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At least 1,063 records · Page 59Linked to original sources
PubMed1993

Modulation of serotonin binding sites in the brain of the Djungarian hamster, Phodopus sungorus, during adaptation to a short photoperiod.

During the physiological adaptation of the Djungarian hamster, Phodopus sungorus, to a short photoperiod in autumn the modulation of specific serotonin (5-HT) binding sites of synaptic membranes was investigated in two brain regions, i.e. cerebral cortex and basal brain (CNS without cerebral cortex, cerebellum, pineal gland, and spinal cord). The radioligands [3H]5-HT and [3H]ketanserin were used to characterize total 5-HT1 and 5-HT2 binding sites, respectively. An increase of 5-HT1 and 5-HT2 binding sites was observed in both brain regions within 14 days after reduction of the photoperiod from a 14:10 h light/dark (l/d) cycle to an 8:16 h l/d cycle. The increase was still present after 56 days of the short photoperiod. Binding kinetics assayed after 4 days of the short photoperiod show that maximal specific binding of [3H]5-HT and [3H]ketanserin was increased, while dissociation constants (KD) were not changed. The membrane anisotropy of synaptic membranes, measured by fluorescence polarization, was reduced transiently during the early part of the adaptation. Neither the phospholipids nor the mole ratio of cholesterol to phospholipids were significantly affected by adaptation to short photoperiod. The results suggest an important role of the central nervous 5-HT system in the physiological adaptation of the Djungarian hamster to a short photoperiod.

Adaptation, Physiological↗
PubMed1996

Accumulation and extrusion of permeant Ca2+ chelators in attenuation of synaptic transmission at hippocampal CA1 neurons.

The effects of extracellularly applied membrane-permeant Ca2+ chelators on field excitatory postsynaptic potentials were determined in the hippocampal CA1 region of rat brain slices. Field excitatory postsynaptic potentials in slices perfused with 0.05-50 microM bis-(-O-aminophenoxy)-ethane-N,N,N,N,-tetraacetic acid acetoxymethyl (BAPTA-AM) for 15 min were reversibly attenuated by 10-45% in a concentration-dependent manner. Attenuation occurred earlier at higher concentrations of BAPTA-AM, thus indicating that the rate of accumulation of BAPTA salt was concentration dependent. Antidromically evoked responses and presynaptic volleys were unaffected by BAPTA-AM. Attenuation of the field excitatory postsynaptic potentials by BAPTA-AM was temporarily eliminated by repetitive stimulation at 1 Hz, suggesting saturation of the chelator's Ca(2+)-binding capacity. The amplitude of field excitatory postsynaptic potentials was unaffected by similar applications of 5'5-dinitro-BAPTA-AM, a low Ca(2+)-affinity BAPTA analogue, and EGTA-AM (5 or 50 microM), a chelator with slow Ca(2+)-binding kinetics, suggesting a dependence of the BAPTA-AM effect on fast Ca2+ binding and high Ca2+ affinity. BAPTA-AM concentrations as low as 0.05 microM were effective provided application was prolonged to 40 min. Probenecid (1 mM), an anion transport inhibitor, accelerated the onset and significantly enhanced the BAPTA-mediated synaptic attenuation caused by low concentrations of BAPTA-AM. These data show that even very low extracellular concentrations of BAPTA-AM can profoundly affect synaptic transmission provided that sufficient chelator accumulates presynaptically. The effectiveness of BAPTA-AM can be increased by procedures which inhibit chelator extrusion.

Animals↗
PubMed1989

A single-site allosteric model of intracellular androgen-receptor interaction.

