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PubMed1979

Co-binding studies on Hb M Iwate. Allostery of a T state haemoglobin.

The mutant haemoglobin Hb M Iwate alpha 2Mmet87His leads to Tyr beta 2, is characterized by a stable T structure and a low ligand affinity. Sigmoidal CO-binding isotherms of symmetrical shape with Hill coefficients of n = 1.4 at pH 6 to n = 1.9 at pH 10 and the differences in the mean affinity (PCO(1/2)) and the affinity of the first ligand-binding beta subunit (1/L1 greater than Pco(1/2)) are the evidence for the cooperativity. The comparison of the Bohr effects of the two valency hybrid states (alpha 2Mmet beta met beta deoxy alpha 2Mmet beta 2deoxy) in the absence of and in the presence of polyphosphates leads to an indirect proof of pH-dependent subunit-subunit interaction. Inositol hexaphosphate-binding suppresses cooperativity in the pH range 5.5-8 (n = 1). Above pH 8 hte cooperativity increases to a final value of n = 1.9 at pH greater than 10, which is identical to that of stripped Hb M Iwate. The CO binding to the first binding site exhibits a Bohr effect. Polyphosphate anions have no influence on the CO binding of the first binding site. The heterotropic effects are discussed as intrachain effects (Bohr effect of the first binding site) and interchain effects (Bohr effect of Pco(1/2); influence of polyphosphates).

Allosteric Regulation↗
PubMed1981

Amphitrite ornata erythrocruorin. II. Molecular controls of function.

In the marine terebellid worm Amphitrite ornata the vascular fluid contains a high molecular weight erythrocruorin, while cells of the coelom contain a monomeric hemoglobin. The structural integrity of the erythrocruorin molecule is known to be dependent on the presence of a minimal concentration of divalent cations (1-3 mM) in the medium. The functional properties of Amphitrite erythrocruorin are also affected by cations. The oxygen affinity tends to increase with increasing cation concentration and the degree of cooperative interactions, expressed in the kinetics and equilibria of ligand binding, goes through a maximum. Maximal Hill coefficients of 3-4 are observed with 50 mM CaCl2, 50 mM MgCl2 or 1 M NaCl in measurements at the physiological pH of 7.75. Only 2 mM CaCl2 is required for maximal cooperativity at pH 8.5. This suggests partial deprotonation of the cation binding site at high pH. It is somewhat unusual that pH effects on cooperativity are reversible, since this is not a common feature of the giant erythrocruorin molecules. The oxygen binding experiments revealed a marked effect of divalent cations of Amphitrite erythrocruorin at high pH and cation concentration. Above pH 8.5, at 50 mM CaCl2 and 12 degrees C, the erythrocruorin will form a polymer upon deoxygenation. This polymerization is readily reversible by bringing the temperature for 12 to 20 degrees C or by oxygenation. Under physiological conditions of pH and cation concentration and at 12 degrees C, the erythrocruorin and the monomeric coelomic hemoglobin require a similar oxygen pressure for half saturation. However, the allosteric regulation of function is absent for the coelomic protein.

Animals↗
PubMed1981

Human serum albumin as an allosteric two-state protein. Evidence from effects of calcium and warfarin on proton binding behaviour.

The proton binding of human serum albumin, and the influence on it of Ca2+ and warfarin (3-(alpha-acetonylbenzyl)-4-hydroxycoumarin), has been studied in the pH region 6 to 9, in order to get more information on the conformational change occurring in serum albumin around neutral pH, the so-called N-B transition. Some of the results for human serum albumin are compared with bovine serum albumin. A two-state model describing this transition is presented. In this model the two states are assumed to be the N state (found at low pH) and the B state (found at high pH). The ligand to be considered is the proton, having the highest affinity for the N conformation. An allosteric constant, L, governs the equilibrium between the two states. Both Ca2+ and warfarin can act as allosteric effectors, thereby increasing L. The model is used to describe results such as: (a) the cooperativity in proton binding, most clearly observable when Ca2+ is present, and the difficulty of measuring experimentally this cooperativity; (b) the different number of protons bound when Ca2+ is present or absent; (c) the fraction of protein found in one of the two conformations; (d) the correspondence between the increase of L due to addition of Ca2+ or warfarin, as predicted from model calculations, and the experimentally found increase of L.

