Mobility in the mitochondrial electron transport chain

The role of lateral diffusion in mitochondrial electron transport has been investigated by measuring the diffusion coefficients for lipid, cytochrome c, and cytochrome oxidase in membranes of giant mitoplasts from cuprizone-fed mice using the technique of fluorescence redistribution after photobleaching (FRAP). The diffusion coefficient of the phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine is dependent on the technique used to remove the outer mitochondrial membrane. A sonication technique yields mitoplasts with monophasic recovery of the lipid probe (D = 6 X 10(-9) cm2/s), while digitonin-treated mitochondria show biphasic recoveries (D1 = 5 X 10(-9) cm2/s; D2 = 1 X 10(-9) cm2/s). Digitonin appears to incorporate into mitoplasts, giving rise to decreased lipid mobility concomitant with increased rates of electron transfer from succinate to oxygen, in a manner reminiscent of the effects of cholesterol incorporation [Schneider, H., Lemasters, J. J., Hochli, M., & Hackenbrock, C. R. (1980) J. Biol. Chem. 255, 3748-3756]. FRAP measurements on tetramethylrhodamine cytochrome c modified at lysine-39 and on a mixture of active morpholinorhodamine derivatives of cytochrome c gave diffusion coefficients of (3.5-7) X 10(-10) cm2/s depending on the assay medium. With morpholinorhodamine-labeled antibodies purified on a cytochrome oxidase affinity column, the diffusion coefficient for cytochrome oxidase was determined to be 1.5 X 10(-10) cm2/s. The results are discussed in terms of a dynamic aggregate model in which an equilibrium exists between freely diffusing and associated electron-transfer components.     

The binding of porphyrin cytochrome c to yeast cytochrome c peroxidase. A fluorescence study of the number of sites and their sensitivity to salt

Porphyrin c, the iron-free derivative of cytochrome c, is a reasonably good model for cytochrome c binding to cytochrome c peroxidase (CcP). It binds with the same stoichiometry but only one-quarter as tightly as cytochrome c. CcP (resting, FeIII) and CcP X CN can both bind up to two molecules of porphyrin c. The binding of the first porphyrin c is tight (kd = 1 X 10(-9) M, pH 6, ionic strength mu = 0, 4 degrees C) and results in quenching of the porphyrin c fluorescence. The binding is sensitive to ionic strength. The binding of the second porphyrin c is looser (Kd unknown) and does not result in quenching of the porphyrin fluorescence. The binding of porphyrin c to the cyano form and the resting forms of CcP cannot be distinguished by our methods. ES is the first acceptor of electrons from c(II) and can bind at least two molecules of porphyrin c. The binding of the first porphyrin c is extremely tight, results in substantial quenching and is insensitive to ionic strength. The binding of porphyrin c to the loose site (Kd = 2 X 10(-9) M, pH 6, 4 degrees C, mu = 0) results, unlike the resting and cyano forms, in quenching of fluorescence of the second porphyrin c. The binding of the second porphyrin c to ES is sensitive to ionic strength. The calculated distances between porphyrin c and the hemes of CcP(FeIII) and ES are approximately 2.5 nm.     

Lateral diffusion of proteins in the periplasm of Escherichia coli.

We have introduced biologically active, fluorescently labeled maltose-binding protein into the periplasmic space of Escherichia coli and measured its lateral diffusion coefficient by the fluorescence photobleaching recovery method. Diffusion of this protein in the periplasm was found to be surprisingly low (lateral diffusion coefficient, 0.9 X 10(-10) cm2 s-1), about 1,000-fold lower than would be expected for diffusion in aqueous medium and almost 100-fold lower than for an equivalent-size protein in the cytoplasm. Galactose-binding protein, myoglobin, and cytochrome c were also introduced into the periplasm and had diffusion coefficients identical to that determined for the maltose-binding protein. For all proteins nearly 100% recovery of fluorescence was obtained after photobleaching, indicating that the periplasm is a single contiguous compartment surrounding the cell. These data have considerable implications for periplasmic structure and for the role of periplasmic proteins in transport and chemotaxis.     

