Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “binding kinetics”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources
PubMed2006

Slow ligand binding kinetics dominate ferrous hexacoordinate hemoglobin reactivities and reveal differences between plants and other species.

Hexacoordinate hemoglobins are found in many living organisms ranging from prokaryotes to plants and animals. They are named "hexacoordinate" because of reversible coordination of the heme iron by a histidine side chain located in the heme pocket. This endogenous coordination competes with exogenous ligand binding and causes multiphasic relaxation time courses following rapid mixing or flash photolysis experiments. Previous rapid mixing studies have assumed a steady-state relationship between hexacoordination and exogenous ligand binding that does not correlate with observed time courses for binding. Here, we demonstrate that this assumption is not valid for some hexacoordinate hemoglobins, and that multiphasic time courses are due to an appreciable fraction of pentacoordinate heme resulting from relatively small equilibrium constants for hexacoordination (K(H)). CO binding reactions initiated by rapid mixing are measured for four plant hexacoordinate hemoglobins, human neuroglobin and cytoglobin, and Synechocystis hemoglobin. The plant proteins, while showing a surprising degree of variability, differ from the others in having much lower values of K(H). Neuroglobin and cytoglobin display dramatic biphasic time courses for CO binding that have not been observed using other techniques. Finally, an independent spectroscopic quantification of K(H) is presented that complements rapid mixing for the investigation of hexacoordination. These results demonstrate that hexacoordination could play a much larger role in regulating affinity constants for ligand binding in human neuroglobin and cytoglobin than in the plant hexacoordinate hemoglobins.

Binding Sites↗
PubMed1987

Different binding kinetics of Serratia 56K protease with plasma alpha 2-macroglobulin and chicken egg white ovomacroglobulin.

We recently reported that Serratia 56K protease is inhibited by plasma alpha 2 macroglobulin (alpha 2M) temporarily and by chicken egg white ovomacroglobulin (ovoM) continuously (Molla, A. et al. (1986) Infect. Immun. 53, 522-529). The inhibition of this protease is almost complete with ovoM whereas it is incomplete with alpha 2M, although these two macroglobulins show homology and many similarities. In the present study we determined the apparent numbers of binding sites and binding constants for the two macroglobulins by means of the fluorescence polarization method using FITC-labeled 56K protease. The time courses of complex formation of 56K protease with alpha 2M and ovoM were different; with ovoM it was complete within 5 min while with alpha 2M 150 min was required. Their apparent molecular volumes were also different; the fluorescence polarization value of the E/I complex was 18.7% larger with ovoM than with alpha 2M. The association constants obtained on Scatchard plot analysis with 56K protease and alpha 2M or ovoM were 0.33 X 10(7) M-1 and 1.09 X 10(7) M-1, respectively. One molecule of each of these macroglobulins binds 1.13 and 1.35 molecules of 56K protease, respectively. Upon E/I complex formation, an increase in amino groups due to proteolysis was noted in both cases, but more progressive proteolysis was observed in the case of alpha 2M. Furthermore, when the 56K protease was inactivated through the depletion of Zn atoms, complex formation did not occur.

Amino Acids↗
PubMed1981

Studies on toxicity and binding kinetics of abrin in normal and Epstein Barr virus-transformed lymphocyte culture-I: experimental results - 2.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Studies of activation and stimulation indices, as well as two new indices; the metabolic self and cross-coupling indices, lead to the prediction that there are three morphologically distinct subpopulations of EBV-transformed lymphocytes with different abrin binding site numbers. This conclusion is supported by SEM morphological differences.

Abrin↗
PubMed1995

Altered muscle calcium channel binding kinetics in autoimmune motoneuron disease.

While skeletal muscle is not apparently affected directly in amyotrophic lateral sclerosis (ALS), immunoglobulin G fractions purified from patients with ALS (ALS IgG) bind dihydropyridine (DHP)-sensitive L-type voltage-gated calcium channel (VGCC) antigen isolated from skeletal muscle in ELISA and Western immunoblot, and alter VGCC function in vitro. To determine whether muscle VGCC properties are altered in ALS, VGCC-enriched subsarcolemmal membrane fractions were prepared from biopsied quadriceps muscle of patients with ALS, with other neurologic diseases, or without apparent muscle disease, and tested for DHP binding with [3H]PN200-110. ALS muscle VGCCs possessed eightfold higher binding affinities for [3H]PN200-110 than did VGCCs from muscle fractions of most other patients, independent of denervation-induced increases in DHP binding site number. Similarly elevated DHP binding affinities were observed in specimens from patients with autoimmune motor neuropathies, suggesting that ALS and immune mediated motoneuron disease share skeletal muscle L-type VGCC alterations.

Amyotrophic Lateral Sclerosis↗
PubMed2000

Imaging serotonin and dopamine transporters with 123I-beta-CIT SPECT: binding kinetics and effects of normal aging.

