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Biomedical subjects

W H Sawyer

Publications and source records attributed to W H Sawyer.

At least 19 recordsLinked to original sources
PubMed1997

Effects of charged lipids on the interaction of cholesteryl ester transfer protein with lipid microemulsions.

This study reports the effects of charged lipids on the transfer of cholesterlyl-1 pyrene decanoate (Py-CE) between apolipoprotein-free microemulsion particles mediated by cholesteryl ester transfer protein (CETP). The surface charge characteristics of microemulsion particles composed of cholesteryl oleate and egg yolk phosphatidylcholine were altered by incorporating phosphatidyl-serine, oleate or stearylamine into the phosphatidylcholine that forms the surface monolayer of the particle. The transfer of Py-CE was measured continuously by following the decrease in excimer fluorescence that accompanies the transfer of the probe from donor to acceptor particles [Rajaram, Chan and Sawyer (1994) Biochem. J. 304, 423-430]. The inclusion of 20 mol% phosphatidylserine relative to the phospholipid in the surface monolayer of the emulsion caused a 64% decrease in the first-order rate constant describing the transfer. An increase in ionic strength caused a partial reversal of this effects, indicating that electrostatic factors are only partially responsible for the interaction with lipid. Complete inhibition of transfer was observed when 10 mol% sodium oleate was incorporated into the surface monolayer. The incorporation of stearylamine into the emulsion caused a 32% increase in the transfer rate. The binding of CETP to the different emulsion surfaces was also examined using a surface plasmon resonance biosensor. The presence of negatively charged lipid (phosphatidylserine or oleic acid) decreased the rate of association of CETP with the emulsion without a significant change in the dissociation rate constant. The presence of the positively charged lipid stearylamine increased the rate of association of CETP with the lipid surface. It is concluded that a negative surface charge on the monolayer decreases the rate of transfer by decreasing the affinity of CETP for these particles.

Apolipoproteins↗
PubMed1996

The effect of self-association on the interaction of the Escherichia coli regulatory protein TyrR with DNA.

The interaction of the Escherichia coli regulatory protein TyrR, with a 42 bp oligonucleotide (42A/42B) containing a centrally located recognition sequence (TyrR box), was examined by analytical ultracentrifugation. The stoichiometry of the binding of oligonucleotide to dimeric TyrR was determined by equilibrium centrifugation of a mixture of fluorescein-5-isothiocyanate-labelled 42A/42B (F-42A/42B) in the presence of an eightfold molar excess of TyrR. The molecular mass (M) of the labelled oligonucleotide was estimated as 148,000, indicating a 1:1 complex composed of oligonucleotide (M = 27,000) and TyrR dimer (M = 113,000). The association constant (Ko,d = 2.8(+/- 0.1) x 10(6) M-1) was determined by a global analysis of sedimentation data, collected at multiple wavelengths between 230 and 285 nm. The presence of 30 microM ATP gamma S enhanced the affinity of TyrR for DNA approximately 3.5-fold, (Ko,d = 9.9(+/- 0.3) x 10(6) M-1). The effect of dimer to hexamer self-association of TyrR on the binding of 42A/42B was also examined. Multiple wavelength sedimentation data fitted a model in which the oligonucleotide could bind to one site on the dimer (Ko,d = 9.9 x 10(6) M-1), and to either one or three sites on the hexamer (Ko,h) = 2.0(+/- 0.1) x 10(6) M-1 and 3.8(+/- 0.1) x 10(6) M-1, respectively). Competitive sedimentation equilibrium and fluorescence anisotropy titrations were performed under stoichiometric conditions to resolve the number of oligonucleotide binding sites per hexamer. In these experiments, 42A/42B was used as a competitor to displace F-42A/42B from the hexamer, which was found to bind the 42mer with a 1:1 stoichiometry. Our data support a model in which ATP increases the affinity of TyrR for the DNA recognition sequence, and tyrosine induced self-association of TyrR generates a hexameric species with a single binding site for the 42A/42B oligonucleotide.

