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PubMedDate not supplied

Insulin receptors in rat brain cortex. Kinetic evidence for a receptor subtype in the central nervous system.

Binding kinetics of porcine 125I-insulin were studied in synaptosomal and microsomal fractions of rat brain cortex. Receptor binding was temperature- and pH-dependent with optimum at 4 degrees C and pH 8.0-8.3. At 15 degrees C, steady state binding was heterogenous, and Scatchard analysis revealed two classes of receptors with Kd of 2 nmol/l and 40 nmol/l in amounts of 50 pmol/g and 200 pmol/g of membrane protein. Dissociation kinetics were biexponential with T1/2 of about 5 min and 180 min, and in contrast to other cell-types, not influenced by negative cooperativity. No receptor-mediated insulin degradation was detectable at 37 degrees C in the presence of bacitracin. Insulin analogues inhibited 125I-insulin binding with potencies relative to porcine insulin (%): human insulin 100, rat insulin (I + II) 71, coypu insulin 47, rat multiplication stimulating activity 8, porcine proinsulin 5, among which the three last values were significantly higher than in rat liver and fat cells. No competition was observed with porcine relaxin and mouse nerve growth factor up to about 1 mumol/l. Receptors were present in all regions of central nervous system with highest concentrations in the cerebral cortex, cerebellum and olfactory bulb, and lowest in the pons, medulla oblongata and spinal cord. In conclusion, insulin receptors in rat brain cortex are functionally different from other tissues regarding the insulin specificity and the absence of negative cooperativity. It is suggested that an insulin receptor subtype in rat brain mediates the growth activity of insulin on nerve cells.

Animals↗
PubMed2003

Binding sites of nitrogenase: kinetic and theoretical studies of cyanide binding to extracted FeMo-cofactor derivatives.

The first kinetic study of a substrate (CN(-)) binding to the isolated active site (extracted FeMo-cofactor) of nitrogenase is described. The kinetics of the reactions between CN(-) and various derivatives of extracted FeMo-cofactor [FeMoco-L; where L is bound to Mo, and is NMF, Bu(t)NC, or imidazole (ImH)] have been followed using a stopped-flow, sequential-mix method in which the course of the reaction is followed indirectly, by monitoring the change in the rate of the reaction of the cofactor with PhS(-). The kinetic results, together with DFT calculations, indicate that the initial site of CN(-) binding to FeMoco-L is controlled by a combination of the electron-richness of the cluster core and lability of the Mo-L bond. Ultimately, the reactions between FeMoco-L and CN(-) involve displacement of L and binding of CN(-) to Mo. These reactions occur with a variety of rates and rate laws dependent on the nature of L. For FeMoco-NMF, the reaction with CN(-) is complete within the dead-time of the apparatus (ca. 4 ms), while with FeMoco-CNBu(t) the reaction is much slower and exhibits first order dependences on the concentrations of both FeMoco-CNBu(t) and CN(-) (k = 2.5 +/- 0.5 x 10(4) dm(3) mol(-1) s(-1)). The reaction of FeMoco-ImH with CN(-) occurs at a rate which exhibits a first order dependence on FeMoco-ImH but is independent of the concentration of CN(-) (k = 50 +/- 10 s(-1)). The results are interpreted in terms of CN(-) binding directly to the Mo site for FeMoco-NMF and FeMoco-ImH, but with FeMoco-CNBu(t) initial binding at an Fe site is followed by movement of CN(-) to Mo. Complementary DFT calculations are consistent with this interpretation, indicating that, in FeMoco-L, the Mo-L bond is stronger for L = ImH than for L = CNBu(t) and the binding of CN(-) to Mo is stronger than to any Fe atom in the cofactor.

Binding Sites↗
PubMed2001

Spectroscopic contributions to the understanding of hemoglobin function: implications for structural biology.