Rapid lateral diffusion of the variant surface glycoprotein in the coat of Trypanosoma brucei

The membrane form of the variant surface glycoprotein (mfVSG) is anchored in the plasma membrane of Trypanosoma brucei by a dimyristoylphosphatidylinositol residue connected via a glycan to the COOH-terminal amino acid. The glycoprotein molecules are tightly packed, forming a coat that is impenetrable to lytic serum components. Lateral diffusion of mfVSG was measured by the fluorescence recovery after photobleaching technique. mfVSG labeled on the cell surface with rhodamine-conjugated anti-VSG Fab fragments showed a diffusion coefficient of 1 X 10(-10) cm2/s at 37 degrees C and of 0.7 X 10(-10) cm2/s at 27 degrees C. About 80% of the molecules were mobile. Affinity-purified mfVSG molecules implanted into the plasma membrane of baby hamster kidney cells exhibited a similar mobility to that found in the trypanosome coat [D = (0.4-0.7) X 10(-10) cm2/s at 4 degrees C]. Phospholipid mobility in the plasma membrane of trypanosomes was characterized by a diffusion coefficient of 2.2 X 10(-9) cm2/s at 37 degrees C. It is concluded that mfVSG mobility in the surface coat of the parasite is rapid and comparable to that of other membrane-bound glycoproteins but slower than that of phospholipids.     

Diffusion, patching, and capping of stearoylated dextrans on 3T3 cell plasma membranes

Fluorescence-labeled trinitrophenylated stearoylated dextrans have been used as controllable analogues of cell membrane proteins on model membranes and on a variety of natural cell membranes. This paper reports their behavior on 3T3 mouse fibroblast plasma membranes. Spatial distribution on the membrane was studied by fluorescence microscopy, and molecular mobility was measured by fluorescence photobleaching recovery. At concentrations from 10(2) to 3 X 10(3) molecules/micron2 essentially homogeneous fluorescence was observed after treatment with these stearoyldextrans in culture. Diffusion coefficients and fractional recovery of fluorescence after photobleaching were cvoncentration independent. For 3 X 10(3) molecules/micron2 we found at 23 degrees C D = (3.0 +/- 1.8) X 10(-10) cm2/s with 65 +/- 17% recovery and at 37 degrees C D = (7.0 +/- 5.0) X 10(-10) cm2/s without a change of the fractional recovery. Cross-linking with antibodies stopped diffusion on a macroscopic scale and sometimes induced patching, mottling (defined as the development of gaps in the fluorescence layer), and capping (defined as the confinement of the fluorescence to less than 50% of the cell). Capping required approximately 3 h at 37 degrees C and was inhibited by metabolic poisons and cytochalasin B. These drugs did not affect stearoyldextran diffusion or fractional recovery. Colchicine, which did not dramatically affect capping, slowed diffusion two- to threefold but did not affect fractional recovery. The antibody inhibition of the diffusion of stearoyldextrans precedent to capping did not affect the diffusion of a lipid probe or fluorescein isothiocyanate labeled membrane proteins. When the trinitrophenylated stearoyldextran was cleared from most of the surface by capping and the surface subsequently relabeled with stearoyldextran, the diffusion coefficient and fractional recovery of the second label were identical with those of the first label prior to capping. Thus, capping does not clear an immobilizing factor from the membrane.     

Lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in the membrane of differentiating HL-60 and U-937 cells assessed with fluorescence recovery after photobleaching (FRAP)

The promyelocytic leukemia cell line HL-60 and the histiocytic cell line U-937 were grown in suspension culture. They were induced to differentiate during 5-d cultivation in the presence of dimethylsulfoxide (DMSO; 1.3% w/v) or phorbol-12-myristate-acetate (PMA; 10(-7) M), which yields granulocyte- and macrophage-like cells, respectively. Differentiation was evidenced by increased capacity to recognize and phagocytize IgG- or complement-coated yeast particles. Aliquots taken from the cultures with and without DMSO (or PMA) were spun down directly on glass microscope slides, washed, labeled with fluoresceinated wheat germ agglutinin (WGA), and directly examined at room temperature for the rate of fluorescence recovery after photobleaching (FRAP). It was found that cultivation of the HL-60 and the U-937 cells in the presence of DMSO, which yields granulocyte-like cells, reduced the average value of lateral diffusion coefficient D (X 10(10] from 1.72 +/- 0.13 cm2s-1 to 0.97 +/- 0.13 cm2s-1 and from 1.77 +/- 0.11 cm2s-1 to 0.82 +/- 0.13 cm2s-1, respectively. U-937 cells grown with PMA also showed a reduction of D(X 10(10] to 0.88 +/- 0.10 cm2s-1. There was a larger immobile fraction of fluorescence in the HL-60 cells than in the U-937 cells, viz., 70-80% compared to 10-50%. The total number of binding sites for WGA was not altered, but the surface density changed, since the HL-60 and the U-937 cells became smaller and larger, respectively, when grown in the presence of DMSO. It is concluded that differentiation reduces the average lateral mobility of the WGA-binding membrane component by a factor around 2.     

Insulin effect on translational diffusion of lipids and proteins in the plasma membrane of isolated rat hepatocytes

The effects of insulin (10(-10)-10(-8) mol/l) on lateral diffusion of three fluorescent lipid probes, 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl phosphatidylcholine (NBD-PC), 5-(N-hexadecanoyl)aminofluorescein (F-C16), 5-(N-dodecanoyl)aminofluorescein (F-C12), and of fluorescein isothiocyanate-labeled proteins in the plasma membrane of intact rat hepatocytes were studied by the technique of fluorescence recovery after photobleaching. The absolute lateral diffusion coefficients of the lipid analogues NBD-PC, F-C16 and F-C12 at 21 degrees C were 2.5 X 10(-9) cm2/s, 5.4 X 10(-9) cm2/s and 19 X 10(-9) cm2/s, respectively. The diffusion coefficient mean of proteins labeled with fluorescein isothiocyanate was 6.4 X 10(-10) cm2/s. Insulin at 10(-9) and 10(-8) mol/l reduced the lateral diffusion coefficient for F-C12- and F-C16-labeled cells by 20% and for NBD-PC-labeled cells by 30% (P less than 0.025). The insulin effect was specific as tested by cell incubation with proinsulin and desoctapeptide insulin (10(-8) mol/l) and was detectable after 7 min of insulin preincubation. In contrast to lateral diffusion of lipid probes, lateral mobility of unselected membrane proteins was not altered by insulin. The observed modulation of lipid dynamics in the plasma membrane of intact hepatocytes, by which a variety of membrane functions can be influenced, may be an important step in the mechanism of insulin action.     

Oxygen diffusion in biological and artificial membranes determined by the fluorochrome pyrene

Quenching of pyrene fluorescence by oxygen was used to determine oxygen diffusion coefficients in phospholipid dispersions and erythrocyte plasma membranes. The fluorescence intensity and lifetime of pyrene in both artificial and natural membranes decreases about 80% in the presence of 1 atm O2, while the fluorescence excitation and emission spectra and the absorption spectrum are unaltered. Assuming the oxygen partition coefficient between membrane and aqueous phase to be 4.4, the diffusion coefficients for oxygen at 37 degrees C are 1.51 X 10(-5) cm2/s in dimyristoyl lecithin vesicles, 9.32 X 10(-6) cm2/s in dipalmitoyl lecithin vesicles, and 7.27 X 10(-6) cm2/s in erythrocyte plasma membranes. The heats of activation for oxygen diffusion are low (less than 3 kcal/degree-mol). A dramatic increase in the diffusion constant occurs at the phase transition of dimyristoyl and dipalmitoyl lecithin, which may result from an increase in either the oxygen diffusion coefficient, partition coefficient, or both. The significance of the change in oxygen diffusion below and above the phase transition for biological membranes is discussed.     

