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At least 973 records · Page 54Linked to original sources
PubMed1999

Quantitative autoradiographic studies of relaxin binding in rat atria, uterus and cerebral cortex: characterization and effects of oestrogen treatment.

The binding characteristics of the relaxin receptor in rat atria, uterus and cortex were studied using a [33P]-labelled human gene 2 relaxin (B33) and quantitative receptor autoradiography. The binding kinetics of [33P]-human gene 2 relaxin (B33) were investigated in slide-mounted rat atrial sections. The binding achieved equilibrium after 60 min incubation at room temperature (23+/-1 degrees C) and dissociated slowly. The association and dissociation rate constants were 4.31+/-0.34x10(8) M(-1) x min(-1) and 1.55+/-0.38x10(-3) min(-1) respectively. Thus, the kinetic dissociation constant was 3.46+/-0.59 pM. Binding was saturable to a single population of non-interacting sites throughout atria, in uterine myometrium and the 5th layer of cerebral cortex. The binding affinities (pK(D)) of [33P]-human gene 2 relaxin (B33) were 8.92+/-0.09 in atrial myocardium and 8.79+/-0.04 in cerebral cortex of male rats, and 8.79+/-0.10 in uterine myometrium. Receptor densities in the cerebral cortex and atria were higher than in uterine myometrium, indicating that relaxin also has important roles in non-reproductive tissues. In male rats, treatment with 17beta-oestradiol (20 microg in 0.1 ml sesame oil s.c., 18-24 h) significantly decreased the density of relaxin receptors in atria and cerebral cortex. Identical treatment in female rats had no significant effect in atria and cerebral cortex, but it significantly increased the density of relaxin receptors in uterine myometrium. Relaxin binding was competitively displaced by porcine and rat native relaxins. Porcine native relaxin binds to the relaxin receptor in male rat atria (8.90+/-0.02), and cerebral cortex (8.90+/-0.03) and uterine myometrium (8.89+/-0.03) with affinities not significantly different from human gene 2 (B33) relaxin. Nevertheless, rat relaxin binds to the receptors with affinities (8.35+/-0.09 in atria, 8.22+/-0.07 in cerebral cortex and 8.48+/-0.06 in uterine myometrium) significantly less than human gene 2 (B33) and porcine relaxins. Quantitative receptor autoradiography is the method of choice for measurement of affinities and densities of relaxin receptor in atria, uterine myometrium and cerebral cortex. High densities were found in all these tissues. 17beta-oestradiol treatment produced complex effects where it increased the densities of relaxin receptors in uterus but decreased those in atria and cerebral cortex of the male rats, and had no effect on the atria and cerebral cortex of the female rats.

Animals↗
PubMed1998

Interaction of human plasma membrane proteins and oligodeoxynucleotides.

Two oligodeoxynucleotide (oligodN) binding proteins of 100-110 kDa on plasma membranes of human cell lines were recently identified by us. These two proteins seemed to play a role in oligodN uptake. In this study, the impact of the chain length and the sequence of the oligodN on the interaction with those two proteins was investigated. Chain length of oligodN was an important determinant, but not the sole determinant for the interaction. Binding affinity of oligodNs was determined predominantly by base composition, where pyrimidine bases but not purine bases were required in the sequence to retain high affinity. The binding kinetics of the homopolymers of deoxycytidine (dC21) and deoxythymidine (dT21) suggests that the proteins may have different binding sites, with one site preferring thymine bases and the other cytosine bases. Moreover, some additional plasma membrane proteins were identified, with an apparent molecular mass ranging from 40 to 58 kDa, which could bind thymine bases but not cytosine bases.

Base Composition↗
PubMed1992

Characterization of receptors for substance P in human astrocytoma cells: radioligand binding and inositol phosphate formation.

