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1.
Studies of mammalian terminal deoxyribonucleotidyltransferase (TdT) are facilitated by use of inhibitors that selectively knock down the activity of the enzyme. We have screened for selective inhibitors of TdT and identified a natural compound with this property in the Japanese vegetable, Arctium lappa. The compound has little effect on the activities of mammalian DNA polymerases, such as alpha, beta, delta or lambda polymerase, and prokaryotic DNA polymerases, such as Taq DNA polymerase, T4 DNA polymerase and Klenow fragment. H1- and C13-NMR spectroscopic analyses showed the compound to be baicalin, a compound previously reported as an anti-inflammatory or antipyretic agent. The IC50 value of baicalin to TdT was 18.6 microM. We also found that genistin, a baicalin derivative known to be antimutagenic, more selectively inhibited TdT activity than baicalin, although its IC50 value was weaker (28.7 microM). Genistin and baicalin also inhibited the activity of truncated TdT (the so-called pol beta core domain) in which the BRCT motif was deleted in its N-terminal region. In kinetic analyses, inhibition by either genistin or baicalin was competitive with the primer and non-competitive with the dNTP substrate. The compounds may, therefore, bind directly to the primer-binding site of TdT and simultaneously disturb dNTP substrate incorporation into the primer. Genistin and baicalin should prove to be useful agents for studying TdT.  相似文献   

2.
Potential bisubstrate analogs, with adenosine and thymidine joined at their 5' positions by polyphosphoryl linkages of varying lengths (ApndT, where n = the number of phosphoryl groups), were examined as inhibitors of cytosolic thymidine kinase from blast cells of patients with acute myelocytic leukemia. Ki values were 1.2 microM for Ap3dT, 0.31 microM for Ap4dT, 0.12 microM for Ap5dT, and 0.19 microM for Ap6dT. The best inhibitor of the cytosolic enzyme, Ap5dT, was somewhat less effective as an inhibitor of the mitochondrial enzyme (Ki = 0.50 microM). In addition to their inhibitory modes of binding by the cytosolic enzyme, these compounds were bound at considerably lower concentrations (Kd = 0.029 microM for Ap4dT, 0.0025 microM for Ap5dT, and 0.0027 microM for Ap4dT), in such a way as to protect the cytosolic enzyme from thermal inactivation at 37 degrees C in the absence of substrates.  相似文献   

3.
Adenosine(5')tetraphospho(5')adenosine-binding protein of calf thymus   总被引:5,自引:0,他引:5  
An adenosine(5')tetraphospho(5')adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high-affinity (Kd = 13 microM) binding of Ap4A with great substrate specificity. The Ap4A binding protein has been isolated in two forms: a 'free', or non-polymerase-bound, form which predominates, and a similar form which copurifies with DNA polymerase alpha, but which can be resolved from it. The free form of Ap4A binding protein contains associated adenosine(5')tetraphospho(5')adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase alpha contains no such activity. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50-100 microM) of Zn2+ without any effect on the Ap4A binding protein activity. This difference in associated Ap4Aase activity between free and polymerase-bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase alpha.  相似文献   

4.
(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate (BVdUTP), known as a specific inhibitor of herpes simplex virus (type 1)-DNA polymerase, was found to be a potent inhibitor of the activity of terminal deoxynucleotidyltransferase (TdT) from calf thymus. BVdUTP was not an efficient substrate of TdT, but it inhibited the incorporation of normal deoxynucleotide substrates in competitive fashion at the nucleotide binding site of TdT molecule. The Ki value for BVdUTP (5 microM) was much less than the Km value for dGTP (83 microM), indicating stronger affinity of the inhibitor to TdT than that of the substrate. These results indicate the usefulness of BVdUTP as a potent inhibitor of TdT for elucidation of the reaction mechanism of this enzyme.  相似文献   

5.
Terminal deoxynucleotidyltransferase (TdT) exhibits strong sensitivity to ATP and its dinucleotide analogues, Ap2A, Ap3A, Ap4A, Ap5A and Ap6A. Similar to ATP, all of the dinucleotides appear to be competitive inhibitors of TdT catalysis with respect to substrate deoxynucleoside triphosphates and effectively block the UV-mediated substrate cross-linking to TdT. Among the various dinucleotides, Ap5A and Ap6A (diadenosine 5'-5' penta- and hexaphosphate, respectively) are significantly more effective than dinucleotides containing 2, 3 or 4 phosphate backbones. Furthermore, Ap5A is found to be the only dinucleotide which has reactivity at both substrate- and primer-binding domains in TdT.  相似文献   

