scholarly journals Interactions of small molecules with phospholipid bilayers. Binding to egg phosphatidylcholine of some uncharged molecules (2-acetylaminofluorene, 4-dimethylaminoazobenzene, oestrone and testosterone) that bind to ligandin and aminoazo-dye-binding protein A

1979 ◽  
Vol 180 (2) ◽  
pp. 319-326 ◽  
Author(s):  
Edward Tipping ◽  
Brian Ketterer ◽  
Lucia Christodoulides
Keyword(s):  
Lipid Bilayers ◽  
Partition Coefficients ◽  
Intracellular Transport ◽  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Protein A ◽  
Molar Ratio ◽  
Intracellular Proteins ◽  
Dye Binding ◽  
Membrane Bound

1. To assess the possible involvement of ligandin and aminoazo-dye-binding protein A in intracellular transport it is necessary to know how their ligands, most of which are molecules with hydrophobic moieties, interact with cellular membranes. To obtain such information we have examined the interactions of 2-acetylaminofluorene, 4-dimethylaminoazobenzene, oestrone and testosterone with aqueous dispersions of egg phosphatidylcholine and egg phosphatidylcholine/cholesterol (1:1, molar ratio) by equilibrium dialysis and spectrophotometry. 2. At 25°C and pH7.4, the partition coefficients for binding to phosphatidylcholine [expressed as (mol of ligand bound/mol of phosphatidylcholine)/unbound ligand concentration] were: for 2-acetylaminofluorene, 5.0×103 litre·mol−1; for 4-dimethylaminoazobenzene, 2.1×104 litre·mol−1; for oestrone, 3.1×103 litre·mol−1; and for testosterone, 4.2×102 litre·mol−1. In the ranges studied these values were independent of concentration. The results for the two steroids confirm those of Heap, Symons & Watkins [(1970) Biochim. Biophys. Acta218, 482–495]. 3. The introduction of cholesterol into the lipid bilayers caused large decreases in the partition coefficients of oestrone and testosterone, but had relatively little effect on the binding of 2-acetylaminofluorene and 4-dimethylaminoazobenzene. 4. By assuming that the interactions with egg phosphatidylcholine bilayers resemble those with the phospholipid components of mammalian intracellular membranes the phosphatidylcholine partition coefficients, together with data for binding to the intracellular proteins ligandin and aminoazo-dye-binding protein A, enable the subcellular distributions of the four compounds to be estimated. For the rat hepatocyte up to 98, 99, 89 and 58% of the total 2-acetylaminofluorene, 4-dimethylaminoazobenzene, oestrone and testosterone respectively may be membrane-bound.

scholarly journals Interactions of small molecules with phospholipid bilayers. Binding to egg phosphatidylcholine of some organic anions (bromosulphophthalein, oestrone sulphate, haem and bilirubin) that bind to ligandin and aminoazo-dye-binding protein A

1979 ◽  
Vol 180 (2) ◽  
pp. 327-337 ◽  
Author(s):  
E Tipping ◽  
B Ketterer ◽  
L Christodoulides
Keyword(s):  
Intracellular Transport ◽  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Protein A ◽  
Molar Ratio ◽  
Oestrone Sulphate ◽  
Dye Binding ◽  
Order Of Magnitude ◽  
Saturation Effects ◽  
Limiting Values

