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1.
The microfluorometric determination of DNA with diaminobenzoic acid in combination with a filter binding assay offers an easy and accurate procedure to study the interaction of proteins with any source of DNA. Using a highly polymerized commercial preparation of calf thymus DNA, the binding curve of histones or protamines changes from hyperbolic to increasingly sigmoidal depending on the length and temperature of incubation. The presence in the DNA preparation of small amounts of contaminating proteases, undetectable by conventional methods, is responsible for this change in the binding curve, since the presence of phenylmethylsulfonylfluoride in the reaction mixture or the removal of the proteases from the DNA produces only hyperbolic curves. 相似文献
2.
The problems of whether the kinetic and regulatory properties of AMP deaminase were modified by formation of a deaminase-myosin complex were investigated with an enzyme preparation from rat skeletal muscle. Results showed that AMP deaminase was activated by binding to myosin. Myosin-bound AMP deaminase showed a sigmoidal activity curve with respect to AMP concentration in the absence of ATP and ADP, but a hyperbolic curve in their presence. Addition of ATP and ADP doubled the V value, but did not affect the Km value. Myosin-bound AMP deaminase also gave a sigmoidal curve in the presence of alkali metal ions, whereas free AMP deaminase gave a hyperbolic curve. GTP abolished the activating effects of both myosin and ATP. 相似文献
3.
(i) The steady-state kinetic data obtained with purified gizzard and uterus smooth muscle myosins indicated the presence of a plateau region on the substrate-saturation curves. Hill plots of these data provided evidence for mixed positive and negative cooperative interactions. In contrast, when gizzard myosin was prepared according to the method of A. Sobieszek and R.D. Bremel (1975, Eur. J. Biochem.55, 49–60), the saturation curve in the presence of CaATP was hyperbolic and no cooperativity of the binding site(s) was discerned. However, in the presence of MgATP although the curve appeared hyperbolic the Hill plot of the data was biphasic with negative cooperativity at low MgATP concentration, (ii) When thiophosphorylated gizzard myosin was used for kinetic analysis, the plateau region in the presence of MnATP was eliminated from the saturation curve and this curve became hyperbolic. However, in the presence of MgATP, although the plateau was almost eliminated, the saturation curve was still biphasic with either no or greatly reduced negative cooperativity of binding sites at low MgATP concentrations but positive cooperativity of binding at high MgATP concentrations. In addition, the thiophosphorylation of myosin also increased the Km and V of MgATP and MnATP, thus indicating weaker affinity for these substrates with thiophosphorylated myosin. (iii) Gizzard myosin also hydrolyzed other nucleotides (the order of rates being CTP = ITP > ATP = UTP > GTP), therefore saturation kinetics using different nucleotides as substrates was also carried out. The saturation curves with each nucleotide were different i.e., hyperbolic with CTP, sigmoid with GTP, hyperbolic with biphasic Hill plot with ITP, and possessing plateau with UTP. In addition, it was observed that the kinetic pattern with each nucleotide was very sensitive to temperature and pH. 相似文献
4.
Acid-soluble proteins were isolated from liver and spleen mitochondria and their ability to form complexes with DNA was investigated. According to electrophoresis data, acid-soluble proteins include about 20 polypeptides ranging in the molecular mass from 10 to 120 kDa. It was found that acid-soluble proteins form stable DNA-protein complexes at a physiological NaCl concentration. Different polypeptides possess different degrees of DNA affinity. There is no significant difference between DNA-binding proteins of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and nDNA. In the presence of 5 microg of DNA most polypeptides were bound to DNA, and further increase in DNA amount affected little the binding of proteins to DNA. There was no distinct difference in DNA-protein complex formation of liver mitochondrial acid-soluble proteins with nDNA or mtDNA. Also, it was detected that with these mitochondrial acid-soluble proteins, proteases that specifically cleave these proteins are associated. It was shown for the first time that these proteases are activated by DNA. DNA-binding proteins including DNA-activated mitochondrial proteases are likely to participate in the regulation of the structural organization and functional activity of mitochondrial DNA. 相似文献
5.