We present a single-site, two-state model for analyzing the effect of time and ligand concentration on the extent and character of 5 alpha-dihydrotestosterone, methyltrienolone or mibolerone binding to the specific androgen receptor within cultured human genital skin fibroblasts. The model has three basic attributes: formation of the initial low-affinity androgen-receptor complex, and its transformation to a higher affinity state are irreversible, first-order processes; and receptors released from complexes in each state not only differ from each other and from their pre-liganded progenitor, but can also reassociate with androgen to yield complexes in their respective parental states. The rate constants of dissociation and apparent equilibrium binding constants of the two affinity states were determined for each of the three androgens within normal cells and those of a transformation-defective mutant. When these values are combined with estimates of the rate constants at which the complexes are formed and transformed, the model accurately simulates time-dependent changes in the slopes and character of experimental Scatchard plots. It can also generate Scatchard plots that are concave, convex or sigmoidal simply by making sequential changes in its formation or transformation constants. Thus, our model can explain complex ligand-receptor binding kinetics that have heretofore been interpreted according to alternate models of transformation and binding-site multiplicity with or without properties of cooperativity, and it supports the notion that receptor recycling involves intermediate receptor states.

Allosteric Regulation↗
PubMed1991

Characterization of recombinant human renin: kinetics, pH-stability, and peptidomimetic inhibitor binding.

The kinetic behavior and pH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P6-P3' renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. The pH-stability profile for renin, measured with the substrate at 25 degrees C, indicated a broad plateau of stability between pH 6.0 and 10.0. Analysis of the pH-activity profile of renin for the substrate indicated a minimum Km (approximately 1.8 microM) at pH approximately 7.4 and a maximum Vm between pH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P2 site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.

Amino Acid Sequence↗
PubMed1995

Evaluation of the rate constants of sugar transport through maltoporin (LamB) of Escherichia coli from the sugar-induced current noise.

LamB (maltoporin) of Escherichia coli outer membrane was reconstituted into artificial lipid bilayer membranes. The channel contains a binding site for sugars and is blocked for ions when the site is occupied by a sugar. The on and off reactions of sugar binding cause an increase of the noise of the current through the channel. The sugar-induced current noise of maltoporin was used for the evaluation of the sugar-binding kinetics for different sugars of the maltooligosaccharide series and for sucrose. The on rate constant for sugar binding was between 10(6) and 10(7) M-1.s-1 for the maltooligosaccharides and corresponds to the movement of the sugars from the aqueous phase to the central binding site. The off rate (corresponding to the release of the sugars from the channel) decreased with increasing number of glucose residues in the maltooligosaccharides from approximately 2,000 s-1 for maltotriose to 180 s-1 for maltoheptaose. The kinetics for sucrose movement was considerably slower. The activation energies of the stability constant and of the rate constants for sugar binding were evaluated from noise experiments at different temperatures. The role of LamB in the transport of maltooligosaccharides across the outer membrane is discussed.

Bacterial Outer Membrane Proteins↗
PubMed1997

Regulation of interleukin-12 receptor beta1 chain expression and interleukin-12 binding by human peripheral blood mononuclear cells.

The interleukin-12 receptor (IL-12R)beta1 chain is an essential component of the functional IL-12R on both human T and natural killer cells. In this report it is shown that activation of human peripheral blood mononuclear cells (PBMC) with anti-CD3 monoclonal antibody (mAb) or phytohemagglutinin resulted in the up-regulation of IL-12Rbeta1 expression and IL-12 binding. Kinetic studies revealed that maximum expression of IL-12Rbeta1 and IL-12 binding occurred on days 3-4. Anti-CD3-induced expression of IL-12Rbeta1 chain and IL-12 binding by PBMC was augmented by anti-CD28 mAb, indicating that the potentiating effect of anti-CD28 on T cell responses to IL-12 could be mediated, at least in part, by the enhancement of IL-12R expression. Among 16 cytokines tested, IL-2, IL-7 and IL-15 markedly induced IL-12Rbeta1 expression and IL-12 binding on resting PBMC, whereas IL-1alpha and tumor necrosis factor-alpha had a minimal enhancing effect. In contrast, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, interferon (IFN)-alpha, IFN-gamma, granulocyte/macrophage colony-stimulating factor and transforming growth factor (TGF)-beta2 had no detectable enhancing effect. Anti-CD3-induced expression of IL-12Rbeta1 and of low-affinity IL-12 binding sites was partially inhibited by TGF-beta2, IL-10 and IL-4; however, TGF-beta2 and IL-10 completely abolished anti-CD3-induced expression of high-affinity IL-12 binding sites. Consistent with the reduction of high affinity IL-12 binding sites, PBMC activated with anti-CD3 mAb in the presence of TGF-beta2 or IL-10 failed to produce IFN-gamma or to proliferate in response to IL-12. These results suggest that Th2 cell-derived cytokines can inhibit IL-12-induced biological functions by inhibiting IL-12R expression and that expression of a second subunit of the IL-12R (IL-12Rbeta2), required for the formation of high-affinity IL-12 binding sites, may be more highly regulated by TGF-beta2 and IL-10 than is expression of IL-12Rbeta1.