UC11 cells, derived from a human astrocytoma, have a high density of functional substance P receptors. Radioligand binding studies were conducted with the highly selective neurokinin-1 receptor ligand [3H][Sar9,Met(O2)11]-substance P. Kinetic binding experiments conducted at 4 degrees C yielded an association rate constant k1 of 1.86 x 10(7) M-1 min-1, a dissociation rate constant k-1 of 0.00478 min-1, and a calculated kinetic KD of 257 pM. Saturation binding experiments yielded average values of KD = 447 +/- 103 pM, Bmax = 862 +/- 93 fmol/mg of protein. This Bmax corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar9,Met(O2)11]-substance P yielded average values of KD = 491 +/- 48 pM and Bmax = 912 +/- 67 fmol/mg of protein. In [3H]inositol-labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20-fold within approximately 15 s of addition of substance P. This inositol trisphosphate formation was transient and had returned to baseline within the first 60-120 s. Inositol monophosphate formation, however, was linear for at least 2 h. Structure activity data on binding and inositol monophosphate formation confirmed the presence of a neurokinin-1 receptor subtype in these cells. Thus, the UC11 cell should be a useful model cell for delineating the physiological role of substance P receptors in astrocytes.

Astrocytoma↗
PubMed1999

Characterization of insulin-like growth factor binding protein-3 (IGFBP-3) binding to human breast cancer cells: kinetics of IGFBP-3 binding and identification of receptor binding domain on the IGFBP-3 molecule.

Insulin-like growth factor binding protein-3 (IGFBP-3) binds to specific membrane proteins located on human breast cancer cells, which may be responsible for mediating the IGF-independent growth inhibitory effects of IGFBP-3. In this study, we evaluated IGFBP-3 binding sites on breast cancer cell membranes by competitive binding studies with IGFBP-1 through -6 and various forms of IGFBP-3, including synthetic IGFBP-3 fragments. Scatchard analysis revealed the existence of high-affinity sites for IGFBP-3 in estrogen receptor-negative Hs578T human breast cancer cells (dissociation constant (Kd) = 8.19 +/- 0.97 x 10(-9) M and 4.92 +/- 1.51 x 10(5) binding sites/cell) and 30-fold fewer receptors in estrogen receptor-positive MCF-7 cells (Kd = 8.49 +/- 0.78 x 10(-9) M and 1.72 +/- 0.31 x 10(4) binding sites/cell), using a one-site model. These data demonstrate binding characteristics of typical receptor-ligand interactions, strongly suggesting an IGFBP-3:IGFBP-3 receptor interaction. Among IGFBPs, only IGFBP-5 showed weak competition, indicating that IGFBP-3 binding to breast cancer cell surfaces is specific and cannot be attributed to nonspecific interaction with glycosaminoglycans. This was confirmed by showing that synthetic IGFBP-3 peptides containing IGFBP-3 glycosaminoglycan-binding domains competed only weakly for IGFBP-3 binding to the cell surface. Rat IGFBP-3 was 20-fold less potent in its ability to compete with human IGFBP-3(Echerichia coli), as well as 10- to 20-fold less potent for cell growth inhibition than human IGFBP-3, suggesting the existence of species specificity in the interaction between IGFBP-3 and the IGFBP-3 receptor. When various IGFBP-3 fragments were evaluated for affinity for the IGFBP-3 receptor, only those fragments that contain the midregion of the IGFBP-3 molecule were able to inhibit 125I-IGFBP-3(Escherichia coli) binding, indicating that the midregion of the IGFBP-3 molecule is responsible for binding to its receptor. These observations demonstrate that specific, high-affinity IGFBP-3 receptors are located on breast cancer cell membranes. These receptors have properties that support the notion that they may mediate the IGF-independent inhibitory actions of IGFBP-3 in breast cancer cells.

Amino Acid Sequence↗
PubMed1995

Kinetic and functional mapping of viral epitopes using biosensor technology.

Some monoclonal antibodies (Mabs) that react with the extremity of the tobacco mosaic virus (TMV) particle containing the 5' end of the RNA are able to block the disassembly of TMV by ribosomes while others are totally devoid of such activity. No correlation could be established between the binding kinetics and affinity of the Mabs and their inhibitory capacity. An epitope map of the Mab binding sites was constructed on the basis of kinetic two-site binding assays with the viral monomeric protein (TMVP) performed using biosensor technology (BlAcore). Mabs possessing inhibitory activity were found to bind to the part of the TMVP surface closest to the central axis in the polymerized particle. As this part of the subunit is known to interact with the viral RNA, it seems that inhibitory Mabs act by sterically preventing the interaction between virus and ribosomes. This study illustrates the advantages of the biosensor technology for locating conformational epitopes in viral proteins.

