Large-ligand adsorption to membranes: II. Disk-like ligands and shape-dependence at low saturation

Large-ligand adsorption to membranes or cells is considered in the absence of cooperative interactions. For the low-saturation regime, a general and exact treatment is given by means of the concept of excluded areas. With the help of this formalism, shape dependence of the adsorption behavior can be discussed quantitatively. In addition, a formalism is presented which allows to calculate binding curves at arbitrary saturation for ligands having a symmetric shape (disks, regular polygons). The underlying model is a modified version of the hard core fluid theory of Andrews (Andrews, F.C. (1976) J. Chem. Phys. 64, 1941–1947). Apart from applications to symmetric ligands, the results can be used to derive limiting conditions for ligands of any shape.     

Large-ligand adsorption to membranes III. Cooperativity and general ligand shapes

In previous reports (Stankowski, S. (1983) Biochim. Biophys. Acta 735, 341–351 and 352–360) the ordinary Scatchard-type analysis has been shown to yield erroneous results when applied to the binding of large molecules to membranes or cells. Formulae have been given to treat the limiting cases of very thin and of very bulky ligands. These results are now extended to include ligands of any shape and cooperative interactions. As an example, data on the cooperative binding of polymyxin to charged lipid bilayers are reevaluated. Adsorption with concomitant incorporation of the large molecule into the membrane is also considered.     

Protein binding studies of luminescent rhenium(I) diimine complexes

Interaction of four luminescent rhenium(I) diimine complexes, [Re(CO)₃(N-N)L]PF₆ ((N-N = 2,2-bipyridine, L = py-3-COOH) 1a, (N-N = 2,2-bipyridine, L = py-3-CONH₂) 1b, (N-N = 1,10-phenanthroline, L = py-3-COOH) 2a, (N-N = 1,10-phenanthroline, L = py-3-CONH₂) 2b with bovine serum albumin (BSA) at physiological pH has been examined using UV-Vis absorption and luminescence spectroscopy, excited state lifetime measurement and circular dichroism (CD). In the presence of BSA, the luminescence of Re(I) complexes is quenched due to the locking-in of the probe into the protein environment. Interestingly the probe is released from the protein environment in the presence of sodium dodecyl sulfate (SDS) resulting in the restoration of the original luminescence along with a red shift in the emission maximum. These observations are explained in terms of binding constants (K_a) of probe with protein and surfactant and the nature of the binding has been investigated from Scatchard plot and Hill’s coefficient (n) value. These studies point out that the interaction between Re(I) complexes and BSA is cooperative in nature.     

Phospholipid composition of dystrophic chicken erythrocyte plasmalemmae I. Isolation of a unique lipid in dystrophic erythrocyte membranes

Several structural and functional properties are characterized in nucleated erythrocyte plasmalemmae of age and sex-matched dystrophic (line 413) and normal (line 412) chickens obtained from the University of California at Davis. Plasmalemma purity is assessed through marker enzymes. Significant differences are observed in the phospholipid content between dystrophic and normal chickens. The dystrophic chicken erythrocyte plasmalemma has an increased concentration of phosphatidylserine and a decreased concentration of phosphatidylethanolamine compared with control birds. Also, a measurable and distinct polar lipid, observed only on thin-layer chromatography (TLC) plates spotted with dystrophic preparations, is visualized adjacent to phosphatidylethanolamine. These abnormalities in the dystrophic chicken erythrocyte may signal a general defect in membrane structure for chicken dystrophy.     

Interaction of Fe(III) with herbicide-carboxylato ligands. Di-, tri- and tetra-nuclear compounds: Structure, antimicrobial study and DNA interaction

The iron complexes with the phenoxyalkanoic acids 2,3-D = 2,3-dichlorophenoxyacetic acid, 3,4-D = 3,4-dichlorophenoxyacetic acid, 2,4,5-T = 2,4,5-trichlorophenoxyacetic acid, and mcpa = 2-chloro-4-methyl-phenoxyacetic acid, in the presence or not of a nitrogen donor heterocyclic ligand, py = pyridine, bipy = 2,2′ bipyridine, phen = 1,10-phenanthroline, were prepared and characterized.The interaction of Fe(III) with phenoxyalkanoic acids and bipy or phen leads to dinuclear neutral complexes, while the presence of py favors tetranuclear neutral forms. The crystal structures of [Fe₂OCl₂(mcpa)₂(bipy)₂] · 0.25(bipy) · 0.8MeCN (1a), and {[Fe₄O₂(mcpa)₆Cl₂(py)₄] · 2MeCN} (3a), have been determined. DNA-Fe(III) complex interaction studies suggest that iron complexes promote the hydrolytic cleavage of double stranded DNA that seems to be oxygen independent, while pDNA shows cross-linking with many molecules of the iron clusters. Antibacterial screening data showed that the presence of chelating agents, bipy or phen, increased the efficiency of iron complexes.     

