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Biomedical subjects

S Kawato

Publications and source records attributed to S Kawato.

54 records · Page 3Linked to original sources
PubMed1982

Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles.

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes.

Animals↗
PubMed1982

Rotation of cytochrome P-450. I. Investigations of protein-protein interactions of cytochrome P-450 in phospholipid vesicles and liver microsomes.

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.

Animals↗
PubMed1982

Protein-protein interactions of cytochrome oxidase in inner mitochondrial membranes. The effect of liposome fusion on protein rotational mobility.

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.

Animals↗
PubMed1982

Selective labeling and rotational diffusion of the ADP/ATP translocator in the inner mitochondrial membrane.

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.

Animals↗
PubMed1982

On the wobbling-in-cone analysis of fluorescence anisotropy decay.

Interpretation of fluorescence anisotropy decay for the case of restricted rotational diffusion often requires a model. To investigate the extent of model dependence, two models are compared: a strict cone model, in which a fluorescent probe wobbles uniformly within a cone, and a Gaussian model, where the stationary distribution of the probe orientation is of a Gaussian type. For the same experimental anisotropy decay, analysis by the Gaussian model predicts a smaller value for the rate of wobbling motion than the strict cone analysis, but the difference is 35% at most; the cone angle obtained by the strict cone analysis agrees closely with the effective width of the Gaussian distribution. The results suggest that, when only two parameters (the rate and the angular range) are extracted from an experiment, the choice of a model is not crucial as long as the model contains the essential feature, e.g., the more-or-less conical restriction, of the motion under study. Model-independent analyses are also discussed.

Fluorescence Polarization↗
PubMed1981

The effect of cytochrome oxidase on lipid chain dynamics. A nanosecond fluorescence depolarization study.

Molecular motions in membranes composed of purified cytochrome oxidase (EC 1.9.3.1) and synthetic lipid (L-alpha-dimyristoylphosphatidylcholine or L-alpha-dioleoylphosphatidylcholine) at various ratios were investigated with a lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Nanosecond fluorescence depolarization kinetics of the probe showed that the rod-shaped probe molecules perform a fast wobbling motion (restricted rotation) in all membranes studied, presumably reflecting the motion of lipid acyl chains. At temperatures where the pure lipid was in the liquid-crystalline phase, presence of cytochrome oxidase reduced the angular range of the wobbling motion, whereas its rate, the wobbling diffusion constant, was unaffected. On the other hand, incorporation of the protein into lipid in the gel phase resulted in the increase in the wobbling diffusion constant while the range of the wobbling motion remained the same. A time-dependent view of lipid dynamics that accounts for the above findings, as well as the results of recent electron spin resonance and nuclear spin resonance studies of protein-lipid interactions, is proposed.

Animals↗
PubMed1981

Rotation of cytochrome oxidase in phospholipid vesicles. Investigations of interactions between cytochrome oxidases and between cytochrome oxidase and cytochrome bc1 complex.

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.

Animals↗
PubMed1981

Nanosecond time-resolved fluorescence investigations of temperature-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicles and solubilized systems.

Intrinsic and lipid phase transition-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicle and solubilized systems were examined by the fluorescence lifetime of N-(1-anilinonaphthyl-4)-maleimide conjugated with the enzyme. The time-dependent fluorescence intensity of N-(1-anilinonaphthyl-4)-maleimide attached to cytochrome oxidase was described as a triple exponential decay. Both the intrinsic and lipid phase transition-induced conformational changes were detectable in plots of the average lifetime against temperature. In most cases a peak occurred at the temperature of the conformational change. The time-dependent emission anisotropy showed that N-(1-anilinonaphthyl-4)-maleimide embedded in cytochrome oxidase in phosphatidylcholine vesicles underwent a rapid restricted wobbling within a cone. The half-angle of the cone was around 30 degrees for cytochrome oxidase in dimyristoyl phosphatidylcholine vesicles.

Animals↗
PubMed1981

Time-dependent absorption anisotropy and rotational diffusion of proteins in membranes.

The decay of flash-induced absorption anisotropy, r(t), of a chromophore in a membrane protein is closely correlated with rotational diffusion of the protein in the membrane. We develop a theory of time-dependent absorption anisotropy which is applicable to both linear chromophores and planar chromophores which have two different absorption moments at right angles to one another. The theory treats two types of rotational diffusion of membrane proteins: one is rotation of the whole protein about the normal to the plane of the membrane, and the other is restricted wobbling of the whole or part of the protein molecule. In the former case, r(t) is determined by a rotational diffusion coefficient and an angle between the absorption moment(s) and the normal to the plane of the membrane. Rotation of rigid transmembrane proteins can be described by this treatment. In the latter case, r(t) is characterized by a wobbling diffusion coefficient and the degree of orientational constraint. This treatment may be applicable to independent wobbling of the hydrophilic part of membrane proteins. We further show that, for linear and circularly degenerate chromophores, the effect of the excitation flash intensity on r(t) can be accounted for by a constant scaling factor.