Wu Z Xing X Bohl CE Wisler JW Dalton JT Bell CE 《The Journal of biological chemistry》2006,281(35):25205-25214
beta protein from bacteriophage lambda promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. beta protein binds most tightly to an intermediate of annealing formed by the sequential addition of two complementary oligonucleotides. Here we have characterized the domain structure of beta protein in the presence and absence of DNA using limited proteolysis. Residues 1-130 form an N-terminal "core" domain that is resistant to proteases in the absence of DNA, residues 131-177 form a central region with enhanced resistance to proteases upon DNA complex formation, and the C-terminal residues 178-261 of beta protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of beta protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of beta protein are protected from biotinylation in the DNA complex, whereas none of the lysine residues in the C-terminal portion are protected. The results lead to a model for the domain structure and DNA binding of beta protein in which a stable N-terminal core and a more flexible central domain come together to bind DNA, whereas a C-terminal tail remains disordered. A fragment consisting of residues 1-177 of beta protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA. 相似文献
6.
The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies. 总被引:10,自引:2,他引:8
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The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction. 相似文献
7.
Purification and preliminary characterization of a macromolecular inhibitor of glucocorticoid receptor binding to DNA 总被引:1,自引:0,他引:1
Rat liver cytosol contains a heat-labile macromolecule that inhibits the binding of the transformed glucocorticoid-receptor complex to nuclei or DNA-cellulose (Milgrom, E., and Atger, M. (1975) J. Steroid Biochem. 6, 487-492; Simons, S. S., Jr., Martinez, H. M., Garcea, R. L., Baxter, J. D., and Tomkins, G. M. (1976) J. Biol. Chem. 251, 334-343. We have developed a quantitative assay for the inhibitor and have purified it 600-700-fold by ammonium sulfate precipitation, ethanol precipitation, and phosphocellulose and Sephacryl S-300 chromatography. The inhibitory activity copurifies with a Mr = 37,000 protein doublet. Under low salt conditions, both the inhibitory activity and the 37-kDa protein doublet behave as high Mr aggregates that subsequently dissociate in the presence of salt. The inhibitor is positively charged at physiological pH, and it is not affected by digestion with several serine proteases or RNase. The inhibitor does not affect the transformation process, and it does not cause the release of steroid-receptor complexes that have been prebound to DNA-cellulose. The inhibitor preparation does not cleave receptors in L-cell cytosol that are covalently labeled with the site-specific affinity steroid [3H]dexamethasone 21-mesylate. If the steroid-receptor complex is first separated from the great majority of cytosol protein by transforming it and binding it to DNA-cellulose, addition of the inhibitor preparation results in receptor cleavage. Under these conditions, cleavage can be blocked with 1-chloro-3-tosylamido-7-amino-L-2-heptanone and antipain, but protease inhibitors do not affect the inhibition of DNA binding that occurs in whole cytosol. The inhibitor acts through an interaction with the receptor, not with DNA. We suggest that the inhibitor may prove to be a useful tool for studying the interaction of the steroid-receptor complex with DNA or nuclei and speculate that it may be important in determining normal events of the receptor cycle as they occur in the intact cell. 相似文献
8.
