Biomedical subjects

M J Clague

Publications and source records attributed to M J Clague.

45 records · Page 3Linked to original sources

PubMed1990

Leakage of internal markers from erythrocytes and lipid vesicles induced by melittin, gramicidin S and alamethicin: a comparative study.

The membrane-disruptive capacities of melittin, derivatised melittins, alamethicin and gramicidin S have been compared for the human erythrocyte membrane and lipid vesicles of three different compositions (phosphatidylcholine, 85% phosphatidylcholine/15% phosphatidylserine, and a lipid analogue of the outer leaflet of the human erythrocyte membrane). The sensitivity to ionic strength, divalent metal ions and polylysine of release of fluorescent markers from liposomes and of haemoglobin from intact erythrocytes has been assayed. Acetyl melittin was found to he more effective than melittin in lysing phosphatidylcholine and phosphatidylcholine/phosphatidylserine vesicles, somewhat less effective in the lipid analogue and markedly less effective in lysing erythrocytes. Succinyl melittin was non-haemolytic, but was able to lyse lipid vesicles at a high concentration. Ca2+ inhibited melittin haemolysis at high ionic strength (150 mM NaCl), but produced a more complex response of stimulation followed by inhibition at low ionic strength. In lipid vesicles, Ca2+ either stimulated melittin lysis or was ineffective. Zn2+ exerted effects similar to Ca2+ with lipid vesicles at approx. 10-fold lower concentration except that a weak inhibition was observed for the erythrocyte membrane lipid analogue at high ionic strength. Polylysine strongly inhibited haemolysis by melittin at low ionic strength, but was ineffective or stimulatory in lipid vesicle lysis. High phosphate concentration also inhibited melittin haemolysis, but again no corresponding effect could he found in any of the lipid vesicle systems. These disparities between effects of melittin on erythrocytes and lipid vesicles support the proposal that melittin-protein interactions are of consequence to its haemolytic action. Similar experiments were performed with gramicidin S and alamethicin in order to compare their lytic properties with those of melittin. It was found that each lysin exhibited its own individual pattern of sensitivity to lipid composition, ionic strength and inhibition by cations. It thus appears likely that the detailed molecular interactions responsible for lysis are significantly different for each of these three agents.

Alamethicin↗

PubMed1990

Effect of erythrocyte transbilayer phospholipid distribution on fusion with vesicular stomatitis virus.

To identify the specific component(s) in the target membrane involved in fusion of vesicular stomatitis virus (VSV), we examined the interaction of the virus with human erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically by the octadecylrhodamine dequenching assay. Fusion of VSV with lipid-symmetric erythrocyte ghosts was rapid at 37 degrees C and low pH, whereas little or no fusion was observed with lipid-asymmetric ghosts. Conversion of phosphatidylserine in the lipid-symmetric ghost membrane to phosphatidylethanolamine by means of the enzyme phosphatidylserine decarboxylase did not alter the target membrane's susceptibility to VSV fusion. Spin-labeled phospholipid analogues with phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine headgroups incorporated into the outer leaflet of lipid-asymmetric erythrocytes did not render those membranes fusogenic. Electron spin resonance spectra showed an increased mobility of a phosphatidylcholine spin-label incorporated into the outer leaflet of lipid-symmetric erythrocyte ghosts as compared to that of lipid-asymmetric ghosts. These results indicate that the susceptibility to VSV fusion is not dependent on any particular phospholipid but rather is related to packing characteristics of the target membrane.

Electron Spin Resonance Spectroscopy↗

PubMed1990

Transient dichroism studies of spectrin rotational diffusion in solution and bound to erythrocyte membranes.

Spectrin was purified from human erythrocytes and labeled with the triplet probe eosin-5-maleimide. Rotational diffusion of spectrin was investigated by observing transient dichroism following flash excitation of the probe. Measurements were performed at 4 degrees C in solutions of varying viscosity and with spectrin rebound to spectrin/actin-depleted erythrocyte membranes. In solution, complex anisotropy decays were observed which could not be satisfactorily fitted by the equations for a rod-shaped molecule of appropriate dimensions. When spectrin was rebound to the erythrocyte membrane, a decay in the anisotropy was still present but was markedly less sensitive to solution viscosity and flatter at longer times. In order to overcome the objection that the cytoskeleton is only partially reconstituted when spectrin is rebound, a method was developed for labeling spectrin with eosin-5-maleimide in situ. Anisotropy decays for these labeled membranes exhibited features similar to those obtained for spectrin labeled in solution and subsequently rebound. Taken together, the results provide good evidence for segmental motion of spectrin when incorporated into the erythrocyte cytoskeleton. Upon increasing the temperature, the initial anisotropy ro for both rebound and in situ labeled spectrin decreases, and above 30 degrees C the measured anisotropies are small. Thus, at physiological temperature the probe is almost completely randomized by motions with correlation times less than 10 microseconds.

Circular Dichroism↗

PubMed1990

Gating kinetics of pH-activated membrane fusion of vesicular stomatitis virus with cells: stopped-flow measurements by dequenching of octadecylrhodamine fluorescence.

To identify the initial stages of membrane fusion induced by vesicular stomatitis virus, we performed stopped-flow kinetic measurement with fluorescently labeled virus attached to human erythrocyte ghosts that contained symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically using an assay based on mixing of the lipid fluorophore octadecylrhodamine. At 37 degrees C and pH values near the threshold for fusion, a lag phase of 2 s was observed. The lag time decreased steeply as the pH decreased, while the initial rate of fusion showed the reverse functional dependence on pH. The observed rapid fluorescence changes resulted from fusion of virus bound to the target, and the time lags were not due to association-dissociation reactions between virus and target. For a given pH value, the temperature dependence of the lag time was similar to that of the initial rate of fusion. The results were fitted to a multistate model similar to that resulting from ion channel gating kinetics. The model allows testing of hypotheses concerning the role of cooperativity and conformational changes in viral spike glycoprotein-mediated membrane fusion.