CD28 Antigens↗
PubMedDate not supplied

Reduced 3H-ouabain binding site (Na,K-ATPase) concentration in ventricular myocardium of dogs with tachycardia induced heart failure.

The present study evaluates 3H-ouabain binding site (Na,K-ATPase) concentration in left ventricular myocardium of dogs with heart failure induced by tachycardia as a result of ventricular pacing. Samples of left ventricle were obtained from 10 dogs exposed to pacing of 240 beats/min for 3 to 4 weeks and eight sham-operated controls. Na,K-ATPase was quantified using vanadate facilitated 3H-ouabain binding to intact samples. At time of sacrifice paced dogs showed clinical signs of heart failure, a significant 257% increase in left ventricular end diastolic pressure and a significant 46% decrease in left ventricular dP/dt compared with control. There was no significant change in left ventricular mass. 3H-ouabain binding concentration was significantly reduced by 16%. Evaluation of 3H-ouabain binding kinetics revealed no significant difference between myocardium from paced and control dogs: Equilibrium binding conditions were at the various concentrations used obtained after similar incubation time; nonspecific uptake and retention of 3H-ouabain was 0.9-0.8% of total uptake and retention obtained in the standard assay; apparent dissociation constant (KD) was 6.5 x 10(-8)-6.6 x 10(-8) mol/l; loss of specifically bound 3H-ouabain during washout at 0 degrees C occurred with a half-life time (T1/2) of 120 and 121 h. Hence, total 3H-ouabain binding site concentration in left ventricular myocardium was (mean +/- SEM) 1110 +/- 56 and 1317 +/- 68 pmol/g wet weight, 8.54 +/- 0.43 and 10.05 +/- 0.52 pmol/mg protein, and the total amount of 3H-ouabain binding sites in the entire left ventricle 121 +/- 6 and 162 +/- 8 nmol in paced (n = 10) and control (n = 8) dogs (p < 0.05), respectively. In conclusion, the present study reports a significant reduction in left ventricular myocardium 3H-ouabain binding site concentration in tachycardia induced heart failure. This observation supports the concept of a relationship between Na,K-ATPase concentration and contractile capacity and may be of pathophysiological importance in tachycardia and heart failure.

Animals↗
PubMed1992

Existence of three subtypes of bradykinin B2 receptors in guinea pig.

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1989

Inhibition of Na-K pump current in guinea pig ventricular myocytes by dihydroouabain occurs at high- and low-affinity sites.

Binding of cardiac glycosides to the Na+,K+-dependent ATPase has been shown to occur at both high- and low-affinity sites. However, recent reports suggest that glycoside-induced inhibition of electrogenic Na-K pump current occurs with simple first-order binding kinetics at relatively low-affinity sites. This implies that high-affinity binding sites have little to do with Na-K pump inhibition during exposure to cardiac glycosides. To better understand the role of the high-affinity site, we investigated the concentration dependence of Ipump inhibition by dihydroouabain (DHO) in guinea pig ventricular myocytes through use of wide-pore patch pipettes to "fix" internal Na+ activity at approximately 30 mM and to voltage clamp at -40 mV (T = 34 degrees C). DHO was found to have no effect on membrane conductance at a holding potential of -40 mV. Holding current was monitored and the difference between steady-state holding current before and during external exposure to nine concentrations (range, 0.01-1,000 microM) of DHO was measured and normalized to cellular membrane capacitance. The concentration dependence of the inhibition of Na-K pump current was biphasic and well fitted to a two-binding site model with inhibitory KD values of 0.05 microM and 64.5 microM. This is consistent with previously reported 3H-ouabain binding studies in guinea pig myocardium. These findings indicate that the electrogenic properties of the Na-K pump can be inhibited by glycoside binding to both high- and low-affinity sites.

