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At least 991 records · Page 55Linked to original sources
PubMed1993

Oxygen-binding properties of extracellular hemoglobin from the leech, Hirudo medicinalis. Effects of pH, cations and temperature.

Electron microscopy of the extracellular hemoglobin from the medicinal leech, Hirudo medicinalis, revealed a typical annelid two-tiered hexagonal structure. The dimensions of the molecule were 30.2 +/- 1.6 nm corner to corner across the hexagon and 18.9 +/- 1.1 nm in height. The oxygenation properties of this high-molecular-weight hemoglobin have been thoroughly studied. The effects of hemoglobin concentration, pH, temperature and concentration of specific cations on the oxygen affinity and the degree of cooperativity have been investigated with special consideration given to physiological conditions. At near-physiological cation concentrations (6 mM Ca2+, 0.125 M Na+), the oxygen affinity and cooperativity tend to increase with increasing alkalinity. At physiological conditions ((pH 7.5) temperature 20 degrees C), they reach P50 values of 5.0 mmHg and n50 = 3.1. An alkaline Bohr effect with considerable magnitude (phi = -0.50) is observed at pH 7.0-8.0. The oxygen-binding properties are also affected by cations. Both oxygen affinity and cooperativity rise with increasing cation concentrations. Divalent cations, such as Ca2+ and Mg2+, bring about larger effects than monovalent ones. They begin to be effective at concentrations as low as 0.1 mM. The magnitude and position of the Bohr effect of H. medicinalis hemoglobin are controlled by cations to such an extent that this Bohr effect can be predominantly ascribable to the oxygen-linked binding of the physiologically available allosteric effectors Ca2+ and Na+. The results obtained indicate that protons and cations are effective allosteric regulators of achaete (leech) extracellular hemoglobins.

Animals↗
PubMedDate not supplied

N-methyl-D-aspartate receptors and Alzheimer's disease.

The results of several studies now suggest that the density of N-methyl-D-aspartate (NMDA) receptors is maintained in many Alzheimer's disease (AD) cases, although loss of these receptors can occur in specific regions as a consequence of severe neuronal loss. Recent findings demonstrate that there are at least two subtypes of the NMDA receptor which are allosterically regulated. To determine the status of the NMDA receptor in AD, studies are required which will examine the activation state of the NMDA receptor and the properties of subtypes in relation to neuronal density and structure.

Alzheimer Disease↗
PubMed1976

Kinetic cooperativity in the concerted model for allosteric enzymes.

The cooperativity of enzyme-substrate interactions is investigated in the concerted allosteric model of Monod, Wyman and Changeux. The general case of K-V systems is considered, in which the two protomer conformational states R and T postulated in the theory differ in catalytic and binding properties. An expression for the Hill coefficient nH defined with respect to the asymptotic velocity V infinity to is analyzed in conditions which exclude substrate inhibition. Kinetic cooperativity is always positive (nH greater than 1) in the case of a dimer enzyme, and in the case of an inactive T state. Slight kinetic negative cooperativity (nH less than 1) occurs under restrictive conditions for larger numbers of protomers when the substrate binds significantly to the less active state of the enzyme, but the phenomenon remains negligible for trimers and tetramers. These conclusions differ from those obtained [A. Goldbeter, J. Mol.Biol.90 (1974) 185] with the Hill coefficient based on the absolute maximum velocity, which may exceed the experimental value V infinity to in K-V systems. The results extend those of Paulus and DeRiel [J. Mol. Biol. 97 (1975) 667] and support the view that in most cases, negative cooperativity is not compatible with a mechanism based on a concerted and conservative allosteric transition. The Hill coefficients for binding and catalysis are compared in K-V systems.

Allosteric Regulation↗
PubMed1990

Fitting abnormal oxygen equilibrium curves of hemoglobin.

A growing number of oxygen equilibrium curves for hemoglobin (Hb) mutants, post-translational modifications, or the binding of potent new effectors of Hb cannot be fitted adequately with the two-state model. Examples are curves showing double maxima in the derivative of the Hill plot, or slopes of less than unity. We present such examples of modified hemoglobins and strong effectors in this study and calculate at which substate level the two-state model differs from the data. Analysis of hemoglobin oxygen equilibrium curves is reconsidered using the two-state model extended to allow variation of the individual substate probabilities. In this way the effect on the equilibrium due to perturbations in energy of each substate can be studied as a diagnostic tool.

