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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.

Ca2+,Mg2+-dependent adenosine 5'-triphosphatase (ATPase) in sarcoplasmic reticulum vesicles is labeled with the triplet probe, 5-iodoacetamidoesin. Rotational mobility of the ATPase is investigated by measuring flash-induced transient dichroism of the eosin probe. The absorption anisotropy measured 20 mus after the exciting flash is found to be small at 37 degrees C but increases considerably with decreasing temperature and upon fixation with glutaraldehyde. A purified Ca2+,Mg2+-dependent ATPase preparation partially depleted of membrane lipids exhibits similar properties. The low value of the anisotropy at 37 degrees C is due to the existence of a fast motion which in part is assigned to independent segmental motion of the protein. This internal flexibility of the ATPase may have considerable significance for the functional properties of the enzyme. At times longer than 20 mus, the anisotropy decays with a time constant which varies from approximately 90 mus at 0 degrees C to approximately 40 mus at 37 degrees C. This decay is assigned to rotation of the ATPase about an axis normal to the plane of the membrane. There is some evidence for self-aggregation of the protein at lower temperatures.

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.

Rotation of cytochrome P-450 was examined in both liver microsomes and reconstituted and phospholipid vesicles. Purified cytochrome P-450 was incorporated into lipid vesicles composed of phosphatidylcholine-phosphatidylethanolamine-Phosphatidylserne. Rotational diffusion was measured by detecting the decay of absorption anisotropy, r(t), after photolysis of the heme. CO complex by a vertically polarized laser flash. No contribution of vesicle tumbling to r(t) was observed over the experimental time range of 0-500 mus for samples in 60% sucrose. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate intermolecular interactions of cytochrome P-450. In vesicles of a high lipid to protein ratio (=30 by weight), the residual time-independent normalized anisotropy, r (infinity)/r (0), reached a limiting low value, implying that all cytochrome P-450 was rotating. The mean rotational relaxation time, phi 1, was about 95 mus. In contrast, about 35% of cytochrome P-450 was immobilized in vesicles of a low lipid to protein ratio (=1), with phi 1 of about 95 mus for the mobile fraction. The immobile fraction is presumably due to self-aggregation of cytochrome P-450. In rat liver microsomes, 0-50% of cytochrome P-450 was mobile with phi 1 of about 120 mus at 20 degrees C, and the rest was immobile. A significant temperature dependence of r(t) was observed in microsomes. All cytochrome P-450 was immobile below 7 degrees C, and about 50% of the enzyme was mobile at 37 degrees C with phi 1 approximately 60 mus. From the limiting value of r(infinity)/r(0) congruent to 0.12, the tilt angle of the heme plane of cytochrome p-450 from the membrane plane was calculated to be about 40 degrees.

Rotational diffusion of bacteriorhodopsin in dimyristoyl phosphatidylcholine vesicles has been measured at different temperatures and lipid; protein ratios by the technique of flash-induced transient linear dichroism. The data are used to evaluate critically the theory of anisotropy decay due to protein rotation in the lipid bilayer. The theoretical model assumes that rotation of the protein occurs only around the membrane normal. Under conditions favoring completely monomeric bacteriorhodopsin, namely at molar lipid; protein ratios greater than or approximately 100 and for temperatures sufficiently above the lipid phase transition, it is found that the theoretical model provides an excellent description of the experimental data. Curve-fitting analyses of the experimental decay curves show that the retinal is oriented at an angle of 78 +/- 2 degrees with respect to the membrane normal. Between 25 and 37 degrees C, the protein rotates with a relaxation time of 15 +/- 5 micros in the lipid liquid crystalline phase, corresponding to the membrane viscosity of 3.7 +/- 1.3 P. The curve analysis also provides a sensitive test for the presence of protein aggregates in the lipid bilayer.

