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At least 775 records · Page 43Linked to original sources
PubMed2000

Age dependent changes of insulin receptors in rat tissues.

Aging is associated with insulin resistance but the exact molecular mechanism is still unknown. Tissue insulin resistance can be evoked by the decreased sensitivity to insulin, the decreased responsiveness to hormone or both. As the first step in insulin action is its binding to alfa subunits of the receptor we, therefore, studied the insulin binding kinetics in plasma membranes of the liver, heart and skeletal muscle in order to establish whether their ability to bind the hormone is altered with aging. Plasma membranes were prepared and purified according to Havrankowa and binding assay was performed using (125I)-iodoinsulin. The kinetic parameters of the hormone-receptor interaction were analysed by the method of Scatchard using the LIGAND-Pc v.3.1. computer program. The binding potency of insulin was calculated as IC50 using ALLFIT-Pc v.2.7. computer program. We have shown that there are striking differences in insulin binding kinetics in newborn and old rats, depending on kind of tissue tested. The liver plasma membranes ability for insulin binding, number of high (HAIR) and low (LAIR) affinity insulin receptors, values of the dissociation constants and products of association constants and number of insulin receptors, were almost the same, being not dependent on age of the rats. By contrast, there is less high affinity insulin receptors in skeletal muscle of the old animals. The most dramatic changes in insulin binding occur in the heart where both high and low affinity insulin receptors are greatly affected by aging. Our results indicate that the response of the three tissues tested to hyperglycemia and hyperinsulinemia, observed in the old rats, has not been identical and probably can be accounted for by the different distribution of insulin receptor isoforms in the liver, heart and skeletal muscles as shown recently by Vidal et al.

Aging↗
PubMed1996

The mechanism of porcine pancreatic alpha-amylase. Kinetic evidence for two additional carbohydrate-binding sites.

Kinetics of inhibition of the two porcine pancreatic alpha-amylase components (PPA I and PPA II) by acarbose were performed using reduced DP18-maltodextrin and amylose as substrates. Similar Line-weaver-Burk primary plots were obtained. Two mixed non-competitive models are proposed. X-ray crystallographic data [Qian, M., Buisson, G., Duée. E., Haser, R. & Payan, F. (1994) Biochemistry 33, 6284-6294] are in support of the mixed non-competitive inhibition model which involves abortive complexes. Secondary plots are different; inhibition of reduced DP18-maltodextrin hydrolysis gives straight-lines plots while amylose gives parabolic curves. These results, confirmed by Dixon-plot analyses, allow us to postulate that, in inhibition of reduced DP18-maltodextrin hydrolysis, one molecule of acarbose is bound/ amylase molecule. In contrast, using amylose as a substrate, two molecules of acarbose are bound. These kinetically determined binding sites might correspond to surface sites found by X-ray crystallography [Qian, M., Haser, R. & Payan, F. (1995) Protein Sci. 4, 747-755]; the glucose site close to the active site and the maltose site, 2 nm away. In conclusion, no significant difference between PPA I and PPA II has been observed, either from molecular mass or from kinetic behaviours; this suggests multiple forms of the enzyme. A general mechanism of PPA action is proposed; in addition to the active site, long-chain substrate hydrolysis requires the glucose-binding site and the maltose-binding site, while only one site is necessary for the hydrolysis of short chain substrate.

Acarbose↗
PubMed2004

Flow cytometry: interesting tool for studying binding behavior of DNA on inorganic layered double hydroxide (LDH).

BACKGROUND: A new method was established to characterize the binding kinetics of DNA toward layered double hydroxides (LDHs). The setup consisted of a newly developed sampling tube that allows the injection of analyte during the flow cytometric measurement. METHODS: Layered double hydroxides consist of cationic metal hydroxide layers and exchangeable interlayer anions. This negatively charged structure permits biomolecules such as DNA to adsorb, and a so-called DNA-LDH hybrid is formed. The hydroxide layers can be removed in acidic media and the DNA will be released. CERATOFIX (a registered trademark of Sud-Chemie AG NA that belongs to the family of LDHs, produced by Sud-Chemie AG). The chemical structure can be summarized as [Mg(2)Al(OH)(6)](CO(3))(0.5). The binding capacity and kinetic characteristics of different types of CERATOFIX NA for a model DNA was determined by flow cytometry. RESULTS: The static binding capacities of the different LDHs were determined after 1- and 16-h incubation with DNA solution, showing different binding patterns between the LDH materials. The binding kinetics were revealed by flow cytometric measurements in short-term and long-term kinetic experiments, showing that the majority of DNA adsorbs within the first 60 s. CONCLUSIONS: DNA removal from cell culture supernatants is one of the major concerns in downstream processing. Due to the anion exchange capabilities of LDHs it seemed a very interesting approach to use these materials for binding of DNA for elimination purposes.