Multidimensional diffusion modes and collision frequencies of cytochrome c with its redox partners

We have determined the modes and rates of cytochrome c diffusion as well as the collision frequencies of cytochrome c with its redox partners at the surface of the isolated, mitochondrial inner membrane over a broad range (0-150 mM) of ionic strengths. Using fluorescence recovery after photobleaching, resonance energy transfer, and direct binding assay, we determined that the diffusion coefficient of cytochrome c is independent of its concentration and quantity bound to the inner membrane, that the distance of cytochrome c from the membrane surface increases with increasing ionic strength, and that there is no significant immobile fraction of cytochrome c on the membrane regardless of ionic strength. The rate of cytochrome c diffusion increases while its mode of diffusion changes progressively from lateral to three-dimensional with increasing ionic strength. At physiological ionic strength (100-150 mM), the diffusion of cytochrome c is three-dimensional with respect to the surface of the inner membrane with a coefficient of 1.0 x 10(-6) cm2/s, and little, if any cytochrome c is bound to the membrane regardless of its concentration. Furthermore, as ionic strength is raised from zero to 150 mM, the cytochrome ckd for the inner membrane increases, its mean occupancy time on the inner membrane to collide with a redox partner (tau) decreases, and its diffusion-based collision frequencies with its redox partners decrease. These data reveal the significance of both diffusion and concentration (affinity) of cytochrome c near the surface of the inner membrane in the control of the collision frequency of cytochrome c with its redox partners.     

Inhomogeneous translational diffusion of monoclonal antibodies on phospholipid Langmuir-Blodgett films.

The translational mobility of fluorescent-labeled monoclonal antibodies specifically bound to supported phospholipid bilayers containing hapten-conjugated phospholipids has been measured as a function of the surface concentration of bound antibodies using fluorescence recovery after photobleaching. Fluorescence recovery curves are fit well by a model that assumes the presence of two populations of antibodies with different lateral diffusion coefficients. The larger diffusion coefficient equals 3.5 x 10(-9) cm2/s, the smaller diffusion coefficient ranges from 1.5 x 10(-9) cm2/s to 2.5 x 10(-10) cm2/s, and the fractional fluorescence recovery associated with the smaller coefficient increases from approximately 0 to approximately 0.7 with increasing concentration of bound antibody. These results suggest that complexes of haptenated phospholipids and antibodies in phospholipid Langmuir-Blodgett films form clusters or domains in a concentration-dependent fashion.     

The use of the fluorescence photobleaching recovery technique to study the self-assembly of tubulin

Fluorescently labeled microtubule-associated proteins or poly-L-lysine (13,000 MW) were prepared by reaction with fluorescein isothiocyanate. The labeled compounds were used as probes of the assembly of calf brain tubulin using fluorescence photobleaching recovery techniques which allow measurement of the diffusion coefficient and percentage mobility of the fluorescent probe. When unfractionated tubulin (defined as material containing tubulin and microtubule-associated proteins) was polymerized at room temperature or 37 degrees C, either probe could be incorporated into microtubules, since the observed diffusion coefficient (approximately 1.7 X 10(-8) cm2/s) was much slower than that for either probe free in solution. The microtubules formed in the presence of labeled microtubule-associated proteins were free to diffuse while those formed in the presence of labeled polylysine were partially immobilized. Thus the fluorescence photobleaching recovery technique can be used to measure crosslinking of microtubules as well as assembly or interactions with other structures. When unfractionated tubulin was incubated with labeled polylysine in the presence of Ca2+ at room temperature, the observed diffusion coefficient (approximately 5.1 X 10(-8) cm2/s) probably represents the formation of rings of tubulin. The effect of mild and vigorous shearing, of cholchicine, and of different Mg2+ concentrations on the properties of the system were examined.     