Animals↗
PubMed1975

The prosence of estrogen receptor in kidneys from normal and androgen-insensitive tfm/y mice.

In vitro and in vivo binding of [3H]estradiol to cytosol proteins and nuclei was studied in kidneys from normal and androgen-insensitive tfm/y mice. The use of tfm/y mice permitted study of the estrogen-receptor complex in the absence of androgen receptor. A high affinity, [3H]estradiol-labeled, 8S molecule was demonstrated in low salt sucrose gradients. This macromolecule sedimented more slowly in gradients containing KC1 (0.5 M). Binding of [3H]estradiol was inhibited by 100-fold excess estrone, estradiol, or diethylstillbestrol but was not affected by testosterone, androstenedione, or progesterone. Studies of equilibrium-binding kinetics for estradiol indicated a Kd of 1.4 X 10(-9) M and 4.4 X 10(-14) mol of binding sites/mg cytosol protein. Furthermore, the binder was an acidic, heat-labile protein (pI = 4.8) which could be precipitated from cytosol with 33% ammonium sulfate, and needed sulfhydryl groups for activity. The demonstration of an estradiol-binding protein in vitro was correlated with specific [3H]estradiol uptake by tfm/y kidney nuclei in vivo. We concluded that the mouse kidney contains an estradiol-binding protein, distinct from that for androgens, which has many of the characteristics of a steroid receptor. The presence of an estrogen receptor in both normal and tfm/y mice indicates that a genetic defect in one receptor does not influence the properties of another. We concluded that androgen and estrogen receptors are under independent genetic control.

Animals↗
PubMed2001

Single-molecule analysis of chemotactic signaling in Dictyostelium cells.

Single-molecule imaging techniques were used to reveal the binding of individual cyclic adenosine 3',5'-monophosphate molecules to heterotrimeric guanine nucleotide-binding protein coupled receptors on the surface of living Dictyostelium discoideum cells. The binding sites were uniformly distributed and diffused rapidly in the plane of the membrane. The probabilities of individual association and dissociation events were greater for receptors at the anterior end of the cell. Agonist-induced receptor phosphorylation had little effect on any of the monitored properties, whereas G protein coupling influenced the binding kinetics. These observations illustrate the dynamic properties of receptors involved in gradient sensing and suggest that these may be polarized in chemotactic cells.

Animals↗
PubMed1980

Nerve growth factor receptors: analysis of the interaction of beta NGF with membranes of chick embryo dorsal root ganglia.

The binding of the beta subunit of Nerve Growth Factor (beta NGF) to membrane preparations of 8-day chick embryo dorsal root ganglia (DRG) has been investigated under conditions similar to those used to study the binding of beta NGF to intact single cell dissociates of DRG. The equilibrium binding data reveal heterogeneityy of binding that is more complex than that seen with intact cells. Binding is not saturable up to 125I beta NGF concentrations of 10(-8) M. Steady-state and kinetic binding data show two sites with dissociation constants similar to those found on DRG cells. In addition, displacement data reveal a binding component with lower affinity (Kd = 10(-6) M) which is not found on intact cells. As with intact cells, the difference in the affinities of the two high affinity sites has been shown to be due to different rate constants of dissociation. The kinetics of dissociation of NGF are slower with membranes than with cells, and dissociation characteristics of 125I beta NGF change with increasing time of exposure to membranes. Degradation of 125I beta NGF during incubation with membranes is minimal and does not complicate the analysis of steady-state binding. Insulin does not bind to either of the two high affinity sites. Heterogeneity of the 125I beta NGF preparation and cooperativity of binding as a cause for the heterogeneity of the binding of NGF has been ruled out. Although there was an apparent increase in the rate of dissociation of 125I beta NGF in the presence of unlabelled NGF, a finding previously interpreted as evidence for negative cooperativity, this was shown to be independent of receptor site occupancy by NGF, and in part due to isotopic dilution within a diffusion barrier around the membranes.