Adenosine Triphosphate↗
PubMed1996

Dynamics and morphology of the in vitro polymeric form of elongation factor Tu from Escherichia coli.

Elongation factor Tu from Escherichia coli is known to polymerize at slightly acidic pH and low ionic strength. The structure and dynamics of these aggregates have been examined using imaging and spectroscopic methodologies. Electron microscopy provides evidence for two-dimensional sheets and bundled filaments of EF-Tu, whereas fluorescence microscopy of EF-Tu covalently labeled with tetramethylrhodamine isothiocyanate showed highly branched polymers of EF-Tu several microns in diameter. These polymers were studied using quasi-elastic light scattering to determine the evolution of the translational diffusion coefficient during the polymerization process. The rotational dynamics of the aggregate were investigated using phosphorescence anisotropy of EF-Tu covalently labeled with erythrosin isothiocyanate. A high infinite-time anisotropy was observed, suggesting a lack of motion or entanglement of EF-Tu polymers. A sub-microsecond motion which was slowed in the presence of glycerol may be due to local flexibility of the polymers. The possible relevance of polymeric EF-Tu to its function in vivo is discussed.

Anisotropy↗
PubMed1996

Evaluation of affinity constants by competitive binding assays: removal of empiricism from the Müller method.

A method is described for the quantitative screening of antigen analogs by competitive radioimmunoassay. This analytical procedure is based on an experimental design developed for the comparison of binding affinities by means of an empirical quantitative expression (Müller, R. (1980) J. Immunol. Methods 92, 589-601). The present analysis, which allows retention of the same general approach without sacrifice of thermodynamic rigor, is illustrated by application to literature results obtained by radioimmunoassay screening of the interactions between antigen analogs and antibodies elicited in response to (i) a scorpion toxin and (ii) 8-hydroxy-2-deoxyguanosine.

Antigen-Antibody Reactions↗
PubMed1996

Structural and functional properties of full-length and truncated human proapolipoprotein AI expressed in escherichia coli.

Utilizing the Escherichia coli/pGex vector expression system incorporating a thrombin cleavage site, full-length (residues -6-243) and truncated forms of proapolipoprotein AI (proapoAI), terminating at amino acid residues 222, 210, 150, and 135, were purified to levels of at least 5 mg/L, after thrombin cleavage. Assessed by circular dichroism, the helical contents of L-alpha-dimyristoylphosphatidylcholine-associated forms of human plasma-derived apolipoprotein AI (apoAI) and recombinant proapoAI were comparable, being 69% and 65%, respectively. Circular dichroism measurements of the lipid-associated complexes of the truncated forms showed that between the sequence of residues 150-222 no additional helicity was gained until the carboxyl-terminal sequence was present in the molecule, indicating that the carboxyl terminus of the protein is required for the formation of helix within this central region. While tryptophan residues were more than 86% accessible, as assessed by iodide quenching, in the two truncated forms, proapoAI-6-135 and proapoAI-6-150, for both free and complexed protein, this figure fell to about 50% for full-length recombinant proapoAI, further indicating the influence of the carboxyl terminus on the structure of the whole protein. While cross-linking human plasma apoAI in solution with dithiobis-(succinimidyl propionate) revealed high molecular weight oligomers by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, recombinant proapoAI did not strongly form complexes larger than trimers. None of the truncated proapoAI molecules formed oligomers larger than trimers. The shortest form, proapoAI-6-135, only dimerized. Initial results from lecithin:cholesterol acyltransferase activation (apoAI peptide concentration 0.2 microM) indicated that truncation of the 21 carboxy-terminal amino acids resulted in a drop of approximately 53% in activation and 33 residues a drop of 67% relative to the full-length protein. Overall these results indicate the important influence of the carboxyl terminus on the structure of apoAI.

Apolipoprotein A-I↗
PubMed1996

Characterisation of lipid-protein interactions using a surface plasmon resonance biosensor.

