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At least 73 records · Page 4Linked to original sources
PubMed1987

Quantitative analysis of the interaction between immune complex and C1q complement subcomponent. The role of interdomain interactions in rabbit IgG in binding of C1q to immune precipitates.

A novel method was developed for the analysis of the interaction of large multivalent ligands with surfaces (matrices) to analyse the binding of complement subcomponent C1q to immune precipitates. Our new evaluation method provides quantitative data characteristic of the C1q-immune-complex interaction and of the structure of the immune complex as well. To reveal the functional role of domain-domain interactions in the Fc part of IgG the binding of C1q to different anti-ovalbumin IgG-ovalbumin immune complexes was studied. Immune-complex precipitates composed of rabbit IgG in which the non-covalent or covalent bonds between the heavy chains had been eliminated were used. Non-covalent bonds were abolished by splitting off the CH3 domains, i.e. by using Facb fragments, and the covalent contact was broken by reduction and alkylation of the single inter-heavy-chain disulphide bond. The quantitative analysis of the binding curves provides a dissociation constant (K) of 200 nM for the interaction between C1q and immune precipitate formed from native IgG. Surprisingly, for immune precipitates composed of Facb fragments or IgG in which the inter-heavy-chain disulphide bond had been selectively reduced and alkylated, stronger binding (K = 30 nM) was observed. In this case, however, changes in the structure of the immune-complex matrix were also detected. These structural changes may account for the strengthening of the C1q-immune-complex interaction, which can be strongly influenced by the flexibility and the binding-site pattern of the immune-complex precipitates. These results suggest that domain-domain interactions in the Fc part of IgG affect the segmental mobility of IgG molecules and the spatial arrangement of the immune-complex matrix rather than the affinity of individual C1q-binding sites on IgG.

Animals↗
PubMed1997

Interaction of the maize and Arabidopsis kinase interaction domains with a subset of receptor-like protein kinases: implications for transmembrane signaling in plants.

The kinase interaction (KI) domain of kinase-associated protein phosphatase (KAPP) interacts with the phosphorylated form of an Arabidopsis thaliana receptor-like protein kinase (RLK). The KI domain may recruit KAPP into an RLK-initiated signaling complex. To examine additional roles that this domain may play in plant signal transduction, a search was conducted for other KI domain-containing proteins. One gene was isolated which encodes a KI domain, the maize homolog of KAPP. To test whether the maize KI domain associates with other maize proteins, it was used as a probe in a protein-protein interaction cloning strategy. A new maize RLK, KI domain interacting kinase 1 (KIK1), was identified by its interaction with the maize KI domain. The maize KI domain and the KIK1 kinase domain association required phosphorylation of the kinase. This work establishes that the KI domain phosphorylation-dependent signaling mechanism is present in both monocots and dicots. Additionally, it was determined that both the maize and Arabidopsis KI domains interact with several but not all of the active RLKs assayed. These multiple associations imply that KAPP may function in a number of RLK-initiated signaling pathways.

Amino Acid Sequence↗
PubMed1995

A novel ubiquitous form of Munc-18 interacts with multiple syntaxins. Use of the yeast two-hybrid system to study interactions between proteins involved in membrane traffic.

Munc-18-1 is a 67-kDa neuronal protein that binds tightly to syntaxin 1 and functions in synaptic vesicle exocytosis (Hata, Y., Slaughter, C.A., and Südhof, T.C. (1993a) Nature 366, 347-351). We have now characterized a new Munc-18 isoform, Munc-18-2, that exhibits 63% amino acid sequence identity with Munc-18-1. Munc-18-2 is expressed in most tissues, whereas Munc-18-1 is primarily expressed in brain. Using recombinant Munc-18-1 and Munc-18-2 produced in COS cells, we show that both forms of Munc-18 bind tightly to syntaxins 1A, 2, and 3 but not to syntaxin 4. In an independent approach to study the binding specificities of Munc-18-1 and Munc-18-2, we used the yeast two-hybrid system. This assay system depends on protein-protein interactions in the cell nucleus. We validated its utility for studying membrane trafficking proteins by testing well characterized interactions between cytosolic proteins that are known to be physiologically important in exocytosis. Strong interactions, such as the binding of syntaxins 1-4 with SNAP-25, were effectively detected by the yeast two-hybrid assay, but weak binding, such as the binding of syntaxins to synaptotagmin or of synaptotagmin to neurexins, was not. Studies on full-length and truncated forms of Munc-18s by the yeast two-hybrid system confirmed their interactions with syntaxins. Both the N and the C terminus of Munc-18 were essential for binding. Munc-18-1 and Munc-18-2 bind only to syntaxins 1A, 2, and 3 but not 4 and 5 by yeast-two hybrid system assays. Our studies demonstrate that neural and non-neural tissues have distinct forms of Munc-18, which may function in different types of exocytosis. The lack of specificity of the interactions between syntaxins and Munc-18s indicates that specificity of membrane trafficking reactions is not dependent on this interaction.