UNLABELLED: [123I]beta-carbomethoxy-3-beta-(4-iodophenyl)-tropane (CIT) is a useful ligand for dopamine transporters (DATs) and serotonin transporters (5-HTTs). Previous SPECT studies have shown a state of sustained equilibrium in the striatum on day 2 after injection that allows quantification of striatal DATs using a simple ratio of specific-to-nondisplaceable binding. The aim of this study was to investigate the kinetics of [123I]beta-CIT uptake in the thalamus, hypothalamus, and midbrain, areas known to contain 5-HTTs in high densities. METHODS: SPECT with a triple-head camera was performed on 16 healthy volunteers (13 women, 3 men; mean age [+/-SD], 32 +/- 11 y) after intravenous bolus injection of 130 +/- 20 MBq (3.5 +/- 0.5 mCi) [123I]beta-CIT. Two individuals were scanned 1, 2, 4, 7, 10, 13, 16, and 24 h after injection, and the remaining 14 were scanned 4, 7, 10, 20, and 24 h after injection. Values from 19 previously examined healthy volunteers (8 women, 11 men; mean age, 52 +/- 20 y) were included in the analysis to study the age dependency of beta-CIT binding in striatal and 5-HTT-rich brain areas in a larger control sample. RESULTS: Peak uptake 4 h after injection, followed by stable uptake until 10 h and a slow decrease until 24 h, was observed in the thalamus-hypothalamus region. Activity in the midbrain-pons region peaked 2 h after injection. Because of a concomitant slow but steady decline of uptake in reference regions starting 4 h after injection, a higher stability of binding ratios for 5-HTT-rich brain areas was observed on day 2, suggesting that a state of transient equilibrium is reached between 20 and 24 h but that conditions are only close to transient equilibrium between 4 and 10 h after injection for 5-HTT-rich brain areas. In addition to an age-related decline of striatal [123I]beta-CIT binding of 6.6% per decade, a significant age-associated decrease of beta-CIT binding of 3-4% per decade was found in 5-HTT-rich brain areas. The decline of beta-CIT binding in these regions may be explained, at least in part, by a loss of monoamine transporters with age but may also be related to age-associated morphologic changes. CONCLUSION: [123I]beta-CIT appears to be a suitable ligand for imaging serotonin transporters with SPECT. However, careful age matching is warranted for [123I]beta-CIT SPECT studies of 5-HTT changes in patients with neuropsychiatric disorders.

Adult↗
PubMed1981

Studies on the toxicity and binding kinetics of abrin in normal and Epstein Barr virus-transformed lymphocyte culture-I: experimental results - 1.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Activation, stimulation and relative toxicity factor indices are studied as well as possible relationships between the DNA and protein synthesis rates, as measured by simultaneous 3H-TdR and 14C-leucine uptake.

Abrin↗
PubMed2002

Inhibition of Na(+) current by imipramine and related compounds: different binding kinetics as an inactivation stabilizer and as an open channel blocker.

Use-dependent block of Na(+) channels plays an important role in the action of many medications, including the anticonvulsants phenytoin, carbamazepine, and lamotrigine. These anticonvulsants all slowly yet selectively bind to a common receptor site in inactivated but not resting Na(+) channels, constituting the molecular basis of the use-dependent block. However, it remains unclear what channel gating process "makes" the receptor, where the receptor is located, and how the slow drug binding rate (to the inactivated channels) is contrived. Imipramine has a diphenyl structural motif almost identical to that in carbamazepine (a dibenzazepine tricyclic compound), as well as a tertiary amine chain similar to that in many prototypical local anesthetics, and has also been reported to inhibit Na(+) channels in a use-dependent fashion. We found that imipramine selectively binds to the inactivated (dissociation constant approximately 1.3 microM) rather than the resting Na(+) channels (dissociation constant >130 microM). Moreover, imipramine rapidly blocks open Na(+) channels, with a binding rate approximately 70-fold faster than its binding to the inactivated channels. Similarly, carbamazepine and diphenhydramine are open Na(+) channel blockers with faster binding rates to the open than to the inactivated channels. These findings indicate that the anticonvulsant receptor responsible for the use-dependent block of Na(+) channels is located in or near the pore (most likely in the pore mouth) and is made suitable for drug binding during channel activation. The receptor, however, continually changes its conformation in the subsequent gating process, causing the slower drug binding rates to the inactivated Na(+) channels.

Adrenergic Uptake Inhibitors↗
PubMed1986

B ring regulation of colchicine binding kinetics and fluorescence.

Several properties of the colchicine-tubulin interaction such as association rate, reversibility, and the promotion of drug fluorescence have been related to the B ring of colchicine. The B ring itself retards the binding rate, and substitution at C-7 leads to further binding rate decreases that appear to be related to both substituent bulk and the presence of a N-acyl group. Thus, the decreasing order of binding rates is 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone greater than deacetamidocolchicine greater than deacetylcolchicine greater than or equal to colcemid greater than colchicine greater than N-benzoyldeacetylcolchicine, etc. The apparent irreversibility of the binding seems more closely related to the presence of an N-acyl group rather than the bulk of the substituent at C-7. Substitution at C-7 also affects the tropolone fluorophore. Thus, amines (deacetylcholchicine, colcemid, or N-methylcolcemid) fluoresce poorly in the presence of tubulin, whereas substitution of the amino group with an acyl group enhances fluorescence. The presence of an N-acyl group at C-7 is essential for enhanced fluorescence. We conclude that, in addition to A- and the C-ring portion of the molecule, the B ring of colchicine is a third determinant recognized by the binding site on tubulin.