Allosteric Regulation↗
PubMed1984

Inhibition of brain hexokinase by a multisubstrate analog results from binding to a discrete regulatory site.

P1-(adenosine-5')-P3-(glucose-6)-triphosphate (Ap3glucose) is a linear uncompetitive inhibitor vs glucose and a linear mixed inhibitor vs ATP of brain hexokinase, an inhibition pattern inconsistent with binding of Ap3glucose to the catalytic site when either the rapid equilibrium random or ordered sequential mechanism, which have been proposed for this enzyme, is considered. It is concluded that inhibition results from binding to a discrete regulatory site. The apparent ability of the regulatory site to accommodate both hexose and nucleotide moieties is consistent with suggestions by previous investigators that the regulatory site on mammalian hexokinases may have evolved from what was originally a catalytic site.

Adenosine Triphosphate↗
PubMed1989

Direct evidence for modulation of porter quaternary structure by transport site ligands.

The transport inhibitor DNDS (4,4'-dinitrostilbene-2,2'-disulfonate) changes the bis(sulfosuccinimidyl)suberate (BS3) crosslinking pattern of band 3 protein from a mixture of dimer-crosslinkable (DC) and tetramer-crosslinkable (TC) states to the TC-state as the exclusive crosslinked product for reactions occurring in membranes of intact human erythrocytes. Pretreatment of cells with DNDS followed by extensive washing restores the original DC to TC proportionality indicating that the two states are reversibly interconvertible. We suggest a model wherein band 3 transport site ligands allosterically modulate the global conformation of a tetrameric porter between two reversibly interconvertible quaternary structures. These transitions in quaternary structure may be important to transmembrane signaling of events between the exofacial ligand binding site and the sites on the porter extension which bind ankyrin and hemoglobin.

Allosteric Regulation↗
PubMed1986

Effects of mono (2-ethylhexyl) phthalate and its straight chain analogues mono-n-hexylphthalate and mono-n-octyl phthalate on lipid metabolism in isolated hepatocytes.

In cultured hepatocytes, as in vivo, mono-2-ethylhexyl phthalate (MEHP) and its straight chain analogues mono-n-hexyl phthalate (MnHP) and mono-n-octyl phthalate (MnOP) each cause accumulation of lipid but only MEHP produces significant induction of peroxisomal fatty acid oxidizing enzymes. To elucidate the mechanisms underlying this lipid accumulation we investigated the effects of these phthalates and the drug clofibric acid on fatty acid metabolism in suspensions of isolated hepatocytes. The effects were found to be markedly dependent on the nutritional state of the animals from which the hepatocytes were isolated. In hepatocytes isolated from animals fasted overnight, or animals fed ab libitum but killed at approximately 2.30 p.m., MEHP, MnHP, MnOP and clofibric acid each caused a marked rapid stimulation of fatty acid oxidation and the synthesis of triglycerides in hepatocytes when incubated in Hanks saline. Export of very low density lipoprotein (VLDL) from the cells was either unchanged or somewhat reduced. In contrast, in hepatocytes isolated from rats fed ad libitum but killed at approximately 9.30 a.m. MEHP and clofibric acid did not alter fatty acid oxidation or triglyceride synthesis, while MnOP and MnHP increased triglyceride synthesis but decreased fatty acid oxidation. The effects of fasting were largely abolished by incubations of the cells in a complete tissue culture medium (Liebowitz L-15). The results suggest that MEHP and its straight chain analogues can, either as the free acid or the CoA ester, mimic the action of fatty acids in the allosteric regulation of fatty acid metabolism.

Animals↗
PubMed1987

Ribonucleotide reductase induced by varicella zoster virus. Characterization, and potentiation of acyclovir by its inhibition.