Structural biology is based on the assumption that structural determinations will explain macromolecular function. To examine the basis of these proposals, the structure/function connections in hemoglobin have been examined. Presently the Monod, Wyman, Changeux (MWC) model of hemoglobin function has great validity. In this model, ligand-binding affinities are linked to quaternary structure, and it has been shown that the model describes the function accurately to a high first approximation. To see how this understanding developed, we review two sets of experimental studies in 1970-71 that supported the applicability of MWC to hemoglobin oxygen binding. One set of data from NMR and ligand binding kinetics supported the quaternary-linked nature of binding required by the MWC model. The other approach, by Perutz, proposed a structural basis for MWC, by suggesting that in one quaternary structure the binding of oxygen broke a salt bridge that caused a lowered quaternary-linked affinity. However, experiments since that time, mostly by X-ray crystallography of deoxygenated hemoglobin, have failed to show salt bridges breaking upon ligation, whereas affinities have remained low. This pattern of results shows that the small energies responsible for ligand-binding affinities and reaction rates have not been identified by discrete structural features. Rather, thermodynamic and kinetic data from a variety of spectroscopic studies have played the central role in establishing the MWC model for hemoglobin.

Crystallography, X-Ray↗
PubMed2005

In vitro binding of purified NahR regulatory protein with promoter Psal.

The NahR regulatory protein activates the naphthalene catabolic operon through binding to the Psal promoter in the presence of salicylate. Here, we investigated in vitro binding interaction between NahR and Psal using purified functional recombinant NahR. The T7-tagged NahR was shown to exist as a monomer in solution. Electrophoretic mobility shift assay (EMSA) showed that purified NahR bound to Psal in 3 different forms, whereas surface plasmon resonance (SPR) showed on an SPR chip at ratios ranging from 1:1 (at 0.42 microM NahR) to 8:1 (at 6.8 microM NahR). The binding was slightly inhibited by salicylate, suggesting that salicylate may not be involved in the binding of NahR to the promoter, but rather may be important in the activation of prebound NahR. An examination of the binding kinetics by SPR for the interaction between NahR and Psal revealed that the equilibrium dissociation constant was approximately 2.44 x 10(-6) M and the association and dissociation rates were 7.82 x 10(4) M(-1) s(-1) and 0.191 s(-1), respectively. These results demonstrate for the first time that purified NahR binds as a monomer to Psal and undergoes multimerization. In addition, we present novel data on the kinetics of NahR binding.

Bacterial Proteins↗
PubMed1996

Simultaneous binding and bending of promoter DNA by the TATA binding protein: real time kinetic measurements.

The binding and bending of tetramethylrhodamine-5'-(GGGCTATAAAAGGG) duplex-3'-fluorescein by native Saccharomyces cerevisiae TATA binding protein (TBP) have been investigated using fluorescence resonance energy transfer. Probability distributions derived from fluorescein emission lifetime measurements show a decrease in the mean 3'-fluorescein-5'- rhodamine distance from 56.5 to 46.8 A upon binding of the oligomer to TBP, consistent with the DNA bend observed by X-ray crystallography. The kinetics, monitored in real time using stopped flow fluorimetry, demonstrate simultaneous binding and bending of a TATA box by TBP with a single second-order rate constant of (2.4 +/- 0.3) x 10(6) M-1 s-1 at 30 degrees C.

Base Sequence↗
PubMed2000

Interaction of the noncovalent molecular adapter, beta-cyclodextrin, with the staphylococcal alpha-hemolysin pore.