Animals↗
PubMed1984

Ligand binding kinetic studies on the hybrid hemoglobin alpha(carp):beta(human): a hemoglobin with a restricted allosteric range.

The preparation of native alpha- and beta-chains of carp hemoglobin and the preparation of the hybrid hemoglobin alpha(carp):beta(human), hybrid I, are described. CO association and dissociation kinetics were determined for the hybrid hemoglobin alpha(carp):beta(human) as a function of fractional saturation by stopped-flow and by flash and laser photolysis. Oxygen dissociation from the liganded distribution of conformers and overall oxygen association were studied by laser photolysis. An allosteric model was used to fit successfully all of the CO and oxygen kinetics and equilibria for this hybrid. In this model, it was assumed that the conformational changes were rapid with respect to ligand binding. A linear free-energy relationship relating rate constants and equilibrium constants was assumed in order to reduce the number of fitting parameters. In the allosteric model itself, it was assumed that L, but not c, varied with pH. For CO, the allosteric parameter c was 0.24; for oxygen, the value was 0.14. At pH 7, L was 80. In hybrid I, the calculated CO association rate constants for the R and T states differ by only a factor of 2. From the equilibrium data, with no assumptions as to the rate of conformational equilibrations , one can show that the allosteric model requires that the measured CO association constant from stopped-flow measurements be assigned to the T state. This state, however, is poised approximately midway between a carp-human T state and a carp hemoglobin R state. In this hybrid, neither chain shows normal R-state behavior; rather, the liganded state is also intermediate between R and T. In this hemoglobin, the low cooperativity appears to be associated with a very restricted range for the R-T conformational change.

Animals↗
PubMed1981

Studies on the toxicity and binding kinetics of abrin in normal and Epstein Barr virus-transformed lymphocyte culture-I: experimental results - 3.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Using data on EBV-transformed lymphocyte cell density as a function of time and dose of abrin, one can demonstrate that the mean number of sites bound/EBV-lymphocyte needed to exert a biological influence upon the cell DNA synthesis lies between 59,264 and 370,000 sites/cell. Using a simple packing model, one can demonstrate that a theoretical estimate places the number of binding sites between 57,600 and 360,000 sites/cells.

Abrin↗
PubMed1992

Surface binding kinetics of prothrombin fragment 1 on planar membranes measured by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy (TIRFM) has been employed to investigate the Ca(2+)-dependent membrane-binding characteristics of fluorescein-labeled bovine prothrombin-fragment 1 (F-BF1). Light scattering measurements demonstrated that F-BF1 bound to small unilamellar phosphatidylserine/phosphatidylcholine (25/75, mol/mol) vesicles with an apparent dissociation constant (1.5 +/- 0.2 microM) similar to that of unlabeled protein (1.1 +/- 0.1 microM). Negatively charged supported planar membranes were constructed by fusing small unilamellar vesicles at quartz surfaces. TIRFM measurements under equilibrium conditions showed that F-BF1 bound to planar membranes with an apparent dissociation constant (0.9 +/- 0.2 microM) approximately equal to that on vesicles. Total internal reflection/fluorescence photobleaching recovery (TIR/FPR) curves for F-BF1 on 25 mol% PS planar surfaces were diffusion-influenced at F-BF1 solution concentrations less than or equal to 5 microM. Fluorescence recovery rates from samples of high F-BF1 concentrations were slowed by increasing the solution viscosity with glycerol, thus providing further support for a diffusion-limited effect at low F-BF1 concentrations. Analysis of the reaction-limited fluorescence recovery curves at F-BF1 solution concentrations greater than or equal to 10 microM gave average association and dissociation kinetic rates of approximately 10(5) M-1 s-1 and approximately 0.1 s-1, respectively. Kinetic association rates increased significantly with increasing PS, whereas kinetic dissociation rates increased only slightly. Fluorescence recovery curves were nonmonoexponential; possible mechanisms for this behavior are described.

Animals↗
PubMed1982

Studies on the toxicity and binding kinetics of abrin in normal and Epstein-Barr virus-transformed lymphocyte culture-I. Experimental results - 4.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein-Barr virus (EBV)-transformed lymphocytes have been previously investigated. Using data on EBV-transformed lymphocyte cell density as a function of both time and dose of abrin, the authors introduced the concept of self- and cross-coupling metabolic variables as a means of understanding how abrin affected DNA and protein uptake. In this paper, the self-coupling constant is studied in more detail and the relationship between DNA and protein synthesis is further expanded. We find that there is a significant linear relationship between DNA and protein synthesis in normal lymphocyte culture as measured by abrin interaction in the culture. We further find that there is a much stronger relationship between these variables in EBV-transformed lymphocyte culture. This relationship is further examined, and possible analytic equations are expressed.

Abrin↗