Ca2+-dependent and phospholipid-independent binding of annexin 2 and annexin 5

Annexins are a family of homologous proteins that associate with anionic phospholipid (aPL) in the presence of Ca2+. Evidence that the function of one annexin type may be regulated by another was recently reported in studies investigating cytomegalovirus—aPL interactions, where the fusogenic function of annexin 2 (A2) was attenuated by annexin 5 (A5). This observation suggested that A2 may bind directly to A5. In the present study, we demonstrated this interaction. The A2—A5 complex was first detected utilizing (covalently linked) fluorescein-labelled A5 (F-A5) as a reporter group. The interaction required concentrations of Ca2+ in the millimolar range, had an apparent dissociation constant [Kd(app)] of 1nM at 2mM Ca2+ and was independent of aPL. A2 bound comparably with F-A5 pre-equilibrated with an amount of aPL that could bind just the F-A5 or to an excess amount of aPL providing sufficient binding sites for all of F-A5 and A2. A2—A5 complex formation was corroborated in an experiment, where [125I]A2 associated in a Ca2+-dependent manner with A5 coated on to polystyrene. Surface plasmon resonance was used as a third independent method to demonstrate the binding of A2 and A5 and, furthermore, supported the conclusion that the monomeric and tetrameric forms of A2 bind equivalently to A5. Together these results demonstrate an A2—A5 interaction and provide an explanation as to how A5 inhibits the previously reported A2-dependent enhancement of virus—aPL fusion.

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Cortical tubular and glomerular dopamine receptors in the rat kidney

AJP Renal Physiology ◽

10.1152/ajprenal.1984.246.5.f557 ◽

1984 ◽

Vol 246 (5) ◽

pp. F557-F568 ◽

Cited By ~ 6

Author(s):

R. A. Felder ◽

M. Blecher ◽

G. M. Eisner ◽

P. A. Jose

Keyword(s):

Adenylate Cyclase ◽

Dopamine Receptors ◽

Binding Sites ◽

Radioligand Binding ◽

Rat Kidney ◽

D2 Dopamine Receptors ◽

Single Class ◽

Apparent Dissociation Constant ◽

Tubular Membranes ◽

Dissociation Constant Kd

Dopamine receptors in glomeruli and renal cortical tubules were characterized using radioligand binding and adenylate cyclase studies. The binding of [3H]haloperidol to glomeruli and tubules was rapid, saturable with time and ligand concentration, reversible, of high affinity, and demonstrated stereoselectivity and antagonist and agonist rank potency for binding to dopamine receptors. Analysis of kinetic data and Rosenthal plots in glomeruli revealed a single class of [3H]haloperidol binding sites with an apparent dissociation constant (Kd) of 6 nM and maximum receptor density (Bmax) of 0.42 pmol/mg protein. In tubules, at least two binding sites were noted, one with an apparent Kd of 38 nM and Bmax of 1.90 pmol/mg protein and another with an apparent Kd of 183 nM and Bmax of 3.50 pmol/mg protein. Dopamine and apomorphine increased adenylate cyclase in tubular membranes while no increases were noted in glomeruli. These studies suggest that glomeruli have D2 dopamine receptors, while renal cortical tubules contain the D1 dopamine receptor.

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The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex

Biochemical Journal ◽

10.1042/bj2630453 ◽

1989 ◽

Vol 263 (2) ◽

pp. 453-462 ◽

Cited By ~ 29

Author(s):

G A Rutter ◽

R M Denton

Keyword(s):

Dissociation Constant ◽

Isocitrate Dehydrogenase ◽

Binding Sites ◽

Tight Binding ◽

Variable Number ◽

Apparent Dissociation Constant ◽

Oxoglutarate Dehydrogenase ◽

Dissociation Constant Kd ◽

Flow Dialysis ◽

Dehydrogenase Complex

1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent.