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles by a cholate dialysis technique. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme X CO complex by a vertically polarized laser flash. All cytochrome P-450 was found to be rotationally mobile when co-reconstituted with equimolar amounts of NADPH-cytochrome P-450 reductase in lipid to cytochrome P-450 ((L/P450)) = 1 (w/w] vesicles. Antibodies against NADPH-cytochrome P-450 reductase were raised. Their specificity was demonstrated by Ouchterlony double diffusion analysis. Antireductase Fab fragments were prepared from antireductase IgG by papain digestion. The N-demethylation of benzphetamine, catalyzed by the proteoliposomes, was significantly inhibited by antireductase IgG and by antireductase Fab fragments. Cross-linking of NADPH-cytochrome P-450 reductase by antireductase IgG resulted in complete immobilization of cytochrome P-450 in L/P450 = 1 vesicles. Antireductase IgG also immobilized cytochrome P-450 in L/P450 = 5 vesicles, although the degree of immobilization was slightly smaller. No immobilization of cytochrome P-450 in L/P450 = 1 vesicles was detected in the presence of antireductase Fab fragments or preimmune IgG. These results further support the proposal of the formation of monomolecular complexes between cytochrome P-450 and NADPH-cytochrome P-450 reductase in liposomal membranes (Gut, J., Richter, C., Cherry, R.J., Winterhalter, K.H., and Kawato, S. (1982) J. Biol. Chem. 257, 7030-7036).

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.

The ADP/ATP translocator was selectively labeled with the triplet probe eosin-5-maleimide (EMA) after pretreatment with N-ethylmaleimide in beef heart mitochondria, as reported previously for submitochondrial particles (Müller, M., Krebs, J. J. R., Cherry, R. J., and Kawato, S. (1982) J. Biol. Chem. 257, 1117-1120). The EMA binding was completely inhibited by carboxyatractylate. 0.7-1.1 molecules of EMA conjugated with 1 molecule of the dimeric translocator with Mr approximately 65,000. The EMA binding decreased [14C]ADP uptake by about approximately 25%. The EMA-labeled translocator bongkrekate complex was purified and reconstituted in liposomes by removing Triton X-100 with Amberlite XAD-2. The liposomes were composed of phosphatidylcholine/phosphatidylethanolamine/cardiolipin and the lipid to protein ratio by weight was (L/P) = 60. Rotational diffusion of the ADP/ATP translocator around the membrane normal was measured in reconstituted proteoliposomes and in the mitochondrial inner membranes by observing the flash-induced absorption anisotropy, r(t), of EMA. In proteoliposomes with L/P = 60, the translocator was rotating with an approximate average rotational relaxation time of phi congruent to 246 microseconds and a normalized time-independent anisotrophy [r3/rr(0)]min congruent to 0.55. In intact mitochondria, values of phi congruent to 405 microseconds and r3/rr(0) congruent to 0.79 were obtained. The higher value of r3/rr(0) in mitochondria compared with proteoliposomes indicates the co-existence of rotating and immobile translocator (phi greater than 20 ms) in the inner mitochondrial membrane. Based on the assumption that all the translocator is rotating in the lipid-rich proteoliposomes, the population of the mobile translocator at 20 degrees C was calculated to be approximately 47%. By removing the outer membrane, the mobile population was increased to approximately 70% in mitoplasts, while approximately 53% of the translocator was rotating in submitochondrial particles. The above results indicate a significant difference in protein-protein interactions of the ADP/ATP translocator in the different types of inner membranes of mitochondria. The immobile population of the translocator could be due to nonspecific protein aggregates caused by the very high concentration of proteins in the inner membrane of mitochondria (L/P approximately 0.4).

Rotational diffusion of cytochrome oxidase in the inner membrane of rat liver mitochondria was measured by detecting the decay of absorption anisotropy after photolysis of the heme a3.CO complex by a vertically polarized laser flash. As in previous experiments with beef heart mitochondria (Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J. (1980) J. Biol. Chem. 255, 5508-5510), co-existence of rotating cytochrome oxidase (mean rotational relaxation time, phi, of 700 to 1400 microseconds) and immobilized cytochrome oxidase (phi greater than 20 ms) was observed in mitochondria and mitoplasts. The effect of lipid/protein ratio by weight (L/P) on the relative proportions of mobile and immobile cytochrome oxidase was investigated following the fusion of soybean phospholipid vesicles with mitoplasts. The fusion procedure yielded four separate fractions upon sucrose density gradient centrifugation with L/P as follows: 0.3 in Pellet, 0.7 in Band 3, 1.5 in Band 2, and 3.0 in Band 1. The percentage of rotationally mobile cytochrome oxidase (phi = 700 to 1000 microseconds) in each of the different bands was found to be 16% in Pellet, 25% in Band 3, 47% in Band 2, and 76% in Band 1 at 37 degrees C. The dependence of the amount of mobile cytochrome oxidase on L/P indicates that the fraction of aggregated protein progressively decreases with decreasing concentration of proteins in the membrane. Thus, the large immobile fraction of cytochrome oxidase in mitochondrial inner membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P. The decrease in the mobile fraction in Pellet compared with mitoplasts was shown to be due to the pH 6.5 incubation used for fusion.

