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At least 559 records · Page 31Linked to original sources
PubMed2003

Limited proteolysis reveals conformational changes in uncoupling protein-1 from brown adipose tissue mitochondria.

Limited proteolytic digestion of uncoupling protein-1 (UCP1) from hamster brown adipose tissue mitochondria was studied. Under optimal conditions, trypsin and chymotrypsin cleave at Lys-292 and at Phe-102, yielding major products 31-kDa T1 and 22-kDa Ch1. Both T1 and Ch1 remained dimers, as in UCP1. Using fluorescent nucleotide derivative 2'-O-dansyl GTP, it is shown that T1 retains the nucleotide binding affinity (K(D)=1 microM for dansyl GTP) while Ch1 does not bind nucleotide. Previously kinetic binding and H(+) transport studies [Biochemistry 35 (1996) 7846] have shown that UCP1 forms tight complexes to varying degrees with nucleotides and their derivatives. Nucleotides strongly protect against tryptic digestion but less against chymotryptic digestion, because the chymotryptic product Ch1 does not bind nucleotide. The nucleotides and derivatives show the same potency profile in protecting against both trypsinolysis and chymotryptic digestion, suggesting that UCP1 undergoes a major conformational change upon nucleotide binding from an initial loose complex into a tight complex, in which the cleavage sites become masked from proteolysis.

Adipocytes↗
PubMed1993

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators.

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.

Animals↗
PubMed2002

PH-induced collapse of the extracellular loops closes Escherichia coli maltoporin and allows the study of asymmetric sugar binding.

LamB (maltoporin) is essential for the uptake of maltose and malto-oligosaccharides across the outer membrane of Escherichia coli. Purified LamB was reconstituted in artificial lipid bilayer membranes forming channels in the permanently open configuration at neutral pH. Almost complete channel closure was observed when the pH on both sides of the membrane was lowered to pH 4. When LamB was added to only one side of the membrane, the cis-side, and the pH was lowered at either side of the membrane, the cis- or the trans-side, the response to pH was asymmetric, suggesting preferential orientation of maltoporin channels and pH- dependent closure of only one side of the channel. In experiments with LamB mutants in which major external loops L4, L6, and/or L9 were deleted, we identified the surface-exposed loops L4 and L6 as the cause of pH-mediated closure. The pH dependence of the LamB channel is consistent with the assumption that it inserts in a preferential orientation into the lipid bilayer. About 70-80% of the reconstituted channels are oriented with the extracellular entrance toward the side to which the protein was added (the cis-side) and with the periplasmic opening on the opposite side (the trans-side). The possibility of closing the channels, which are oriented in the reverse direction by low pH at the trans-side, allowed the deduction of channel asymmetry with respect to carbohydrate binding kinetics. Whereas maltose binding was found to be almost symmetric with respect to the channel orientation, the sucrose and trehalose binding to LamB was asymmetric. The results are discussed in respect to possible physiological function of the pH-dependent closure of maltoporin.

Bacterial Outer Membrane Proteins↗
PubMed1987

Kinetics of binding of lucensomycin to natural and artificial membranes.

The binding of the polyenic antibiotic lucensomycin to native or modified human erythrocyte ghosts and to model membranes has been studied by monitoring the absorbance variations of the polyene at 320 nm. The non-steroidal components of the membranes (such as proteins and phospholipids) seem to affect the rate of the individual reaction steps leading to the formation of cholesterol-lucensomycin complexes rather than the ratio among these heterologous aggregates at equilibrium.

Antifungal Agents↗
PubMed2002

Endocytosis of adeno-associated virus type 5 leads to accumulation of virus particles in the Golgi compartment.

Among the adeno-associated virus (AAV) serotypes which are discussed as vectors for gene therapy AAV type 5 (AAV5) represents a candidate with unique advantages. To further our knowledge on AAV5-specific characteristics, we studied the entry pathway of wild-type virus in HeLa cells in the absence of helper virus by immunofluorescence and electron microscopy and by Western blot analysis. We found virus binding at the apical cell surface, especially at microvilli and, with increasing incubation time, virus accumulation at cell-cell boundaries. The different binding kinetics suggest different binding properties at apical versus lateral plasma membranes. Endocytosis of viruses was predominantly by clathrin-coated vesicles from both membrane domains; however, particles were also detected in noncoated pits. AAV5 particles were mainly routed to the Golgi area, where they could be detected within cisternae of the trans-Golgi network and within vesicles associated with cisternae and with the dictyosomal stacks of the Golgi apparatus. These data suggest that AAV5 makes use of endocytic routes that have hitherto not been described as pathways for virus entry.

Blotting, Western↗
PubMed2001

Mutations at the boundary of the hinge and ligand binding domain of the androgen receptor confer increased transactivation function.