The binding of proteins to lipid surfaces is of fundamental importance when considering the mechanisms of lipases and lipid transfer proteins. Current procedures for determining the binding constants suffer from problems related to poor sensitivity, poor reproducibility and to the use of restrictive assumptions in the analysis. We demonstrate here the use of the surface plasmon resonance biosensor to determine the association and dissociation kinetics of binding of cholesteryl ester transfer protein to the surface of emulsion particles of defined size and composition. Uncertainty about the emulsion valency, that is, the number of sites on the lipid particle occupied by the immobilised transfer protein on the biosensor surface, means that the association kinetics can only be characterised in terms of the product of the association rate constant and the concentration of sites, whereas the dissociation rate constant can be obtained unambiguously. The technique will prove useful in characterising the binding of proteins to lipid surfaces.

Apolipoproteins↗
PubMed1995

Interaction between the Escherichia coli Regulatory protein TyrR and DNA: a fluorescence footprinting study.

The Escherichia coli regulatory protein TyrR controls the expression of eight transcription units that encode proteins involved in the biosynthesis and transport of aromatic amino acids. It is a homodimer of 57 600 subunit molecular weight and has a binding site for ATP and weak ATPase activity. In the presence of ATP, TyrR binds tyrosine, which induces self-association of TyrR from a dimer to a hexamer. This report examines the interaction of TyrR with a 42 bp DNA oligonucleotide containing a centrally located binding site for TyrR (TyrR box). Replacement of a thymidine residue with an aminouridine residue at positions 7, 9, 13, 15, 19, 22, and 26 from one end of the 42mer enables labeling with fluorescein and successive placement of the label along the major groove of the DNA. The fluorescence footprinting of the oligonucleotide was followed using steady-state and time-resolved fluorescence methods. Binding of the TyrR dimer caused significant changes in the fluorescent properties of the labels attached to positions 13, 15, and 26, suggesting the involvement of these bases in the binding of the protein. Except for the position 15 conjugate, binding of the TyrR dimer caused little change in fluorescence intensity. Therefore, fluorescence anisotropy was used to follow the binding equilibrium. The fluorescence of the position 15 conjugate increased 1.6-fold on binding TyrR, suggesting that the fluorophore was in close contact with the protein. For all conjugates, the addition of tyrosine at the end of the titration with TyrR increased the anisotropy markedly, suggesting that the hexameric form of TyrR could bind the oligonucleotide. Two rotational correlation times were found for the labeled conjugates: one reflecting the motion of the probe at its point of attachment to the DNA (220-290 ps), the other reflecting the global tumbling of the labeled oligonucleotide (14-21 ns). On binding TyrR, changes in the correlation times and their associated amplitudes and changes in the range of angular motion of the probe depended on the position of the label. Evidence is presented that the binding of the TyrR hexamer, but not the TyrR dimer, affects regions that flank the binding sequence. The results support the hypothesis that the binding of the TyrR hexamer is responsible for interaction between tandem TyrR boxes in the tyrR regulon.

Base Sequence↗
PubMed1995

Synthesis and characterisation of fluorescent oligonucleotides. Effect of internal labelling on protein recognition.

Fluorescently labelled 42 base pair DNA duplexes were synthesised to examine the interaction between the TyR repressor protein of Escherichia coli and its DNA recognition sequence. An Fmoc-protected 5-(3-aminoprop-l-yn-l-yl)-2'-deoxyuridine phosphoramidite was synthesised and incorporated into oligonucleotides using standard beta-cyanoethyl phosphoramidite chemistry. Oligonucleotides containing the 3-aminopropynyl nucleotide at internal positions were reacted with fluorescein isothiocyanate to generate fluorescent DNA molecules useful for characterising interactions between DNA and proteins. Short DNA duplexes were investigated with respect to their melting temperatures and their ability to bind TyrR. Oligonucleotides containing a TyrR binding site were labelled in the central region of the recognition sequence or near the 5' edge of the recognition sequence. Fluorescein-labelled oligonucleotides could hybridise to form duplex DNA, and gel retardation experiments showed that the presence of the dye did not alter the binding affinity for the TyrR protein significantly. Fluorescence anisotropy measurements were used to examine the binding equilibrium in low and high salt buffers. A dissociation constant of 200-500 nM was obtained for the interaction of the TyrR dimer with a 42 bp duplex containing a centrally located 22 bp TyrR binding site.