1. To assess the possible involvement of ligandin and aminoazo-dye-binding protein A in intracellular transport it is necessary to know how their ligands, most of which are molecules with hydrophobic moieties, interact with cellular membranes. To obtain such information we examined the interactions of bromosulphophthalein, oestrone sulphate, haem and bilirubin with aqueous dispersions of egg phosphatidylcholine and egg phosphatidylchone/cholesterol (1:1, molar ratio) by equilibrium dialysis and spectrophotometry. 2. In all four cases, saturation effects were observed. Values of Vmax (v = mol of compound bound/mol of lipid phosphorus) at 25 degrees C were: for bromosulphophthalein, approximately 0.1; for oestrone sulphate, approximately 0.25; for haem, approximately 0.25 (all at pH 7.4); and for bilirubin 0.1–0.2 (at pH 8.2). 3. Limiting values of v/c (c = unbound concentration) as v leads to 0 at 25 degrees C and pH 7.4 are: for bromosulphophthalein, 6.25 × 10(4) litre-mol-1; for oestrone sulphate, 7.8 × 10(2) litre-mol-1; for haem, 4.5 × 10(5) litre-mol-1; and for bilirubin, approximately 1.2 × 10(4) litre-mol-1. For haem the result depends on the assumption that only the monomeric form binds to the lipid. 4. The binding of each compound was decreased by cholesterol; bromosulphophthalein and oestrone sulphate were affected more than haem and bilirubin. 5. Bromosulphophthalein at saturating concentration decreased the limiting values of v/c of the other three compounds by approximately one order of magnitude. 6. By assuming that the interactions with egg phosphatidylcholine resemble those with the phospholipid components of mammalian intracellular membranes the binding data for phosphyatidylcholine, together with data for binding to the intracellular proteins ligandin and aminoazo-dye-binding protein A, enable the subcellular distributions of the four compounds to be estimated. For the rat hepatocyte up to 92, 51, 98 and 47% of the total bromosulphophthalein, oestrone sulphate, haem and bilirubin respectively may be membrane-bound.

Binding of Al by protein in plasma of patients on maintenance hemodialysis.

1985 ◽  
Vol 31 (8) ◽  
pp. 1314-1316 ◽  
Author(s):  
M Cochran ◽  
D Patterson ◽  
S Neoh ◽  
B Stevens ◽  
R Mazzachi
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Human Albumin ◽  
Hemodialysis Patients ◽  
Single Peak ◽  
Molar Ratio ◽  
Maintenance Hemodialysis ◽  
Mass Fractions

Abstract Gel filtration of plasma from hemodialysis patients, with use of reagents and apparatus with carefully minimized background Al concentrations, reproducibly showed a single peak for Al, corresponding exactly to the elution position of transferrin. The Al/transferrin molar ratio in adjacent fractions was constant (mean 0.126, SE 0.006) in replicate experiments. In contrast, the association of Al with albumin varied. Using both equilibrium dialysis and gel-filtration techniques, in the presence and absence of calcium or phosphate, we could demonstrate no significant binding of Al by human albumin at Al concentrations of 1 to 12 mumol/L. We saw no Al peak in pooled, concentrated, low-molecular-mass fractions of plasma gel-filtered on Sephadex G-50. Evidently, transferrin is the sole Al-binding protein in plasma of hemodialysis patients.

scholarly journals The interactions of haem with ligandin and aminoazo-dye-binding protein A.

1976 ◽  
Vol 157 (2) ◽  
pp. 461-467 ◽  
Author(s):  
E Tipping ◽  
B Ketterer ◽  
L Christodoulides ◽  
G Enderby
Keyword(s):  
Association Constant ◽  
Protein Complexes ◽  
Binding Protein ◽  
Protein A ◽  
Association Constants ◽  
Dye Binding ◽  
Cotton Effects ◽  
Haem Protein ◽  
Haem Iron ◽  
Soret Region

1. The interactions of ferriprotoporphyrin IX with ligandin and aminoazo-dye-binding protein A result in absorption spectra in the Soret region characteristic of the ligand in its monomeric state. 2. Both proteins are able to bind ferrous as well as ferric haem. 3. Ferriprotoporphyrin IX is bound at a single site on both proteins. At pH7.0, I 0.16M, difference-spectrophotometric measurements gave association constants of 10(7) and 4 × 10(6) LITRE/MOL FOR LIGANDIN AND PROTEin A respectively. Under the same conditions fluorescence-quenching experiments gave an association constant of 2 × 10(7) litre/mol for ligandin. 4. Bilirubin, bromosulphophthalein and oesterone sulphate each compete with haem for binding by the two proteins. 5. Ferriprotoporphyrin IX bound to both ligandin and protein A is able to form co-ordination complexes with CN-, but not, to any measurable extent, with either N3- or F-. From these results it is suggested that binding by the two proteins may not involve the haem iron atom. 6. Both haem-protein complexes give rise to measurable extrinsic Cotton effects in the Soret region. 7. The formation and properties of the ligandin- and protein A-haem complexes are compared with those of haem-albumin, haemoglobin, myoglobin and other haemoproteins.