Antibodies, Monoclonal↗
PubMed1997

Theoretical analysis of protein concentration determination using biosensor technology under conditions of partial mass transport limitation.

The theory of a method for determining active concentration of nonpurified protein samples using the BIAcore biosensor technology has been developed. The method relies on change in binding rate with varying flow rate at high ligand concentration where mass transport from bulk to surface with immobilized ligand becomes partially rate limiting. Prior study of binding kinetics is not necessary. If the molecular weight and the diffusion coefficient of the analyte protein are known, no standard with a known concentration is required. From numeric computer simulations a simple analytical expression for the mass transport coefficient derived at totally mass transport limiting conditions is shown to be applicable for conditions of partial mass transport limitation. Simple one to one association is assumed in deriving the method, but it is applicable even with more complex kinetics due to a bivalent analyte or heterogeneity of analyte or ligand.

Algorithms↗
PubMed1996

[3H]dofetilide binding: biological models that manifest solely the high or the low affinity binding site.

Dofetilide is a Class III antiarrhythmic agent known to selectively block the rapid component of the delayed rectifier K+ current (IKr). [3H]Dofetilide binds to a low and a high affinity sites on guinea-pig myocytes. The purposes of this study were: (1) to find biological models which express solely the high or the low [3H]dofetilide binding sites; (2) to characterize the single binding site models; and (3) to establish which of the high or the low affinity binding sites is associated with IKr. We compared and characterized the [3H]dofetilide binding on guinea-pig myocytes, neonatal mouse ventricular homogenate and untransfected CHO cells. These tissue preparations were selected since the neonatal mouse tissue expresses IKr while this current is absent from CHO cells. We compared the IC50 concentrations of dofetilide and two other known IKr blockers E-4031 and sotalol, on [3H]dofetilide binding to these three preparations. Using steady-state and kinetic binding techniques, we characterized the interaction of E-4031 and sotalol with the high and the low [3H]dofetilide binding sites. We found that neonatal mouse ventricle manifest solely the high affinity site (Kd 20 +/- 4 nmol/l, Bmax 18 +/- 4 fmol/mg) while CHO cells manifest solely the low affinity binding site (Kd 1.6 +/- 0.1 mumol/l, Bmax 5.8 +/- 0.8 pmol/mg). We demonstrated that the high and low affinity binding sites present on guinea-pig myocytes show characteristics similar to the single high affinity site expressed on neonatal mouse homogenate and to the single low affinity site expressed on CHO cells, respectively. Class III antiarrhythmic drugs inhibited binding to the high affinity site at concentrations similar to those required to inhibit 50% of IKr current in electrophysiologic studies. In contrast, dofetilide and E-4031 inhibited [3H]dofetilide binding to the low affinity site only at supra-pharmacologic concentrations. We next demonstrated that Class III drugs interact in a competitive manner with the high affinity site on neonatal mouse tissue while they interact with a site allosterically coupled to the low binding site on CHO cells. These data suggest that dofetilide interacts with the high and low affinity sites in a fundamentally different manner. We defined biological models which express solely the high or low [3H]dofetilide binding sites. Only the high affinity site is related to IKr.

Animals↗
PubMed2004

Ellipsometric approach for the real-time detection of label-free protein adsorption by second harmonic generation.

Second harmonic generation (SHG) was performed using a novel ellipsometric detection approach to selectively probe the real-time surface binding kinetics of an unlabeled protein. The coherence of nonlinear optical processes introduces new possibilities for exploiting polarization that are unavailable with incoherent methods, such as absorbance and fluorescence. Adsorption of bovine serum albumin (BSA) at silica/aqueous solution interfaces resulted in changes in the polarization state of the frequency-doubled light through weak, dynamic interactions with a coadsorbed nonlinear optical probe molecule (rhodamine 6G). Using a remarkably simple instrumental approach, signals arising exclusively from surface interactions with BSA were spatially isolated and selectively detected with high signal-to-noise. The relative intensities acquired during the kinetics experiments using both circularly and linearly polarized incident beams were in excellent agreement with the responses predicted from SHG ellipsometry polarization measurements. Analysis of the polarization-dependent SHG generated during BSA adsorption at glass/aqueous solution interfaces provided direct evidence for slow conformational changes within the protein layer after adsorption, consistent with protein denaturation. This polarization selection approach is sufficiently general to be easily extended to virtually all coherent nonlinear optical processes and a variety of different surface interactions and architectures.