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.

The effect of mast-cell-triggering peptides on the rotational properties of band 3, a protein component of the human erythrocyte membrane, was measured by observing flash-induced transient dichroism of the triplet probe eosin maleimide. In the presence of melittin, polylysine and five synthetic peptides, varying degrees of retardation in the rotational motion of band 3 were produced. When placed in order of band 3 immobilizing activity, the peptides formed a series identical with their order of efficacy in releasing 5-hydroxytryptamine from rat peritoneal mast cells. The correspondence in the abilities to immobilize band 3 in the erythrocyte and trigger mast cells is significant because structure-activity analyses of the peptides show both processes to have the same cationic, hydrophobic and stereochemical requirements. Probably, the immobilization of band 3 proteins reflects an ability of the basic peptides to aggregate anionic surface moieties, and therefore a similar mechanism is implied in mast-cell triggering.

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.

Experiments have been performed to test the validity of measurements of slow rotational diffusion using eosin isothiocyanate as a probe. When eosin-protein conjugates are covalently bound to Sepharose 4B, the dichroism of the flash-induced absorption transient does not decay with time. The magnitude of the dichroism is smaller than for eosin in solid solution. These results, together with measurements with highly viscous solutions, demonstrate the existence of a rapid but restricted independent motion of the eosin probe. This causes a partial loss of pf dochroism leaving a residual dichroism whose time dependence is determined by the rotational motion of the protein. Rotational correlation times in the microsecond-millisecond time range have been determined for eosinprotein conjugates dissolved in glycerol-water mixtures of varying viscosity. The measured values are in good agreement with theoretical predictions and show the correct dependence on the viscosity of the medium and the size and state of association of the protein.

Incorporation of bacteriorhodopsin (BR), derived from the purple membrane by solubilization with detergents, into phosphatidylcholine-(PC-) vesicles resulted in a disperse distribution of the protein particles in the lipid bilayer as revealed by freeze-fracture electron microscopy. Rotational diffusion measurements showed a high rotational mobility when the system is kept above the lipid phase transition temperature (Tc). Below the Tc the particles are immobilized as observed by flash photolysis while in electron micrographs, segregation of the protein from the lipids and particle aggregation can be seen. In some cases a hexagonal lattice is observed. Solubilization of the purple membrane with LDAO (lauryl-dimethyl-amine oxide) followed by centrifugation through a sucrose density gradient resulted in BR depleted by 80% of bacterial lipids. Incorporation of this lipid-depleted BR into PC-vesicles did not reveal any significant differences in its mobility of its aggregation behaviour. Incorporation of cholesterol into the vesicle system resulted in a marked immobilization of BR. Freeze-fracture electron micrographs show typical segregation phenomena above the Tc of the pure lipid. In the sample with 30 mole% cholesterol, aggregation of the protein appears to be accompanied by lattice formation.

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.

A daily hand injury review clinic takes place at East Birmingham Hospital. The purpose of this study is to determine whether this clinic is beneficial in reducing the morbidity of neglected hand injuries, and to compare the running of the clinic with the original criteria laid down at its inception as a "once only' screening clinic. We conclude that such a clinic is highly beneficial, and recommended that it should be standard practice in other hospitals.

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.

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.

The rotational mobility of band 3, a protein constituent of the human erythrocyte membrane, was measured by observing the flash-induced transient dichroism of the triplet probe eosin maleimide. In the presence of melittin, a pharmacologically active polypeptide from honey bee (Apis mellifera) venom, a dose-dependent loss of rotational mobility was detected. With acetylated melittin, the ability to immobilize is reduced. Succinylated melittin, however, is devoid of immobilizing activity. The possible relevance of these findings to the normal mode of action of melittin was examined by comparing the relative abilities of the native, acetylated and succinylated melittins to lyse erythrocytes and synergize with phospholipase A2, another constituent of bee venom. For both these properties, the order of effectiveness is native melittin greater than acetyl melittin greater than succinyl melittin = 0, the same as their order of effectiveness in immobilizing band 3. A mechanism is proposed in which melittin is anchored in the membrane by its hydrophobic N-terminus, while its cationic C-terminal moiety binds to negatively charged residues on membrane proteins. This leads either directly or indirectly to protein aggregation and hence loss of mobility. From a detailed comparison of the different effects of the melittin derivatives, it is concluded that melittin may function in vivo by aggregating membrane proteins in order to allow phospholipase A2 to gain access to the membrane bilayer and commence catalysis.