Cyclodextrins act as noncovalent molecular adapters when lodged in the lumen of the alpha-hemolysin (alphaHL) pore. The adapters act as binding sites for channel blockers, thereby offering a basis for the detection of a variety of organic molecules with alphaHL as a biosensor element. To further such studies, it is important to find conditions under which the dwell time of cyclodextrins in the lumen of the pore is extended. Here, we use single-channel recording to explore the pH- and voltage-dependence of the interaction of beta-cyclodextrin (betaCD) with alphaHL. betaCD can access its binding site only from the trans entrance of pores inserted from the cis side of a bilayer. Analysis of the binding kinetics shows that there is a single binding site for betaCD, with an apparent equilibrium dissociation constant that varies by >100-fold under the conditions explored. The dissociation rate constant for the neutral betaCD molecule varies with pH and voltage, a result that is incompatible with two states of the alphaHL pore, one of high and the other of low affinity. Rather, the data suggest that the actual equilibrium dissociation constant for the alphaHL. betaCD complex varies continuously with the transmembrane potential.

Bacterial Toxins↗
PubMed1982

Effects of reduction and alkylation on ligand binding and cation transport by Torpedo californica acetylcholine receptor.

The effects of sulfhydryl group modification on ligand binding and functional properties of the membrane-bound acetylcholine receptor from Torpedo californica have been investigated. Agonist binding kinetics were monitored by changes in fluorescence of the probe 5-(iodoacetamido)-salicylic acid which was covalently bound to the receptor after reduction of a reactive disulfide bond(s) by low concentrations of dithiothreitol. These labeling procedures did not affect either the equilibrium binding constant for [3H]acetylcholine or the number of high-affinity binding sites measured in centrifugation experiments. Further reduction of these labeled receptor preparations by higher concentrations of dithiothreitol and subsequent alkylation by excess iodoacetamide resulted in a more than 10-fold decrease in the affinity of the receptor for [3H]acetylcholine. This reduction and alkylation did not, however, radically alter the observed kinetics of acetylcholine binding. The fluorescence signal change on binding consisted of at least three phases similar to those observed for the control preparations, and the ligand concentration dependencies of the measured rate constants could be described by the same kinetic mechanism involving sequential binding of two ligand molecules and three conformational changes. Variation in the values of some of the kinetic parameters describing the formation of the monoliganded complex adequately accounted for the measured decrease in affinity for [3H]acetylcholine. Stopped-flow fluorescence experiments showed that extensive reduction and alkylation resulted in an apparent loss of the ability of the acetylcholine receptor to mediate agonist-induced cation flux. These results show that reduction of disulfide bonds by high concentrations of dithiothreitol followed by alkylation with iodoacetamide seriously perturbs receptor function although the receptor can still undergo its characteristic conformational changes on the binding of acetylcholine but with altered concentration dependence accounting for the reduced affinity for agonist.

Acetylcholine↗
PubMed2005

QZ1 and QZ2: rapid, reversible quinoline-derivatized fluoresceins for sensing biological Zn(II).

QZ1, 2-[2-chloro-6-hydroxy-3-oxo-5-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, and QZ2, 2-[6-hydroxy-3-oxo-4,5-bis-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, two fluorescein-based dyes derivatized with 8-aminoquinoline, have been prepared and their photophysical, thermodynamic, and zinc-binding kinetic properties determined. Because of their low background fluorescence and highly emissive Zn(II) complexes, QZ1 and QZ2 have a large dynamic range, with approximately 42- and approximately 150-fold fluorescence enhancements upon Zn(II) coordination, respectively. These dyes have micromolar K(d) values for Zn(II) and are selective for Zn(II) over biologically relevant concentrations of the alkali and alkaline earth metals. The Zn(II) complexes also fluoresce brightly in the presence of excess Mn(II), Fe(II), Co(II), Cd(II), and Hg(II), offering improved specificity for Zn(II) over di(2-picolyl)amine-based Zn(II) sensors. Stopped-flow kinetic investigations indicate that QZ1 and QZ2 bind Zn(II) with k(on) values of (3-4) x 10(6) M(-1) s(-1), compared to (6-8) x 10(5) M(-1) s(-1) for select ZP (Zinpyr) dyes, at 4.3 degrees C. Dissociation of Zn(II) from QZ1 and QZ2 occurs with k(off) values of 150 and 160 s(-1), over 5 orders of magnitude larger than those for ZP probes, achieving reversibility on the biological (millisecond) time scale. Laser scanning confocal and two-photon microscopy studies reveal that QZ2 is cell-permeable and Zn(II)-responsive in vivo. Because of its weaker affinity for Zn(II), QZ2 responds to higher concentrations of intracellular Zn(II) than members of the ZP family, illustrating that binding affinity is an important parameter for Zn(II) detection in vivo.