Adsorption↗
PubMed1991

Excitatory amino acid receptors in primary cultures of glial cells from the retina.

Binding of 3H-L-aspartate to membranes from retinal glial cells in primary culture was characterized. Binding kinetics showed a saturable, reversible binding to three populations of sites with KB = 40, 200, and 1,300 nM. The first two were present at 1 day in vitro (DIV), whereas the latter two were observed at 12 DIV. The possibility of the 40 nM site being neuronal cannot be discarded, since some neurons are present at 1 DIV. In 12 DIV cultures, the presence or absence of sodium determined two different pharmacological patterns, comparable to those described for electrogenic glutamate transport in Müller cells, and QA metabotropic receptors in astrocytes, respectively. Results suggest that, as has been shown for some receptors in nerve tissue, the properties of glial cell receptors undergo age-dependent changes. In turn, this could be related to changes in the function of neurotransmitter substances during development.

Animals↗
PubMed1999

The novel Mrf-2 DNA-binding domain recognizes a five-base core sequence through major and minor-groove contacts.

Recent NMR studies of the purified Mrf-2 DNA-binding domain peptide have shown that its structure differs significantly from previously characterized classes of DNA-binding domains. Here we report biochemical studies of the DNA-binding properties of this peptide. Binding interference and binding site selection assays indicated that Mrf-2 requires the core sequence AATA(C/T) for high affinity binding. Kinetic analyses of several selected sequences indicated that the core sequence alone is not sufficient for high affinity binding, however. Kinetic analyses were also performed using a series of synthetic oligonucleotides with single base analogues at each position in the core sequence. Base analogues that altered the major groove structure reduced or eliminated Mrf-2 binding when present in the second, third, and fourth base-pairs of the core sequence, but had little or no effect in the first and fifth positions. These results suggest that Mrf-2 contacts both the major and minor grooves of its target sequences.

Base Sequence↗
PubMed2000

Speeding molecular recognition by using the folding funnel: the fly-casting mechanism.

Protein folding and binding are kindred processes. Many proteins in the cell are unfolded, so folding and function are coupled. This paper investigates how binding kinetics is influenced by the folding of a protein. We find that a relatively unstructured protein molecule can have a greater capture radius for a specific binding site than the folded state with its restricted conformational freedom. In this scenario of binding, the unfolded state binds weakly at a relatively large distance followed by folding as the protein approaches the binding site: the "fly-casting mechanism." We illustrate this scenario with the hypothetical kinetics of binding a single repressor molecule to a DNA site and find that the binding rate can be significantly enhanced over the rate of binding of a fully folded protein.

Kinetics↗
PubMed2004

Structural dynamics in the active site of murine neuroglobin and its effects on ligand binding.

We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4-11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His64 and Lys67. His64 interferes with exogenous ligand access to the heme iron. Lys67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.

Animals↗
PubMed1997

Binding affinities of insulin-like growth factor-I (IGF-I) fusion proteins to IGF binding protein 1 and IGF-I receptor are not correlated with mitogenic activity.

In this report, comparisons between molecular affinities and cellular proliferation activities have been made for insulin-like growth factor-I (IGF-I) and two IGF-I fusion proteins in order to evaluate fusion proteins as tools for receptor binding studies. Binding affinities and growth promoting effects of the N-terminal fusion Z-IGF-I and the C-terminal fusion IGF-I-Z, and native recombinant human IGF-I, were analyzed. Binding kinetic properties of the three IGF-I variants were analyzed using BIAcore kinetic interaction analysis testing for binding to both human IGF binding protein 1 (IGFBP-1) and a soluble form of the human IGF type I receptor extracellular domains (sIGF-IR). The growth promoting effects on SaOS-2 human osteosarcoma cells of the different fusion proteins were analyzed. A comparison of receptor binding affinities and growth promoting effects shows that the fusion protein receptor affinity does not correlate with proliferative potential. The IGF-I-Z fusion, with the lowest receptor affinity, shows similar proliferative potential to native IGF-I. However, the Z-IGF-I fusion protein, with twice the receptor affinity of IGF-I-Z, displays only about 70% of the IGF-I-Z growth promoting activity. Both IGF-I fusion proteins possess similar affinity to IGFBP-1. These results indicate that determinants other than the receptor affinity could be involved in the regulation of IGF-I proliferative action. This study demonstrates that ligand fusion proteins may be useful to study mechanisms of ligand induced receptor activation.