6.
5-(Hydroxymethyl)-2-furfural (HMF), a pyrolysate of carbohydrate isolated from instant coffee (Coffea arabica L.), selectively inhibits the activities of mammalian DNA polymerase lambda (pol lambda) and terminal deoxynucleotidyltransferase (TdT) which are family X pols, in vitro. The compound influenced neither the activities of replicative DNA polymerases such as alpha, delta, and epsilon, nor even the activity of pol beta which is from the same family and thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of HMF such as furan, furfuryl alcohol, and 2-furaldehyde did not influence the activities of any enzymes tested, the substituted form of furan with a hyroxymethyl group and a formyl group might be important for the inhibition of pol lambda and TdT. The inhibitory effect of HMF on intact pol lambda (i.e., residues 1-575), a truncated pol lambda lacking the N-terminal BRCA1 C-terminus domain (133-575, del-1 pol lambda) and another truncated pol lambda lacking the N-terminal proline-rich region (245-575, del-2 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 26.1, 10.3, and 4.6 microM, respectively. The IC(50) value of HMF for TdT was the same as that for del-2 pol lambda (5.5 microM). The HMF-induced inhibition of both pol lambda and TdT activities was competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, HMF was suggested to bind to the pol beta-like region of pol lambda and TdT.  相似文献   

7.
The polymerase and 3'-5'-exonuclease activities of the Klenow fragment of DNA polymerase I are located on separate structural domains of the protein, separated by about 30 A. To determine whether a DNA primer terminus can move from one active site to the other without dissociation of the enzyme-DNA complex, we carried out reactions on a labeled DNA substrate in the presence of a large excess of unlabeled DNA, to limit observations to a single enzyme-DNA encounter. The results indicated that while Klenow fragment is capable of intramolecular shuttling of a DNA substrate between the two catalytic sites, the intermolecular pathway involving enzyme-DNA dissociation can also be used. Thus, there is nothing in the protein structure or the reaction mechanism that dictates a particular means of moving the DNA substrate. Instead, the use of the intermolecular or the intramolecular pathway is determined by the competition between the polymerase or exonuclease reaction and DNA dissociation. When the substrate has a mispaired primer terminus, DNA dissociation seems generally more rapid than exonucleolytic digestion. Thus, Klenow fragment edits its own polymerase errors by a predominantly intermolecular process, involving dissociation of the enzyme-DNA complex and reassociation of the DNA with the exonuclease site of a second molecule of Klenow fragment.  相似文献   

8.
Di(adenosine-5')oligophosphate nucleotides of general structure ApnA (n = 3-6) inhibited the protein kinase activity of homogeneous phorboid receptor. These nucleotides did not affect the phorboid binding activity. Ap4A competed for an ATP binding site on the phorboid receptor. Km for ATP was increased from 0.5 to 2 microM in the presence of 0.2 mM of Ap4A. KI was calculated to be approximately 0.1 mM. Ap4A-elicited inhibition of phorboid receptor kinase activity was independent of receptor concentration as well as of phosphoacceptor substrate concentration.  相似文献   

9.
10.
11.
Binding of adenosine(5')tetraphospho(5')adenosine (Ap4A) to histones of calf thymus was investigated by non-equilibrium dialysis. Histone H1 interacts with the dinucleotide via two strong sites and competes with Mg2+ ions. Intrinsic dissociation constants were 1.6 +/- 0.1 microM and 11 +/- 1 microM for zero and 0.4 mm-Mg2+ concentration respectively. Binding of poly(dT) and of other nucleotides to histone H1 was measured in an [3H]Ap4A-competition assay. The tendency to form complexes among nucleotides was highest for bisnucleoside tetraphosphates and decreased in the order poly(dT) greater than or equal to Ap4A approximately Gp4G greater than Ap4 much greater than Ap3A approximately Ap5A greater than or equal to ATP, GTP and dTTP. The co-ordination complex derived from Ap4A and cis-diammine-dichloroplatinum(II) was not reactive. The other histones of calf thymus also bound Ap4A with affinities decreasing in the order H4 approximately H3 greater than H1 greater than H2b greater than H2a. Ap4A stimulated the exchange of histone H1 between nucleosomes, but this effect was referred to ionic strength. It did not bind to assembled nucleosomes. Binding of Ap4A to histone H1 was decreased by salt (NaCl). At physiological saline concentration the value of the dissociation constant is commensurable with the value of the Ap4A concentration in the nucleus and thus indicative of complex-formation in vivo.  相似文献   