A purified RAR/RXR-DeltaAB heterodimer was obtained by production of His-tagged RAR and untagged RXR in Escherichia coli, followed by combined purification on a Ni(2+) affinity column using excess RXR extract, and finally a gel filtration chromatography step to isolate a pure heterodimer. The purified heterodimer preparation bound 9-cisRA at a level of 0.85-0.95 mol of binding sites per mole of protein monomer. Titration of a 26 kDa fluorescent labeled fragment of the SRC-1 coactivator protein with the purified heterodimer in the presence of the agonist 9-cisRA yielded a binding affinity near 300 nM, whereas no binding was observed in the absence of agonist. Binding of the purified heterodimer to a DR5 target was identical in the absence of ligand and in the presence of 9-cisRA. Competition by unlabeled specific and nonspecific DNA allowed us to demonstrate that the binding curve was bimodal. The first phase of binding was highly specific and of high affinity. This phase also exhibited a high degree of cooperativity in the binding profile. Nonspecific DNA efficiently competes for the second phase. Thus, the first phase of binding likely corresponds to the formation of the specific heterodimer complex in which heterodimerization is energetically coupled to DNA binding. While agonist binding had no effect on the apparent affinity of the heterodimer for DR5, a series of antagonists significantly destabilized the heterodimer-DR5 complex, either through a direct decrease in the affinity of the protein for the DNA or through destabilization of the heterodimer itself. Impeding the interaction between the heterodimer and DNA appears as an additional mechanism of antagonist action of varying efficiency, depending upon the chemical structure of the antagonist. 相似文献
9.
S C Kowalczykowski 《Biochemistry》1986,25(20):5872-5881
The binding and cross-linking of the ATP photoaffinity analogue 8-azidoadenosine 5'-triphosphate (azido-ATP) with recA protein have been investigated, and through cross-linking inhibition studies, the binding of other nucleotide cofactors to recA protein has also been studied. The azido-ATP molecule was shown to be a good ATP analogue with regard to recA protein binding and enzymatic function by three criteria: first, the cross-linking follows a simple hyperbolic binding curve with a Kd of 4 microM and a cross-linking efficiency ranging from 10% to 70% depending on conditions; second, ATP, dATP, and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) specifically inhibit the cross-linking of azido-ATP to recA protein; third, azido-ATP is a substrate for recA protein ATPase activity. Quantitative analysis of the cross-linking inhibition studies using a variety of nucleotide cofactors as competitors has shown that the binding affinity of adenine-containing nucleotides for recA protein decreases in the following order: ATP-gamma-S greater than dATP greater than ATP greater than adenylyl beta,gamma-imidodiphosphate (AMP-PNP) much greater than adenylyl beta,gamma-methylenediphosphate (AMP-PCP) approximately adenine. Similar competition studies also showed that nearly all of the other nucleotide triphosphates also bind to recA protein, with the affinity decreasing in the following order: UTP greater than GTP approximately equal to dCTP greater than dGTP greater than CTP. In addition, studies performed in the presence of single-stranded DNA demonstrated that the affinity of ATP, dATP, ATP-gamma-S, and AMP-PNP for recA protein is significantly increased. These results are discussed in terms of the reciprocal effects that nucleotide cofactors have on the modulation of recA protein--single-stranded DNA binding affinity and vice versa. In addition, it is demonstrated that nucleotide and DNA binding are necessary though not sufficient conditions for ATPase activity. The significance of this result in terms of the possible requirement of critically sized clusters of 15 or more recA protein molecules contiguously bound to DNA for ATPase activity is discussed. 相似文献
10.
We have constructed a series of clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. All fragments exhibit DNA-dependent ATPase activity. Fragment 1-420 shows hyperbolic dependence of ATPase on DNA concentration, whereas fragment 1-453 shows hyperstimulation at low ratios of DNA to enzyme, a phenomenon found previously with the full-length enzyme. The minimum length of DNA found to stimulate the ATPase activity was approximately 10 bp; fragments >or=32 bp manifest the hyperstimulation phenomenon. Molecular mass studies show that fragment 1-453 is a monomer in the absence of nucleotides and a dimer in the presence of nucleotide triphosphate. The results are consistent with the role of the N-terminal domain of topoisomerase II as an ATP-operated clamp that dimerises in the presence of ATP. The hyperstimulation effect can be interpreted in terms of a "piggy-back binding" model for protein-DNA interaction. 相似文献
11.