Amino Acid Sequence↗
PubMed1973

Cell interactions between histoincompatible T and B lymphocytes. II. Failure of physiologic cooperative interactions between T and B lymphocytes from allogeneic donor strains in humoral response to hapten-protein conjugates.

Several experimental approaches, designed specifically to circumvent the possible contribution of a complicating "allogeneic effect," have been successfully used to answer the question of physiologic cooperative interactions between histoincompatible T and B lymphocytes in antibody responses to hapten-protein conjugates. This was accomplished for in vivo cell transfer studies by using an F(1) hybrid host as the recipient of irradiated, carrier-primed T lymphocytes from one parent and 2,4-dinitrophenyl (DNP)-primed B lymphocytes from the opposite strain. Under these conditions, very good T-B cell cooperative interactions were observed to occur between T and B lymphocyte populations derived from syngeneic donors, whereas no cooperative response was obtained when T cells were derived from one parental strain and B cells from the other. Corroborative experiments were performed in a totally in vitro system in which DNP-primed B cells developed good secondary anti-DNP antibody responses in vitro to soluble DNP-keyhole limpet hemocyanin (KLH) when cultured in the presence of irradiated KLH-primed T cells derived from syngenic donors but not from allogeneic donors. The failure of histoincompatible T and B lymphocytes to effect physiologic cooperative interactions has important implications for our understanding of how such interactions normally occur. The possibility that these results reflect the existence of a "block" of some sort to cell-cell interaction by virtue of the presence of a foreign major histocompatibility antigen on the surface of either cell has been definitively ruled out in the present studies. These observations demonstrate that the gene(s) that conditions the capability for physiologic T-B cell cooperation must be shared in common by the respective cell types, and suggest, furthermore, that this gene (or genes) belongs to the major histocompatibility system of the mouse. These findings, together with other relevant phenomena described previously, have led us to postulate that there exists on the B lymphocyte surface an "acceptor" molecule either for the putative active T cell product or for the T cell itself. The important genetic considerations and the possible sequence of events surrounding the actual T-B cell interaction implied by these postulates are discussed in detail.

Animals↗
PubMed1996

Interaction of 5,7-dihydroxy-4'-methoxyflavone with a multisubunit protein, carmin: thermodynamics and kinetics of interaction.

Acacetin (5,7-dihydroxy-4'-methoxy flavone) is a flavone intrinsically present in the seeds of Carthamus tinctorius. Carmin is a multimeric, high molecular weight protein from the seeds of Carthamus tinctorius. The association constant of interaction of acacetin and carmin is maximum at 37.2 degrees C with a value of (3.96 +/- 0.61) x 10(4) M-1 as measured by fluorescence quenching. Acacetin has at least two binding sites on carmin. The interaction follows pseudo-first-order kinetics with a reaction rate constant of 3.4 +/- 0.4 s-1. The titration calorimetric data suggest that binding sites for acacetin and its structural analogue, biochanin A, are conserved. The interaction does not affect the association-dissociation equilibrium of the protein. Also, the binding does not induce any significant conformational changes in the protein as monitored by circular dichroic spectra. Biochanin A (5, 7-dihydroxy-4'-methoxyisoflavone), a structural analogue, interacts with carmin with an association constant of (9.33 +/- 1.44) x 10(4) M-1 at 36.9 degrees C. This indicates that the stereochemistry of the ligand plays an important role in the binding process of flavone to protein. Interaction studies of chemically modified lysyl and tryptophanyl groups separately, and lysyl and tryptophanyl groups sequentially, in the protein carmin with the ligands reveal the involvement of tryptophanyl residues in the binding process and show that it is predominantly an entropically driven hydrophobic interaction.

