template and the primer. This process, however, doesn't depend on the complementary interaction of the nucleotide base with the template. The Kd values of the complexes between the different nucleotides and DNA polymerase alpha were estimated. The affinity of Im-dTMP was determined from the dependence of the Kapp of the enzyme inactivation rate on the reagent concentration. The Kd values for dNMP, dNDP, dNTP were estimated using the protective effect of these nucleotides under the enzyme modification by Im-dTMP. The comparison of the interaction efficiency between the polymerase and dNMP, dNDP, dNTP (complementary or non-complementary to the template) allow to conclude that the nucleotide discrimination occurs on the dNTP level, i. e. dNMP and dNDP upon forming the complex with the enzyme, don't interact complementarily with the template. The additional contacts between the enzyme and the nucleotide terminal phosphate were supposed to form only for the complementary dNTP. The studies allowed to put forward a hypothetical model of the template complementary dNTP binding to the polymerases. The role of the hydrophobic interaction of the nucleotides with the enzyme as well as the possible influence of the nucleotide gamma-phosphate group on the template--dNTP complement formation. The Watson-Crick bound formation of the nucleotide with the template was supposed to be followed by the additional conformational rearrangement of the nucleotide triphosphate chain. The latter process leads to the formation of additional contacts between the enzyme and the nucleotide gamma-phosphate.

Catalysis↗
PubMed2005

Intense exercise induces the degradation of adenine nucleotide and purine nucleotide synthesis via de novo pathway in the rat liver.

The purpose of this study was to investigate the influence of intense exercise on the metabolism of adenine nucleotides in the liver. In the first experiment, to determine the degradation of adenine nucleotides, hepatic adenine nucleotides of rats were labeled by an intraperitoneal administration of 15N-labeled adenine the day before treadmill running to exhaustion. In the second experiment, to determine the de novo synthesis of purine nucleotides after intense exercise, 14C-glycine was intraperitoneally administered to rats performing intense running on a treadmill. In the first experiment, hepatic levels of ATP and total adenine nucleotides showed a reduction immediately after exercise. In contrast, hepatic levels of AMP, adenosine, hypoxanthine and uric acid showed an increase immediately after exercise. The hepatic 15N level continued to decline during the recovery period after exercise. Urinary excretion of 15N-urate was 40% higher in the exercised rats than in the control rats. In the second experiment, the radioactivity of 14C detected in the fraction of hepatic urate and allantoin was approximately 300% higher in the exercised rats than in the control rats. 14C-radioactivity that excreted into urine as urate and allantoin was approximately 200% higher in the exercised rats. Intense exercise led to the degradation of hepatic adenine nucleotides, which were not utilized for the re-synthesis of nucleotide and further degraded to hypoxanthine or uric acid. Intense exercise induced the synthesis of purine nucleotides in the liver via a de novo pathway and these synthesized nucleotides were also degraded to nucleosides and excreted into urine.

Adenine Nucleotides↗
PubMed2000

Kinetic mechanism of nucleotide cofactor binding to Escherichia coli replicative helicase DnaB protein. stopped-flow kinetic studies using fluorescent, ribose-, and base-modified nucleotide analogues.

The kinetic mechanism of binding nucleotide cofactors to the Escherichia coli primary replicative helicase DnaB protein has been studied, using the fluorescence stopped-flow technique. The experiments have been performed with fluorescent ATP and ADP analogues bearing the modification on the ribose, MANT-AMP-PNP and MANT-ADP, and on the base, epsilonAMP-PNP and epsilonADP. Association of the DnaB helicase with nucleotide cofactors is characterized by four relaxation times that indicate that the binding occurs by a minimum of four-steps. The simplest mechanism which can describe the data is a four-step sequential process where the bimolecular binding step is followed by three isomerization steps. This mechanism is described by the following equation: [equation in text]. The binding mechanism is independent of the location of the nucleotide cofactor modification and is an intrinsic property of the DnaB helicase-nucleotide system. Quantitative amplitude analyses, using the matrix projection operator technique, allowed us to determine specific fluorescence changes accompanying the formation of all intermediates relative to the fluorescence of the free nucleotide. It shows that the major conformational change of the DnaB helicase-nucleotide complex occurs in the formation of the (H-N)(1). Moreover, the value of the bimolecular rate constant, k(1), is 3-4 orders of magnitude lower than the value expected for the diffusion-controlled reaction. These results indicate that the determined first step includes formation of the collision and an additional transition of the enzyme-nucleotide complex. The obtained results provide evidence of profoundly different conformational states of the ribose and base regions of the nucleotide-binding site in different intermediates. The sequential nature of the mechanism of the nucleotide binding to the DnaB helicase indicates the lack of the existence of a kinetically significant conformational equilibrium of the helicase protomer and the DnaB hexamer prior to the binding. The significance of these results for the functioning of the DnaB helicase is discussed.

Adenosine Diphosphate↗
PubMed1971

Interconversion of cyclic nucleotide-activated and cyclic nucleotide-independent forms of a protein kinase from beef heart.

A protein kinase activated by cyclic nucleotides was purified from beef heart. Upon exposure to adenosine 3':5'-cyclic monophosphate (cyclic AMP) during gel filtration on Sephadex G-200, the protein kinase dissociated into a cyclic nucleotide-independent protein kinase and a cyclic nucleotide-binding protein. A similar or identical cyclic nucleotide-independent protein kinase could be obtained in highly purified form by clution from a DEAE-cellulose column with 10(-6) M cyclic AMP; the cyclic AMP-binding protein was apparently retained by the resin. The addition of cyclic nucleotide-binding protein to cyclic nucleotide-independent protein kinase resulted in the reappearance of cyclic nucleotide-dependent protein kinase, which could be isolated by filtration on Sephadex G-200 in the absence of cyclic AMP. These results confirm and extend previous suggestions that cyclic nucleotides activate protein kinases by dissociating them from inhibitory, cyclic nucleotide-binding proteins.

Adenine Nucleotides↗