A tof-like protein that has 434-specific DNA binding activity has been copurified with the 434 tof protein from lambda imm434cI dv carrier cells. The apparent molecular weight of the new 434-specific DNA binding protein is 9,000 to 9,500, a little higher than that of the 434 tof protein, as estimated by SDS gel electrophoresis. Amino acid analysis revealed the protein to be an arginine-rich component whereas the 434 tof protein is a lysine-rich component. The specific binding reaction of the new protein to lambda imm434dv DNA is distinct from that of the 434 tof protein in respect to the sigmoid shape of the binding curve and to the temperature dependency. This suggests that the specific binding to lambda imm434dv DNA observed with the new protein is due not to a trace of the 434 tof protein contaminating the new protein preparation but rather to the new protein itself. The NH2-terminal 11 residues of the new 434-specific DNA binding protein were sequenced by manual Edman degradation. This technique revealed that the new protein is not a fragment of the 434 tof, cII, or O protein or an NH2-terminal fragment of the cI repressor. The origin and the physiological roles of the new 434-specific DNA binding protein remain unknown. 相似文献
12.
Partial purification and properties of an exonuclease inhibitor induced by bacteriophage Mu-1. 总被引:7,自引:0,他引:7
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From an induced lysogen of bacteriophage Mu-1, we partially purified a substance of high molecular weight that blocks the action of several exonucleases on double-stranded DNA. The presence of the inhibitor in cell-free extracts is dependent on induction of a Mu prophage. The Mu-related inhibitor acts by binding to double-stranded DNA rather than by interacting with the DNase. The inhibitor protects linear duplex DNA of Mu, P22, and phi X174am3 from exonucleolytic degradation by recBC DNase and lambda exonuclease. Single-stranded DNA, however, is not protected by the inhibitor from degradation by either recBC DNase or exonuclease I. The inhibitor preparation contains a protein that binds to linear duplex DNA, but not to circular duplex DNA; ends are required for binding to occur. Single-stranded DNA is not a substrate for the binding protein. These and other results suggest that the binding protein and the inhibitor are the same activity. 相似文献
13.
DNA and RNA binding by the mitochondrial lon protease is regulated by nucleotide and protein substrate 总被引:8,自引:0,他引:8
Liu T Lu B Lee I Ondrovicová G Kutejová E Suzuki CK 《The Journal of biological chemistry》2004,279(14):13902-13910
The ATP-dependent Lon protease belongs to a unique group of proteases that bind DNA. Eukaryotic Lon is a homo-oligomeric ring-shaped complex localized to the mitochondrial matrix. In vitro, human Lon binds specifically to a single-stranded GT-rich DNA sequence overlapping the light strand promoter of human mitochondrial DNA (mtDNA). We demonstrate that Lon binds GT-rich DNA sequences found throughout the heavy strand of mtDNA and that it also interacts specifically with GU-rich RNA. ATP inhibits the binding of Lon to DNA or RNA, whereas the presence of protein substrate increases the DNA binding affinity of Lon 3.5-fold. We show that nucleotide inhibition and protein substrate stimulation coordinately regulate DNA binding. In contrast to the wild type enzyme, a Lon mutant lacking both ATPase and protease activity binds nucleic acid; however, protein substrate fails to stimulate binding. These results suggest that conformational changes in the Lon holoenzyme induced by nucleotide and protein substrate modulate the binding affinity for single-stranded mtDNA and RNA in vivo. Co-immunoprecipitation experiments show that Lon interacts with mtDNA polymerase gamma and the Twinkle helicase, which are components of mitochondrial nucleoids. Taken together, these results suggest that Lon participates directly in the metabolism of mtDNA. 相似文献
14.
Treatment of T7 DNA ligase with a range of proteases generates two major fragments which are resistant to further digestion. These fragments, of molecular weight 16 and 26 kDa, are derived from the N- and C-termini of the protein, respectively. The presence of ATP or a non-hydrolysable analogue, ADPNP, during limited proteolysis greatly reduces the level of digestion. The N-terminal 16 kDa region of the intact T7 ligase is labelled selectively in the presence of [alpha-32P]ATP, confirming that it contains the active site lysine residue. In common with the intact enzyme, the C-terminal portion of the protein retains the ability to band shift DNA fragments of various lengths, implicating it in DNA binding. It can also inhibit ligation by the intact protein, apparently by competing for target sites on DNA. We conclude that the N-terminal region, which contains the putative active site lysine, plays a role in the transfer of AMP from the enzyme-adenylate complex to the 5'phosphate at the nick site, while the C-terminal 26 kDa fragment appears to position the enzyme at the target site on DNA. 相似文献
15.