Amides↗
PubMed1995

Effects of a D-Cys6/L-Cys6 interchange in nonselective and selective vasopressin and oxytocin antagonists.

We report the solid-phase synthesis of the D-Cys6 analogues of arginine-vasopressin (AVP), peptide 1, of the selective AVP vasopressor (V1a receptor) antagonist [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O-methyltyrosine]arginine-vasopressin (d(CH2)5[Tyr(Me)2]-AVP, (A)), peptide 2, of the three nonselective antidiuretic/vasopressor (V2/V1a receptor) AVP antagonists d(CH2)5[Tyr(Et)2]VAVP (B), d(CH2)5[D-Tyr(Et)2]VAVP (C), and d(CH2)5[D-Phe2]VAVP (D) (where V = Val4), peptides 3-5, of the nonselective oxytocin (OT) antagonists d(CH2)5-[Tyr(Me)2]OVT (E) and d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT (F) (where OVT = ornithine-vasotocin), peptides 6 and 7, and of the selective OT antagonists desGly-NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT (G) and d(CH2)5]D-Trp2,Thr4]OVT (H), peptides 8 and 9. We also present the repeat syntheses of the previously reported d(CH2)5[D-Trp2]AVT (peptide 10) and its D-Cys6 analogue (peptide 11) (where AVT = arginine-vasotocin). Peptides 1-11 were assayed for agonistic and antagonistic activities in in vivo V1a, V2, and oxytocic assays and in in vitro oxytocic assays without and with 0.5 mM Mg2+. With V2 and V1a agonistic potencies of 0.82 and 0.41 units/mg, [D-Cys6]AVP has retained less than 0.3% of the V2 and V1a potencies of AVP. It exhibits no oxytocic activity and is an in vitro OT antagonist. pA2 = 6.67 (no Mg2+); pA2 = 5.24 (0.5 mM Mg2+). By contrast, with one or two exceptions, a D-Cys6/L-Cys6 interchange in antagonists 2-9, although resulting in reductions of antagonistic potencies in all assays for virtually all peptides 2-9 relative to A-H, has been well tolerated. For peptides 2-5, the anti-V2 and anti-V1a pA2 values range from approximately 5.54 to 7.33 and from 7.19 to 8.06, respectively; the range of in vitro anti-OT pA2 values (no Mg2+) is 7.35-7.87; with 0.5 mM Mg2+, the range is 7.24-8.21. Peptides 2 and 4 have in vivo anti-OT pA2s = 6.60 and 7.16, respectively. For peptides 6-9, the range of in vitro anti-OT pA2 values (no Mg2+) is 7.65-7.96; with 0.5 mM Mg2+, the range is 7.41-7.65, and the in vivo anti-OT pA2 values range from 6.85 to 7.33. With an in vivo anti-OT pA2 = 7.33, peptide 6 is equipotent with its parent E. The in vivo anti-OT potencies of peptides 7-9 are significantly reduced relative to those of F-H. The in vitro anti-OT (0.5 mM Mg2+) pA2 values of 10 and 11 are 7.54 and 7.50, both significantly lower than those previously reported.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Advances in the design of selective antagonists, potential tocolytics, and radioiodinated ligands for oxytocin receptors.

Despite intensive efforts over three decades in many laboratories, attempts to design peptide antagonists of oxytocin (OT) which are more selective for OT uterine receptors than for vasopressin (AVP), vasopressor V1a receptors, have met with only limited success. We will review the current status of the field and report on studies in our laboratories which have led to the design of highly potent non-selective and selective OT antagonists. Virtually all are more potent (2-6 fold) and a number are more selective (10-12 fold) than Atosiban, currently in clinical trial as a tocolytic agent. Many of these new published and unpublished OT antagonists are thus promising candidates for development as potential tocolytic agents for the prevention of pre-term labor. We also report on promising new radioiodinatable ligands for OT receptors. All the new OT antagonists are valuable new tools for studies on the physiological roles of OT and as probes for OT and AVP receptors.