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Kinetic Analysis Of Prothrombin Activation By Des (l-44) Factor Xa

10.1055/s-0038-1651976 ◽

1981 ◽

Author(s):

W F Skogen ◽

C T Esmon ◽

A C Cox

Keyword(s):

Binding Sites ◽

Factor Xa ◽

Factor X ◽

Apparent Dissociation Constant ◽

Phospholipid Analysis ◽

Factor Va ◽

Gla Domain ◽

Dissociation Constant Kd ◽

Prothrombin Activation

The region of factor X containing the gla residues was released by mild chymotryptic digestion and the resulting Des (1-44) factor X was converted to its activated form with the Russell’s viper venom factor X activator as described by others. The modified enzyme, factor Xa(-gd), retains its ability to activate prothrombin, the activation was still accelerated by factor Va, but the activation was no longer accelerated by phospholipid. The interaction between factor Va and either factor Xa or factor Xa(-gd) was studied kinetically. The rate of prothrombin activation was measured as a function of increased factor Va concentration in reaction mixtures where the factor Xa or factor Xa(-gd), prothrombin, and Ca2+ concentrations were constant. In the absence of phospholipid, analysis of the above data indicated an apparent dissociation constant (Kd’) of factor Xa for factor Va of 3.6 x 10-8 M. The removal of the gla domain lowered the apparent affinity of factor Xa for factor Va to 2.3 x 10-6 M indicating a role of the gla domain in the factor Xa-factor Va interaction even in the absence of phospholipid. Although the affinity was lowered, the V max of the reaction was identical for factdr Xa and factor Xa(-gd) in the presence of saturating concentrations of factor Va. With the addition of phospholipid, the Kd’ of factor Xa for factor Va was 4.7 x 10-10 M and V max increased some 297 fold. Phospholipid had no effect on the Kd’ of the factor Xa(-gd)-factor Va complex (Kd’ s 2.3 x 10-6 M) and V max was unaltered. These results demonstrate that phospholipid has little or no effect on factor Va function when factor Xa has lost its gla mediated Ca2+ binding sites.

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Renal receptors for atrial and C-type natriuretic peptides in the rat

AJP Renal Physiology ◽

10.1152/ajprenal.1992.263.1.f89 ◽

1992 ◽

Vol 263 (1) ◽

pp. F89-F96 ◽

Cited By ~ 3

Author(s):

J. Brown ◽

Z. Zuo

Keyword(s):

Atrial Natriuretic Peptide ◽

Natriuretic Peptide ◽

Binding Sites ◽

Wistar Rats ◽

Apparent Dissociation Constant ◽

High Affinity ◽

Vasa Recta ◽

Dissociation Constant Kd ◽

Specific Ligand

Receptors for alpha-atrial natriuretic peptide (alpha-ANP) and C-type natriuretic peptide [CNP-(1-22)] were quantified in kidneys from adult Wistar rats by in vitro autoradiography. 125I-labeled alpha-ANP (100 pM) bound reversibly to glomeruli, outer medullary vasa recta, and inner medulla with an apparent dissociation constant (Kd) of 3–6 nM. The presence of 10 microM des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4– 23) (C-ANP), a specific ligand of the ANPR-C subtype of alpha-ANP receptor, inhibited approximately 50% of the glomerular binding of 125I-alpha-ANP, and this moiety of glomerular binding was also inhibited by CNP-(1–22) with an apparent inhibitory constant (Ki) of 10.47 +/- 7.59 nM. C-ANP and CNP-(1–22) showed little affinity for the medullary binding sites of alpha-ANP. 125I-[Tyr0]CNP-(1–22) (110 pM) bound solely to glomeruli and was competitively displaced by increasing concentrations of [Tyr0]CNP-(1–22) with an apparent Kd of 1.42 +/- 0.48 nM. Binding of increasing concentrations (25 pM to 1 nM) of 125I-[Tyr0]CNP-(1–22) in the presence or absence of 1 microM [Tyr0]CNP-(1–22) also demonstrated a high affinity (Kd of 0.41 +/- 0.07 nM) for the glomerular binding of 125I-[Tyr0]CNP-(1–22). Bound 125I-[Tyr0]CNP-(1–22) could be displaced by excess alpha-ANP and excess CNP-(1–22), both with high affinities. The glomerular binding of 125I-[Tyr0]CNP-(1–22) was also prevented by 10 microM C-ANP. Guanosine 3',5'-cyclic monophosphate produced by isolated glomeruli was measured by radioimmunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)