The androgen receptor (AR), a member of the steroid receptor superfamily of nuclear transcription factors, mediates androgen signaling in diverse target tissues. Here we report AR gene mutations identified in human prostate cancer and the autochthonous transgenic adenocarcinoma of the mouse prostate model that colocate to residues (668)QPIF(671) at the boundary of the hinge and ligand-binding domain, resulting in receptors that exhibit 2- to 4-fold increased activity compared with wild-type AR in response to dihydrotestosterone, estradiol, progesterone, adrenal androgens, and the AR antagonist, hydroxyflutamide, without an apparent effect on receptor levels, ligand binding kinetics, or DNA binding. The expression of these or similar variants could explain the emergence of hormone refractory disease in a subset of patients. Homology modeling indicates that amino acid residues (668)QPIF(671) form a ridge bordering a potential protein-protein interaction surface. The naturally occurring AR gene mutations reported in this study result in decreased hydrophobicity of this surface, suggesting that altered receptor-protein interaction mediates the precocious activity of the AR variants.

Adenocarcinoma↗
PubMed1989

[Modification of selective adsorption of muscarinic antagonists in cerebral membranes during chlorophos poisoning].

Using radioligand assay it was demonstrated that chlorophos intoxication produced inhibiting effect on the kinetics of membrane binding of 3H3-quinuclidinylbenzilate and 14C-cyclosil in the brain. Cholinesterase reactivator dipyroxime was shown to induce normalization of the cyclosyl specific binding kinetics. It seems to be justified to propose that cooperative action of cholinolytic agents and cholinesterase reactivator at the level of m-cholinoreceptors in the course of intoxication may be one of the mechanisms of the potentiation of therapeutic effects of these drugs.

Animals↗
PubMed1981

Interactions between photoexcited rhodopsin and GTP-binding protein: kinetic and stoichiometric analyses from light-scattering changes.

In rod outer segments, photoexcited rhodopsin (R*) activates a cyclic GMP phosphodiesterase through a sequence of reactions involving a GTP-binding protein. By measuring light-scattering changes above 700 nm, we have studied the kinetics and stoichiometry of the association of R* with this protein and of the dissociation of the complex upon GDP/GTP exchange. Two light-scattering signals were obtained upon photoexcitation of rhodopsin in bovine rod outer segment membranes as well as in a reconstituted system consisting of purified GTP-binding protein and washed disc membranes; both signals depended specifically on the presence of GTP-binding protein. A "binding signal" that was observed in the absence of gTP as an increase in turbidity became saturated when a number of rhodopsin molecules equal to the number of GTP-binding protein molecules present (congruent to 10% in rod outer segments) has been bleached, suggesting that the protein binds to R* in a 1:1 complex. A "dissociation signal" of opposite sign, observed in presence of GTP at greater than or equal to 1 microM, is half maximal at 0.04% bleaching and saturated at 0.5% bleaching; it is interpreted as reflecting the dissociation of GTP-binding protein-R* complexes after GDP/GTP exchange on the GTP-binding protein, one R* being able to interact sequentially with about 100 GTP-binding protein molecules. The early time course of the binding signal is faster than that of the dissociation signal, and both signals take place in the 100-msec range at 20 degrees C.

Animals↗
PubMed1995

Fluorescent nucleotide derivatives as specific probes for the uncoupling protein: thermodynamics and kinetics of binding and the control by pH.

Fluorescent 2'-O-dansylated (DANS) purine nucleotides were synthesized. The fluorescence of the nucleotide derivatives is quenched in aqueous solutions but strongly enhanced on binding to the uncoupling protein (UCP) from brown adipose tissue mitochondria. The fluorescence enhancement was 30-, 10-, and 10-fold for DANSGTP, DANSATP, and DANSADP. One mole of DANS nucleotide binds to 1 mol of dimeric UCP. The binding affinity ranges from 10(5) to 10(8) M-1, similar to that of the unsubstituted nucleotides, while dansylation of AMP increases the affinity 50-fold. The pH dependence in the pKD/pH plots for the DANS nucleotides is basically similar to that for the unsubstituted nucleotides, i.e., for nucleoside diphosphates the slope delta pKD/delta pH < -1 at pH 5-6.5, = -1 at pH > 6.8, and only for triphosphates = -2 at pH > 7.2. Two different protonation sites with a pKH approximately 4 (Asp/Glu) and pKH approximately 7.2 (His), only for nucleoside triphosphates, are suggested to be involved in binding. The higher affinity of DANSGTP indicates additional participation in binding of the C-6 oxygen on the guanine. The binding as measured with the anion exchange method agrees with the fluorescence measurement for DANSGTP, whereas for the more loosely binding DANSATP it is 40% lower. This is interpreted in terms of tight/loose UCP-nucleotide complexes, 100% tight complex for DANSGTP (as well GTP or ATP) but 40% loose complex for DANSATP. By measuring the rapid kinetics using the fluorescence signal, the binding rate is found to be fast and fairly constant for the various nucleotides, whereas the dissociation is slow and strongly nucleotide dependent. The rates are pH dependent with delta pkon/delta pH = 1 for all the nucleotides and delta pkoff/delta pH = -1 for DANSNTP but more weakly with delta pkoff/delta pH < -0.5 for DANSADP and DAN-ATP. The pH dependence of the binding rate corresponds to a protonation at the carboxylate group (Glu/Asp). The high pH dependence of the dissociation rate only for DANSNTP is explained by deprotonation at the HisH+ which is involved only in nucleoside triphosphate binding. This is in line with the very strong pH dependence of nucleoside triphosphate affinity above pH 7 with a delta pKD/delta pH = -2 as an important regulatory mechanism for the H+ transport activity of UCP. The differences of the DANS nucleotides versus the DAN and unsubstituted nucleotides as well as the nucleoside tri- versus diphosphate are rationalized in a specific H+ dependent regulatory mechanism at the binding site.