Amino Acid Sequence↗
PubMed1994

Effect of unesterified cholesterol on the activity of cholesteryl ester transfer protein.

Cholesteryl ester transfer protein (CETP) catalyses the transfer of cholesteryl ester from high-density lipoprotein to triacylglycerol-rich lipoproteins and the transfer of triacylglycerols in the reverse direction. The activity of CETP has been studied using a continuous fluorescence assay which measures the excimer fluorescence of cholesteryl 1-pyrene decanoate in a synthetic donor microemulsion as the indicator of cholesteryl ester transfer. Emulsions were composed of cholesteryl oleate and egg phosphatidylcholine and had an average particle size of 14 +/- 1 nm as calculated from the molar volume of the components. The effect of changing the physical state of the emulsion surface was examined by including unesterified cholesterol in the donor and acceptor particles. The rate of CETP-induced transfer of the fluorescent cholesteryl ester between microemulsion particles increased when unesterified cholesterol was present at concentrations up to 17 mol% relative to phospholipid. The presence of cholesterol also changed the exchange kinetics from an apparent single-exponential to a double-exponential phenomenon. Binding of CETP to the emulsion surface was accompanied by an enhancement of fluorescence which was used to measure the binding equilibria. The enhancement of exchange due to the presence of cholesterol did not correlate with any increased binding of CETP to the emulsion surface. The presence of unesterified cholesterol in the donor did not affect the rate of transfer of the fluorescent cholesteryl ester when unlabelled emulsion was replaced by high-density lipoprotein as the acceptor. The studies demonstrate the use of microemulsions of defined size and composition for the study of the mechanism of action of CETP.

Carrier Proteins↗
PubMed1994

Interactions between fatty acids and lipoprotein lipase: specific binding and complex formation.

Lipoprotein lipase is the extrahepatic lipase responsible for the hydrolysis of triglycerides in chylomicrons and very low-density lipoproteins. Its enzymic activity and its location on the surface of endothelial cells are affected by the presence of fatty acids, implying that the protein possesses binding sites for that ligand. In this study, we examine the binding of fatty acids to LpL and describe factors that must be considered when the dissociation constant of the acceptor-ligand equilibrium is close to the critical micelle concentration of the fatty acid. The interaction of fatty acids with lipoprotein lipase (LpL) was studied by two methods. A new direct method, based on the LpL-induced increase in the apparent critical micelle concentration of the sodium soap of the fatty acid, indicates the presence of multiple high-affinity binding sites. In the second method, the specific binding of fatty acids to LpL was measured by quantitating the blue shift in the tryptophan fluorescence of LpL that occurs upon binding the ligand. Both methods suggest the existence of 4-6 fatty acid binding sites on LpL with a dissociation constant on the order of 10(-6)-10(-7) M. Further analysis of the blue shift indicates that at higher concentrations of fatty acid, large complexes are formed consisting of 260-310 molecules of fatty acid per LPL monomer. In contrast, no large complexes are formed with fatty acids that form crystals above their solubility limit.

Animals↗
PubMed1994

A spectroscopic and equilibrium binding analysis of cationic detergent-protein interactions using soluble and insoluble recombinant porcine growth hormone.