12.
An assay of adenosine(5')tetraphospho(5')adenosine (Ap4A), based on the luciferin/luciferase method for ATP measurement, was developed, which allows one to determine picomolar amounts of unlabeled Ap4A in cellular extracts. In eukaryotic cells this method yielded levels of Ap4A varying from 0.01 microM to 13 microM depending on the growth, cell cycle, transformation, and differentiation state of cells. After mitogenic stimulation of G1-arrested mouse 3T3 and baby hamster kidney fibroblasts the Ap4A pools gradually increased 1000-fold during progression through the G1 phase reaching maximum Ap4A concentrations of about 10 microM in the S phase. Quiescent 3T3 cells reach a high level of Ap4A (1 microM) in a 'committed' but prereplicative state if exposed to an external mitogenic stimulant (excess of serum) and simultaneously to a synchronizer which inhibits entry into the S phase (hydroxyurea). When the block for DNA replication was removed at varying times after removal of the stimulant decay of commitment to DNA synthesis was found correlated with a shrinkage of the Ap4A pool. Cells lacking a defined G1 phase (V79 lung fibroblasts, Physarum) possess a constitutively high base level of Ap4A (about 0.3 microM) even during mitosis. From this high level, Ap4A concentration increases only about tenfold during the S phase. Temperature-down-shift experiments, using chick embryo cells infected with transformation-defective temperature-sensitive viral mutants(td-ts), have shown that the expression of the transformed state at 35 degrees C is accompanied by a tenfold increase of the cellular Ap4A pool. Treatment of exponentially growing human cells with interferon leads, concomitantly with an inhibition of DNA syntheses, to a tenfold decrease in intracellular Ap4A levels within 20 h. The possibility of Ap4A being a 'second messenger' of cell cycle and proliferation control is discussed in the light of these results and those reported previously demonstrating that Ap4A is a ligand of mammalian DNA polymerase alpha, triggers DNA replication in quiescent mammalian cells and is active in priming DNA synthesis.  相似文献   

13.
Comparative inhibition kinetics with natural dNTP end products (dNp3) and new synthetic bisubstrate-type analogs, dNp4A (deoxynucleoside 5'-adenosine 5'-P1,P4-tetraphosphate), have been studied with their target deoxynucleoside kinases from Lactobacillus acidophilus. Analysis of inhibition specificity, inhibition patterns, and Ki(app) under various conditions has revealed the following conclusions. Both dNTP and dNp4A bind to the active site of the corresponding kinase through multiple binding determinants. The deoxynucleoside moiety of dNTP fits optimally at the deoxynucleoside binding site and provides the basis for its inhibition specificity, whereas the triphosphate group interacts with the ATP binding site, reinforcing the affinity of the molecule as a potent end product inhibitor (Ki = 0.4-3 microM). The adenosine moiety of dNp4A does not contribute to the binding of this compound, whereas the tetraphosphate portion is the second binding determinant, just as in the model developed for dNTP. dNTP and dNp4A proved to be useful tools for distinguishing the kinetic mechanisms of kinases which follow sequential pathways, i.e. the rapid equilibrium Random Bi Bi for dCyd and dGuo kinases and the steady state Ordered Bi Bi mechanism for two dAdo kinases associated either with dCyd kinase or with dGuo kinase on different multifunctional proteins.  相似文献   

14.
Conyers GB  Wu G  Bessman MJ  Mildvan AS 《Biochemistry》2000,39(9):2347-2354
Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.  相似文献   

15.
16.
D J Allen  P L Darke  S J Benkovic 《Biochemistry》1989,28(11):4601-4607
Fluorescent derivatives of short oligonucleotides of defined sequence were prepared by the incorporation of 5-(propylamino)uridine via current phosphoramidite chemistry, followed by derivatization of the propylamine function with mansyl chloride. These oligomers, annealed to complementary oligomers, yielded short duplex DNA fluorescently labeled at a specific base. The fluorescence emission from this labeled duplex increases upon binding to the Klenow fragment of DNA polymerase I (KF) at specific positions within the duplex DNA. By varying the position of the label within the duplex DNA and observing the emission, points of strong enzyme-DNA interactions were elucidated. A similar fluorescent derivative of a deoxynucleoside triphosphate (dNTP), 5-[[[[[[(5- sulfonaphthalenyl)amino]ethyl]amino]carbonyl]- methyl]thio]-2'-deoxyuridine 5'-triphosphate (AEDANS-S-dUTP), was synthesized, whose emission also was increased upon binding to KF. The change in emission intensities between unbound and bound substrates enabled the measurements of KDs for the DNA and dNTP derivative, which were found to be 0.15 nM and 2.9 microM, respectively. Stopped-flow measurements on these species yielded association and dissociation rates for each. Anisotropy measurements of the labeled base at various positions in the duplex yielded values that support the measurements made by observing the emission intensities.  相似文献   