Allosteric Regulation↗
PubMed1990

The energetics of ligand-linked subunit assembly in hemoglobin require a third allosteric structure.

For partially ligated cyanomet hemoglobins, Smith and Ackers (Proc. Natl. Acad. Sci. U.S.A. 71 (1985) 4312) determined the free energies of dimer-tetramer assembly for all of the partially ligated species using a combination of kinetic and equilibrium methods. They found a third apparent cooperative free energy level in addition to those of deoxy- and cyanomethemoglobin. Using cryogenic methods, Perrella et al. (Biophys. Chem. 35 (1990) 97) confirmed the existence of the third cooperative free energy level, but found a different energy level assignment for one of the species. These combined studies have yielded a solid data base for considering mechanistic issues. The number of cooperative free energies delta Gc can, in principle, be different from the number of molecular forms which have unique free energies of heme-heme interaction, since delta Gc can be an average over conformational subspecies. Furthermore, since the delta Gc values are determined from free energies of dimer-tetramer assembly, it is necessary to evaluate possible contributions from dimeric properties, and from quaternary constraint (or enhancement) effects associated with subunit assembly. In this paper we analyze the observed distributions of apparent delta Gc values among the various ligation states in terms of mechanisms based on two interconvertible molecular forms (R and T) under the most general conditions in which (i) dimers may be cooperative, (ii) ligand affinities of alpha-subunits may be different within tetramers and dimers, and the same for beta-subunit affinities, and (iii) dimers need not be halves of R-state tetramers. It is found that the experimental distributions are inconsistent with even the most general model of the two-state class; thus, at least three molecular forms of tetramer are required, each with an individually different value of cooperative free energy (heme-heme interaction). This result implies the existence of at least three corresponding molecular structures; while a degeneracy of multiple structures into only a few dominant free energy levels is frequently to be expected, the reverse situation is extremely unlikely.

Allosteric Regulation↗
PubMed1987

Purification and some properties of liver adenylylsulfate kinase.

Adenylylsulfate kinase (ATP:adenylylsulfate 3'-phosphotransferase, EC 2.7.1.25) has been purified over 1300-fold from rat liver in 10% yield. The enzyme has a molecular weight of 58,000 and is composed of four subunits of equal molecular weight. ATP is an allosteric activator of adenylylsulfate kinase, with a Hill coefficient of 2.2 and a K0.5 of 2.5 mM. Adenosine phosphosulfate is a potent inhibitor of adenylylsulfate kinase, but the adenosine phosphosulfate concentration for maximal reaction is dependent on the ATP concentration. At the physiological levels of ATP the inhibition by adenosine phosphosulfate is not likely to play a role, while the allosteric regulation of adenylylsulfate kinase by ATP may be operative.

Adenosine Phosphosulfate↗
PubMed1990

Age dependent changes of a biochemical rhythm--the glycolytic oscillator of the blowfly Phormia terraenovae.

1. By monitoring changes of fluorescence of NADH the frequencies, amplitudes and maximum slopes of the glycolytic oscillator of Phormia were analyzed in 5, 9, 15 and 21-day-old male flies. 2. In order to evaluate the possible existence of circadian rhythms within the oscillatory system, all determinations were repeated eight times/day. 3. In addition, the activities of three key enzymes of glycolysis, PFK, GAPDH and PK, which are central to the glycolytic oscillator were measured with respect to age and day time. 4. With increasing age the amplitudes of oscillations increased together with the maximum slopes of the oscillatory waves. The frequency appeared to be independent of age. 5. Variations of enzyme activities over the day indicated an age dependent circadian rhythm which, due to the simultaneous activity changes of the three measured enzymes, was not reflected in the whole oscillatory system. 6. The results suggest that modifications in the allosteric regulation of enzymes are responsible for the age dependent changes of the glycolytic oscillator.

Aging↗
PubMed1993

A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase gene is highly expressed in the limbic system of the rat brain.