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Baboon erythrocyte ghosts contain beta-adrenergic receptors

AJP Cell Physiology ◽

10.1152/ajpcell.1985.249.1.c15 ◽

1985 ◽

Vol 249 (1) ◽

pp. C15-C19 ◽

Cited By ~ 1

Author(s):

E. E. Susanni ◽

F. P. Ross ◽

D. R. Scriven ◽

C. Rosendorff

Keyword(s):

Binding Sites ◽

Adrenergic Receptors ◽

Dependent Manner ◽

Erythrocyte Ghosts ◽

Beta Adrenergic Receptors ◽

Concentration Dependent Manner ◽

Apparent Dissociation Constant ◽

Beta Adrenergic ◽

Adrenergic Antagonist ◽

Number Of Binding Sites

We have used the beta-adrenergic antagonist [3H]dihydroalprenolol [( 3H]DHA) to identify binding sites on the erythrocyte membrane of the primate Papio ursinus. Analysis of the saturation isotherm revealed binding to be saturable with a maximal number of binding sites of 499 fmol/mg protein. [3H]DHA binds specifically to the erythrocyte ghosts with an apparent dissociation constant (Kd) of 0.57 +/- 0.06 nM. A similar value for Kd (0.46 +/- 0.07 nM) was evaluated from the rate constants of association (0.013 +/- 0.003 X nM-1 X min-1) and dissociation (0.006 +/- 0.001 X min-1). beta-adrenergic agonists compete for the binding sites with an order of potency (dl-isoproterenol greater than l-epinephrine greater than l-norepinephrine) typical of a beta 2-adrenergic receptor. Binding was shown to be stereospecific with l-stereoisomers being more potent than their corresponding d-stereoisomers in causing half-maximal inhibition. Isoproterenol stimulated the production of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) in a concentration-dependent manner, maximal levels (1.130 +/- 0.358 pmol cAMP/10(8) cells) being four times the basal levels. The results demonstrate the existence of a large number of beta-adrenergic receptors on baboon erythrocyte ghosts.

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Mot3, a Zn Finger Transcription Factor That Modulates Gene Expression and Attenuates Mating Pheromone Signaling in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/149.2.879 ◽

1998 ◽

Vol 149 (2) ◽

pp. 879-892 ◽

Cited By ~ 1

Author(s):

Anatoly V Grishin ◽

Michael Rothenberg ◽

Maureen A Downs ◽

Kendall J Blumer

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Signaling Pathway ◽

G Protein ◽

Binding Sites ◽

Dependent Manner ◽

Pheromone Response ◽

Mating Pheromone ◽

Pheromone Signaling ◽

Yeast Genes

Abstract In the yeast Saccharomyces cerevisiae, mating pheromone response is initiated by activation of a G protein- and mitogen-activated protein (MAP) kinase-dependent signaling pathway and attenuated by several mechanisms that promote adaptation or desensitization. To identify genes whose products negatively regulate pheromone signaling, we screened for mutations that suppress the hyperadaptive phenotype of wild-type cells overexpressing signaling-defective G protein β subunits. This identified recessive mutations in MOT3, which encodes a nuclear protein with two Cys2-His2 Zn fingers. MOT3 was found to be a dosage-dependent inhibitor of pheromone response and pheromone-induced gene expression and to require an intact signaling pathway to exert its effects. Several results suggested that Mot3 attenuates expression of pheromone-responsive genes by mechanisms distinct from those used by the negative transcriptional regulators Cdc36, Cdc39, and Mot2. First, a Mot3-lexA fusion functions as a transcriptional activator. Second, Mot3 is a dose-dependent activator of several genes unrelated to pheromone response, including CYC1, SUC2, and LEU2. Third, insertion of consensus Mot3 binding sites (C/A/T)AGG(T/C)A activates a promoter in a MOT3-dependent manner. These findings, and the fact that consensus binding sites are found in the 5′ flanking regions of many yeast genes, suggest that Mot3 is a globally acting transcriptional regulator. We hypothesize that Mot3 regulates expression of factors that attenuate signaling by the pheromone response pathway.