17.
A Basu  P Kedar  S H Wilson  M J Modak 《Biochemistry》1989,28(15):6305-6309
Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase beta. Kinetic studies indicate that the mechanism of PLP inhibition is complex. In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested. Reduction of the PLP-treated enzyme with sodium [3H]borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active. This protective effect is not observed in the absence of template-primer. Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification. Of these four sites, only one is protected by dNTP from pyridoxylation. Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation. These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate.  相似文献   

18.
Stimulation of a Ca(2+)-induced Ca(2+)-release channel from skeletal muscle sarcoplasmic reticulum by various adenosine(5')oligophospho(5')adenosines (ApnA, n = 2-6) by a rapid quenching technique using radioactive calcium was studied. Ap4A, Ap5A and Ap6A, as well as adenosine 5'-[beta, gamma-methylene]triphosphate (AdoPP [CH2]P), a non-hydrolyzable ATP analogue, stimulated the Ca(2+)-release channel, whereas Ap2A and Ap3A had no effect. At a concentration of 0.5 mM, the order of stimulation was AdoPP[CH2]P less than Ap4A less than Ap5A much less than Ap6A. As well as having the highest affinity (0.44 mM for half-maximal stimulation), Ap6A showed an extraordinarily high Hill coefficient of 3.3 (1.9 for AdoPP[CH2]P, 2.1 for Ap5A). The stimulating effect of Ap6A was reversible, yet its dissociation proceeded very slowly. Stimulation of Ca2+ release by Ap6A was counteracted by Mg2+ and ruthenium red. A 2',3'-dialdehyde derivative of Ap6A, which is a chemical probe for amino groups, stimulated irreversibly the Ca(2+)-release channel and modified some high-molecular-mass sarcoplasmic reticulum proteins, possibly including the channel protein. Our data suggest that Ap6A stimulates the Ca2+ channel by binding to the activation site of the channel subunit and simultaneously preventing the spontaneous decay of the Ca2+ channel by keeping together two of the four channel subunits by bridging them with its two adenosine groups.  相似文献   

19.
Pyridoxal(5')diphospho(1)-alpha-D-glucose has been tested as an inhibitor of native glycogen phosphorylases a and b. Its inhibition patterns with respect to substrate, glucose 1-phosphate, and activator, adenosine monophosphate, show it to be a potent (Ki = 40 microM) R-state inhibitor of phosphorylase b, mimicking the binding of glucose-1-phosphate, and, as predicted for an R-state inhibitor, its binding to AMP-activated phosphorylase a is even tighter (Ki = 10 microM). Moreover, it is demonstrated that its binding does not involve covalent imine formation from the pyridoxal aldehyde to an active-site lysine residue. It thus represents the tightest binding R-state inhibitor reported to date, and a 31P NMR study of the effects of binding of this inhibitor upon 31P resonances for the coenzyme phosphate and that of the nucleotide activator is presented. Results obtained are essentially identical to those obtained previously using glucose cyclic 1,2-phosphate, corroborating the previous conclusions. A rationale for the tightness of the binding is presented, as are other possible uses of this compound in studies on glycogen phosphorylase and other similar enzymes.  相似文献   

20.
3'-NH2-BV-dUrd, the 3'-amino derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, was found to be a potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) replication. 3'-NH2-BV-dUrd was about 4-12 times less potent but equally selective in its anti-herpes activity as BV-dUrd. Akin to BV-dUrd, 3'-NH2-BV-dUrd was much less inhibitory to herpes simplex virus type 2 than type 1. It was totally inactive against a thymidine kinase-deficient mutant of HSV-1. The 5'-triphosphate of 3'-NH2-BV-dUrd (3'-NH2-BV-dUTP) was evaluated for its inhibitory effects on purified herpes viral and cellular DNA polymerases. Among the DNA polymerases tested, HSV-1 DNA polymerase and DNA polymerase alpha were the most sensitive to inhibition by 3'-NH2-BV-dUTP (Ki values 0.13 and 0.10 microM, respectively). The Km/Ki ratio for DNA polymerase alpha was 47, as compared with 4.6 for HSV-1 DNA polymerase. Thus, the selectivity of 3'-NH2-BV-dUrd as an anti-herpes agent cannot be ascribed to a discriminative effect of its 5'-triphosphate at the DNA polymerase level. This selectivity most probably resides at the thymidine kinase level. 3'-NH2-BV-dUrd would be phosphorylated preferentially by the HSV-1-induced thymidine kinase (Ki 1.9 microM, as compared with greater than 200 microM for the cellular thymidine kinase), and this preferential phosphorylation would confine the further action of the compound to the virus-infected cell.  相似文献   

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