Cyclic AMP and cyclic GMP serve as second messengers in a variety of neural cells, modulating their metabolic and electrical activity. The cyclic GMP-stimulated cyclic nucleotide phosphodiesterase, an enzyme whose hydrolytic activity is allosterically regulated by cyclic GMP in peripheral tissues, could play an important role in the regulation of cyclic nucleotide levels in the brain. To study the presence and distribution of cyclic GMP-stimulated phosphodiesterase in the rat brain, we cloned a portion of rat liver cyclic GMP-stimulated phosphodiesterase complementary DNA by polymerase chain reaction, using degenerate phosphodiesterase-specific oligonucleotide primers. Northern blot analysis of rat tissues reveals abundant expression of cyclic GMP-stimulated phosphodiesterase messenger RNA in the brain. Northern blot analysis of brain subregions shows especially strong expression in hippocampus and cortex, modest expression in the remainder of the forebrain and in the midbrain, and little expression in cerebellum and hindbrain. In situ hybridization studies with cyclic GMP-stimulated phosphodiesterase riboprobes confirm these northern blot results, and delineate cell groups with high levels of expression. Medial habenular nucleus is intensely labeled, as is hippocampus in the vicinity of pyramidal and granule cell bodies in areas CA1, CA2, CA3, and dentate gyrus. Other elements of the limbic system also contain cyclic GMP-stimulated phosphodiesterase messenger RNA, including olfactory and entorhinal cortices, subiculum, and amygdala. Additional cortical regions show more diffuse expression of cyclic GMP-stimulated phosphodiesterase messenger RNA, as do the basal ganglia. Cerebellum, thalamus, and hypothalamus do not show appreciable specific labeling. These studies demonstrate the presence of cyclic GMP-stimulated phosphodiesterase messenger RNA in specific regions of the rat brain, and suggest that the cyclic GMP-stimulated phosphodiesterase might modulate neuronal activity by regulating intracellular cyclic AMP levels in response to changes in intracellular cyclic GMP levels.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

The 'cerebral diabetes' paradigm for unipolar depression.

Unipolar depression, alcoholism and suicide have become more common over the past decades. Genetic studies have attempted to link (bipolar) affective disorder to the short arm of chromosome 11 (where the loci for insulin, insulin growth factor (IGF), tyrosine hydroxylase (TH) and h-ras-oncogene are located) but these have failed. Since TH and the insulin receptor require phosphorylation by protein kinases, then a defect of the h-ras-oncogene or its products (p21) could disorder both these systems and compromise catecholaminergic transmission in neurones and energy flow in glial cells. This could lead not only to a predisposition to depression ('trait markers') but to neurotoxic damage, predisposed by inadequate cytosol Mg2+ levels of hypometabolism. Tyrosine, tryptophan and phenylalanine hydroxylases all require tetrahydrobiopterin (BH4) which allosterically regulates its own activity as well as that of these enzymes. Anything which impairs this cofactor could lead to overt depression in predisposed individuals, and the heterocyclic amines are being increasingly implicated. These substances are derived from fried and broiled meats, azo food dyes, soft drinks and hard candies, but particularly from cigarette and petroleum fumes. The heterocyclic amines can inhibit aromatic-l-amino-acid-decarboxylase (AADC) as well as the hydroxylases reversibly, but BH4 is inhibited noncompetitively. Thus, susceptible individuals (those with inherited defective protein kinase phosphorylation) might be 'tipped over' by chronic exposure to these neurotoxins. The rising incidence of unipolar depression-associated morbidity could be significantly linked to increasing levels of heterocyclic amines in the developed nations.

Amines↗
PubMed1988

Analysis of the functional domains of biosynthetic threonine deaminase by comparison of the amino acid sequences of three wild-type alleles to the amino acid sequence of biodegradative threonine deaminase.

The nucleotide sequence of the gene, ilvA, for biosynthetic threonine deaminase (Tda) from Salmonella typhimurium was determined. The deduced amino acid sequence was compared with the deduced amino acid sequences of the biosynthetic Tda from Escherichia coli K-12 (ilvA) and Saccharomyces cerevisiae (ILV1) and the biodegradative Tda from E. coli K-12 (tdc). The comparison indicated the presence of two types of blocks of homologous amino acids. The first type of homology is in the N-terminal portion of all four isozymes of Tda and probably indicates amino acids involved in catalysis. The second type of homology is found in the C-terminal portion of the three biosynthetic isozymes and presumably is involved in either (i) the binding or interaction of the allosteric effector isoleucine with the enzyme, or (ii) subunit interactions. The sites of amino acid changes of two E. coli K-12 ilvA alleles with altered response to isoleucine are consistent with the conclusion that the C-terminal portion of biosynthetic Tda is involved in allosteric regulation.