Flavones↗
PubMed1989

Dual Mg2+ control of formyl-peptide-receptor--G-protein interaction in HL 60 cells. Evidence that the low-agonist-affinity receptor interacts with and activates the G-protein.

In neutrophils and several other phagocytic cell types, a pertussis- and cholera-toxin-sensitive form of the guanine-nucleotide-binding protein (G-protein) Gp couples receptors for N-formylmethionine-containing chemotactic peptides to stimulation of phospholipase C. Using membranes of myeloid differentiated HL 60 cells, we have examined the role of Mg2+ and guanine nucleotides in regulating (a) the interaction of the formyl-peptide receptor with the chemotactic agonist N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and (b) the receptor-mediated activation of Gp. Mg2+ markedly enhanced the number of receptors with high affinity for the radiolabeled oligopeptide fMet-Leu-[3H]Phe. At the same time, Mg2+ largely increased the potency of guanosine-5'-(3-O-thio)triphosphate, but not of GDP or guanosine-5'-(2-O-thio)diphosphate, to inhibit binding of the peptide. Comparison of the potency of Mg2+ in eliciting these two effects and analysis of the specificities of the relevant divalent cation sites revealed that Mg2+ interacts with at least two independent sites on the receptor-Gp complex. One site is specific for Mg2+ and exhibits affinity in the micromolar range, the other site interacts with millimolar concentrations of several divalent cations in a non-selective fashion. It is suggested that the former site is located on Gp and that interaction of Mg2+ with this site is necessary for the receptor-mediated G-protein activation, whereas interaction of divalent cations with the latter site is necessary for high affinity agonist binding. The regulation of the formyl-peptide receptor binding properties by guanine nucleotides is independent of Gp activation, since inhibition of peptide binding is achieved by addition of both guanine nucleoside diphosphates and triphosphates and is readily seen both in the presence and in the absence of Mg2+. The latter finding, together with the observation that, at micromolar concentrations of Mg2+, high-affinity GTPase activity is stimulated by fMet-Leu-Phe primarily via low affinity receptors, suggests that, contrary to widely held opinions, (a) divalent cations are not required for a functional receptor--G-protein interaction and (b) high-affinity agonist binding is not a prerequisite for the receptor-mediated activation of the G-protein.

Binding Sites↗
PubMed1985

Interaction of opiates with opioid binding sites in the bovine adrenal medulla: II. Interaction with kappa sites.

In this study we examined the interaction of opiates with kappa binding sites in the bovine adrenal medulla. [3H]Ethylketocyclazocine (EKC), [3H]etorphine, and [3H]bremazocine stereoselective bindings were used to assay these interactions. The kappa sites were found to be heterogeneous: [3H]bremazocine identified with high affinity all subtypes of these sites. [3H]EKC, in the presence of saturating concentrations of [D-Ala2, D-Leu5]-enkephalin (DADLE) (5 microM), was used to identify kappa 1 sites, on which dynorphin A (1-13) bound with high affinity. Either [3H]EKC or [3H]etorphine in the presence of 5 microM DADLE identified the kappa 2 subtype. This subtype was found to interact with beta-endorphin and especially with the octapeptide Met5-enkephalyl-Arg6-Gly7-Leu8. Furthermore, [3H]etorphine identified in the bovine adrenal medulla a third high-affinity component, in the presence of 5 microM DADLE. This residual interaction was found to be equally stereoselective and presenting kappa selectivity. Met5-enkephalyl-Arg6-Phe7 interacted preferentially with this site. The three kappa subtypes interacted differentially with monovalent (Na+, K+, and Li+) and divalent (Ca2+, Mg2+, and Mn2+) ions by modification of the apparent concentration of the accessible sites and/or by changes of the apparent KD for radioligands. Modifying agents (proteolytic enzymes, thiol-modifying reagents, and A2-phospholipase) produced different effects on each subtype of the kappa site, suggesting a different protein (or protein-lipid?) composition.