Adsorption↗
PubMed2005

Glycodelin-S in human seminal plasma reduces cholesterol efflux and inhibits capacitation of spermatozoa.

Tight control of sperm capacitation is important for successful fertilization. Glycodelin-S is one of the most abundant glycoproteins in the human seminal plasma. However, its function is unclear. We investigated the role of glycodelin-S on capacitation of human spermatozoa. Binding kinetics experiments demonstrated the presence of two saturable and reversible binding sites of glycodelin-S on human spermatozoa. Differently glycosylated other isoforms of glycodelin, glycodelin-A and -F, did not compete with glycodelin-S for these binding sites, suggesting that the glycodelin-S binding sites are different from those of the other isoforms. Indirect immunofluorescent staining revealed specific binding of glycodelin-S around the sperm head. This immunoreactivity was greatly reduced in spermatozoa that had migrated through the cervical mucus surrogates. Glycodelin-S at physiological concentrations significantly reduced the bovine serum albumin and cyclodextrin-induced cholesterol efflux and down-regulated the adenylyl cyclase/protein kinase A/tyrosine kinase signaling pathway, resulting in suppression of capacitation. Deglycosylation abolished glycodelin-S binding and the effect of glycodelin-S on bovine serum albumin-induced capacitation. This indicates that the carbohydrate moiety of glycodelin-S is critical for the function of the molecule. It is concluded that glycodelin-S in seminal plasma maintains the uncapacitated state of human spermatozoa.

Acrosome↗
PubMed2000

Cytokines and endotoxin induce cytokine receptors in skeletal muscle.

Proinflammatory cytokines are important factors in the regulation of diverse aspects of skeletal muscle function; however, the muscle cytokine receptors mediating these functions are uncharacterized. Binding kinetics (dissociation constant = 39+/-4.7 x 10(-9) M, maximal binding = 3.5+/-0.23 x 10(-12) mol/mg membrane protein) of muscle tumor necrosis factor (TNF) receptors were obtained. Skeletal muscle was found to express mRNAs encoding interleukin-1 type I and II receptors, interleukin-6 receptor (IL-6R), and interferon-gamma receptor by RT-PCR, but these receptors were below limits of detection of ligand-binding assay (> or =1 fmol binding sites/mg protein). Twenty-four hours after intraperitoneal administration of endotoxin to rats, TNF receptor type II (TNFRII) and IL-6R mRNA were increased in skeletal muscle (P<0.05). In cultured L6 cells, the expression of mRNA encoding TNFRII and IL-6R receptors was induced by TNF-alpha, and all six cytokine receptor mRNA were induced by a mixture of TNF-alpha, IFN-gamma, and endotoxin (P<0.05). This suggests that the low level of cytokine receptor expression is complemented by a capacity for receptor induction, providing a clear mechanism for amplification of cytokine responses at the muscle level.

Animals↗
PubMed1990

Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody.

Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.

Amino Acid Sequence↗
PubMed1994

Differential expression and ligand-induced modulation of the human interleukin-6 receptor on interleukin-6-responsive cells.