Bacteriorhodopsin (BR) was incorporated into large unilamellar dimyristoyl- and dipalmitoylphosphatidylcholine vesicles (100--300-nm radius). The effect of this intrinsic membrane protein on the order and dynamics of the lipid and on the cooperativity and transition temperature (Tc) of the gel to liquid-crystalline phase transition was investigated as a function of the lipid:protein ratio (L/BR). The lipid phase transition induces protein segregation. Above Tc, bacteriorhodopsin is in the monomeric state. Below Tc, BR is aggregated in the same hexagonal lattice as in the purple membrane (PM). In this reconstituted system, BR has a photochemical cycle similar to that in the PM and is active as a light-driven proton pump. The lipid phase transition which was monitored by using the steady-state anisotropy of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) broadens with decreasing L/BR but occurs at approximately the same Tc. Below Tc, the fluorescence anisotropy of DPH is quite high (0.35) and independent of the L/BR. Above Tc, however, the anisotrophy increases markedly with decreasing L/BR. It was recently pointed out that the fluorescence anisotropy of probes like DPH contains information not only on the dynamics (correlation times) but also on the order parameters of the lipids [Heyn, M. P. (1979) FEBS Lett. 108, 359--364]. The most likely explanation of the observed increase in anisotropy above Tc is that the perturbation of the lipid bilayer caused by the incorporation of BR leads both to an increase in order and to a slowing of the rotational diffusion of the lipids (increased viscosity). In agreement with this latter dynamical effect, the rotational diffusion constant of BR itself decreases above Tc with decreasing L/BR. Above Tc, the membrane viscosity as determined from the rotational diffusion constant of BR is at least 1.5 times larger than that obtained from the fluorescence depolarization of DPH. The formation of the BR lattice as a function of temperature was followed by using the circular dichroism (CD) exciton effect together with measurements of the rotational diffusion of BR. Both methods show similar transition curves for the protein crystallization whose midpoints, however, occur several degrees below Tc.

Lateral diffusion of bacteriorhodopsin and a lipid analogue has been measured in dimyristoylphosphatidylcholine bilayers as a function of temperature, phospholipid/protein (mol/mol; L/P) ratio, and aqueous phase viscosity. The protein lateral diffusion coefficients measured above the temperature at which the lipid gel-liquid/crystalline phase transition occurs (Tc) are combined with previously determined rotational diffusion coefficients to provide a test of the Saffman-Delbrück equations [Saffman, P. G. & Delbrück, M. (1975) Proc. Natl. Acad. Sci. USA 72, 3111-3113]. Insertion of the diffusion coefficients into these equations enables the protein diameter to be calculated. The value of 4.3 +/- 0.5 nm so obtained is in reasonable agreement with the known structure of bacteriorhodopsin. A 12-fold increase in the viscosity of the aqueous phase reduces protein lateral diffusion coefficients by 50%, which is also consistent with the Saffman-Delbrück equations. Both protein and lipid lateral diffusion coefficients decrease with decreasing L/P ratio above the Tc. It is argued that, at a high L/P ratio, this effect is probably due to changes in membrane viscosity while, at a low L/P ratio, "crowding" effects (steric restrictions) and protein aggregation become important. When comparing diffusion measurements made in different systems, it is important to take the effect of the L/P ratio into account. When this is done, other published measurements of freely diffusing membrane proteins are in good agreement with the present results and the predictions of the Saffman-Delbrück equations. Below the Tc, the presence of protein enhances diffusion rates. The overall effect is to smooth out the large change in diffusion coefficient that occurs at the Tc.

Fusion of influenza virus with human erythrocytes at pH 5.2 was followed by fluorescence microscopy using a cooled slow-scan CCD camera. The high sensitivity of the CCD permits repetitive digital imaging of the same cells with minimal photobleaching. The experimental conditions were such that only a small number of virus particles were adsorbed per cell. Quantitative analysis of the data indicated that for most cells only a single fusion event took place. This was, however, sufficient to cause haemolysis within 30 min at 20-22 degrees C for about 60% of cells. There was a highly variable time lag between fusion and haemolysis. The lateral diffusion coefficient of virus particles on the cell surface when bound at pH 7.4 was less than 2 x 10(-13) cm2.s-1. The technique should be of value for more detailed studies of the dynamics of viral and other membrane fusion events.