Adrenal Medulla↗
PubMedDate not supplied

Hydrophilic interactions between charged amino acids and the effect of ions on the strength of interaction.

The interaction between oppositely charged amino acids was studied by charge-transfer reversed-phase thin-layer chromatography. The dependence of the lipophilicity of Arg, Lys and Orn on the concentration of Glu, Asp, Gln and Asn in the eluent was considered to be related to the strength of interaction. The interaction of dibasic amino acids with Glu and Asp was stronger than with Gln and Asn. Mono- (Li+, Na+, K+, Rb+ and Cs+) and divalent (Mg2+ and Ca2+) cations decreased the strength of interaction suggesting the electrostatic character of the interaction. Their inhibitory effect mainly depended on their concentration and to a lesser extent on the ion charge and hydrated ion radii. Stepwise regression analysis proved that the strength of interaction depends on the polarity parameters of amino acids and is independent of their chemical structure.

Amino Acids↗
PubMed1976

Beta-Adrenergic receptor interactions. Direct comparison of receptor interaction and biological activity.

Iodohydroxybenzylpindolol (I-HYP) is a chemically defined, high affinity, high specific activity beta-adrenergic antagonist that interacts with a single site on the turkey erythrocyte membrane. Study of the interaction of agonists, antagonists, and congeners with this site and concomitant alterations in adenylate cyclase activity have been carried out in the presence of high or low concentrations of guanine nucleotide. The results help clarify the relationship between binding and activation or inhibition of adenylate cyclase and the role of guanine nucleotides in modulating this interaction. There is a close correlation between binding constants (KD) for inhibitors determined by analysis of competitive displacement of 125I-HYP from receptor, and apparent affinities (Ki) for inhibition of adenylate cyclase. For activators, however, there is up to a 10-fold difference between KD and apparent affinity (KDapp) for adenylate cyclase activation at low guanine nucleotide concentration (10(-6) M guanylylimidodiphosphate). This difference is virtually abolished by employing higher nucleotide concentrations (10(-5) M guanylylimidodiphosphate) without significantly altering receptor affinity. This suggests that guanine nucleotides act by modulating receptor-enzyme interactions rather than hormone-receptor interactions. Moreover, several beta-adrenergic analogs previously shown to have no effect on adenylate cyclase in the absence of nucleotide, are partial agonists in the presence of 10(-5) M guanylylimidodiphosphate. Parallel analyses for a series of agonists and antagonists for adenylate cyclase activation and receptor interaction show affinities for levorotatory isomers generally 100-fold greater than for dextrorotatory isomers. Thus stereoconfiguration at the beta carbon clearly influences affinity of agonists or antagonists. Affinity is also importantly influenced by the nature of the aromatic ring as well as the N-alkyl group. The complexity of structure-function relationships for these compounds requires a redefinition of structural requirements for beta-adrenergic activity.

Adenylyl Cyclases↗
PubMed1991

Interaction of vitamin K-dependent protein Z with thrombin. Consequences for the amidolytic activity of thrombin and the interaction of thrombin with phospholipid vesicles.

Protein Z is a vitamin K-dependent protein of unknown function present in normal bovine plasma at a concentration of approximately 0.1 microM. Quantitative affinity chromatographic studies using diisopropylphosphoryl (DIP)-thrombin-Affi-Gel 10 as the affinity matrix and free DIP-thrombin as the competitor demonstrated that protein Z interacts with DIP-thrombin with a dissociation constant of 0.15 +/- 0.05 microM. Binding was independent of Ca2+. Protein C and factor IX, other vitamin K-dependent clotting proteins with the same domain structure as that of protein Z, did not interact with immobilized DIP-thrombin under these conditions; and factor X interacted with an affinity 20-fold lower than that for protein Z. The Michaelis constant, Km, for hydrolysis of pyro-Glu-Pro-Arg-p-nitroanilide by thrombin was increased 1.8-fold, from 130 to 230 microM, as a result of the binding of protein Z and the Km for H-Val-Leu-Arg-p-nitroanilide 1.4-fold, from 390 to 560 microM. From these kinetic studies, a dissociation constant of 0.11 +/- 0.04 microM was calculated for the binding of protein Z to alpha-thrombin. Protein Z bound to large phospholipid vesicles (25% phosphatidylserine, 75% phosphatidylcholine) with a dissociation constant of 0.39 +/- 0.16 microM at a phospholipid to protein ratio of 82 mol of phospholipid/mol of protein Z at saturation. In the presence of protein Z thrombin associated with phospholipid vesicles, whereas thrombin did not interact with phospholipid vesicles in the absence of protein Z. These studies, therefore, demonstrate a physiologically relevant interaction between protein Z and thrombin. They also suggest a mechanism whereby thrombin is localized to an injury site by virtue of its interaction with protein Z bound to phospholipid surfaces.