Alleles↗
PubMed1992

Sequence analysis of a DNA fragment from Buchnera aphidicola (an endosymbiont of aphids) containing genes homologous to dnaG, rpoD, cysE, and secB.

The aphid, Schizaphis graminum, contains a prokaryotic, obligately intracellular endosymbiont, Buchnera aphidicola, which is necessary for the survival of the host. A recent study of Bu. aphidicola 16S rRNA has indicated that it is a member of the gamma-3 subdivision of the eubacterial class, Proteobacteria, which includes Escherichia coli. In order to further characterize the endosymbiont and establish its similarity to free-living eubacteria and/or organelles, we have cloned and sequenced a 4534-bp DNA fragment containing dnaG-rpoD-cysE-secB. The deduced amino acid (aa) sequence identity to the homologous E. coli proteins ranged from 47 to 80%. The close proximity of the pair, dnaG-rpoD, to the pair, cysE-secB, on the Bu. aphidicola DNA, differed from E. coli, in which these two pairs of genes are 14 min apart on the bacterial chromosome. The results of past physiological studies of the endosymbiont were consistent with the presence and function of DNA primase (DnaG), sigma factor (RpoD) and components of the secretory system (SecB). Comparison of the deduced aa sequence of Bu. aphidicola CysE (serine acetyltransferase, a key allosterically regulated enzyme in cysteine biosynthesis) with the E. coli wild-type enzyme and a mutant defective in feedback inhibition suggested that the endosymbiont CysE may not be regulated. By analogy with E. coli, the lack of feedback inhibition may lead to overproduction of cysteine by the endosymbiont. The results of this and previous investigations indicate that Bu. aphidicola has many of the properties of free-living bacteria and not of organelles.

Amino Acid Sequence↗
PubMed1995

Cloning, sequencing and functional expression of a cDNA encoding a NADP-dependent malic enzyme from human liver.

This work reports the structure of a cDNA (ME) encoding a human malic enzyme (ME) (malate NADP oxidoreductase, EC 1.1.1.40) elucidated by joining several overlapping fragments amplified by PCR from human hepatic cDNA or from cDNA libraries. The full-length cDNA has an open reading frame (ORF) of 1719 bp that encodes a 572-amino-acid protein of 64 113 Da, similar to the native monomeric, cytosolic, NADP-dependent ME isolated from human liver. The comparison of the structure of this cDNA with that of the human mitochondrial NAD(P)-dependent ME (EC 1.1.1.39) shows a homology of 63%, suggesting that these two forms originated from the same gene. The expression of the cDNA in Escherichia coli as a translational fusion (glutathione S-transferase::ME) protein yielded a product of the predicted mass. The recombinant protein shows NADP-dependent malate oxidoreductase activity and is virtually inactive with NAD. It also shows other distinct features of the native cytosolic NADP-dependent ME, like Mn2+ dependence, similar substrate (Km = 117 microM) and cofactor affinity (Km = 2 microM) constants, and a lack of allosteric regulation. In human proliferative cells, the NADP-dependent ME activity is poorly expressed and barely inducible by thyroid hormones.

Amino Acid Sequence↗
PubMed1989

Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor.

We have stably expressed in fibroblasts different pairs of alpha and non-alpha subunits of the mouse muscle nicotinic acetylcholine receptor (AChR). The gamma and delta, but not the beta, subunits associated efficiently with the alpha subunit, and they extensively modified its binding characteristics. The alpha gamma and alpha delta complexes formed distinctly different high affinity binding sites for the competitive antagonist d-tubocurarine that, together, completely accounted for the two nonequivalent antagonist binding sites in native AChR. The alpha delta complex and native AChR had similar affinities for the agonist carbamylcholine. In contrast, although the alpha gamma complex contains the higher affinity competitive antagonist binding site, it had an affinity for carbamylcholine that was an order of magnitude less than that of the alpha delta complex or the AChR. The comparatively low agonist affinity of the alpha gamma complex may represent an allosterically regulated binding site in the native AChR. These data support a model of two nonequivalent binding sites within the AChR and imply that the basis for this nonequivalence is the association of the alpha subunit with the gamma or delta subunit.

Animals↗
PubMed1993

Evidence that protein kinase C alpha has reduced affinity towards 1,2-dioctanoyl-sn-glycerol: the effects of lipid activators on phorbol ester binding and kinase activity.

