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Biomedical subjects

R J Cherry

Publications and source records attributed to R J Cherry.

At least 91 records · Page 5Linked to original sources
PubMed1981

Rotational diffusion of erythrocyte membrane proteins.

Rotational diffusion of band 3 proteins in human erythrocyte membranes is measured by observing flash-induced transient dichroism of the triplet probe, eosin-maleimide. At physiological temperature, both fast and slowly rotating populations of band 3 are present in the membrane. Rotational motion of band 3 is the same in membranes from young and old erythrocytes and is unchanged when the cholesterol:phospholipid mole ratio is varied from 1.34 to 1.66. Antibodies against glycophorin A immobilize band 3, indicating an association between these two integral membrane proteins. However, glycophorin A has little effect on the rotational motion of the complex, since band 3 rotation in En(a-) membranes (which lack glycophorin A) is similar to that observed in normal membranes. Cleavage of the cytoplasmic segment of band 3 by trypsin produces a considerable enhancement of band 3 rotational mobility. A similar effect is seen following extraction of bands 2.1 and 4.1 by sequential low salt-high salt treatment. It is concluded that up to 40% of band 3 has restricted rotational mobility due to interaction with the erythrocyte cytoskeleton.

Blood Proteins↗
PubMed1981

Rotational diffusion of membrane proteins: measurements with bacteriorhodopsin, band-3 proteins and erythrocyte oligosaccharides.

Rotational diffusion of membrane proteins may be measured by detecting flash-induced transient dichroism of triplet probes or intrinsic chromophores. The method is illustrated by the following applications. 1. Anisotropy decay curves have been measured for bacteriorhodopsin incorporated into lipid vesicles. The data are used to critically test a theoretical model in which it is assumed that protein rotation occurs only around the membrane normal. 2. Rotational diffusion of eosin-labelled band-3 proteins in the human erythrocyte membrane has been investigated. The measurements illustrate the value of protein rotation measurements for studying protein-protein associations in membranes. In particular, it is shown that up to 40% of band-3 protein has a restricted mobility at 37 degrees C due to interaction with the erythrocyte cytoskeleton. 3. Oligosaccharide chains on the surface of human erythrocytes have been labelled with eosin-TSC. The motion of these chains has been investigated by combining transient dichroism studies in the microsecond time range with picosecond fluorescent depolarization measurements.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1980

Rotational diffusion of band 3 proteins in membranes from En(a-) and neuraminidase-treated normal human erythrocytes.

Recent experiments have demonstrated an association between band 3 and glycophorin A in the human eythrocyte membrane (Nigg, E.A., Bron, C., Girardet, M. and Cherry, R.J. (1980) Biochemistry 19, 1887-1893). Here, the influence of sialoglycoproteins on the rotational diffusion of band 3 in the human erythrocyte membrane was investigated by studying membranes from En(a-) and neuraminidase-treated erythrocytres. Rotational diffusion was measured by observing flash-induced transient dichroism of eosin-labeled band 3. Although erythrocytes of the rare phenotype En(a-) lack the major sialoglycoprotein, glycophorin A, no significant difference in band 3 rotation at pH 7.4 and 37 degrees C could be detected between En(a-) and normal erythrocyte membranes. Band 3 rotation at pH 7.4 was also insensitive to the enzymatic removal of sialic acid from the surface of normal erythrocytes. Moreover, the existence of an essentially similar temperature-dependent equilibrium between band 3 proteins with different mobilities was observed in normal, En(a-) and neuraminidase-treated erythrocytes. From these results it is concluded that glycophorin A contributes less than 15% to the cross-sectional diameter of the band 3-glycophorin A complex in the plane of the normal membrane. The rotation of the complex at pH 7.4 is not significantly influenced by either steric or electrostatic interactions involving the oligosaccharide moiety of glycophorin A.

Diffusion↗
PubMed1980

Cytochrome oxidase rotates in the inner membrane of intact mitochondria and submitochondrial particles.