Animals↗
PubMed1982

Relevance of ferritin-binding sites on isolated mitochondria to the mobilization of iron from ferritin.

Iron can be released from ferritin and utilized by isolated rat liver mitochondria for the synthesis of heme. Mobilization of iron from ferritin is initiated by the binding of ferritin to the mitochondria in an manner compatible with binding sites or receptors for ferritin on the mitochondria. The binding completes rapidly, it is independent of temperature, saturable, reversible and enhanced by K+ and Mg2+. The amount of ferritin binding sites is approx. 0.8 pmol/mg mitochondrial protein, and the affinity constant is 6.4 . 10(6)M-1. The binding kinetics correlate well with the functional features of the ferritin-mitochondrial interaction: i.e. mobilization of iron from ferritin followed by insertion of the iron into heme. The results support the concept of ferritin as a possible donor of iron to the mitochondria.

Animals↗
PubMed2000

Kinetic scheme for intermolecular RNA cleavage by a ribozyme derived from hepatitis delta virus RNA.

A minimal kinetic mechanism for a trans-acting ribozyme derived from the HDV antigenomic RNA self-cleaving element was established from steady-state, pre-steady-state, single-turnover, and binding kinetics. Rate constants for individual steps, including substrate binding and dissociation, cleavage, and product release and binding, were measured at 37 degrees C at pH 8.0 in 10 mM Mg(2+) using oligonucleotides as either substrates, noncleavable analogues or 3' product mimics. A substrate containing a normal 3',5'-linkage was cleaved with a first-order rate constant (k(2)) of 0.91 min(-)(1). The association rate constant for the substrate to the ribozyme (2.1 x 10(7) M(-)(1) min(-)(1)) was at the lower range of the expected value for RNA duplex formation, and the substrate dissociated with a rate constant (1.4 min(-)(1)) slightly faster than that for cleavage. Thus the binary complex was not at equilibrium with free enzyme and substrate prior to the cleavage step. Following cleavage, product release was kinetically ordered in that the 5' product was released rapidly (>12 min(-)(1)) relative to the 3' product (6.0 x 10(-)(3) min(-)(1)). Rapid 5' product release and lack of a demonstrable binding site for the 5' product could contribute to the difficulty in establishing the ribozyme-catalyzed reverse reaction (ligation). Slow release of the 3' product was consistent with the extremely low turnover under steady-state conditions as 3' product dissociation was rate-limiting. The equilibrium dissociation constant for the substrate was 24-fold higher than that of the 3' cleavage product. A substrate with a 2',5'-linkage at the cleavage site was cleaved with a rate constant (k(2)) of 1.1 x 10(-)(2) min(-)(1). Thus, whereas cleavage of a 3',5'-linkage followed a Briggs-Haldane mechanism, 2', 5' cleavage followed a Michaelis-Menten mechanism.

Amino Acid Sequence↗
PubMed2002

The proline-rich domain of dynamin-2 is responsible for dynamin-dependent in vitro potentiation of endothelial nitric-oxide synthase activity via selective effects on reductase domain function.