The effect of 1,2-diacylglycerols on specific binding of [3H]phorbol 12,13-dibutyrate to cytosolic protein kinase C (PKC) was investigated in tissues reported to contain different proportions of PKC isoforms. In lung, frontal cerebral cortex and cerebellum cytosols (enriched in PKC alpha, beta and gamma, respectively) displacement of specific binding by phorbol 12,13-dibutyrate or diacylglycerols containing unsaturated acyl chains was of similar potency for each tissue. A range of 1,2-diacylglycerols containing saturated acyl chains exhibited varying affinities for [3H]phorbol 12,13-dibutyrate binding sites in each tissue; defining an optimal acyl chain length of around 14 carbons in each case. However, the affinities of saturated diglycerides were consistently lower in lung cytosol than in frontal cerebral cortex and cerebellum cytosols, with the greatest differences occurring at lower acyl chain lengths, especially with 1,2-dioctanoyl-sn-glycerol. Furthermore, a mixed micelle assay of PKC activity showed that 1,2-dioctanoyl-sn-glycerol displayed reduced potency at PKC alpha partially-purified from COS 7 cell cytosol compared to the mixture of PKC isoforms present in rat midbrain cytosol. Both low potency of 1,2-dioctanoyl-sn-glycerol as a displacer of [3H]phorbol 12,13 dibutyrate binding and the ability of arachidonic acid to act as an allosteric enhancer of binding, correlated with the proportional PKC alpha content of a range of tissues reported in the literature. In PKC enzyme activity assays, 1,2-dioctanoyl-sn-glycerol, but not phorbol 12,13-dibutyrate, was correspondingly a much poorer activator of PKC alpha from COS 7 cells than of the broad consensus of isoforms in rat midbrain. When alpha and beta isoforms were extensively-purified on DEAE-cellulose then hydroxyapatite, both the low affinity of 1,2-dioctanoyl-sn-glycerol for [3H]phorbol 12,13-dibutyrate binding sites and their allosteric regulation by arachidonic acid were confirmed to be characteristic of the alpha rather than the beta isoforms.

Amino Acid Sequence↗
PubMed1993

Interaction of La3+ with GABAA receptors in rat cerebrocortical membranes as detected with [35S]t-butylbicyclophosphorothionate binding.

Lanthanides at micromolar concentrations stimulated binding of [35S]t-butylbicyclophosphorothionate (TBPS), a highly sensitive marker for allosteric modulation of the chloride channel of GABAA receptors, to purified rat cerebrocortical synaptosomal membranes in the absence of GABA. This trivalent cation effect appeared to reflect a specific and direct interaction with GABAA receptors, and could not be mimicked by divalent metal ions including Ca2+, Cd2+, Co2+, Sr2+, Mg2+ and Mn2+. On the other hand, Zn2+, a known allosteric regulator of GABAA receptors, inhibited TBPS binding, but its presence could not prevent stimulation of TBPS binding by La3+. We further examined the lanthanide binding site by monitoring La3(+)-induced changes in TBPS binding. La3+ reduced the Kd for TBPS without affecting Bmax. Stimulation of TBPS by La3+ was not additive with the increased TBPS binding induced by other agonists, including GABA (at nanomolar concentrations), neurosteroids or benzodiazepines (in the absence of GABA). GABA at micromolar concentrations inhibited TBPS binding and its inhibitory effect was potentiated by neurosteroids and benzodiazepines, but not by La3+. Thus, GABA, in the presence of La3+, inhibited TBPS binding in a monophasic manner with a Hill coefficient of approximately 2 and an IC50 of 6 microM. In the absence of La3+, the dose-response profile for GABA became biphasic with an appearance of a stimulatory phase at concentrations of GABA lower than 1 microM. Analysis of the initial stimulation yielded a Hill coefficient of 1 and an EC50 of 88 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

From analysis to synthesis: new ligand binding sites on the lactate dehydrogenase framework. Part I.

In Part I of this article, the naturally evolved protein framework of lactate dehydrogenase is investigated by genetically introduced modifications which reveal the structural basis of its catalytic and substrate-binding properties. In Part II (to be published in the April issue of TIBS), this analytical information is exploited in the design of two modified forms of the enzyme; one which is specific for a new substrate and one which lacks allosteric regulation.

Binding Sites↗