Amidohydrolases↗
PubMed1990

High affinity interaction between C4b-binding protein and vitamin K-dependent protein S in the presence of calcium. Suggestion of a third component in blood regulating the interaction.

The anticoagulant vitamin K-dependent protein S interacts with the complement regulatory protein C4b-binding protein (C4BP), both in purified systems and in plasma. The concentrations of these proteins in plasma are approximately equimolar (0.3 microM) and 30-40% of protein S in plasma is found in the noncomplexed state. Only the uncomplexed form of protein S displays anticoagulant activity and studies have shown that patients with a selective deficiency of free protein S have a high incidence of thrombosis. In this study, we report that the protein S-C4BP interaction is at least 100-fold tighter in the presence of Ca2+ than in EDTA. The KD in the presence of Ca2+ was estimated with a gel filtration technique to be less than 5 x 10(-10) M, whereas in the presence of EDTA, it was approximately 100-fold higher. Ca2+ titration experiments suggested that the Ca2+ sites which function in the protein S-C4BP interaction are of high affinity which, in turn, suggests that they may be independent of the gamma-carboxyglutamic acid region and may be present in the epidermal growth factor-like domains of protein S. The high affinity of the protein S-C4BP interaction in the presence of Ca2+ suggested that virtually all of the protein S in whole blood should be complexed with C4BP. However, even though the protein S-C4BP interaction in Ca2(+)-containing serum was shown to have the same high affinity as in purified systems, approximately 30-40% of the protein S in serum was free. These results appear best explained by the presence of a third component in whole blood which regulates the protein S-C4BP interaction, keeping approximately 30-40% of circulating protein S in its free, functionally anticoagulant form. It is speculated that persons with little free protein S may be deficient in this hypothetical third component.

Calcium↗
PubMed1989

Multiplicative interaction between intrathecally and intracerebroventricularly administered mu opioid agonists but limited interactions between delta and kappa agonists for antinociception in mice.

Simultaneous action of morphine on supraspinal and spinal sites produces a multiplicative interaction for antinociception which may be important for the analgesia produced by systemically administered morphine. The purpose of this study was to see whether other agonists with more receptor selective opioid actions than morphine would also produce this multiplicative interaction. DAMPGO (Tyr-D-Ala2-Gly-NMePhe4-Gly-ol5), DPDPE (D-Pen2, D-Pen5, enkephalin) and U50-488H, opioid agonists highly selective for mu, delta and kappa receptors, respectively, were administered alone i.c.v. or intrathecally (i.t.) or in combination (i.c.v. plus i.t.) to determine ED50 values for the tail-flick response in mice. These ED50 values were examined isobolographically in relation to the theoretical additive ED50 values by the potency ratio method. First, DAMPGO given i.cv and i.t. was similar to morphine, indicating that simultaneous supraspinal and spinal mu agonist administration produce the multiplicative interaction. Second, concurrent administration of DPDPE or U50,488H, i.c.v. and i.t., as well as cross-over combinations of DPDPE at one and U50,488H at the other site, produced additive interactions only. The multiplicative interaction was a property characteristic of mu but not delta and kappa agonists. Based on the similarity between morphine and DAMPGO, it was postulated that both mu agonists act on redundant descending pain inhibitory pathways to produce multiplication. A second mechanism for multiplicative interaction was based on the difference between DAMPGO and morphine.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1994

Multiple interactions of aluminum with neurofilament subunits: regulation by phosphate-dependent interactions between C-terminal extensions of the high and middle molecular weight subunits.