Lateral diffusion of gramicidin C in phospholipid multibilayers. Effects of cholesterol and high gramicidin concentration

We have measured the lateral diffusion coefficient (D), of active dansyl-labeled gramicidin C (DGC), using the technique of fluorescence photobleaching recovery, under conditions in which the cylindrical dimer channel of DGC predominates. In pure, hydrated, dimyristoylphosphatidylcholine (DMPC) multibilayers (MBL), D decreases from 6 X 10(-8) cm2/s at 40 degrees C to 3 X 10(-8) cm2/s at 25 degrees C, and drops 100-fold at 23 degrees C, the phase transition temperature (Tm) of DMPC. Above Tm, addition of cholesterol decreases D; a threefold stepwise drop occurs between 10 and 20 mol %. Below Tm, increasing cholesterol increases D; a 10-fold increase occurs between 10 and 20 mol % at 21 degrees C, between 20 and 25 mol % at 15 degrees C, and between 25 and 30 mol % at 5 degrees C. In egg phosphatidylcholine (EPC) MBL, D decreases linearly from 5 X 10(-8) cm2/s at 35 degrees C to 2 X 10(-8) cm2/s at 5 degrees C; addition of equimolar cholesterol reduces D by a factor of 2. Thus this transmembrane polypeptide at low membrane concentrations diffuses quite like a lipid molecule. Its diffusivity in lipid mixtures appears to reflect predicted changes of lateral composition. Increasing gramicidin C (GC) in DMPC/GC MBL broadened the phase transition, and the diffusion coefficient of the lipid probe N-4-nitrobenzo-2-diazole phosphatidylethanolamine (NBD-PE) at 30 degrees C decreases from 8 X 10(-8) cm2/s below 5 mol % GC to 2 X 10(-8) cm2/s at 14 mol % GC; D for DGC similarly decreases from 4 X 10(-8) cm2/s at 2 mol % GC to 1.4 X 10(-8) cm2/s at 14 mol % GC. Hence, above Tm, high concentrations of this polypeptide restrict the lateral mobility of membrane components.     

Lateral diffusion of epidermal growth factor complexed to its surface receptors does not account for the thermal sensitivity of patch formation and endocytosis

The patching and endocytosis of EGF (epidermal growth factor) bound to A-431 cells (a human epidermoid carcinoma line) are temperature-sensitive processes which are completely inhibited at 4 degrees C. Receptor-mediated endocytosis generally occurs through coated regions, and EGF bound to its membrane receptor must diffuse laterally to these points of internalization. In this work we investigated the thermal sensitivity of the lateral diffusion of EGF receptor complexes and the thermal sensitivity of the patching and endocytosis of the hormone receptor complexes. Using the fluorescence photobleach recovery technique, we measured the lateral diffusion coefficients of a fluorescent derivative of EGF as a function of temperature. The lateral diffusion coefficient (D) increased gradually from 2.8 X 10(-10) cm2/s at 5 degrees C to 8.5 X 10(-10) cm2/s at 37 degrees C, and no phase transition was detected. Neither was a phase transition detected when we measured the diffusion coefficient of fluorescent lipid probes over this temperature range. From a calculation of the collision frequency of the occupied EGF receptors with coated regions using our measured values of D at 5 and 37 degrees C, we conclude that diffusion is not the rate-limiting step for either endocytosis or patching.     