Binding Sites↗
PubMed2000

Conformational modulation of human cytochrome P450 2E1 by ethanol and other substrates: a CO flash photolysis study.

The alcohol-inducible cytochrome P450 2E1 is a major human hepatic P450 which metabolizes a broad array of endogenous and exogenous compounds, including ethanol, low-molecular weight toxins, and fatty acids. Several substrates are known to stabilize this P450 and inhibit its cellular degradation. Furthermore, ethanol is a known modulator of P450 2E1 substrate metabolism. We examined the CO binding kinetics of P450 2E1 after laser flash photolysis of the heme-CO bond, to probe the effects of ethanol and other substrates on protein conformation and dynamics. Ethanol had an effect on the two kinetic parameters that describe CO binding: it decreased the rate of CO binding, suggesting a decrease in the protein's conformational flexibility, and increased the photosensitivity, which indicates a local effect in the active site region such as strengthening of the heme-CO bond. Other substrates decreased the CO binding rate to varying degrees. Of particular interest is the effect of arachidonic acid, which abolished photodissociation in the absence of ethanol but had no effect in the presence of ethanol. These results are consistent with a model of P450 2E1 whereby arachidonic acid binds along a long hydrophobic binding pocket and blocks exit of CO from the heme region.

Arachidonic Acid↗
PubMed1983

The effect of intercalating drugs on the kinetics of the B to Z transition of poly(dG-dC).

We have measured the ability of the intercalating drugs proflavine, ethidium bromide, actinomycin D, and bismethidiumspermine to inhibit the salt induced transition of poly(dG-dC) from the B to the Z form. While all of the drugs studied slowed the B to Z transition, the effectiveness of the drugs correlates much better with their DNA binding kinetics than their DNA binding constants. In studies where the binding densities of ethidium and actinomycin were varied we have found that high levels of ethidium, more than 1 per 20 base pairs, were required to inhibit the B to Z transition while low levels of actinomycin, less than 1 per 450 base pairs, reduced the transition rate. Studies of the B to Z transition in the presence of both actinomycin and ethidium suggest that the drugs inhibit the transition by different mechanisms. The results are interpreted in terms of a modification of the kinetic model proposed by Pohl and Jovin in which, depending on the DNA binding kinetics of the drug, the drug may inhibit nucleation and/or propagation of the B to Z transition.

Dactinomycin↗
PubMed2000

Characterization of interactions among the heme center, tetrahydrobiopterin, and L-arginine binding sites of ferric eNOS using imidazole, cyanide, and nitric oxide as probes.

Endothelial nitric oxide synthase (eNOS) is a self-sufficient P450-like enzyme. A P450 reductase domain is tethered to an oxygenase domain containing the heme, the substrate (L-arginine) binding site, and a cofactor, tetrahydrobiopterin (BH(4)). This "triad", located at the distal heme pocket, is the center of oxygen activation and enzyme catalysis. To probe the relationships among these three components, we examined the binding kinetics of three different small heme ligands in the presence and absence of either L-arginine, BH(4), or both. Imidazole binding was strictly competitive with L-arginine, indicating a domain overlap. BH(4) had no obvious effect on imidazole binding but slightly increased the k(on) for L-arginine. L-Arginine decreased the k(on) and k(off) for cyanide by two orders, indicating a "kinetic obstruction" mechanism. BH(4) slightly enhanced cyanide binding. Nitric oxide (NO) binding kinetics were more complex. Increasing the L-arginine concentration decreased the NO binding affinity at equilibrium. In both BH(4)-abundant and BH(4)-deficient eNOS, half of the NO binding sites showed a sizable decrease of the binding rate by L-arginine, with the rate of NO binding at the other half of the sites remaining essentially unaltered by L-arginine, implying that the two heme centers in the eNOS dimer are functionally distinct.

Animals↗
PubMed1979

Patterns of transport and binding of sex steroids.

The seminal plasma-sex steroid binding kinetic was evaluated by measuring the amount of labeled hormone extractable after 5 sec to 100 min of incubation. For testosterone in normal subjects, a peak of binding was reached after 30 sec of incubation. In subjects with severe oligoasthenospermia the kinetic pattern for testosterone was different but similar to the binding kinetic between seminal plasma of normal men and estradiol. The phenomena did not occur with frozen plasma samples.

Estradiol↗
PubMed2004

Kinetic exclusion assay technology: characterization of molecular interactions.