An enzyme that catalyzes the conversion of CDP to 2'-dCDP in the presence of dithiothreitol (DTT) was detected in ammonium sulfate fractionated-extracts of varicella zoster virus (VZV)-infected cells. This ribonucleotide reductase was antigenically distinguishable from the isofunctional eucaryotic enzyme as well as the ribonucleotide reductases induced by herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). The VZV-induced enzyme was purified to the extent that most of the contaminating enzymes, which would significantly deplete the substrate, were removed. The VZV-induced ribonucleotide reductase exhibited maximum activity in the absence of ATP and/or magnesium and was only weakly inhibited by 2'-deoxynucleoside triphosphates. Furthermore, ADP, UDP and GDP competitively inhibited CDP reduction with Ki (Km) values of 15, 20, 1.8 and 0.88 microM, respectively. These kinetic properties were very similar to those of the correspondingly purified ribonucleotide reductases induced by HSV-1 [Averett et al., J. biol. Chem. 258, 9831 (1983)] and HSV-2 [Averett et al., J. Virol. 52, 981 (1984)] and were dissimilar to the allosterically regulated mammalian enzyme. A723U, an inactivator of HSV-1 ribonucleotide reductase that potentiates the anti-HSV-1 activity of acyclovir [Spector et al., Proc. natn. Acad. Sci. U.S.A. 82, 4254 (1985)], also appeared to inactivate this VZV-induced ribonucleotide reductase and to potentiate the anti-VZV activity of acyclovir.

Acyclovir↗
PubMed1986

Phosphatidylserine inhibition of monoamine oxidase in platelets of schizophrenics.

Phosphatidylserine (PS) has recently been reported to be a specific inhibitor of B-type monoamine oxidase (MAO-B). The effect of added PS liposomes on platelet MAO-B activity was examined in two schizophrenic groups (paranoid and a mixture of residual/undifferentiated) and in normal controls. PS was a potent partial-mixed-uncompetitive inhibitor of the platelet enzyme, whereas other phospholipids tested were without effect. The PS concentration required for 50% inhibition was significantly higher for the paranoid relative to the residual/undifferentiated group and controls. This correlated with a lower mean basal MAO-B activity in this group. Hill plots, as a measure of the concentration dependence of PS sensitivity, however, revealed a similarity between the two schizophrenic groups in regard to inhibitor binding properties. Mean Hill coefficients for both groups were significantly different from the controls. The results were consistent with an in vivo role for PS as an allosteric regulator of platelet MAO-B.

Adult↗
PubMed1987

Inhibition of [3H]diazepam binding in primary cultures of astrocytes by atrial natriuretic peptide and by a cyclic GMP analog.

Atrial natriuretic peptide (ANP) was found potently (IC50 = 88 nM) to inhibit [3H]diazepam binding to astrocytes in primary cultures but not to a corresponding preparation of neurons. Based upon this finding and literature data demonstrating a pronounced correlation between the distribution of 'peripheral-type' benzodiazepine receptors and ANP receptors, it is suggested that ANP may be an allosteric regulator of the astrocytic benzodiazepine binding site. Since ANP is known to increase the level of cGMP it was also investigated whether 8-bromoguanosine 3':5' cyclic monophosphate, a permeable cGMP analog, displaced diazepam binding. This was the case, suggesting that cGMP might be involved in the ANP effect on the astrocytic benzodiazepine receptor.

Animals↗
PubMed1991

Stereoselective inhibition by D- and L-alanine of phencyclidine-induced locomotor stimulation in the rat.

Bilateral injection of D-alanine, but not L-alanine, (10-100 micrograms per side for each amino acid) into the lateral ventricle reduced the increasing effect of phencyclidine (PCP, 10 mg/kg, given intraperitoneally) on locomotor activity in the rat in a dose dependent manner. This stereoselectivity agrees with the potency of these agents as agonists for the strychnine-insensitive glycine receptor in the N-methyl-D-aspartate receptor complex, suggesting that stimulation of the allosteric regulation site may antagonize the ability of PCP to produce hyperactivity.

Alanine↗
PubMed1994

Steroid modulation of GABAA receptors in an amphibian brain.