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Identification, characterization and cloning of myr 1, a mammalian myosin-I.

The Journal of Cell Biology ◽

10.1083/jcb.120.6.1393 ◽

1993 ◽

Vol 120 (6) ◽

pp. 1393-1403 ◽

Cited By ~ 71

Author(s):

C Ruppert ◽

R Kroschewski ◽

M Bähler

Keyword(s):

Amino Acid ◽

Light Chain ◽

Brush Border ◽

Binding Sites ◽

Neuronal Development ◽

Amino Acid Sequences ◽

Dependent Manner ◽

Tail Domain ◽

Myosin I ◽

Splice Forms

We have identified, characterized and cloned a novel mammalian myosin-I motor-molecule, called myr 1 (myosin-I from rat). Myr 1 exists in three alternative splice forms: myr 1a, myr 1b, and myr 1c. These splice forms differ in their numbers of putative calmodulin/light chain binding sites. Myr 1a-c were selectively released by ATP, bound in a nucleotide-dependent manner to F-actin and exhibited amino acid sequences characteristic of myosin-I motor domains. In addition to the motor domain, they contained a regulatory domain with up to six putative calmodulin/light chain binding sites and a tail domain. The tail domain exhibited 47% amino acid sequence identity to the brush border myosin-I tail domain, demonstrating that myr 1 is related to the only other mammalian myosin-I motor molecule that has been characterized so far. In contrast to brush border myosin-I which is expressed in mature enterocytes, myr 1 splice forms were differentially expressed in all tested tissues. Therefore, myr 1 is the first mammalian myosin-I motor molecule with a widespread tissue distribution in neonatal and adult tissues. The myr 1a splice form was preferentially expressed in neuronal tissues. Its expression was developmentally regulated during rat forebrain ontogeny and subcellular fractionation revealed an enrichment in purified growth cone particles, data consistent with a role for myr 1a in neuronal development.

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Covalent labelling of ligand binding sites of human placental S-adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP

Biochemical Journal ◽

10.1042/bj2320643 ◽

1985 ◽

Vol 232 (3) ◽

pp. 643-650 ◽

Cited By ~ 10

Author(s):

V N Aiyar ◽

M S Hershfield

Keyword(s):