Lateral diffusion and patch formation of H-2Kk antigens on mouse spleen lymphocytes

We have studied the diffusion and aggregation of H-2Kk antigens labeled with a fluorescent anti-H-2Kk monoclonal antibody (IgG) on mouse splenic lymphocytes, employing fluorescence photobleaching recovery and fluorescence microscopy. The H-2Kk antigens were initially distributed homogeneously on all lymphocytes. Upon antibody binding, sub-micron patches were formed on 50-60% of the cells. A lateral diffusion coefficient, D, of 7.1 X 10(-10) cm2/s and a mobile fraction of 0.73 were found for H-2Kk antigens on diffusely-labeled cells, while these antigens were immobile (D less than or equal to 5 X 10(-12) cm2/s) on patched cells. The patched and nonpatched sub-populations did not correspond to B- and T-lymphocytes. Subjection to low temperature or treatment with NaN3 or cytoskeleton-disrupting drugs did not affect the diffusion or patching of H-2Kk, indicating no involvement of metabolic energy or drug-sensitive cytoskeletal components. These findings could be related to the interactions of H-2 antigens on the cell surface, and to the different susceptibilities of various cells to lysis by cytotoxic T-cells.     

Effect of fatty acids on the membrane fluidity of cultured chick dorsal root ganglion measured by fluorescence photobleaching recovery

The effect of fatty acids on the membrane fluidity in tissue cultured chick embryo dorsal root ganglion was studied by fluorescence recovery method. Lateral motion of the lipid was measured by observing the fluorescent probe, 5-(octadecylthiocarbamoylamino) fluorescence, F18. The effective lateral diffusion coefficient of the membrane was around 0.30 X 10(-8) cm2/sec in control cells, 0.42 X 10(-8) cm2/sec in 2-decenoic acid treated cells, and 0.35 X 10(-8) cm2/sec in valeric acid treated cells. From these results it is concluded that effective mobilities of the membrane complex increased about 40% by the external application of 2-decenoic acid, while valeric acid increased it only 12%. From the physiological results that 2-decenoic acid inhibits the Na-channel, it is suggested that this increase in the membrane fluidity might affect the Na-channel.     

High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion

Rhodamine-labelled epidermal growth factor (Rh-EGF) was shown to bind to A431 cells grown at low density both to a small number of high affinity receptors (KD = 2.8 X 10(-10) M; fraction of total binding sites approximately 0.12) and also to a large number of low affinity receptors (KD = 4 X 10(-9) M; fraction of total binding sites approximately 0.88). Measurements of the lateral diffusion of EGF receptors on the cell surface were made using Rh-EGF and the technique of fluorescence photobleaching recovery. The high affinity receptors (labelled with 1.6 X 10(-10) M Rh-EGF, 5% of EGF binding sites occupied) did not show lateral mobility over the temperature range 3 degrees-37 degrees C. The low affinity receptors (labelled with 2.4 X 10(-7) M Rh-EGF, 90% of EGF sites occupied) showed at least 75% fluorescence recovery after photobleaching, and lateral diffusion coefficients of approximately 2 X 10(-10) cm2/s. These results show that the two populations of EGF receptors defined by binding studies differ in their freedom to diffuse laterally. The observation that the high affinity receptors are immobile indicates that lateral diffusion of receptors, at least over a distance of a few hundred nanometres or more, may not be required for the action of low concentrations of EGF.     

The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching.

Hyaluronan (HA) is a highly hydrated polyanion, which is a network-forming and space-filling component in the extracellular matrix of animal tissues. Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to investigate intramolecular hydrogen bonding and electrostatic interactions in hyaluronan solutions. Self and tracer lateral diffusion coefficients within hyaluronan solutions were measured over a wide range of concentrations (c), with varying electrolyte and at neutral and alkaline pH. The free diffusion coefficient of fluoresceinamine-labeled HA of 500 kDa in PBS was 7.9 x 10(-8) cm(2) s(-1) and of 830 kDa HA was 5.6 x 10(-8) cm(2) s(-1). Reductions in self- and tracer-diffusion with c followed a stretched exponential model. Electrolyte-induced polyanion coil contraction and destiffening resulted in a 2.8-fold increase in self-diffusion between 0 and 100 mM NaCl. Disruption of hydrogen bonds by strong alkali (0.5 M NaOH) resulted in further larger increases in self- and tracer-diffusion coefficients, consistent with a more dynamic and permeable network. Concentrated hyaluronan solution properties were attributed to hydrodynamic and entanglement interactions between domains. There was no evidence of chain-chain associations. At physiological electrolyte concentration and pH, the greatest contribution to the intrinsic stiffness of hyaluronan appeared to be due to hydrogen bonds between adjacent saccharides.     