Exposure of individual purified neurofilament (NF) proteins to AlCl3 alters their electrophoretic properties in a time- and concentration-dependent manner, as visualized by their failure to migrate into SDS gels. Co-incubation of purified high (NF-H) and middle (NF-M) but not low (NF-L) molecular weight NF subunits prevents this AlCl3-induced alteration in electrophoretic migration. This latter finding suggested that specific interactions between NF-H and NF-M other than filament formation influenced their interaction with AlCl3. Co-incubation of the 160 kDa alpha-chymotryptic cleavage product of NF-H (corresponding to the highly phosphorylated C-terminal sidearm domain) with native NF-M prevented alteration in subunit electrophoretic migration by AlCl3. By contrast, intact, dephosphorylated NF-H subunits were unable to prevent AlCl3-induced alteration of native NF-M electrophoretic migration. Taken together, these findings suggest that phosphate-dependent interactions between the sidearm extensions of NF-H and NF-M diminish the ability of AlCl3 to associate with either subunit in a manner that alters their electrophoretic migration. This interaction of NF-H and NF-M sidearms is SDS-sensitive, while AlCl3-induced alteration in electrophoretic migration of individual subunits is SDS-resistant. Addition of SDS to mixtures of NF-H and NF-M subunits disrupted the protective effect, and promoted AlCl3-induced alterations in subunit electrophoretic migration. These findings support and extend the current hypothesis that the ability of aluminum to interact with NF subunits is a function of subunit phosphorylation, assembly, and extent of neurofilament-neurofilament cross-linking.

Aluminum↗
PubMed1995

The optimization of protein-solvent interactions: thermostability and the role of hydrophobic and electrostatic interactions.

Protein-solvent interactions were analyzed using an optimization parameter based on the ratio of the solvent-accessible area in the native and the unfolded protein structure. The calculations were performed for a set of 183 nonhomologous proteins with known three-dimensional structure available in the Protein Data Bank. The dependence of the total solvent-accessible surface area on the protein molecular mass was analyzed. It was shown that there is no difference between the monomeric and oligomeric proteins with respect to the solvent-accessible area. The results also suggested that for proteins with molecular mass above some critical mass, which is about 28 kDa, a formation of domain structure or subunit aggregation into oligomers is preferred rather than a further enlargement of a single domain structure. An analysis of the optimization of both protein-solvent and charge-charge interactions was performed for 14 proteins from thermophilic organisms. The comparison of the optimization parameters calculated for proteins from thermophiles and mesophiles showed that the former are generally characterized by a high degree of optimization of the hydrophobic interactions or, in cases where the optimization of the hydrophobic interactions is not sufficiently high, by highly optimized charge-charge interactions.

Bacterial Proteins↗
PubMed1995

Combinatorial interaction of human bcl-2 related proteins: mapping of regions important for bcl-2/bcl-x-s interaction.

Human bcl-2 family of genes including bcl-2, bcl-x-l, bcl-x-s, and bax has been shown to be functionally involved in apoptosis. We applied a yeast two-hybrid system to demonstrate that bcl-2, bcl-x-l, bcl-x-s, and bax proteins can interact with each other directly. All bcl-2 family members except bax were shown to be capable of homo-interactions. By using deletion and point mutations, the interaction domains for bcl-2 and bcl-x-s were elucidated. The BH1 domain and the membrane anchoring region at the C-terminal half of the bcl-2 protein are required for its interaction with bcl-x-s. On the other hand, the N-terminal region consisting of codons 24 to 78 of bcl-x-s interacts with bcl-2.

Base Sequence↗
PubMed1979

Stoichiometry of GABA-receptor interactions: GABA modulates the glycine-receptor interaction allosterically in a vertebrate neuron.