scholarly journals The structure of lactoferrin-binding protein B fromNeisseria meningitidissuggests roles in iron acquisition and neutralization of host defences

2014 ◽  
Vol 70 (10) ◽  
pp. 1312-1317 ◽  
Author(s):  
Cory L. Brooks ◽  
Elena Arutyunova ◽  
M. Joanne Lemieux
Keyword(s):  
Binding Protein ◽  
Iron Acquisition ◽  
Protein A ◽  
Transport Systems ◽  
Cationic Peptide ◽  
Moraxella Bovis ◽  
Component Systems ◽  
Two Component Systems ◽  
Membrane Bound ◽  
Protein B

Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the generaNeisseriaandMoraxellahave evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein. While the mechanism behind iron acquisition from transferrin is well understood, relatively little is known regarding how iron is extracted from lactoferrin. Here, the crystal structure of the N-terminal domain (N-lobe) of the accessory lipoprotein lactoferrin-binding protein B (LbpB) from the pathogenNeisseria meningitidisis reported. The structure is highly homologous to the previously determined structures of the accessory lipoprotein transferrin-binding protein B (TbpB) and LbpB from the bovine pathogenMoraxella bovis. Docking the LbpB structure with lactoferrin reveals extensive binding interactions with the N1 subdomain of lactoferrin. The nature of the interaction precludes apolactoferrin from binding LbpB, ensuring the specificity of iron-loaded lactoferrin. The specificity of LbpB safeguards proper delivery of iron-bound lactoferrin to the transporter lactoferrin-binding protein A (LbpA). The structure also reveals a possible secondary role for LbpB in protecting the bacteria from host defences. Following proteolytic digestion of lactoferrin, a cationic peptide derived from the N-terminus is released. This peptide, called lactoferricin, exhibits potent antimicrobial effects. The docked model of LbpB with lactoferrin reveals that LbpB interacts extensively with the N-terminal lactoferricin region. This may provide a venue for preventing the production of the peptide by proteolysis, or directly sequestering the peptide, protecting the bacteria from the toxic effects of lactoferricin.

scholarly journals Engineering and overexpression of periplasmic forms of the penicillin-binding protein 3 of Escherichia coli

1994 ◽  
Vol 298 (1) ◽  
pp. 189-195 ◽  
Author(s):  
C Fraipont ◽  
M Adam ◽  
M Nguyen-Distèche ◽  
W Keck ◽  
J Van Beeumen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Binding Protein ◽  
Molar Ratio ◽  
Amino Acid Residues ◽  
Penicillin Binding Protein ◽  
Operator Expression ◽  
Membrane Bound ◽  
Terminal Amino ◽  
Laci Repressor

Replacement of the 36 and 56 N-terminal amino acid residues of the 588-amino-acid-residue membrane-bound penicillin-binding protein 3 (PBP3) of Escherichia coli by the OmpA signal peptide allows export of F37-V577 PBP3 and G57-V577 PBP3 respectively into the periplasm. The modified ftsI genes were placed under the control of the fused lpp promoter and lac promoter/operator; expression of the truncated PBP3s was optimized by varying the copy number of the recombinant plasmids and the amount of LacI repressor, and export was facilitated by increasing the SecB content of the producing strain. The periplasmic PBP3s (yield 8 mg/l of culture) were purified to 70% protein homogeneity. They require the presence of 0.25 M NaCl to remain soluble. Like the membrane-bound PBP3, they undergo processing by elimination of the C-terminal decapeptide I578-S588, they bind penicillin in a 1:1 molar ratio and they catalyse hydrolysis and aminolysis of acyclic thioesters that are analogues of penicillin. The membrane-anchor-free PBP3s have ragged N-termini. The G57-V577 PBP3, however, is less prone to proteolytic degradation than the F37-V577 PBP3.

scholarly journals Spectroscopic studies of the binding of bilirubin by ligandin and aminoazo-dye-binding protein A

1976 ◽  
Vol 157 (1) ◽  
pp. 211-216 ◽  
Author(s):  
E Tipping ◽  
B Ketterer ◽  
L Christodoulides ◽  
G Enderby
Keyword(s):  
Association Constant ◽  
Fluorescence Spectra ◽  
Binding Protein ◽  
Protein A ◽  
Spectroscopic Studies ◽  
Absorption And Fluorescence Spectra ◽  
Quantitative Studies ◽  
Dye Binding ◽  
Cotton Effects ◽  
Unbound Bilirubin