Characterization of intermolecular interactions in terms of affinity, binding kinetics, stoichiometry, specificity, and thermodynamics can facilitate the selection of lead compounds in the discovery and development of protein therapeutics. KinExA (Sapidyne Instruments, Inc., Boise, ID) is a relatively new technology that is gaining use in characterizing molecular interactions, particularly with respect to antibody therapeutics. KinExA offers a platform that allows the measurement of true equilibrium binding affinity and kinetics using unmodified molecules in solution phase. This is accomplished by using a solid-phase immobilized molecule to probe for free concentration of one interaction component after allowing sufficient time to reach equilibrium (affinity measurements), or under pre-equilibrium conditions (kinetics). In this review, the theory behind KinExA technology is discussed, and examples of applying this technology to antibody characterization are provided. Finally, a comparison among KinExA, Biacore (surface plasmon resonance), and isothermal titration calorimetry is presented, and potential future improvements and applications of KinExA are discussed.

Algorithms↗
PubMed2001

Homeostasis in mRNA initiation: wheat germ poly(A)-binding protein lowers the activation energy barrier to initiation complex formation.

Previous kinetic binding studies of wheat germ protein synthesis eukaryotic initiation factor iso4F (eIFiso4F) and its subunit, eIF4E, with m(7)GTP and mRNA analogues indicated that binding occurred by a two-step process with the first step being too fast to measure by stopped-flow techniques (). Further equilibrium studies showed that poly(A)-binding protein (PABP) enhanced the cap binding of eIFiso4F about 40-fold. The kinetic effects of PABP on cap binding and the temperature dependence of this reaction were measured and compared. Fluorescence stopped-flow studies of the PABP.eIFiso4F protein complex with cap show a concentration-independent conformational change. PABP did not significantly increase the rate of the conformational change, and because the initial second-order binding is essentially diffusion-controlled, the enhancement of cap affinity must reside in the dissociation rate. The dissociation rate was more than 5-fold slower in the presence of PABP. The temperature dependence of the cap binding reaction was markedly reduced in the presence of PABP. The reduced energy barrier for formation of a cap.eIFiso4F complex suggests a more stable platform for further initiation complex formation and a possible means of adapting to varying temperature conditions.

Eukaryotic Initiation Factor-4E↗
PubMed1988

Kinetics of calcium binding to calbindin mutants.

The kinetics of calcium dissociation from wild-type bovine calbindin D9k (the smallest protein known with a pair of EF-hand calcium-binding sites) and five mutants with single amino-acid substitutions and/or deletions has been studied by stopped-flow fluorescence methods, using the calcium chelator Quin 2. The modifications are confined to the N-terminal half of the molecule, at or near the first calcium-binding site (I). Substitutions and deletions of amino acids in the calcium-binding loop of site I primarily affect the rate of Ca2+ dissociation from this site with only minor effects on the dynamic properties of the C-terminal calcium-binding site (II). This finding corroborates and extends previous kinetic results obtained from 43Ca-NMR studies on the same set of mutants. By contrast, removal of the hydrogen bond between Tyr-13 and Glu-35, an interaction linking the two alpha-helices flanking site I, through replacement of Tyr-13 with Phe, has no observable effect on the rate of Ca2+ dissociation from the protein. Comparison of this kinetic data with binding-constant data, previously obtained in our laboratories, shows that the decrease in Ca2+-affinity of site I, observed in most mutants, is predominantly due to an increased off-rate from this site. At low ionic strengths the second-order rate constants for Ca2+-binding to both Ca2+ sites of calbindin D9k are calculated to be of the order of 10(9) M-1 s-1 for all proteins studied. At higher ionic strengths (0.1 M KCl) the rates of Ca2+ dissociation from both sites are increased by a factor of three or more, suggesting a transition state which is ionic in nature.

Amino Acids↗
PubMed1979

Rate and equilibrium constants for binding of apo-E HDLc (a cholesterol-induced lipoprotein) and low density lipoproteins to human fibroblasts: evidence for multiple receptor binding of apo-E HDLc.