Animals↗

PubMed1989

Cytoskeletal restraints of band 3 rotational mobility in human erythrocyte membranes.

The interaction of band 3 with cytoskeletal proteins was investigated in erythrocyte membranes by measuring the rotational mobility of band 3 using the method of transient dichroism. It was found that selective proteolysis of ankyrin, a protein known to link band 3 to the spectrin-actin network, had no significant effect on band 3 rotation. Incubating ghosts to 70 degrees C, at which temperature ankyrin is expected to be denatured, also had no effect. It thus appears probable that linkage of band 3 to the cytoskeleton via ankyrin does not act as a restraint on band 3 rotational motion. It is suggested that this is a consequence of flexibility in the cytoskeletal structure. In further investigations of the effect of heat treatment, a large enhancement of band 3 rotational mobility was found to result from incubation of intact cells for 1 h at 50 degrees C. This effect was not observed if ghosts were subjected to the same treatment, nor did it occur if the incubation of cells was performed at 47 degrees C. These findings, in combination with previous studies of band 3 rotational mobility, indicate that the interactions which restrain band 3 are likely to be more complex than commonly envisaged.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1989

A comparative study of band 3 aggregation in erythrocyte membranes by melittin and other cationic agents.

The technique of laser flash-induced transient dichroism has been used to measure the rotational diffusion of eosin-labelled band 3 proteins in erythrocyte ghosts. A retardation in the mobility of band 3, measured subsequent to the addition of a variety of polyvalent cationic species, has been interpreted to reflect aggregation or 'clustering' of the protein in the plane of the membrane. A comparative study is reported between three such aggregators: melittin, polylysine and Zn2+, wherein their respective abilities to induce aggregation have been measured under varying conditions. Unlike that for melittin, band 3 aggregation by polylysine and Zn2+ is shown to be sensitive to proteolytic degradation of the membrane and to the ionic strength of the surrounding medium. Studies with fragments of melittin derived from its chymotryptic cleavage show the hydrophilic C-terminal 20-26 section to possess independent aggregating ability, but also the requirement of the 1-19 hydrophobic section to be attached in order to prevent reversibility by high ionic strength buffers. Melittin is also shown to have a unique ability to aggregate bacteriorhodopsin reconstituted into DMPC vesicles, which is partially retained by its 1-19 but not by its 20-26 fragment.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1988

Comparison of p25 presequence peptide and melittin. Red blood cell haemolysis and band 3 aggregation.

The 25 residue presequence (p25) for subunit IV of yeast cytochrome oxidase had previously been shown to possess structural and behavioural characteristics in common with the bee venom polypeptide, melittin. The present study extends the results of leakage experiments on model-membrane systems to the haemolysis of human erythrocytes, which both peptides are shown to accomplish in a manner sensitive to membrane potential. In addition, the laser flash-induced transient dichroism technique for measuring protein rotational diffusion has been used to show that both peptides aggregate band 3, the major integral membrane protein of the erythrocyte. Aggregation cannot be reversed by high ionic strength; this serves to differentiate these peptides from other positively charged species such as polylysine that aggregate band 3 at low ionic strength. These results suggest that aggregation of membrane proteins may possibly prove to be a feature of the interaction of p25 signal peptide with mitochondrial membranes.

Amino Acid Sequence↗

PubMedDate not supplied

Regulation of endosome fusion.

Homotypic fusion between early endosomes can be reconstituted in vitro. By using wortmannin and LY294002, inhibitors of phosphatidylinositol (Pl) 3-kinase, a requirement for this activity has been established in order for fusion to proceed efficiently. It has been shown that Pl 3-kinase activity is required downstream of rab5 activation, although a large excess of activated rab5 can overcome wortmannin inhibition. A series of experiments have also been performed which indicate a role for early endosomal autoantigen 1 (EEA1) in determining fusion efficiency. EEA1 dissociates from membranes following wortmannin treatment. It is proposed that the requirement of endosome fusion for Pl 3-kinase activity is to promote the association of EEA1 with endosomes.

Androstadienes↗

PubMedDate not supplied

Role of target membrane structure in fusion with influenza virus: effect of modulating erythrocyte transbilayer phospholipid distribution.

To study the role of the target membrane in influenza virus fusion we chose erythrocyte membranes whose phospholipid arrangement can readily be modified. The phospholipids of normal erythrocytes are arranged asymmetrically across the plasma membrane; phosphatidylcholine (PC) and sphingomyelin are predominantly on the outer surface, whereas others such as phosphatidylserine (PS) and phosphatidylethanolamine (PE) are predominantly restricted to the inner leaflet. However, erythrocytes can be lyzed and resealed under conditions where the asymmetric distribution of phospholipids is lost or retained. Low pH-induced fusion of the A/PR 8 strain of influenza virus, monitored spectrofluorometrically by the octadecylrhodamine dequenching assay, was more rapid with lipid-symmetric erythrocyte ghosts than with lipid-asymmetric ghosts or intact erythrocytes. Neither conversion of PS in the lipid-symmetric ghost membrane to PE by means of the enzyme PS decarboxylaze, nor incorporation of spin-labeled phospholipid analogs with PS, PC or PE headgroups into the outer leaflet of lipid-asymmetric erythrocytes altered rates or extents of fusion of A/PR 8 with the modified target. These results indicate that effects on influenza virus fusion are not associated with any particular phospholipid headgroup, but rather related to the packing characteristics of the target membrane.

Calcium↗