Ligandin and aminoazo-dye-binding protein A both bind bilirubin at a single site. Quantitative studies of the interactions using difference spectrophotometry show that at pH 7.0, protein A binds the tetrapyrrole with an association constant (K) greater than or equal to 2 × 10(7) litre/mol, whereas binding by ligandin is slightly weaker (K = 7 × 10(6) litre/mol) at this pH. The protein-bilirubin complexes give rise to absorption and fluorescence spectra quite different from those of unbound bilirubin and also to large Cotton effects. It appears that on binding to both proteins, the ligand is forced into a rigid twisted configuration in a hydrophobic environment. Ligandin and protein A resemble serum albumin in their interactions with bilirubin.

scholarly journals The influence of soluble binding proteins on lipophile transport and metabolism in hepatocytes

1981 ◽  
Vol 195 (2) ◽  
pp. 441-452 ◽  
Author(s):  
E Tipping ◽  
B Ketterer
Keyword(s):  
Small Molecules ◽  
Intracellular Transport ◽  
Binding Proteins ◽  
Binding Protein ◽  
Protein A ◽  
Model Systems ◽  
Fatty Acid Binding ◽  
Order Of Magnitude ◽  
Intracellular Binding ◽  
Z Protein

A theory is presented that deals with the involvement of the intracellular binding proteins ligandin and aminoazodye-binding protein A (otherwise known as Z-protein or fatty-acid-binding protein) on the uptake and intracellular transport and metabolism of their ligands. Equations are derived that combine steady-state diffusional fluxes of small molecules that are (a) free in the aqueous phase of the cell, (b) bound to the two proteins and (c) partitioned into intracellular membranes, for model systems that resemble conditions in the rat hepatocyte. These equations are then combined with expressions for the enzyme-catalysed metabolic reactions undergone by these small molecules to assess the influence of diffusion rats on the overall metabolic rates. It is concluded that ligandin and protein A can enhance the rate of intracellular of their ligands by an order of magnitude or more and that this could make the hepatocyte several times more efficient in metabolizing these ligands. Various ways of testing this theory are discussed.

The kidney in vitamin B12and folate homeostasis: characterization of receptors for tubular uptake of vitamins and carrier proteins

2006 ◽  
Vol 291 (1) ◽  
pp. F22-F36 ◽  
Author(s):  
Henrik Birn
Keyword(s):  
Intracellular Transport ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Folate Receptor ◽  
Protein A ◽  
Intrinsic Factor ◽  
Vitamin B ◽  
Carrier Proteins ◽  
Folate Binding Protein ◽  
Renal Tubular

Over the past 10 years, animal studies have uncovered the molecular mechanisms for the renal tubular recovery of filtered vitamin and vitamin carrier proteins. Relatively few endocytic receptors are responsible for the proximal tubule uptake of a number of different vitamins, preventing urinary losses. In addition to vitamin conservation, tubular uptake by endocytosis is important to vitamin metabolism and homeostasis. The present review focuses on the receptors involved in renal tubular recovery of folate, vitamin B12, and their carrier proteins. The multiligand receptor megalin is important for the uptake and tubular accumulation of vitamin B12. During vitamin load, the kidney accumulates large amounts of free vitamin B12, suggesting a possible storage function. In addition, vitamin B12is metabolized in the kidney, suggesting a role in vitamin homeostasis. The folate receptor is important for the conservation of folate, mediating endocytosis of the vitamin. Interaction between the structurally closely related, soluble folate-binding protein and megalin suggests that megalin plays an additional role in the uptake of folate bound to filtered folate-binding protein. A third endocytic receptor, the intrinsic factor-B12receptor cubilin-amnionless complex, is essential to the renal tubular uptake of albumin, a carrier of folate. In conclusion, uptake is mediated by interaction with specific endocytic receptors also involved in the renal uptake of other vitamins and vitamin carriers. Little is known about the mechanisms regulating intracellular transport and release of vitamins, and whereas tubular uptake is a constitutive process, this may be regulated, e.g., by vitamin status.

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

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