Competitive binding assays have demonstrated that a cholesterol-induced canine lipoprotein containing only the E apoprotein (apo-E HDL(c)) binds to the same cell surface receptors of human fibroblasts as human low density lipoproteins (LDL). However, the apo-E HDL(c) have a much greater binding activity than LDL. Equilibrium and kinetic binding studies were conducted at 4 degrees C to determine the mechanism for this enhanced receptor binding activity. Based on the data, the binding of both LDL and apo-E HDL(c) appears to be a simple bimolecular receptor interaction, and no heterogeneity of binding sites or cooperative effects among the receptor sites were observed. Equilibrium dissociation constants determined by Scatchard analysis of the equilibrium binding data for apo-E HDL(c) (K(d) = 0.12 x 10(-9) M) and LDL (K(d) = 2.8 x 10(-9) M) revealed a 23-fold greater affinity of HDL(c) for the receptors. Association and dissociation rate constants for the lipoprotein-receptor complex were determined from the time course of binding at various lipoprotein concentrations. The equilibrium dissociation constants calculated from these kinetic data confirmed that apo-E HDL(c) had a much higher affinity for the receptor than LDL. Furthermore, the kinetic studies indicated that apo-E HDL(c) bound more rapidly than LDL with rates of association of 18.0 x 10(4) and 5.5 x 10(4) M(-1) sec(-1), respectively. The rate of dissociation of the apo-E HDL(c)-receptor complex (1.7 x 10(-5) sec(-1)) was slower than that of the LDL receptor complex (6.3 x 10(-5) sec(-1)). An additional important difference between the binding of apo-E HDL(c) and LDL was that 4 times (3.6 +/- 0.4) as many LDL particles as HDL(c) particles were required for saturation of the receptors at maximal binding. These data indicate that each HDL(c) particle binds to multiple cell surface receptors at a ratio of 4:1 for LDL receptor binding.

Apolipoproteins↗
PubMed1999

Estrogen receptors (ERalpha/ERbeta) in normal and pathological growth of the human myometrium: pregnancy and leiomyoma.

The distributions of the mRNAs for estrogen receptors (ERalpha and ERbeta) and their binding properties in myometria of pregnant and nonpregnant women and in leiomyoma were studied. RT-PCR analysis indicated that the term pregnancy myometria had little ERalpha mRNA, whereas the amounts of ERbeta mRNAs in pregnant or nonpregnant myometria appeared to be similar. Both ERalpha and ERbeta mRNA were greater in certain leiomyoma than in normal nonpregnant myometria. The binding kinetics revealed that two specific binding sites (with high or low affinity) for 17beta-estradiol were present in the nonpregnant myometrium. Only the low-affinity binding sites were detectable in late-pregnancy myometria and in leiomyoma, and their capacities were increased two- to threefold (P < 0.001) in leiomyoma. The pregnancy- and leiomyoma-related changes in myometrial ER status, especially the low concentration of ERalpha mRNA and the lack of high-affinity ER in pregnant women, plus the increased ERalpha and ERbeta mRNAs and the increased low-affinity ER in some leiomyoma, suggest that the redistribution of ER subtypes is associated with the pathological and/or normal growth of the myometrium.

Female↗
PubMed1996

Kinetics of factor VIII-von Willebrand factor association.

The binding of factor VIII to von Willebrand factor (vWF) is essential for the protection of factor VIII against proteolytic degradation in plasma. We have characterized the binding kinetics of human factor VIII with vWF using a centrifugation binding assay. Purified or plasma vWF was immobilized with a monoclonal antibody (MoAb RU1) covalently linked to Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden). Factor VIII was incubated with vWF-RU1-Sepharose and unbound factor VIII was separated from bound factor VIII by centrifugation. The amount of bound factor VIII was determined from the decrease of factor VIII activity in the supernatant. Factor VIII binding to vWF-RU1-Sepharose conformed to the Langmuir model for independent binding sites with a Kd of 0.46 +/- 0.12 nmol/L, and a stoichiometry of 1.3 factor VIII molecules per vWF monomer at saturation, suggesting that each vWF subunit contains a binding site for factor VIII. Competition experiments were performed with a recombinant vWF (deltaA2-rvWF), lacking residues 730 to 910 which contain the epitope for MoAB RU1. DeltaA2-rvWF effectively displaced previously bound factor VIII, confirming that factor VIII binding to vWF-RU1-Sepharose was reversible. To determine the association rate constant (k(on)) and the dissociation rate constant (k(off)), factor VIII was incubated with vWF-RU1-Sepharose for various time intervals. The observed association kinetics conformed to a simple bimolecular association reaction with k(on) = 5.9 +/- 1.9 x 10(6) M(-1) s(-1) and k(off) = 1.6 +/- 1.2 x 10(-3) s(-1) (mean +/- SD). Similar values were obtained from the dissociation kinetics measured after dilution of preformed factor VIII-vWF-RU1-Sepharose complexes. Identical rate constants were obtained for factor VIII binding to vWF from normal pooled plasma and to vWF from plasma of patients with hemophilia A. The kinetic parameters in this report allow estimation of the time needed for complex formation in vivo in healthy individuals and in patients with hemophilia A, in which monoclonally purified or recombinant factor VIII associates with endogenous vWF. Using the plasma concentration of vWF (50 nmol/L in monomers) and the obtained values for K(on) and K(off), the time needed to bind 50% of factor VIII is approximately 2 seconds.

Antibodies, Monoclonal↗