Cytochrome oxidase was incorporated into lipid vesicles composed of phosphatidylethanolamine-phosphatidylcholine-cardiolipin. Large proteoliposomes of 1,000-15,000 A diameter were prepared by calcium-induced fusion of small vesicles. Rotational diffusion of cytochrome oxidase was measured by detecting the decay of the absorption anisotropy, r(t), after photolysis of the heme a3.CO complex by a vertically polarized laser flash. Because of the large size of the proteoliposomes, there was no contribution of vesicle-tumbling to r(t) over the experimental time range of 5 ms for samples in 60% sucrose. Analysis of r(t) curves was based on a "rotation-about-membrane normal" model. The measurements were used to investigate intermolecular interactions between cytochrome oxidases and between cytochrome oxidase and cytochrome bc1 complex co-reconstituted in the above lipid vesicles. In vesicles of a high lipid to protein ratio (congruent to 27), nearly all cytochrome oxidase molecules are rotating with an approximate rotational relaxation time, phi 1, on the order of 500 microseconds. In contrast, about 20% of cytochrome oxidase is immobile in vesicles with a relatively low lipid to protein ratio (congruent to 5), although phi 1 of the mobile population remains about 500 microseconds. In contrast, about 20% of cytochrome oxidase is immobile in vesicles with a relatively low lipid to protein ratio (congruent to 5), although phi 1 of the mobile population remains about 500 microseconds. The immobilized fraction is presumably due to nonspecific self-aggregation of cytochrome oxidase. The presence of cytochrome bc1 complex does not change r(t) curves significantly, either in the presence or absence of cytochrome c. Previously, we have observed the co-existence of mobile and immobile populations of cytochrome oxidase in bovine heart and rat heart mitochondria (Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J. (1980) J. Biol. Chem. 255, 5508-5510). The present results suggest that the immobile population of about one-half of cytochrome oxidase could be simply due to nonspecific protein aggregation resulting from the high concentration of enzymes in the inner mitochondrial membrane (lipid to protein ratio, less than or equal to 0.5). We also conclude that there is no specific interaction between cytochrome oxidase and cytochrome bc1 complex in the above large lipid vesicles. A lateral collision-controlled model for electron transfer from cytochrome bc1 complex to cytochrome oxidase through cytochrome c is discussed based on the above results.

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.

Slow rotational diffusion may be investigated by measuring the decay of dichroism of flash-induced transient absorption changes of suitable probes. The preparation of the covalent ""triplets" probe eosin isothiocyanate is described together with investigations of spectroscopic properties of eosin-protein conjugates. Triplet state lifetimes of air-equilibrated solutions of eosin-protein conjugates are in the order of 10 mus, demonstrating that the probe is protected from oxygen quenching by the protein. Experiments with the sucrase-isomaltase complex from small intestine show that its enzymatic activity is little affected by binding up to 2 mol of eosin/mol of protein.

Band 3, the anion transporter of the human erythrocyte membrane, has been purified and reconstituted into phospholipid vesicles of varying composition. Rotational diffusion of band 3 in these vesicles was measured by observing flash-induced transient dichroism of an eosin triplet probe covalently bound to the protein. In egg phosphatidylcholine vesicles of high lipid/protein ratio at temperatures well above the gel to liquid-crystalline phase transition, the absorption anisotropy decays to a constant value of 12 +/- 1% (expressed as a percentage of the initial anisotropy). However, higher values of the residual anisotropy and a slower decay are observed upon decreasing the temperature, even though the lipids remain in a liquid-crystalline phase. A similar effect is observed upon decreasing the lipid/protein ratio at constant temperature. It is concluded that self-association of band 3 occurs which is dependent on temperature and protein concentration in the bilayer. It is very probable that similar effects occur in the erythrocyte membrane, where a strong temperature dependence of band 3 rotational mobility is also observed. Reconstitution of band 3 into dioleoylphosphatidylcholine vesicles yields results similar to those obtained with egg phosphatidylcholine. When dimyristoylphosphatidylcholine is used for reconstitution, band 3 is immobilized below the lipid phase transition and remains partially associated above the transition. A precise analysis of the anisotropy decay curves is hampered by the presence of multiple rotating species.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Submitochondrial particles were labeled with the triplet probe eosin-5-maleimide (EMA) after pretreatment with N-ethylmaleimide. On sodium dodecyl sulfate-polyacrylamide gels, eosin fluorescence occurred in a single band of Mr approximately 30,000. The labeled band was identified as the ADP/ATP translocator, since EMA binding was completely inhibited by carboxyatractylate. Furthermore, the EMA-labeled polypeptide had the same molecular weight as the purified carboxyatractylate-bound translocator and the purified EMA-labeled translocator. Rotational diffusion of the translocator around the membrane normal in submitochondrial particles was measured by observing flash-induced absorption anisotropy of EMA. The translocator rotates with a time constant which varied from approximately 240 microseconds at 5 degrees C to approximately 100 microseconds at 37 degrees C. However, it is likely that only a fraction of the translocator rotates, the remainder being immobile over the measurement time of 500 microseconds. The mobile fraction of the translocator decreased with decrease in temperature. The observed fluorescence anisotropy of 0.24 indicates that EMA undergoes subnanosecond rapid wobbling in the binding site of the ADP/ATP translocator.