1. Measurement of steady-state conductance changes provides a reliable method for determination of transmitter-receptor stoichiometry in general and for GABA and its receptor interactions in particular. Log-log plotting of steady-state conductance changes as a function of decreasing transmitter concentration gives the molecularity of the interaction as a limiting slope. 2. Suitable measurements of GABA action in locust muscle show a molecularity of 3 with strong positive cooperativity. One molecule of picrotoxin is sufficient to block this reaction. Kinetic studies reveal the presence of occult desensitization. Thermodynamic studies reveal strong negative heats of interaction compatible with conformational changes in a multi-subunit receptor. 3. Measurements in the lateral dendrite of the goldfish Mauthner cell reveal that glycine has a more powerful action than GABA. Both interactions, however, utilize 4 molecules of amino acid with strong positive cooperativity to activate its receptors. The receptors are apparently distinct and there appears to be a higher glycine-receptor density. 4. In addition to its action on its own receptor, GABA allosterically modulates the glycine-receptor interaction in the Mauthner cell by lowering the energy barrier for the binding of the first glycine molecule, thereby increasing the affinity of glycine for its receptor.

Allosteric Regulation↗
PubMed1992

High affinity DNA-microtubule interactions: evidence for a conserved DNA-MAP interaction involving unusual high CsCl density repetitious DNA families.

We have examined high affinity interactions of chick brain microtubule proteins with 35S labelled tracer DNAs from chick, mouse and D. melanogaster under equilibrium conditions by the nitrocellulose filter binding technique. Ternary reaction mixtures of the above two components and a third component, an excess of unlabelled competitor DNA from either E. coli., mouse, D. melanogaster or chick, were used to measure small fractions of DNA in each case (1-4%) bound to microtubule protein under high stringency- large competitor DNA concentration and 0.5 M NaCl. As seen in part previously (Marx, K.A. and Denial, T. (1985) in The Molecular Basis of Cancer, 172B, 65-75 (Rein, ed), A. Liss, N.Y.) the measured order of competitor DNA strengths was identical for all three tracer DNAs. That is: chick > mouse > D. melanogaster > E. coli competitor DNA. Since the homologous interaction, chick competitor DNA with chick brain microtubule protein, is always the strongest interaction measured, we interpret this as evidence for a conserved protein-DNA sequence interaction. 35S chick DNA tracer sequences, isolated from nitrocellulose filters following the stringent binding in the presence of 0.9 mM-1 E. coli. competitor DNA, was used in driven reassociation reactions with total chick driver DNA. This fraction was found to be significantly enriched in repetitive chick DNA sequences. Since we have observed a similar phenomenon in mouse, we then compared the stringent binding mouse sequences and showed that the bulk of these sequences did not cross-hybridize with total chick DNA. Finally, all three 35S tracer DNAs binding to nitrocellulose were isolated and sedimented to equilibrium on CsCl density gradients. The CsCl density distributions from all three DNAs showed significant (100-fold) enrichment in classical satellite DNAs as well as higher enrichment in two very unusual high CsCl density families of DNA (1.720-1.740 g/cm3; 1.750-1.765 g/cm3). These families are never observed as distinct bands in total DNA CsCl gradients, nor could we isolate them in purified tubulin control binding experiments. This apparently general phenomena may be identifying some of the sequence families involved in the high affinity microtubule interaction, which appears to be conserved in evolution.

Animals↗
PubMed1994

Nuclear proteins interacting with DNA and tubulin. Study of the interaction of the High Mobility Group protein 1 with tubulin.

Fractionation of a 0.2 M NaCl nuclear extract from rat liver cells by both tubulin and DNA affinity chromatography steps allowed us to find three polypeptides interacting in vitro with both DNA and tubulin. A 22 kDa polypeptide was identified as a proteolytic fragment of High Mobility Group proteins 1 or 2 (HMG 1 or 2). Purified rat liver HMG 1 immobilized on nitrocellulose was found to bind radioiodinated dimeric tubulin through its central B domain. The C domain of HMG 1 appeared to play a negative role in this association process. Soluble HMG 1 depleted of its C-terminal domain interacted with tubulin immobilized on an agarose gel and with microtubules formed from purified tubulin. In contrast, undigested HMG 1 did not interact with tubulin in these conditions. The modification of HMG 1 with amine by 1-ethyl-3-(dimethylaminopropyl)carbodiimide which caused the neutralization of the C domain carboxyl groups restored the ability of HMG 1 to interact with microtubules. These results show that: (a) HMG 1, through its central B domain, binds to both assembled and non-assembled tubulin in vitro and (b) the C-terminal domain of HMG 1 exerts a negative regulatory action on the interaction.

Animals↗