Site binding as a local equilibrium. Mn2+ binding to poly(acrylic acid) as a function of the degree of ionization

Mn2+ binding to poly(acrylic acid) at different degrees of ionization, alpha, has been studied from the frequency dependence of the water protons' relaxation rates T1(-1) and T2(-1). Site binding is treated as an equilibrium with the concentration of free ions at the immediate vicinity (CIV) of the polyion. The CIV is calculated as the solution of the Poisson-Boltzmann equation at the surface of the cylindrical polyion. A single value of K is shown to fit the results at all values of alpha. The amount of site binding is higher than the total amount of condensed divalent counterions predicted for a finite polyion concentration in the presence of monovalent counterions by Manning's theory.     

Estimating diurnal to annual ecosystem parameters by synthesis of a carbon flux model with eddy covariance net ecosystem exchange observations

We performed a synthetic analysis of Harvard Forest net ecosystem exchange of CO₂ (NEE) time series and a simple ecosystem carbon flux model, the simplified Photosynthesis and Evapo‐Transpiration model (SIPNET). SIPNET runs at a half‐daily time step, and has two vegetation carbon pools, a single aggregated soil carbon pool, and a simple soil moisture sub‐model. We used a stochastic Bayesian parameter estimation technique that provided posterior distributions of the model parameters, conditioned on the observed fluxes and the model equations. In this analysis, we estimated the values of all quantities that govern model behavior, including both rate constants and initial conditions for carbon pools. The purpose of this analysis was not to calibrate the model to make predictions about future fluxes but rather to understand how much information about process controls can be derived directly from the NEE observations. A wavelet decomposition enabled us to assess model performance at multiple time scales from diurnal to decadal. The model parameters are most highly constrained by eddy flux data at daily to seasonal time scales, suggesting that this approach is not useful for calculating annual integrals. However, the ability of the model to fit both the diurnal and seasonal variability patterns in the data simultaneously, using the same parameter set, indicates the effectiveness of this parameter estimation method. Our results quantify the extent to which the eddy covariance data contain information about the ecosystem process parameters represented in the model, and suggest several next steps in model development and observations for improved synthesis of models with flux observations.     

H NMR spectroscopy as a tool to determine accurate photoisomerization quantum yields of stilbene-like ligands coordinated to rhenium(I) polypyridyl complexes

In this work, the use of proton nuclear magnetic resonance, ¹H NMR, was fully described as a powerful tool to follow a photoreaction and to determine accurate quantum yields, so called true quantum yields (Φ_true), when a reactant and photoproduct absorption overlap. For this, Φ_true for the trans-cis photoisomerization process were determined for rhenium(I) polypyridyl complexes, fac-[Re(CO)₃(NN)(trans-L)]⁺ (NN = 1,10-phenanthroline, phen, or 4,7-diphenyl-1,10-phenanthroline, ph₂phen, and L = 1,2-bis(4-pyridyl)ethylene, bpe, or 4-styrylpyridine, stpy). The true values determined at 365 nm irradiation (e.g. Φ_NMR = 0.80 for fac-[Re(CO)₃(phen)(trans-bpe)]⁺) were much higher than those determined by absorption spectral changes (Φ_UV-Vis = 0.39 for fac-[Re(CO)₃(phen)(trans-bpe)]⁺). Φ_NMR are more accurate in these cases due to the distinct proton signals of trans and cis-isomers, which allow the actual determination of each component concentration under given irradiation time. Nevertheless when the photoproduct or reactant contribution at the probe wavelength is negligible, one can determine Φ_true by regular absorption spectral changes. For instance, Φ_313 nm for free ligand photoisomerization determined both by absorption and ¹H NMR variation are equal within the experimental error (bpe: Φ_UV-Vis = 0.27, Φ_NMR = 0.26; stpy: Φ_UV-Vis = 0.49, Φ_NMR = 0.49). Moreover, ¹H NMR data combined with electronic spectra allowed molar absorptivity determination of difficult to isolate cis-complexes.     

Computation of electrostatic complements to proteins: a case of charge stabilized binding.