The human interleukin-6 receptor (IL-6R) was differentially expressed on IL-6-dependent (U266 and SKO-007) and -independent (RPMI8226) myeloma cells as well as melanoma cells (A375-C6) that are growth-inhibited by IL-6. U266 and SKO-007 cells expressed four distinct IL-6R complexes (molecular masses of 100, 120, 145, and 165 kDa) as revealed by affinity cross-linking of iodinated IL-6. RPMI8226 and A375-C6 cells primarily expressed the 165-kDa complex relative to the others. Immunoprecipitation and antibody competition studies showed that the 100- and 120-kDa complexes contained the gp80 subunit, whereas the 145- and 165-kDa complexes contained the gp130 subunit of the IL-6R. Assaying solubilized U266 plasma membrane proteins by affinity cross-linking or ligand blotting revealed that only gp80 bound IL-6 specifically. Induction of an IL-6 response was associated with ligand-induced down-regulation of gp130 and was inhibited by neutralizing anti-IL-6 antibodies. Furthermore, the relative ratios of gp80 to gp130 determined the binding kinetics of the IL-6R, yielding high- and low-affinity binding sites by Scatchard plots. Our data imply that distinct IL-6 bioactivities are based upon the differential expression and regulation by IL-6 of its ligand-binding (gp80) and signal-transducing (gp130) receptor subunits.

Binding, Competitive↗
PubMed1996

Kinetics of the interaction between DNA and the type IC restriction enzyme EcoR124II.

Optical waveguide mode spectroscopy was used to determine the binding constants characterizing the interaction of EcoR124II, a type IC restriction modification enzyme from Salmonella typhimurium, with DNA. The DNA is immobilized on the surface of an optical waveguide, and the enzyme is introduced in bulk solution flowing over the DNA under controlled hydrodynamic conditions. The binding kinetics of the protein to the DNA can be directly observed and the number of bound protein molecules per base pair determined to a high accuracy. Dissociation of the protein was measured by switching flowing protein to protein-free buffer. Binding to two different kinds of DNA, with and without the specific sequence recognized by EcoR124II, was investigated. Protein binding and dissociation ("nonspecific" binding), quantified by association and dissociation rate coefficients ka and kd, were the same for both types, but the DNA carrying the recognition site showed an additional process, "irreversible" association (i.e. dissociation was not observed on the time scale of the experiments) of the protein, quantified by a rate coefficient ks. Some inferences regarding the mechanism of base pair searching are made from the measured ka, kd, and ks values.

Base Composition↗
PubMed1985

Receptor dynamics of closely related ligands: "fast' and "slow' interferons.

Two related human alpha interferons with 83% homology in their primary sequences show a similar specific activity on nonhuman cells, but a striking difference on human cells, on which alpha-1 shows 1-5% of the specific molar activity displayed by alpha-2. Both interferons were labelled with 125I, and their binding kinetics followed on growing cultures of the human Burkitt line Daudi. Binding of alpha-1 showed slower rates of association and faster rates of dissociation implying that differences in apparent binding affinity were responsible for the differences in specific molar activity. However, binding was shown to reach steady-state rather than an equilibrium, so differences in the dynamics of the ligand-receptor complexes may represent amplification of differences in the initial binding constant. alpha-2, but not alpha-1, induces a marked loss of binding sites leading to a high affinity steady-state binding. Inhibition of cell multiplication by both interferons depends on a continued stimulation by free ligands at steady-state. It is proposed that the differences in specific molar activity are, in the main, kinetic and cause alpha-1 and alpha-2 to behave respectively as "slow' and "fast' interferons.

Animals↗
PubMed2005

Characterisation and internalisation of recombinant humanised HMFG-1 antibodies against MUC1.

The humanised HMFG-1 immunoglobulin has been extensively developed as a clinical immunotherapeutic agent for MUC1 expressing tumours. We have constructed a single-chain Fv (scFv) and Fab fragment from this antibody and shown that both these species retain their specificity for MUC1. The scFv was less stable and less soluble than the Fab. Detailed analyses of the binding kinetics of the whole IgG and Fab fragment show that the affinity for MUC1 synthetic peptides is low (approximately 100 nM for the IgG and 10 muM for the Fab), with particularly low but similar dissociation rate constants (0.031-0.095 s(-1)). Binding to native antigen on the cell surface is over two orders of magnitude better. Confocal immunofluorescence microscopy shows that both the IgG and Fab are internalised rapidly (the IgG is internalised within 15 min) and colocalise to early endosomes. This work provides an appreciation of the binding, internalising and trafficking kinetics, important for the development of future therapeutics based on this antibody.