Steroids can modulate gamma-aminobutyric acid (GABAA) receptor function in rat brains, but the physiological relevance of this mechanism is still unclear. To determine whether this phenomenon is widespread among vertebrates, we investigated steroid modulation of GABAA receptors in amphibian brain tissue. Equilibrium binding parameters for t-butylbicyclophosphorothionate ([35S]TBPS) and [3H]flunitrazepam were similar in Taricha granulosa and mammalian brains, as was the allosteric regulation of [35S]TBPS and [3H]flunitrazepam binding by GABA. The rank order and absolute potencies of steroids to inhibit [35S]TBPS binding and enhance [3H]flunitrazepam binding were also similar in Taricha and rat brains. As in mammalian studies, physiological concentrations of corticosterone had no effect on ligand binding or GABA-stimulated Cl- uptake. In autoradiographic studies, 3 alpha-hydroxy-5 alpha-pregnan-20-one inhibited [35S]TBPS binding sites in all brain regions examined, whereas corticosterone had no effect on [35S]TBPS binding. These studies suggest that the steroid recognition sites on GABAA receptors have been highly conserved through vertebrate evolution and thus portend physiologically important functions. However, the pharmacological profiles for the GABAA receptor and the high-affinity corticosteroid receptor are apparently different, suggesting there are multiple types of steroid recognition sites on neuronal membranes.

Animals↗
PubMed1989

Diltiazem enhances and flunarizine inhibits nimodipine's antiseizure effects.

The dihydropyridine calcium channel antagonist, nimodipine has antiepileptic and anticonvulsive properties that are thought to be mediated through neuronal calcium channel blockade. The dihydropyridine binding site can be positively and negatively allosterically regulated by the benzothiazepines and the phenylalkylamines/piperazines, respectively. We investigated this binding interaction at the physiologic level by examining the effects of diltiazem (a benzothiazepine) and flunarizine (a piperazine) on the antiseizure activity of nimodipine. Seizures were induced with pentylenetetrazole in awake rats with chronically implanted EEG electrodes. Calcium channel antagonists were administered intracerebroventricularly 30 min after pentylenetetrazole at doses given at 15 min intervals. Diltiazem and flunarizine alone lacked antiseizure properties. The calculated ED50 values for nimodipine were: nimodipine alone = 135 micrograms; nimodipine + diltiazem (100 micrograms) = 67 micrograms. Nimodipine + flunarizine (10 micrograms) completely suppressed nimodipine's antiseizure activity. These findings may reflect the interaction observed among these agents at binding sites associated with the calcium channel and supports the idea that dihydropyridines mediate their antiseizure actions through neuronal calcium channel antagonism.

Animals↗
PubMed1987

Deoxyribonucleoside triphosphate metabolism and the mammalian cell cycle. Effects of hydroxyurea on mutant and wild-type mouse S49 T-lymphoma cells.

DNA precursor synthesis can be blocked specifically by the drug hydroxyurea (HU) which has therefore been used for anticancer therapy. High concentrations of HU, however, affect other processes than DNA synthesis; nevertheless, most studies on the biological action of HU have been made with concentrations at least one order of magnitude higher than those needed for cell-growth inhibition. In this study we characterized the effects of low concentrations of HU (i.e. concentrations leading to 50% inhibition of cell growth in 72 h) on cell cycle kinetics and nucleotide pools in mouse S49 cells with various defined alterations in DNA precursor synthesis. The effect of 50 microM HU on deoxyribonucleoside triphosphate pools was a 2-3-fold decrease in the dATP and dGTP pools, with no change in the dCTP pool and a certain increase in the dTTP pool. Addition of deoxycytidine or thymidine led to a partial reversal of the growth inhibition and cell-cycle perturbation caused by HU, and was accompanied by an increased level of the deoxyribonucleoside triphosphates. Addition of purine deoxyribonucleoside gave no protection, indicating that salvage of these nucleosides could not supply precursors for DNA synthesis in T-lymphoma cells. We observed a higher sensitivity to HU of cells lacking purine nucleoside phosphorylase or with a ribonucleotide reductase with altered allosteric regulation. Cells lacking thymidine kinase or deoxycytidine kinase were just as sensitive as wild-type cells.

Animals↗