Aminoquinolines↗
PubMed2001

Disabling erbB receptors with rationally designed exocyclic mimetics of antibodies: structure-function analysis.

Overexpression of the HER2 receptor is observed in about 30% of breast and ovarian cancers and is often associated with an unfavorable prognosis. We have recently designed an anti-HER2 peptide (AHNP) based on the structure of the CDR-H3 loop of the anti-HER2 rhumAb 4D5 and showed that this peptide can mimic some functions of rhumAb 4D5. The peptide disabled HER2 tyrosine kinases in vitro and in vivo similar to the monoclonal antibody (Park, B.-W. et al. Nat. Biotechnol. 2000, 18, 194--198). AHNP has been shown to selectively bind to the extracellular domain of the HER2 receptor with a submicromolar affinity in Biacore assays. In the present paper, we demonstrate that in addition to being a structural and functional mimic of rhumAb 4D5, AHNP can also effectively compete with the antibody for binding to the HER2 receptor indicating a similar binding site for the peptide and the parental antibody. To further develop AHNP as an antitumor agent useful for preclinical trials and as a radiopharmaceutical to be used for tumor imaging, a number of derivatives of AHNP have been designed. Structure--function relationships have been studied using surface plasmon resonance technology. Some of the AHNP analogues have improved binding properties, solubility, and cytotoxic activity relative to AHNP. Residues in the exocyclic region of AHNP appear to be essential for high-affinity binding. Kinetic and equilibrium analysis of peptide-receptor binding for various AHNP analogues revealed a strong correlation between peptide binding characteristics and their biological activity. For AHNP analogues, dissociation rate constants have been shown to be better indicators of peptide biological activity than receptor-binding affinities. This study demonstrates a possibility of mimicking the well-documented antibody effects and its applications in tumor therapy by much smaller antibody-based cyclic peptides with potentially significant therapeutic advantages. Strategies used to improve binding properties of rationally designed AHNP analogues are discussed.

Animals↗
PubMed1979

Structural relationships between the chick oviduct progesterone receptor A and B proteins.

The chick oviduct contains two distinct forms of the progesterone receptor, termed progestophilins A (Mr = 79,000) and B (Mr = 117,000). Although these two hormone-binding proteins differ significantly in physico-chemical characteristics, a good deal of similarity exists between these two molecules. Thus, both proteins display identical hormone-binding kinetics and steroid specificity. The hormone-binding fragments obtained after the action of an endogenous oviduct Ca2+-activated protease are indistinguishable as analyzed by gel filtration chromatography. Both the A and B proteins are capable of binding to DNA-cellulose, although they elute at very different salt concentrations when subjected to gradient elution. In spite of these similarities all attempts to demonstrate the conversion of progestophilin B to A or to show a common precursor to both have been unsuccessful. Therefore, either the A and B receptor proteins are separate products of closely related genes or the conversion of one form to another occurs very rapidly either in vivo or in vitro.

Animals↗
PubMed1983

A new approach to the measurement of sigmoid curves with enzyme kinetic and ligand binding data.