Recent evidence suggests that the net effect of electrostatics is generally to destabilize protein binding due to large desolvation penalties. A novel method for computing ligand-charge distributions that optimize the tradeoff between ligand desolvation penalty and favorable interactions with a binding site has been applied to a model for barnase. The result is a ligand-charge distribution with a favorable electrostatic contribution to binding due, in part, to ligand point charges whose direct interaction with the binding site is unfavorable, but which make strong intra-molecular interactions that are uncloaked on binding and thus act to lessen the ligand desolvation penalty.     

Silver(I) complexes with heterocyclic thiones and tertiary phosphines as ligands. Part 4. Dinuclear complexes of silver(I) bromide: the crystal structure of bis[bromo-(pyrimidine-2-thione)(triphenylphosphine)silver(I)]

Mixed-ligand complexes of the formula [Ag(PPh₃)(L)Br]₂ were obtained by treatment of various heterocyclic thiones L {L=pyridine-2-thione (py2SH), pyrimidine-2-thione (pymtH), benz-1,3-imidazoline-2-thione (bzimtH₂), benz-1,3-thiazoline-2-thione (bztztH), 1-methyl-1,3-imidazoline-2-thione (meimtH) and 5-methoxy-benz-1,3-imidazoline-2-thione (5MeObzimtH₂)} with equivalent quantities of silver(I) bromide and triphenylphosphine in dry acetone. The compounds were characterized by their IR, far-IR, UV–Vis and ¹H NMR spectroscopic data. The crystal structure of [Ag(PPh₃)(pymtH)Br]₂ was determined by single-crystal X-ray diffraction methods. The complex exhibits a planar Ag₂Br₂ moiety in which each of the doubly bromine-bridged Ag(I) centres is further bonded to one phosphine P and one thione S atom.     

Phospholipid composition of dystrophic chicken erythrocyte plasmalemmae II. Characterization of a unique lipid from dystrophic erythrocyte membranes as ethanolamine plasmalogen

The phospholipid content of normal (line 412) and dystrophic (line 413) chicken erythrocyte plasmalemmae has been quantified on a developmental basis using sex matched controls. A specific minor phospholipid component, ethanolamine plasmalogen, is identified from dystrophic erythrocyte membrane preparations. To arrive at this identification, data from studies utilizing gas-liquid chromatography, thin-layer chromatography, [¹⁴C]ethanolamine incorporation, and biochemical assay for specific organic moieties were correlated. This phospholipid has the potential to alter and regulate membrane fluidity and thus membrane function. The possible presence of significant concentrations of plasmalogen in human dystrophic tissues may serve as a marker for dystrophy and thus be of clinical importance.     

The interactions of benzo(a)pyrene with cell membranes: uptake into Chinese hamster ovary (CHO) cells and fluorescence studies with isolated membranes.

The interactions of benzo(a)pyrene (B(a)P) with the cell surface membrane were studied by measuring B(a)P uptake into intact mammalian cells and by determining B(a)P fluorescence in the presence of isolated cell surface membranes. It was found that 0.19 mu-g B(a)P were taken up by 10-6 Chinese hamster ovary (CHO) cells after 30 min exposure to a solution containing 0.59 mu-g/ml. Culture conditions were found to markedly alter B(a)P uptake. Low cell culture densities resulted in a four-fold increase in rate of B(a)P uptake per cell relative to confluent monolayer cultures. The uptake rate of B(a)P was reduced in the presence of bovine serum (BS) and, under some conditions, perylene. This information should be considered in the design of experiments on the biological effects of B(a)P. Another aspect of B(a)P membrane interaction was that the binding of B(a)P to cell surface membranes could be measured by fluorescence. The additional B(a)P fluorescence, found in the presence of cell surface membranes, was sufficiently large that the methods of data treatment used in the study of fluorescent probe-membrane interactions could be applied to get quantitative information on B(a)P-membrane interactions. It was found that 0.6 x 10-8 moles B(a)P were bound per mg membrane protein and that the apparent statistical dissociation constant for the complex was 3.8 x 10-7 M. The data suggest that the mechanism of uptake of B(a)P is probably passive diffusion.     

Interaction of antitumor drugs with human erythrocyte ghost membranes and mastocytoma P815: a spin label study.

The cytotoxic and mutagenic properties of antitumor drugs such as adriamycin, acridines, diacridine, actinomycin D and Pt compounds are related to their interaction with nucleic acids and inhibition of protein synthesis. We have examined their interaction with human erythrocyte ghost membranes and murine mastocytoma cells using spin labeling techniques. These drugs induce changes in electron spin resonance of the spin labeled ghost membranes and in the mastocytoma cells. These alterations suggest that these drugs induce changes in protein conformation of the membranes. The membrane binding properties of these drugs may be important in their mechanism of action.     