A transient dichroism is detected after photolysis by a linearly polarized laser flash of the cytochrome oxidaseCO complex in bovine heart mitochondria, rat heart mitochondria, and bovine heart submitochondrial particles. A decay in the absorption anisotropy is characterized by a time constant of about 300 to 400 mus in both mitochondria and submitochondrial particles. Since vesicle tumbling in the time range less than 5 ms can be excluded in these experiments, we conclude that cytochrome oxidase rotates in the mitochondrial membrane with a relaxation time of several hundred microseconds. However, it is likely that only about one-half of cytochrome oxidase contributes to the observed decay, the remainder being relatively immobile.

Animals↗
PubMed1980

Band 3-glycophorin A association in erythrocyte membrane demonstrated by combining protein diffusion measurements with antibody-induced cross-linking.

A new approach to the study of molecular protein interactions in biological membranes is presented. The technique is based on measuring the rotation of a membrane protein in the presence and absence of specific antibodies directed toward a purported complex partner. As a first illustration of the method, the putative association of band 3 with glycophorin A in the human erythrocyte membrane was investigated. The rotational diffusion of band 3 was strongly reduced following cross-linking of glycophorin A with divalent antibodies. However, little or no effect on band 3 rotation was produced by monovalent antiglycophorin A Fab fragments, antispectrinor nonspecific antibodies, ruling out major effects on band 3 mobility due to steric hindrance, unspecific antibody adsorption, or transmembrane interactions involving spectrin. It is concluded that immobilization of band 3 by antiglycophorin A antibodies is directly caused by cross-linking of a preexisting band 3-glycophorin A complex in the human erythrocyte membrane.

Antibodies↗
PubMed1980

Lateral segregation of proteins induced by cholesterol in bacteriorhodopsin-phospholipid vesicles.

Bacteriorhodopsin in dimyristoylphosphatidylcholine vesicles is randomly distributed in the plane of the membrane and exhibits rotational diffusion above the gel to liquid-crystalline phase transition. Incorporation of cholesterol results in loss of rotational mobility of bacteriorhodopsin, which on the basis of electron microscopy and CD measurements can be assigned to the formation of protein aggregates. It is concluded that bacteriorhodopsin is soluble in the fluid phosphatidylcholine phase but segregates when cholesterol is present in the lipid bilayer.

Bacteriorhodopsins↗
PubMed1980

Oligosaccharide motion in erythrocyte membranes investigated by picosecond fluorescence polarization and microsecond dichroism of an optical probe.

Oligosaccharide chains on the surfce of human erythrocytes were labeled with the probe eosin 5-thiosemicarbazide. The probe was conjugated to aldehydes produced by oxidation of sialic acid and galactose residues. The probe is associated mostly with glycophorin A after sialic acid labeling, whereas multiple components, including band 3 and lipids, are labeled after galactose oxidation. Fast molecular motion was studied by measuring steady-state and picosecond time-resolved fluorescence depolarization. Slower motions were investigated by observing flash-induced transient dichroism. It was found that both eosin-labeled sialic acid and galactose residues exhibit a rapid motion with correlation time of approximately 3 nsec. This motion is assigned to independent motion of the probe, possibly in conjunction with a short segment of the oligosaccharide chain. The order parameter of the fast motion is 0.8.-0.9, demonstrating that its angular amplitude is highly restricted. For eosin-labeled sialic acid, the order parameter in the microsecond time range is 0.2-0.3. It is deduced that a second, slower rotational motion is present, which is assigned to a cooperative motion of the oligosaccharide chains. The correlation time of this motion is in the range 10(-7)-(10-5) sec. Some eosin-labeled galactose residues may have a similar slow motion, but most appear to be remarkably immobile over the time range 10(-8)-10(-3) sec.

Eosine Yellowish-(YS)↗
PubMed1980

Anchorage of a band 3 population at the erythrocyte cytoplasmic membrane surface: protein rotational diffusion measurements.