Antibodies, Monoclonal↗
PubMed2004

Selectivity, cooperativity, and reciprocity in the interactions between the delta-opioid receptor, its ligands, and G-proteins.

A better understanding of signal transduction mechanisms is of critical importance. Methodologies that allow studies to be done while receptors are incorporated into lipid bilayers are advantageous. One such technique is plasmon-waveguide resonance (PWR) spectroscopy, which can follow changes in conformation accompanying protein-ligand, protein-protein, and protein-lipid interactions occurring in G-protein-coupled receptors in real time with high sensitivity and without the need for molecular labeling. Here we investigated several aspects of human delta-opioid receptor (hDOR)-G-protein interactions: 1) the effect of different types of agonists on the interaction with individual G-protein subtypes; 2) the affinities of the separate G-protein alpha and betagamma subunits to different ligand-occupied states of the receptor; and 3) the effect of the presence of the G-protein on the interactions of the ligand with the receptor. To accomplish this we have incorporated the receptor into a solid supported lipid bilayer in the presence of ligand or G-protein and monitored the PWR spectral changes induced by the reciprocal G-protein or ligand interactions. We found a high degree of selectivity in the interactions of different agonist-bound states of the receptor with the different G-protein subtypes. This has important implications for agonist-directed trafficking and selective drug design. Studies with the separated alpha and betagamma subunits show that cooperativity exists in these interactions. The high affinities of the separated subunits to the receptor point to the possibility of independent promotion of specific signaling events. The presence of G-proteins increased the affinity of agonists to the hDOR, and caused faster binding kinetics and different ligand-induced conformational changes. Because ligand also influences G-protein binding, reciprocity exists between these two binding processes.

Animals↗
PubMed2002

Detection of a very rapid first phase in complex formation of DnaK and peptide substrate.

Complex formation of the Hsp70 chaperone DnaK with the fluorescence-labeled peptide ALLLSAPRR shows a very rapid first phase that has as yet not been observed with other peptides. This first phase is completed within the dead time (1-2 ms) of the stopped-flow instrument and corresponds to two thirds of the total increase in fluorescence. It occurs both in the presence and in the absence of ATP and is followed by a fast, a slow and a very slow step. These binding kinetics that are vastly different from those observed with other peptides might indicate the existence of a second substrate-binding site of DnaK.

Adenosine Triphosphate↗
PubMed1992

Minimal kinetic mechanism for misincorporation by DNA polymerase I (Klenow fragment).

The minimal kinetic mechanism for misincorporation of a single nucleotide (dATP) into a short DNA primer/template (9/20-mer) by the Klenow fragment of DNA polymerase I [KF(exo+)] has been previously published [Kuchta, R. D., Benkovic, P., & Benkovic, S.J. (1988) Biochemistry 27, 6716-6725]. In this paper are presented refinements to this mechanism. Pre-steady-state measurements of correct nucleotide incorporation (dTTP) in the presence of a single incorrect nucleotide (dATP) with excess KF-(exo+) demonstrated that dATP binds to the KF(exo+)-9/20-mer complex in two steps preceding chemistry. Substitution of (alpha S)dATP for dATP yielded identical two-step binding kinetics, removing nucleotide binding as a cause of the elemental effect on the rate of misincorporation. Pyrophosphate release from the ternary species [KF'(exo+)-9A/20-mer-PPi] was found to occur following a rate-limiting conformational change, with this species partitioning equally to either nucleotide via internal pyrophosphorolysis or to misincorporated product. The rate of 9A/20-mer dissociation from the central ternary complex (KF'-9A/20-mer-PPi) was shown to be negligible relative to exonucleolytic editing. Pyrophosphorolysis of the misincorporated DNA product (9A/20-mer), in conjunction with measurement of the rate of dATP misincorporation, permitted determination of the overall equilibrium constant for dATP misincorporation and provided a value similar to that measured for correct incorporation. A step by step comparison of the polymerization catalyzed by the Klenow fragment for correct and incorrect nucleotide incorporation emphasizes that the major source of the enzyme's replicative fidelity arises from discrimination in the actual chemical step and from increased exonuclease activity on the ternary misincorporated product complex owing to its slower passage through the turnover sequence.

Base Sequence↗