Solubility and diffusion coefficient of adenosine 3':5'-monophosphate.

Several previously unavailable parameters of adenosine 3':5'-monophosphate have been determined. The molar extinction coefficient at pH 7.0 is 1.38 X 10(-4), the aqueous solubility at pH 7.0 is 0.0236 M and the diffusion coefficient is 4.44 X 10(-6) cm2/s.     

Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching

The translational diffusion of bovine rhodopsin, the Ca2+-activated adenosinetriphosphatase of rabbit muscle sarcoplasmic reticulum, and the acetylcholine receptor monomer of Torpedo marmorata has been examined at a high dilution (molar ratios of lipid/protein greater than or equal to 3000/1) in liquid-crystalline phase phospholipid bilayer membranes by using the fluorescence recovery after photobleaching technique. These integral membrane proteins having molecular weights of about 37 000 for rhodopsin, about 100 000 for the adenosinetriphosphatase, and about 250 000 for the acetylcholine receptor were reconstituted into membranes of dimyristoylphosphatidylcholine (rhodopsin and acetylcholine receptor), soybean lipids (acetylcholine receptor), and a total lipid extract of rabbit muscle sarcoplasmic reticulum (adenosinetriphosphatase). The translational diffusion coefficients of all the proteins at 310 K were found to be in the range (1-3) X 10(-8) cm2/s. In consideration of the sizes of the membrane-bound portions of these proteins, this result is in agreement with the weak dependence of the translational diffusion coefficient upon diffusing particle size predicted by continuum fluid hydrodynamic models for the diffusion in membranes [Saffman, P. G., & Delbrück, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113]. Lipid diffusion was also examined in th same lipid bilayers with the fluorescent lipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dimyristoylphosphatidylethanolamine. The translational diffusion coefficient for this lipid derivative was found to be in the range (9-14) X 10(-8) cm2/s at 310 K. In consideration of the dimensions of the lipid molecule, this value for the lipid diffusion coefficient is in agreement with the continuum fluid hydrodynamic model only if a near-complete slip boundary condition is assumed at the bilayer midplane. Alternatively, kinetic diffusion models [Tr?uble, H., & Sackmann. E. (1972) J. Am. Chem. Soc. 94, 4499-4510] may have to be invoked to explain the lipid diffusion behavior.     

Translational diffusion of bovine prothrombin fragment 1 weakly bound to supported planar membranes: measurement by total internal reflection with fluorescence pattern photobleaching recovery.

Previous work has shown that bovine prothrombin fragment 1 binds to substrate-supported planar membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a Ca(2+)-specific manner. The apparent equilibrium dissociation constant is 1-15 microM, and the average membrane residency time is approximately 0.25 s-1. In the present work, fluorescence pattern photobleaching recovery with evanescent interference patterns (TIR-FPPR) has been used to measure the translational diffusion coefficients of the weakly bound fragment 1. The results show that the translational diffusion coefficients on fluid-like PS/PC planar membranes are on the order of 10(-9) cm2/s and are reduced when the fragment 1 surface density is increased. Control measurements were carried out for fragment 1 on solid-like PS/PC planar membranes. The dissociation kinetics were similar to those on fluid-like membranes, but protein translational mobility was not detected. TIR-FPPR was also used to measure the diffusion coefficient of the fluorescent lipid NBD-PC in fluid-like PS/PC planar membranes. In these measurements, the diffusion coefficient was approximately 10(-8) cm2/s, which is consistent with that measured by conventional fluorescence pattern photobleaching recovery. This work represents the first measurement of a translational diffusion coefficient for a protein weakly bound to a membrane surface.