1. It is shown that the extent of sigmoidicity in kinetic or binding curves can always be characterized by two parameters, omega and delta, even when substrate inhibition or other causes of deviations from hyperbolic character are present. 2. The parameter omega is defined in such a way as to measure the fraction of the vertical span of the curve that is sigmoid. 3. The parameter delta is defined in such a way as to measure the S-shaped character in the sigmoid region. 4. It is shown that limits exist to the maximum values of omega and delta for degree n : n and so any individual v(S) or y(x) curve can be described as being barely sigmoid or very sigmoid by a comparison of measured omega and delta values with the limiting values. 5. Monte Carlo simulations of 12 kinetic mechanisms and 6 binding models were performed and the probability density functions and cumulative distribution functions for omega and delta were calculated. 6. An empirical study was performed on the delta values required before experimentalists can recognize a set of data points with error as being sigmoid. 7. The probability with which representative kinetic mechanisms or binding schemes give rise to complex curve shape features has been estimated before. Here using the parameters omega and delta, we calculate the conditional probabilities that sigmoid curves can occur in physiological ranges of substrate or ligand concentration and be sufficiently exaggerated to be recognized as S-shaped. 8. It is shown that some mechanisms, e.g. the random bi bi one, are very unlikely to give strongly sigmoid curves. Such exaggerated curves were found to be more typical of simple sequential kinetic schemes and binding models. 9. It is shown that omega and delta values measured experimentally can sometimes be used in model discrimination and fixing the minimum degree of rate equations. 10. A very powerful result is that in saturation functions of order n the maximum height of the curve that can be sigmoid is (n - 1)/n, and a possible evolutionary significance for this is suggested.

Enzymes↗
PubMed2005

Integrated insights from simulation, experiment, and mutational analysis yield new details of LacI function.

Protein structural change underlies many signal transduction processes. Although end-state structures are known for various allosteric proteins, intermediates are difficult to observe. Recently, targeted molecular dynamics simulation (TMD) was used to examine the conformational transition and predict relevant intermediates for wild-type lactose repressor (LacI). A catalog of involved residues suggests that the transition of this homodimer is asymmetric and that K84 is a prominent participant in the dynamic N-subdomain interface. Previous experiments indicated that hydrophobic substitutions at position 84 engender slowed, biphasic inducer binding kinetics, which might reflect the same phenomena observed in TMD. Here, we report biochemical confirmation that DNA and inducer binding remain allosterically linked in K84A and K84L, albeit with a differential smaller than that found in wild-type LacI. Other features of these mutant proteins are consistent with an allosteric conformational shift that approximates that of the wild type. As a consequence, these repressors can be utilized to explore an unanswered question about LacI function: How many inducers (one or two per dimer) are required to diminish operator affinity? The biphasic natures of the K84L and K84A inducer association rates allow direct correlation between the two distinct inducer binding events and operator release. Indeed, the kinetics of operator release for the K84A and K84L closely parallel those for the second inducer binding event. Together with implications from previous equilibrium results for wild-type and mutant proteins, these kinetic data demonstrate that binding of two inducers per dimeric DNA binding unit is required to release the operator in these variant LacI proteins.

Amino Acid Substitution↗
PubMed1988

Affinities of Treponema pallidum for human lactoferrin and transferrin.

The acquisition of lactoferrin and transferrin by live Treponema pallidum organisms was examined. Saturation binding kinetics were obtained for virulent treponemes with increasing amounts of radioiodinated lactoferrin but not with transferrin. Furthermore, lactoferrin bound up to 100 times more effectively than transferrin. Only unlabelled lactoferrin stoichiometrically completed with iodinated lactoferrin binding. Time course kinetics showed maximum lactoferrin acquisition within the first five minutes at 34 degrees C. Optimum iron accumulation, however, was achieved by T pallidum in 30 minutes at 34 degrees C, and amounts of iron were six times greater than the equivalent amount of lactoferrin bound. Interestingly, iron uptake was also detected in the presence of transferrin, despite the minimal amounts of transferrin acquired by live treponemes. These observations reinforce the possibility that the associations of T pallidum with host molecules, such as plasma proteins, are essential for survival of the parasite within host environments.