Animals↗
PubMed1990

Receptor kinetics differ for endothelin-1 and endothelin-2 binding to Swiss 3T3 fibroblasts.

The equilibrium binding, kinetics of ligand-receptor interactions, and biological activity of endothelin-1 and -2 have been studied in Swiss 3T3 fibroblasts. Scatchard analyses of saturation binding data for ET-1 and -2, performed at 4 degrees C to prevent internalization of the occupied receptor, revealed similar affinity constants and numbers of binding sites for endothelin-1 and -2. Experiments designed to determine ligand-induced effects on 45Ca efflux demonstrated no qualitative or quantitative differences between the two endothelin isoforms. In contrast, kinetic studies resulted in different rates of dissociation for the two isoforms and different extents of dissociation. Specifically, only 40% of the bound [125I]endothelin-1 was dissociated at 4 h following the addition of excess unlabeled ligand, whereas 85-90% of the bound [125I]endothelin-2 was dissociated under the same conditions. Endothelin-1 and -2 also differed in the percent of specific cell-associated ligand bound after a 2 h incubation at 37 degrees C following an initial equilibration at 4 degrees C. The differences in dissociation rates and association or internalization rates at 37 degrees C are the first data that differentiate between the two isoforms. It is suggested that isoform-specific differences in the rate of dissociation from cell surface endothelin receptors influence the level of cell-associated endothelin and may be important in determining physiologic responses in vivo.

Animals↗
PubMed1999

Function of estrogen receptor tyrosine 537 in hormone binding, DNA binding, and transactivation.

The human estrogen receptor (hER) is a ligand-activated transcription factor which, like many other members of the nuclear receptor protein family, exhibits a dimerization-dependent transcriptional activation. Several previous reports have provided evidence of the phosphorylation of the hER at tyrosine 537 (Y537). However, the exact function of a putative phosphorylation at this site remains controversial. Using a yeast transactivation assay, and in vitro biochemical approaches, we show that phosphorylation of tyrosine 537 is not required for the hER to bind hormone, or to activate transcription. An hER tyrosine 537 to phenylalanine (Y537F) mutant retains 70-75% of the transactivation potential of wild type hER in a yeast reporter system. Furthermore, the mutated receptor exhibits wild type hormone and DNA binding affinities. However, this mutation results in a decrease in receptor stability as measured by a decrease in the extent of hormone binding over time. The most striking difference between the wild type and Y537F hER is in the estradiol binding kinetics. Whereas the off-rate for estradiol exhibits a two-state binding mechanism, the Y537F mutant hER exhibits a monophasic estradiol off-rate. On the basis of these data and other reports describing the structure and activity of Y537 mutations, as well as knowledge of the three-dimensional structure of the hER ligand binding domain, we propose an alternate model wherein Y537F mutation favors an "open" pocket conformation, affecting the estrogen binding kinetics and stability of the hormone-bound, transcriptionally active "closed" pocket conformation. Although its phosphorylation is not essential for function of the hER, Y537 is nevertheless a critical residue intricately involved with the conformational changes of the hER and its ability to activate transcription.

Amino Acid Sequence↗
PubMed1985

Comparison of lipid binding and kinetic properties of normal, variant, and gamma-carboxyglutamic acid modified human factor IX and factor IXa.

