Shared and unique determinants of the erythropoietin (EPO) receptor are important for binding EPO and EPO mimetic peptide.

We have shown previously that Phe93 in the extracellular domain of the erythropoietin (EPO) receptor (EPOR) is crucial for binding EPO. Substitution of Phe93 with alanine resulted in a dramatic decrease in EPO binding to the Escherichia coli-expressed extracellular domain of the EPOR (EPO-binding protein or EBP) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe93 forms extensive contacts with a peptide ligand in the crystal structure of the EBP bound to an EPO-mimetic peptide (EMP1), suggesting that Phe93 is also important for EMP1 binding. We used alanine substitution of EBP residues that contact EMP1 in the crystal structure to investigate the function of these residues in both EMP1 and EPO binding. The three largest hydrophobic contacts at Phe93, Met150, and Phe205 and a hydrogen bonding interaction at Thr151 were examined. Our results indicate that Phe93 and Phe205 are important for both EPO and EMP1 binding, Met150 is not important for EPO binding but is critical for EMP1 binding, and Thr151 is not important for binding either ligand. Thus, Phe93 and Phe205 are important binding determinants for both EPO and EMP1, even though these ligands share no sequence or structural homology, suggesting that these residues may represent a minimum epitope on the EPOR for productive ligand binding.     

Functional analysis of the chicken PPARγ gene 5'-flanking region and C/EBPα-mediated gene regulation

Peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα) are the master regulators of adipogenesis. The regulatory mechanism of PPARγ and C/EBPα gene expression is clear in mammals, however, little is known in chicken. The aim of the present study was to characterize chicken PPARγ promoter and investigate whether PPARγ could be regulated by C/EBPα in chickens. A 2-kb nucleotide sequence upstream of the start codon of chicken PPARγ gene was cloned and characterized by using bioinformatics and experimental approaches. This 2-kb promoter region exhibited strong promoter activity in DF1 cells. The reporter gene assay showed that the chicken C/EBPα could activate PPARγ gene promoter. Further study by electrophoretic mobility shift assay and mutational analysis revealed that the chicken C/EBPα could directly bind to and regulate the PPARγ gene promoter. Our results demonstrate that PPARγ can be directly regulated by C/EBPα in chickens.     

Investigation of enantioselective ligand–protein binding and displacement interactions using capillary electrophoresis

When a protein such as human serum albumin is added to the separation buffer in capillary electrophoresis, the mobility of solutes which bind to that protein may be altered. The change in mobility of the solute is a function both of the strength of the binding interaction, and the difference in mobility between the free solute and protein additive. By adding other ligands which themselves bind to the protein, the strength of the solute–protein binding may be modified, leading to a measurable change in the mobility of the solute. These effects are particularly striking for chiral compounds, where enantioselectivity may be completely lost on addition of a competitive ligand. Capillary electrophoresis with human serum ablumin as a buffer additive was used to separate the enantiomers of benzoin and three phenothiazine derivatives. A comparison of the binding of (S)-benzoin to human serum albumin as determined by capillary electrophoresis and by ultrafiltration was made. A variety of other ligands were then added to the buffer along with the protein, and the effects on mobility and enantioselectivity were studied. The displacers included (R)- and (S)-oxazepam hemisuccinate, (R)- and (S)-warfarin, nitrazepam, phenylbutazone, and octanoic acid. From the results obtained, it seems that capillary electrophoresis may be a useful, rapid method to screen for drug–drug interactions. There are some advantages of using this technique to study protein–ligand interactions: Only very small amounts of ligand are needed (useful when dealing with metabolites); for chiral compounds, if protein binding is stereoselective, then the method is also stereoselective, so single enantiomers are not needed; finally, measurements are obtained in solution, without the need for immobilization of the protein. A disadvantage is that the ligand and protein must have significantly different electrophoretic mobilities. © 1994 Wiley-Liss, Inc.     

Binding of basic peptides to acidic lipids in membranes: effects of inserting alanine(s) between the basic residues.

We studied the binding of peptides containing five basic residues to membranes containing acidic lipids. The peptides have five arginine or lysine residues and zero, one, or two alanines between the basic groups. The vesicles were formed from mixtures of a zwitterionic lipid, phosphatidylcholine, and an acidic lipid, either phosphatidylserine or phosphatidylglycerol. Measuring the binding using equilibrium dialysis, ultrafiltration, and electrophoretic mobility techniques, we found that all peptides bind to the membranes with a sigmoidal dependence on the mole fraction of acidic lipid. The sigmoidal dependence (Hill coefficient greater than 1 or apparent cooperativity) is due to both electrostatics and reduction of dimensionality and can be described by a simple model that combines Gouy-Chapman-Stern theory with mass action formalism. The adjustable parameter in this model is the microscopic association constant k between a basic residue and an acidic lipid (1 less than k less than 10 M-1). The addition of alanine residues decreases the affinity of the peptides for the membranes; two alanines inserted between the basic residues reduces k 2-fold. Equivalently, the affinity of the peptide for the membrane decreases 10-fold, probably due to a combination of local electrostatic effects and the increased loss of entropy that may occur when the more massive alanine-containing peptides bind to the membrane. The arginine peptides bind more strongly than the lysine peptides: k for an arginine residue is 2-fold higher than for a lysine residue. Our results imply that a cluster of arginine and lysine residues with interspersed electrically neutral amino acids can bind a significant fraction of a cytoplasmic protein to the plasma membrane if the cluster contains more than five basic residues.     