The rotational diffusion of bacteriorhodopsin reconstituted into dimyristoylphosphatidylcholine vesicles was measured by the technique of flash-induced transient dichroism. In the presence of melittin, a cell lysing peptide from honey bee (Apis mellifera) venom, dose-dependent loss of rotational mobility was observed. Chemically modified melittin derivatives, in which free amine groups were either acetylated or succinylated, were impaired in their ability to induce immobilisation of bacteriorhodopsin. Bacteriorhodopsin reconstitutions of differing lipid/protein ratio were tested and it was found that the bacteriorhodopsin immobilisation phenomena depended on the melittin/protein ratio, not the melittin/lipid ratio. This suggests that melittin produces its effect via direct interaction with bacteriorhodopsin. A mechanism is proposed in which the aggregation of bacteriorhodopsin is induced by electrostatic attraction between its anionic surface moieties and the highly cationic C-terminal segment of melittin.

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.

Influenza virus (strain X-47) was labeled with the triplet probe, eosin 5-isothiocyanate. Most of the label was found to be associated with haemagglutinin, the major glycoprotein of the viral envelope. Rotational diffusion of the glycoprotein was investigated by measuring flash-induced transient dichroism of the eosin probe. The anisotropy decay curves showed that mobility of haemagglutinin measured at pH 7.3 increased considerably with temperature with the greatest change occurring over the range 20-30 degrees C. However, at pH 5.2 no mobility was detectable over the time range of the experiment. The activity of the virus was determined by assaying haemolysis of human erythrocytes. The haemolytic activity showed an optimum at pH 5.2 and increased markedly with temperature, being negligible below 20 degrees C. In addition, inactivation of the virus by incubation at pH 5.2 was also strongly temperature dependent. A 15 min incubation at pH 5.2 inactivated the virus above 30 degrees C but had no effect below 20 degrees C. On the basis of these results, it is proposed that mobility of haemagglutinin is significant for its functional properties. When the pH is reduced from 7.3 to 5.2, the mobility observed at higher temperatures is required for the molecular rearrangements which accompany the fusion event. In the absence of an apposing membrane, these rearrangements result in irreversible aggregation of haemagglutinin in the viral membrane, and hence loss of mobility and activity.

The rotational mobility of Sendai virus glycoprotein spikes was measured by flash-induced transient dichroism of eosin triplet probes. The possible importance of this molecular motion for function was investigated by parallel assays of hemagglutination and fusion with erythrocytes. For mobility measurements, the glycoproteins were labeled on amino groups with eosin-5-isothiocyanate and on the galactose residues of the oligosaccharide chains with eosin-5-thiosemicarbazide. The decay of the absorption anisotropy of both probes, which has a time constant of about 100-200 musec at 37 degrees is attributed to the rotation of the proteins about an axis normal to the plane of the membrane. This motion was inhibited by crosslinking of the spike proteins with glutaraldehyde or by the specific binding of human erythrocyte glycophorin (a virus receptor) to the HN glycoprotein. Low values of the initial anisotropy for both probes indicate the existence of a second, faster motion. This is attributed to segmental motion of the glycoproteins. Segmental motion is inhibited by crosslinking with glutaraldehyde but appears to be little affected by interaction with glycophorin. The temperature dependence of the segmental and rotational motion of the proteins revealed a pronounced increase in mobility in the range of 30-35 degrees which was not paralleled by the lipid motion of the Sendai virus envelope membrane. Since the temperature dependence of virus-induced hemolysis has a similar characteristic, the mobility of glycoproteins appears to be correlated with the fusion activity. The hemagglutination activity, however, is not dependent on the mobility of the glycoprotein spikes.

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.