Humans↗
PubMed2001

Hybridization of 2'-ribose modified mixed-sequence oligonucleotides: thermodynamic and kinetic studies.

In this study, we characterize the thermodynamics of hybridization, binding kinetics and conformations of four ribose-modified (2'-fluoro, 2'-O-propyl, 2'-O-methoxyethyl and 2'-O-aminopropyl) decameric mixed-sequence oligonucleotides. Hybridization to the complementary non-modified DNA or RNA decamer was probed by fluorescence and circular-dichroism spectroscopy and compared to the same duplex formed between two non-modified strands. The thermal melting points of DNA-DNA duplexes were increased by 1.8, 2.2, 0.3 and 1.3 degrees C for each propyl, methoxyethyl, aminopropyl and fluoro modification, respectively. In the case of DNA-RNA duplexes, the melting points were increased by 3.1, 4.1 and 1.0 degrees C for each propyl, methoxyethyl and aminopropyl modification, respectively. The high stability of the duplexes formed with propyl-, methoxyethyl- and fluoro-modified oligonucleotides correlated with high preorganization in these single-strands. Despite higher thermodynamic duplex stability, hybridization kinetics to complementary DNA or RNA was slower for propyl- and methoxyethyl-modified oligonucleotides than for the non-modified control. In contrast, the positively-charged aminopropyl-modified oligonucleotide showed rapid binding to the complementary DNA or RNA.

Base Pairing↗
PubMed1991

High-affinity binding sites for bombesin on mouse colonic mucosal membranes.

Bombesin (BBS) has specific biological effects on colonic mucosal cells, but the presence of BBS receptors on colonic mucosa have not been described to-date. In the present study we examined the mouse colonic mucosal membranes for the presence of specific binding sites for BBS/gastrin releasing peptides (GRP), and characterized the binding kinetics and molecular weight of the specific binding proteins. The radiolabeled ligand (125I-Tyr4-BBS), in the absence or presence of a 1000-fold excess of BBS, was used to establish the optimal binding assay conditions of time, pH and temperature for measuring the maximum number of specific binding sites for BBS related peptides. Under the optimal binding assay conditions, BBS displaced the binding of 125I-Tyr4-BBS in a dose-related manner. A single class of high-affinity binding sites (Kd = 0.23 +/- 0.02 nM) for BBS were measured, with a binding capacity of 27.3 +/- 4.6 fmoles/mg membrane protein. The binding sites were specific for binding BBS/GRP related peptides, since all structurally related peptides inhibited the binding of 125I-Tyr4-BBS in a dose-dependent manner, while structurally unrelated peptides did not compete for the 125I-Tyr4-BBS binding sites. The relative binding affinity (RBA) of BBS/GRP related peptides was determined to be in the order of GRP (14-27) = GRP (18-27) greater than GRP (1-27) greater than neuromedin B greater than BBS. The BBS-receptor antagonists, [Leu13-psi-(CH2NH) Leu14]-BBS (LL-BBS) and D-Phe6, BN(6-13) propylamide (D-Phe6, BN(6-13)-PA), inhibited the specific binding of 125I-Tyr4-BBS to colonic mucosal membranes in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Voltammetric studies of the interaction of quinacrine with DNA.

The binding interaction of the antimalarial drug quinacrine with herring sperm deoxyribonucleic acid (DNA) has been studied by square wave voltammetry. The binding parameters, the binding constant K and the binding site size s, were obtained simultaneously by the analysis of bound and free quinacrine concentration corresponding to the limits of slow and fast binding kinetics compared to the experimental timescale. The binding constant and the binding site size for the interaction of quinacrine with DNA were K = 1.59 (+/-0.18)x10(5) M(-1) and s = 7.1 (+/-0.15) base pairs and K = 7.35 (+/-0.83)x10(5) M(-1), s = 6.2 (+/-0.02) base pairs for the limiting conditions of static and mobile binding equilibrium respectively. The standard Gibbs free energy change (Delta G0 = - RT ln K) is approximately -29.67 kJ/mol at 25 degrees C, which highlights the spontaneity of the binding of quinacrine with DNA. The binding of quinacrine to herring sperm DNA results in peak potential shifts in voltammetric and a red shift in UV-absorption measurements. The ionic strength dependence of the binding constant is not large. Furthermore, the relative viscosity of DNA increases in the presence of quinacrine. These characteristics strongly support the intercalation of quinacrine into DNA. The results also show that the intercalation of quinacrine into DNA may occur at approximately every seventh base pair.