Overexpression of cloned eukaryote genes in bacteria often leads to the formation of insoluble refractile bodies which require solubilization by harsh denaturants or detergents. We describe the conformational changes associated with the binding of a surfactant, cetyltrimethylammonium chloride (CTAC) to recombinant porcine growth hormone (PGH). The stoichiometry of binding by CTAC to the soluble and insoluble forms of recombinant PGH was also assessed. Optimum CTAC binding and protein solubilisation were obtained at 50 degrees C and at extreme pH. Increased ionic strength and changes in pH towards the isoelectric point of PGH (pH 6) decreased both the binding of CTAC and the efficiency of solubilising PGH from inclusion bodies. The positive charge on the quaternary ammonium head group of CTAC was found to be critical in the binding of CTAC to PGH and for the subsequent solubilisation of inclusion bodies. The binding of CTAC to the soluble form of PGH caused appreciable changes to the tertiary structure of the protein but did not significantly alter secondary structure, or cause complete unfolding. These observations help to explain earlier results which demonstrate that urea, guanidine hydrochloride and CTAC solubilized recombinant PGH molecules behave differently during in vitro refolding (Puri, N.K., Crivelli, E.C., Cardamone, M., Fiddes, R., Bertolini, J., Ninham, B. and Brandon, M.R. (1992) Biochem. J. 285, 871-879.).

Anilino Naphthalenesulfonates↗
PubMed1994

Vasopressin-binding sites in the pig pituitary gland: competition by novel vasopressin antagonists suggests the existence of an unusual receptor subtype in the anterior lobe.

Arginine vasopressin (AVP) acts in the pituitary gland, in synergy with corticotrophin-releasing factor, to induce ACTH release in response to stressful stimuli. Pituitary AVP receptors in the rat are coupled to phospholipase C, as are the so-called V1-type AVP receptors. The present study examined [3H]AVP binding in membranes prepared from the anterior lobe of the pituitary gland of the pig. [3H]AVP, alone or in competition with analogues, bound to sites in the pig anterior lobe which are pharmacologically similar to those described previously by others in the rat pituitary gland. For comparison, the same competition studies were performed on membrane preparations from the rat liver which contain the classic V1-type AVP receptor. Pituitary and liver AVP-binding sites were dissimilar; both cyclic and linear V1 antagonists had, in general, a much lower affinity for pituitary AVP-binding sites than for those in the liver. Thus, Phaa-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 (Phaa = phenylacetyl) has a 2500-fold greater affinity for the latter (negative logarithm of inhibition constant (pKi) = 9.64) than for the former (pKi = 6.22). One linear antagonist, Pa-D-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2 (Pa = propionyl) had about equal affinities for liver and pituitary membranes (pKi = 6.39 and 6.53 respectively). Another compound, Phaa-D-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2 had the highest affinity found to date for binding to AVP sites in the pituitary (pKi = 7.43). These findings suggest some ideas for the design of more potent and/or selective AVP analogues acting in the pituitary gland.

Amino Acid Sequence↗
PubMed1993

The problem of partial competition in the quantitative characterization of interactions by competitive binding assays.

Binding expressions are derived and analytical procedures developed for the quantitative characterization of inhibitor binding that is only partially competitive with the interaction between an acceptor and the ligand that is being monitored. Two such situations are considered: (i) that in which the partial competition reflects binding of inhibitor to fewer acceptor sites than available to ligand; and (ii) that in which the partial competition reflects binding of inhibitor to acceptor sites in addition to those occupiable by ligand. The potential efficacy of the suggested analyses is then explored by their application to simulated data that span the likely range of experimental behavior. Quantitative analysis of the displacement of [3H]nitrobenzylthioinosine from cultured leukemic cells by an adduct of 5'-S-(2-amino-ethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine with fluorescein-5-isothiocyanate is used to establish that the cells possess 6% fewer sites (150,000 cf. 159,000 sites/cell) for the fluorescent adduct than for the tritiated ligand, and that the binding is 10-fold weaker (binding constant of 0.28 cf. 2.8 nM-1). Corresponding analysis of results obtained with bovine aorta endothelial cells indicates that a 3-fold weaker interaction (binding constant of 1.1 cf. 3.3 nM-1) occurs between the fluorescent adduct and 79% of the cell sites accessible to the tritiated ligand. The present analytical procedures extend the utility of competitive binding assays for the quantitative screening of potential inhibitors by removing the inherent limitation of existing analyses that all acceptor sites be accessible to both the competing solute and the indicator ligand.

Adenosine↗