The abilities of normal and three abnormal factor IXa molecules to activate factor X and to bind to phospholipid membranes have been compared to define the contributions of protein-lipid interactions and factor IXa light chain-heavy chain interactions to the functioning of this protein. The abnormal proteins studied had altered amino acid residues in their light chains. The heavy-chain regions, containing the active site serine and histidine residues, were normal in the abnormal proteins on the basis of titration by antithrombin III. The binding constants (Kd) for normal (N), variant [Chapel Hill (CH) and Alabama (AL)], and gamma-carboxyglutamic acid (Gla) modified (MOD) factors IX and IXa to phosphatidylserine (PS)/phosphatidylcholine (PC) small, unilamellar vesicles (SUV) were measured by 90 degrees light scattering. The Kd values for factor IXN binding were quite sensitive to the PS content of the membrane but less sensitive to Ca2+ concentrations between 0.5 and 10 mM. The zymogen and activated forms of both normal and abnormal factor IX bound with similar affinities to PS/PC (30/70) SUV. In the cases of factor IXaN and factor IXaAL, but not factor IXaCH or factor IXaMOD, irreversible changes in scattering intensity suggested protein-induced vesicle fusion. Since the activation peptide is not released from factor IXaCH, the normal interaction of factor IXa with a membrane must require the release of the activation peptide and the presence of intact Gla residues. The rate of factor X activation by normal and abnormal factor IXa was obtained by using a chromogenic substrate for factor Xa in the presence of PS/PC (30/70) SUV and 5 mM Ca2+.

1-Carboxyglutamic Acid↗
PubMed1989

Purification of cytochrome-c oxidase retaining its pulsed form.

A new purification procedure for cytochrome-c oxidase from bovine heart mitochondria is described. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxyapatite and gel chromatography. The preparation was highly pure and active. The subunit composition and steady-state kinetics were found to be the same as those reported for other preparations. In contrast to most of the previously published protocols the method presented here resulted in a preparation which had a rapid intramolecular electron transfer from cytochrome a to cytochrome a3, i.e. it was found to have retained its pulsed state. This correlated with monoexponential cyanide-binding kinetics. The formation of resting kinetics and biphasic cyanide-binding kinetics was shown to be induced by a short incubation at pH 5.0.

Animals↗
PubMed2004

Transfer of human alpha- to beta-hemoglobin via its chaperone protein: evidence for a new state.

The alpha-hemoglobin-stabilizing protein (AHSP), a small protein of 102 amino acids, is synthesized in red blood cell precursors. It binds specifically to alpha-hemoglobin (alpha-Hb) subunits acting as a chaperone protein, preventing the formation of alpha-hemoglobin-cytotoxic precipitates. We have engineered recombinant AHSP in a pGEX vector to study the functional consequence of interaction between AHSP and alpha-Hb. By in vitro binding assays, we have isolated the complexes glutathione S-transferase-AHSP.alpha-Hb and AHSP.alpha-Hb. The latter assembles as a heterodimer based on size-exclusion chromatography. These complexes exhibited monophasic CO binding kinetics, as observed for isolated alpha- and beta-subunits of hemoglobin. However, the rate of CO (or oxygen) binding to alpha-hemoglobin bound to its chaperone is three times slower than that observed for isolated alpha-hemoglobin, demonstrating a form that is intermediate to the R- and T-hemoglobin states. The physiologically relevant replacement of the chaperone by beta-hemoglobin chains could be detected by both ligand binding kinetics and tryptophan fluorescence quenching.

Blood Proteins↗
PubMed2004

Mechanical measurement of single-molecule binding rates: kinetics of DNA helix-destabilization by T4 gene 32 protein.

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein, and is essential for DNA replication, recombination and repair. While gp32 binds preferentially and cooperatively to ssDNA, it has not been observed to lower the thermal melting temperature of natural double-stranded DNA (dsDNA). However, in single-molecule stretching experiments, gp32 significantly destabilizes lambda DNA. In this study, we develop a theory of the effect of the protein on single dsDNA stretching curves, and apply it to the measured dependence of the DNA overstretching force on pulling rate in the presence of the full-length and two truncated forms of the protein. This allows us to calculate the rate of cooperative growth of single clusters of protein along ssDNA that are formed as the dsDNA molecule is stretched, as well as determine the site size of the protein binding to ssDNA. The rate of cooperative binding (ka) of both gp32 and of its proteolytic fragment *I (which lacks 48 residues from the C terminus) varies non-linearly with protein concentration, and appears to exceed the diffusion limit. We develop a model of protein association with the ends of growing clusters of cooperatively bound protein enhanced by 1-D diffusion along dsDNA, under the condition of protein excess. Upon globally fitting ka versus protein concentration, we determine the binding site size and the non-cooperative binding constants to dsDNA for gp32 and I. Our experiment mimics the growth of clusters of gp32 that likely exist at the DNA replication fork in vivo, and explains the origin of the "kinetic block" to dsDNA melting by gene 32 protein observed in thermal melting experiments.

Bacteriophage T4↗