Cyclic Amp ◽

Binding Sites ◽

Catalytic Mechanism ◽

Periodate Oxidation ◽

Regulatory Subunit ◽

Adenosylhomocysteine Hydrolase ◽

Dependent Protein ◽

Covalently Linked ◽

The Relationship ◽

Derivatives Of

S-Adenosylhomocysteine hydrolase (AdoHcyase) has previously been identified as a cytoplasmic adenosine and cyclic AMP binding protein. In order to examine the relationship between the adenosine and cyclic AMP binding sites on this enzyme we have explored the use of 8-azido analogues of adenosine and cyclic AMP as photoaffinity reagents for covalently labelling AdoHcyase purified from human placenta. 8-Azidoadenosine (8-N3-Ado), like adenosine, inactivated AdoHcyase, and the rate of inactivation was greatly increased by periodate oxidation. In addition, 8-N3-Ado was found to participate in the first step in the catalytic mechanism for AdoHcyase, resulting in conversion of enzyme-bound NAD+ to NADH, although it was not a substrate for the full enzyme-catalysed reaction. Radioactively labelled 8-N3-Ado, its periodate-oxidized derivative and 8-azidoadenosine 3′, 5′-phosphate (8-N3-cAMP) bound specifically to adenosine binding sites on AdoHcyase and, after irradiation, became covalently linked to the enzyme. Photoaffinity-labelled enzyme could be precipitated by monoclonal antibody to human AdoHcyase. Two observations suggested that cyclic AMP and adenosine bind to the same sites on AdoHcyase. First cyclic AMP and adenosine each blocked binding of both radioactively labelled 8-N3-Ado and 8-N3-cAMP, and second, digestion with V8 proteinase generated identical patterns of peptides from AdoHcyase that had been photolabelled with [32P]8-N3-cAMP and [3H]8-N3-Ado. Binding sites for cyclic AMP on AdoHcyase were found to differ functionally and structurally from cyclic AMP binding sites on the R1 regulatory subunit of cyclic AMP-dependent protein kinase.

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Neuropilin-1 Mediates Collapsin-1/Semaphorin III Inhibition of Endothelial Cell Motility

The Journal of Cell Biology ◽

10.1083/jcb.146.1.233 ◽

1999 ◽

Vol 146 (1) ◽

pp. 233-242 ◽

Cited By ~ 225

Author(s):

Hua-Quan Miao ◽

Shay Soker ◽

Leonard Feiner ◽

José Luis Alonso ◽

Jonathan A. Raper ◽

...

Keyword(s):

Binding Sites ◽

Aortic Ring ◽

Dependent Manner ◽

Vascular Endothelial ◽

Binding Assays ◽

Angiogenesis Assay ◽

Neuropilin 1 ◽

Endothelial Growth Factor ◽

Neuronal Guidance

Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65–75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

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Inhibition of angiotensinogen production by angiotensin II analogues in human hepatoma cell line

AJP Cell Physiology ◽

10.1152/ajpcell.1989.257.5.c888 ◽

1989 ◽

Vol 257 (5) ◽

pp. C888-C895 ◽

Cited By ~ 3

Author(s):

E. Coezy ◽

I. Darby ◽

J. Mizrahi ◽

B. Cantau ◽

M. H. Donnadieu ◽

...

Keyword(s):

Angiotensin Ii ◽

Cell Line ◽

Binding Sites ◽

Inhibitory Effect ◽

Hepatoma Cell Line ◽

Dependent Manner ◽

Human Hepatoma ◽

Ang Ii ◽

Hep G2 ◽

Dose Dependent

The aim of this study was to examine in Hep G2, a human hepatoma-derived cell line, the presence of angiotensin II (ANG II) receptors and the effect of ANG II and its analogues on angiotensinogen production. The presence of ANG II receptors was demonstrated using a long-acting ANG II analogue, 125I-labeled [Sar1]ANG II. A single class of specific binding sites was identified in these cells with a dissociation constant (Kd) of 2 nM. The number and affinity of these binding sites were not changed by [Sar1]ANG II treatment over 24 h. ANG II showed an inhibitory effect on angiotensinogen production. [Sar1]ANG II also exhibited a similar inhibitory effect as that of ANG II but to a greater extent and therefore was used throughout these studies. [Sar1]ANG II inhibited angiotensinogen production in a dose-dependent manner, exhibiting a half-maximal inhibitory concentration (IC50) of 2 nM. Other ANG II analogues showed similar effects on angiotensinogen production. In order of decreasing ability, they were [Sar1]ANG II greater than [Sar1-Ala8]ANG II greater than [Sar1-Val8]ANG II greater than [Sar1-Val5-(Br5)-Phe8]ANG II greater than [Sar1-Val5-DPhe8]ANG II. Results of these studies show that the Hep G2 cell possesses specific ANG II receptors and that [Sar1]ANG II induces a dose-dependent inhibition of angiotensinogen production in this system.

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