It has been known for some time that ATP inhibits the DNA relaxation activity of human DNA topoisomerase I. However, the underlying mechanism of this inhibitory effect remains largely unknown. Using filter binding assays, the binding of human DNA topoisomerase I to DNA was decreased in the presence of ATP. This result suggests that the inhibition of DNA relaxation activity of human DNA topoisomerase I by ATP is at the binding step rather than at the nicking or resealing step. DNA topoisomerase I cleavage assay further supports this notion. ATP-agarose binding and UV cross-linking assays also demonstrate that ATP directly and specifically binds human DNA topoisomerase I. To address whether the ATP binding results in conformational changes in human DNA topoisomerase I, various proteases were employed for detecting potential protein conformational changes. Our results indicated that the proteolytic susceptibilities of trypsin and chymotrypsin were altered in the presence of ATP. The result suggests that the conformation of human DNA topoisomerase I was altered upon ATP binding. In addition, the binding between ATP and human DNA topoisomerase I was also reduced by increasing concentrations of DNA. Our data suggests that human DNA topoisomerase I exhibits at least two incompatible conformations. One conformation is in the form of a topoisomerase I-ATP complex, which inhibits DNA relaxation activity of human DNA topoisomerase I, and the other, a topoisomerase I-DNA complex, which exerts DNA relaxation activity. Our studies identify the role of ATP in the regulation of human DNA topoisomerase I and provide a substantial implication of how human DNA topoisomerase I compromises its versatile functions. 相似文献
16.
—The binding of [14C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [14C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [14C]GABA/μg of protein, with an apparent Kd= 30 μm . The binding is competitively inhibited by bicuculline with a Ki= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle. 相似文献
17.
18.
We used a spin-labeled ATP analog, SL-ATP, to study nucleotide binding to highly purified human multidrug resistance protein
3, MRP3, which had been expressed in the yeast Pichia pastoris. SL-ATP was shown to be a good substrate analog and is hydrolyzed by MRP3 at about 10% of the Vmax for normal ATP. ESR titrations
showed that 2 mol of SL-ATP readily bound per mole of MRP3 with a dissociation constant of about 100 μM in the presence of
Mg2+ ions. The binding curve was easily fitted for a hyperbolic binding relationship. SL-ATP also bound readily to MRP3 in the
absence of divalent ions and presence of EDTA. The resulting binding curve, however, could not be satisfactorily fitted using
the equation for hyperbola. Analysis showed that a good fit was only obtained with the Hill equation using a Hill coefficient
of 4 or close to 4. Lower Hill coefficients resulted in lower goodness of the fit. Such cooperative binding may be explained
by a dimerization event triggered in the absence of divalent ions and a close communication of nucleotide binding sites of
the interacting dimers. These findings may be of great importance for the overall mechanism and regulation of multidrug resistance
proteins. 相似文献
19.
20.
Conditions are reported under which ATP protects membrane-bound coupling factor 1 against sodium bromide inactivation. The presence of Mg2+ was found to be obligatory for this protection. ADP and GTP also protected the enzyme against salt inactivation but to a much smaller extent. Other nucleotides tested were ineffective. At low ATP concentrations ADP prevented the effect of ATP and modified the saturation curve for ATP from hyperbolic to sigmoidal. Treatment of chloroplasts with 0.4 M MgCl2 or 2 M LiCl resulted in inactivation of photophosphorylation. In contrast to NaBr-depleted particles the MgCl2 or LiCl-depleted chloroplasts can be reconstituted by purified coupling factor 1. A binding site for Mg2+ and two different sites for ATP upon the coupling factor 1 are suggested to explain the mechanism of their protection against salt inactivation. 相似文献