The GTPase dynamin-2 (dyn-2) binds and positively regulates the nitric oxide-generating enzyme, endothelial nitric-oxide synthase (eNOS) (Cao, S., Yao, Y., McCabe, T., Yao, Q., Katusic, Z., Sessa, W., and Shah, V. (2001) J. Biol. Chem. 276, 14249-14256). Here we demonstrate, using purified proteins, that this occurs through a selective influence of the dyn-2 proline-rich domain (dyn-2 PRD) on the eNOS reductase domain. In vitro studies demonstrate that dyn-2 PRD fused with glutathione S-transferase (GST) binds recombinant eNOS protein specifically and with binding kinetics comparable with that observed between dyn-2 full-length and eNOS. Additionally, GST-dyn-2 PRD binds the in vitro transcribed (35)S-eNOS reductase domain but not the (35)S-eNOS oxygenase domain. Furthermore GST-dyn-2 PRD binds a (35)S-labeled eNOS reductase domain fragment (amino acids 645-850) that partially overlaps with the FAD binding domain of eNOS. A recombinant form of the SH3-containing protein Fyn competes the binding of recombinant eNOS protein with dyn-2 PRD, thereby implicating the SH3-like region contained within this reductase domain fragment as the dyn-2 binding region. Mammalian two-hybrid screen corroborates these interactions in cells as well. Functional studies demonstrate that dyn-2 PRD selectively potentiates eNOS activity in a concentration-dependent manner in an order of magnitude similar to that observed with dyn-2 full-length and in a manner that requires calmodulin. Although dyn-2 PRD does not influence eNOS oxygenase domain function or ferricyanide reduction, it does potentiate the ability of recombinant eNOS to reduce cytochrome c, supporting an influence of dyn-2 PRD on electron transfer between FAD and FMN. (These data indicate that the binding domains of dyn-2 and eNOS reside within the dyn-2 PRD domain and the FAD binding region of the eNOS reductase domains, respectively, and that dyn-2 PRD is sufficient to mediate dyn-2-dependent potentiation of eNOS activity, at least in part, by potentiating electron transfer.)

Amino Acid Sequence↗
PubMed1992

The human class II MHC protein HLA-DR1 assembles as empty alpha beta heterodimers in the absence of antigenic peptide.

We have produced the human class II histocompatibility protein, HLA-DR1, as a soluble, secreted glycoprotein in insect cells infected with baculoviruses carrying truncated alpha and beta subunit genes. The peptide-binding site is empty, and the empty molecules are fully competent to bind antigenic peptide. We used the empty molecules to measure an intrinsic rate for peptide association, and to investigate the role of peptide in stabilizing the class II structure. Peptide binding kinetics for the empty molecule are only 10-fold faster than for peptide exchange into an occupied site, suggesting that a conformational change may accompany peptide binding. The native alpha beta heterodimer assembles in the absence of antigenic peptide, but peptide binding stabilizes the empty heterodimer against aggregation and against SDS-induced denaturation.

Amino Acid Sequence↗
PubMed2006

Evolutional design of a hyperactive cysteine- and methionine-free mutant of Escherichia coli dihydrofolate reductase.

We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine- and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher k(cat) value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-angstroms resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-L-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.

Binding Sites↗
PubMed1986

Dopamine denervation does not alter in vivo 3H-spiperone binding in rat striatum: implications for external imaging of dopamine receptors in Parkinson's disease.

Striatal particulate preparations, both from rats with lesion-induced striatal dopamine (DA) loss and from some striatal dopamine (DA) loss and from some patients with Parkinson's disease, exhibit increased 3H-neuroleptic binding, which is interpreted to be the mechanism of denervation-induced behavioral supersensitivity to dopaminergic compounds. After intravenous 3H-spiperone (3H-SP) administration to rats with unilateral nigral lesions, we found no differences in accumulation of total or particulate-bound 3H-SP in dopamine-denervated compared with intact striata. 3H-SP in vivo binds to less than 10% of striatal sites labeled by 3H-SP incubated with striatal particulate preparations in vitro. Quantitative autoradiography of 3H-SP binding to striatal sections in vitro also failed to reveal any effects of dopamine denervation. 3H-SP bound to striatal sites in vivo dissociates more slowly than that bound to striatal particulate preparations labeled in vitro. Striatal binding properties of 3H-SP administered in vivo are quite different from the same kinetic binding parameters estimated in vitro using crude membrane preparations of striatum. In addition, striatal binding of in vivo-administered 3H-SP is not affected by prior lesion of the substantia nigra, which results in profound ipsilateral striatal dopamine depletion. Thus, behavioral supersensitivity to dopaminergic compounds may not be associated with altered striatal binding properties for dopamine receptor ligands in vivo.

Animals↗