Direct physical evidence for the linkage of a band 3 population to the cytoskeleton in the erythrocyte ghost membrane is presented. The rotational diffusion of band 3 proteins was mesured by observing flash-induced transient dichroism of a covalently bound eosin probe. After proteolytic release of a 40,000-dalton cytoplasmic segment of band 3 by trypsin, a considerable enhancement in the decay of the absorption anisotropy was observed. Analysis of the data indicates that proteolytic cleavage of band 3 produces a mobile band 3 population which has restricted mobility in the unperturbed membrane due to protein-protein interactions involving the cytoplasmic band 3 moiety. Band 2.1 (ankyrin) or 4.1 or both are likely to be involved in this interaction because a similar effect on band 3 mobility is observed after low-salt/high-salt extraction of these components. Quantitatively, it is estimated that up to 40% of band 3 may be linked to the cytoskeleton. Because the ankyrin-band 3 dimer stoichiometry in the membrane is approximately 1:5, only about 20% of band 3 dimers can be directly linked to ankyrin. The remainder could be explained by the existence of higher oligomers of band 3 linked to single ankyrin polypeptides or by linkages involving other components such as band 4.1 or 4.2.

Diffusion↗
PubMed1979

Influence of temperature and cholesterol on the rotational diffusion of band 3 in the human erythrocyte membrane.

Band 3 rotation in the human erythrocyte membrane is measured by observing flash-induced dichroism of eosin probes. The decay of the absorption anisotropy is found to be strongly dependent on temperature. The results are analyzed on the assumption that rotation of band 3 only occurs about the membrane normal. It is deduced that both fast and slowly rotating forms of band 3 coexist in the membrane. The equilibrium between these forms is temperature dependent, the slowly rotating species becoming increasingly dominant as the temperature is reduced. Plots of the fractional distribution of the different species against temperature show a marked change of slope at around 37--40 degrees C. The effects are essentially reversible over the range 1--45 degrees C and independent of the presence of the spectrin--actin network. The results could be due to temperature-dependent protein--protein associations mediated either by a protein conformational change or by lipid phase segregation. In further experiments, the cholesterol content of the erythrocyte membrane is varied by incubation with lipid vesicles. No significant changes in the rotational diffusion of band 3 are observed following variation of membrane cholesterol/phospholipid mole ratios over the range 0.34--1.66. This is a surprising result in view of the well-known effects of cholesterol on lipid fluidity.

Actins↗
PubMed1979

Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements: inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase.

The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate exchange in intact erythrocytes. 50% inhibition is obtained by binding 4.7 . 10(5) or 6.0 . 10(5) molecules/cell for eosin-NCS and IA-eosin, respectively. Both eosin probes are irreversibly bound and occupy common binding sites with 4,4-diisothiocyano-1,2-diphenyl-ethane-2,2'disulfonic acid (H2DIDS), although other sites are labeled as well. The inhibition of anion transport is light independent and can therefore not be attributed to a photosensitizing action of the eosin probes. Both eosin derivatives, however, inactivate acetylcholinesterase upon illumination of air-equilibrated samples of hemoglobin-free labeled ghosts. The inactivation of the enzyme is accompanied by the formation of protein aggregates as visualized by polyacrylamide gel electrophoresis. These effects are not observed when intact erythrocytes are illuminated in the presence of eosin probes suggesting a protective effect of hemoglobin during the labeling procedure. Protection of ghosts from photo-oxidation is achieved by displacing air with argon. These results are discussed in relation to the use of these and similar probes to measure protein diffusion in membranes.

Biological Transport, Active↗
PubMed1979

Rotational diffusion of band 3 in the red cell membrane: measurements using triplet probes.

Rotational diffusion of band 3 in the human erythrocyte membrane has been investigated using the technique of flash photolysis. Membranes are labeled with the triplet probe eosin isothiocyanate; most of the label is found to be associated with band 3. Rotation is measured by observing the decay of dichroism of flash-induced absorbance changes arising from the eosin probe. The results indicate that band 3 rotates about an axis normal to the plane of the membrane with a diffusion coefficient in the order of 1,000 sec-1. Removal of spectrin has no observable effect on the rotation of band 3 at pH 7.6. Essentially similar results are obtained with a different probe, iodoacetamidoeosin. Both eosin derivatives are strong inhibitors of anion transport. Illumination of eosin-labeled ghosts causes a rapid loss of acetylcholinesterase activity but this can be prevented by prior displacement of oxygen in the sample by argon.

Acetylcholinesterase↗