Copper(I) complexes with Schiff base and 1,2-bis(diphenylphosphino)ethane as ligands: Synthesis, structure and catalytic properties for the amination of aryl halide

Some copper(I) complexes of the type [Cu(L)(dppe)]X (1-4) [where L = (3-trifluoromethylphenyl)pyridine-2-ylmethylene-amine; dppe = 1,2-bis(diphenylphosphino)ethane; X = Cl⁻, CN⁻, ClO₄⁻ and BF₄⁻] have been synthesized by the condensation of 3-aminobenzotrifluoride with 2-pyridinecarboxaldehyde followed by the reaction with CuCl, CuCN, [Cu(MeCN)₄]ClO₄ and [Cu(MeCN)₄]BF₄ in presence of dppe. The complexes 1-4 were then characterized on the basis of elemental analysis, IR, UV-Vis and ¹H NMR spectral studies. The representative complex of the series 4 has been characterized by single crystal X-ray diffraction which reveal that in complex the central copper(I) ion assumes the irregular pseudo-tetrahedral geometry. The catalytic activity of the complexes was tested and it was found that all the complexes worked as effective catalyst in the amination of aryl halide.     

Insulin action in isolated fat cells: II. Effects of divalent cations on stimulation by insulin of protein synthesis,on inhibition of lipolysis by insulin,and on the binding of 125I-labelled insulin to isolated fat cells

The effects of omission of Ca²⁺ and Mg²⁺ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca²⁺ + Mg²⁺)-free incubation medium incorporation of L-[¹⁴C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[¹⁴C]-leucine incorporation only in the presence of added CaCl₂ or MgCl₂. Incubation of the cells in the (Ca²⁺ + Mg²⁺)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of ¹²⁵I-labelled insulin to the fat cells was reduced in the absence of Ca²⁺ and Mg²⁺ but was not abolished, even in the presence of EDTA. Ca²⁺ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg²⁺, Sr²⁺ and Ba²⁺.Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

Development of image analysis techniques as a tool to detect and quantify morphological changes in anaerobic sludge: I. Application to a granulation process

Image analysis techniques were developed and applied to quantify the process of anaerobic granulation in an expanded granular sludge blanket reactor (EGSB) fed with a synthetic substrate based on glucose [60-30% COD (chemical oxygen demand)] and volatile fatty acids (40-70% COD) over 376 days. In a first operation period that lasted 177 days, the aggregation of dispersed sludge was quantitatively monitored through the recognition and quantification of aggregates and filaments. A parameter defined as the ratio between the filaments' length and the aggregates projected area (LfA) has proven to be sensitive to detect changes in the aggregation status of the anaerobic sludge. The aggregation time-defined as the moment when a balance between filaments' length and aggregates' size was established-was recognized through the LfA. The percentage of projected area of aggregates within three size ranges (0.01-0.1 mm, 0.1-1 mm, and >1 mm, equivalent diameter) reflected the granular size spectrum during the aggregation process. When sudden increases on the upflow velocity and on the organic loading rate were applied to the previously formed granules, the developed image analysis techniques revealed to be good indicators of granular sludge stability, since they were sensitive to detected filaments release, fragmentation, and erosion that usually leads to washout. The specific methanogenic activities in the presence of acetate, propionate, butyrate, and H(2)/CO(2) increased along the operation, particularly relevant was the sudden increase in the specific hydrogenophilic activity, immediately after the moment recognized as aggregation time.     

Specific photoaffinity crosslinking of [125I]cholecystokinin to pancreatic plasma membranes. Evidence for a disulfide-linked Mr 76 000 peptide in cholecystokinin receptors

In rat pancreatic plasma membranes, preincubated with [125I]cholecystokinin-33 (CCK-33) and washed free of unbound tracer, the irradiation by UV light induced the irreversible binding of radioactivity to high molecular weight peptides as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and autoradiography. This was not observed when the membranes were preincubated in the simultaneous presence of [125I]CCK-33 and of either an excess of unlabelled CCK-8 or of guanosine 5'-(beta, gamma-imido)-triphosphate. The radioactivity was mostly crosslinked with a Mr 96,000 peptide and peptide species of Mr greater than 200,000, after SDS solubilization in the absence of beta-mercaptoethanol. Peptide reduction with beta-mercaptoethanol converted the high molecular weight radioactive species into a Mr 76,000 peptide that contained as much as 65% of the radioactivity crosslinked. The Mr 76,000 peptide appears, therefore, to be a disulfide-linked constituent of rat pancreatic cholecystokinin receptors.