Animals↗
PubMed1976

The binding properties and regional ontogeny of receptors for alpha-bungarotoxin in chick brain.

The subcellular distribution, binding kinetics, pharmacologic properties and regional ontogeny of binding sites for alpha-[125I]bungarotoxins (alpha-[125I]BT) were studied in preparations of chick brain. Almost 95% of the specific binding was found in a 20,000 X g pellet. Two binding components were found, one with high (half-saturation at 1.5 X 10(-9) M) and another with low affinity for the toxin. The rate constants of association and dissociation of the toxin for the high-affinity site were found to be 3.5 X 10(4) M-1 - sec-1 and 3.8 X 10(-5) sec-1 (tau1/2 = 5.1 h, respectively). These data yielded a K alpha value of 1.1 X 10(-9) M. Nicotinic, but not muscarinic ligands were potent inhibitors of toxin binding at the high-affinity site. Thus, as in muscle, the binding site for toxin in chick brain appears to have the properties of a nicotinic acetylcholine (ACh) receptor. The ontogeny of the alpha-BT/ACh receptor was studied using homogenates of both whole brain and of discrete brain regions. In brain stem, optic lobe and cerebellum specific binding reached maximum levels in ovo and then decreased by maturity to values of 10,22 and 4.5 fmoles/mg protein, respectively. In contrast, whole brain and cerebral hemispheres reached plateau levels in ovo of 17 and 10 fmoles/mg protein, respectively. Major increases in total receptor number per region appeared in ovo between days 12 and 19 in brain stem and cerebellum, days 12 and 15 in optic lobes, and days 15 and 19 in hemispheres. The hemispheres were the only region to show a continued increase in total binding after hatching (3-fold increase by maturity). These findings are interpreted with respect to the development of chick brain and a model is suggested for the relationship between receptor ontogeny and neuronal differentiation and maturation.

Age Factors↗
PubMed1987

L-aspartate and L-glutamate binding sites in developing normal and 'nervous' mutant mouse cerebellum.

This study concerns the ontogeny and the cellular localization of L-aspartate and L-glutamate binding sites in normal and 'nervous' mutant mouse cerebellar membranes. The binding kinetics revealed for L-aspartate a single binding system (Kd = 750 nM) and for L-glutamate also a single binding component of higher affinity (Kd = 344 nM). The pharmacological study, using various amino acid analogues, revealed a differential specificity for the binding sites of the two amino acids. The developmental study showed that the binding sites of both amino acids appear mainly during the second and third week of life, a period when parallel and climbing fiber synaptogenesis occurs, but they follow a slightly different developmental pattern. The study using 'nervous', mutant mouse cerebellum showed an age-dependent decrease of L-aspartate and L-glutamate binding, which coincides in time with the Purkinje cell degeneration in this mutant, indicating a cellular localization of these binding sites on the Purkinje cell membranes. These results suggest that L-aspartate and L-glutamate binding sites may be respectively associated with the postsynaptic target of climbing and parallel fibers on the Purkinje cell dendrites. However, the decrease of specific binding in 'nervous' mutant mouse cerebellum was about 50% for L-aspartate and 60% for L-glutamate, implying that a significant number of L-aspartate and L-glutamate binding sites are located on cerebellar elements other than the Purkinje cell membranes.

Aging↗