Antibodies to FMRF amide, and the related pentapeptide LPLRF amide, reveal two groups of immunoreactive peptides in chicken brain

A radioimmunoassay has been developed for the chicken brain peptide, Leu-Pro-Leu-Arg-Phe-amide (LPLRF amide); this peptide was originally discovered because it reacts with antibodies to the molluscan neuropeptide FMRF amide. The present antibody to LPLRF amide reacts about twenty times less well with FMRF amide compared with LPLRF amide. Using radioimmunoassays employing antibodies raised against LPLRF amide and FMRF amide we have separated by gel filtration and HPLC several different immunoreactive peptides in acid alcohol extracts of chicken brain. When LPLRF amide was used as the assay standard one group of peptides reacted similarly with the two types of antibody; the other group, which was represented by a single major component, reacted at least 50 times better with FMRF amide antibodies compared with LPLRF amide antibodies. It seems, therefore, that in the avian central nervous system, and probably other vertebrates, there are several different groups of peptides immunochemically related to FMRF amide.     

Various properties of cardiac troponin C tryptic peptides

Using chromatography and preparative polyacrylamide gel electrophoresis, tryptic peptides TP 1 (residues 47-83), TP 2 (residues 84-118) and TP 3 (residues 119-161) were isolated in a highly homogeneous state from cardiac troponin C. Peptides TP 1, TP 2 and TP 3 were found to contain isolated cation-binding sites II, III and IV of cardiac troponin C. The interaction of these peptides with troponins I and T was studied. It was found that only peptide TP 2 could interact with troponin I. Neither of the peptides isolated interacted with troponin T. The cation-binding properties and structural peculiarities of peptide TP 1 were investigated. It was shown that despite its small size (37 amino acid residues), peptide TP 1 retained its ability to bind Ca2+ which caused conformational changes in the peptide structure. This was accompanied by changes in the electrophoretic mobility and absorption of TP 1 on phenyl-Sepharose.     

The CEC behaviour of several synthetic peptides related to the activin betaA-betaD subunits.

The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin betaA-betaD subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n-octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge-state-specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n-octadecyl-silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro-osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electro-osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, kappacec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their kappacec values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. > 40% (v/v) and nonpolar interactions with the Hypersil n-octadecyl-silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the ln kappacec values and the volume fractions, psi, of the organic modifier over a limited range of psi-values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.     

Binding of peptides with basic residues to membranes containing acidic phospholipids.

There are clusters of basic amino acids on many cytoplasmic proteins that bind transiently to membranes (e.g., protein kinase C) as well as on the cytoplasmic domain of many intrinsic membrane proteins (e.g., glycophorin). To explore the possibility that these basic residues bind electrostatically to monovalent acidic lipids, we studied the binding of the peptides Lysn and Argn (n = 1-5) to bilayer membranes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). We made electrophoretic mobility measurements using multilamellar vesicles, fluorescence and equilibrium binding measurements using large unilamellar vesicles, and surface potential measurements using monolayers. None of the peptides bound to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but all bound to vesicles formed from PC/PS or PC/PG mixtures. None of the peptides exhibited specificity between PS and PG. Each lysine residue that was added to Lys2 decreased by one order of magnitude the concentration of peptide required to reverse the charge on the vesicle; equivalently it increased by one order of magnitude the binding affinity of the peptides for the PS vesicles. The simplest explanation is that each added lysine binds independently to a separate PS with a microscopic association constant of 10 M-1 or a free energy of approximately 1.4 kcal/mol. Similar, but not identical, results were obtained with the Argn peptides. A simple theoretical model combines the Gouy-Chapman theory (which accounts for the nonspecific electrostatic accumulation of the peptides in the aqueous diffuse double layer adjacent to the membrane) with mass action equations (which account for the binding of the peptides to greater than 1 PS). This model can account qualitatively for the dependence of binding on both the number of basic residues in the peptides and the mole fraction of PS in the membrane.     

Electrophoretic detection of reversible chlorpromazine · HCl binding at the human erythrocyte surface

The binding of chlorpromazine · HCl at the human erythrocyte surface has been detected through its effect on cellular electrophoretic mobility. Incubation of erythrocytes (approx. 5 · 10⁶/ml) in 23 μM chlorpromazine · HCl resulted in a reduction of negative electrophoretic mobility from the control value of $\text{?1.11 ± 0.01}$ (μm · s^?1)/(V · cm^?1) to $\text{?1.00 ± 0.02}$ (μm · s^?1)/(V · cm^?1) (pH 7.2, ionic strength 0.155). This mobility change was completely reversed when chlorpromazine · HCl was removed by centrifugal washing. Increasing the drug concentration to 70μM did not affect the mobility change, indicating saturation of the electrophoretically detectable drug binding sites over chlorpromazine · HCl concentration range studied here. The effect of the 23 μM chlorpromazine · HCl on electrophoretic mobility was also measured in isotonic media of reduced ionic strength. The drug-induced reduction in negative surface charge density was found to be independent of ionic strength over the range 0.155 (Debye length, 0.8 nm) to 0.00310 (Debye length, 5.7 nm).Fixation of erythrocytes with glutaraldehyde affected neither the normal electrophoretic mobility of discocytes nor the reduced electrophoretic mobility of chlorpromazine · HCl-induced stomatocytes. When these stomatocytes were first fixed with glutaraldehyde, then washed free of chlorpromazine · HCl, they retained the stomatocyte form while regaining a normal control electrophoretic mobility. Conversely, when discocytes fixed in that form were treated with chlorpromazine · HCl, they showed the same mobility change as did fixed or unfixed stomatocytes. The drug-induced mobility change is therefore independent of the shape change, but reflects a contribution to cellular surface charge density from the membrane-bound chlorpromazine · HCl molecules. From the charge reduction, it is estimated that about 10⁶ chlorpromazine · HCl molecules are bound at the electrokinetic cell surface and occupy approximately 0.4% of the total surface area.