Kinetics↗
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

Fluorescent probe study of temperature-induced conformational changes in cytochrome oxidase in lecithin vesicle and solubilized systems.

A protein-bound label, N-(1-anilinonaphthyl-4)-maleimide (ANM), was used to investigate conformational changes in bovine heart cytochrome oxidase. The fluidity of cytochrome oxidase vesicles was monitored by a lipophilic probe, 1,6-diphenyl-1,3,5-hexatriene. The fluroescence intensity and emission anisotropy of these probes were examined between 4 and 60 degrees C in enzyme--dipalmitoyllecithin vesicles, in enzyme--dimyristoyllecithin vesicles, in enzyme--dioleoyllecithin vesicles, and in the soluble enzyme. The temperature-dependent changes in these quantities indicated that there were two types of conformational changes in oxidized cytochrome oxidase: one was attributed to an intrinsic enzyme conformation change which occurred around 20 degrees C, and the other was attributed to a conformational change induced by the lipid phase transition. Although ANM-reactive subunits of cytochrome oxidase in these four lecithin vesicle and solubilized systems were different from each other, subunit I always reacted with ANM in preference to other subunits.

Animals↗
PubMed1979

Effects of temperature on cytochrome oxidase activity in solubilized form and in lipid vesicle systems.

Isolated mammalian cytochrome oxidase gave an Arrhenius plot with a break (Tb) at about 20 degrees C when assayed in a medium containing Emasol. The activation energies above and below 20 degrees C were 9.3 (EH) and 18.9 kcal/mol (EL), respectively. Isolated cytochrome oxidase was also incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC, phase transition temperature Tt = 40 degrees C), dimyristoyl phosphatidylcholine (DMPC, Tt = 23 degrees C) and dioleoyl phosphatidylcholine (DOPC, Tt = -22 degrees C). The DPPC system showed a nearly linear Arrhenius plot between 9 and 36 degrees C with E = 22.8 kcal/mol. When cytochrome oxidase was resolubilized from the DPPC vesicles and assayed in solution a biphasic plot was obtained again. Cytochrome oxidase-DOPC was more active than the solubilized enzyme and exhibited a biphasic Arrhenius plot with Tb = 23 degrees C. EH and EL were 6.6 and 15.8 kcal/mol, respectively. The plot for the oxidase-DMPC also showed a break (Tb = 26 degrees C) with EH = 6.6 and EL = 26.6 kcal/mol. These results indicate that the break in the Arrhenius plot reflects primarily a structural transition in the cytochrome oxidase molecule between the "hot" and "cold" conformations, as proposed previously. This transition, as well as the molecular state of cytochrome oxidase, is affected by the physical state of the membrane lipids as reflected by changes in the kinetic properties.

Animals↗
PubMed1978

Effect of cholesterol on the molecular motion in the hydrocarbon region of lecithin bilayers studied by nanosecond fluorescence techniques.

Effects of cholesterol on the dynamic structure of the hydrocarbon region of dipalmitoyllecithin vesicles were examined. Decays of the emission anisotropy and the fluorescence intensity of 1,6-diphenyl-1,3,5-hexatriene embedded in lecithin-cholesterol vesicles were measured over a temperature range of 10--60 degrees C. The emission anisotropy decreased rapidly with time and then leveled off. The rotational motion of the probe was analyzed by a model of wobbling diffusion confined in a cone. Cholesterol (10--50 mol%) decreased the cone angle in the liquid-crystalline phase and increased it in the gel phase. In the presence of 33 mol% cholesterol, the wobbling diffusion constant increased in the gel phase and changed little in the liquid-crystalline phase. The viscosity in the cone decreased in the gel phase and remained almost unchanged in the liquid-crystalline phase in the presence of 33 mol% cholesterol. The total fluorescence intensity followed a singel exponential decay independently of the cholesterol content 0--50 mol%.

Chemical Phenomena↗
PubMed1977

Dynamic structure of lipid bilayers studied by nanosecond fluorescence techniques.

Molecular motions in liposomes of dipalmitoyl-phosphatidylcholine (DPPC) were studied by nanosecond fluorescence techniques. As a fluorescent probe for the hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used. Time courses of fluorescence intensity IT(t) and emission anisotropy r(t) of DPH embedded in DPPC liposomes were measured at various temperatures. The value of the fluorescence lifetime tau obtained froma single exponential decay of IT(t) was somewhat higher than that in liquid paraffin below the transition temperature Tt and decreased above Tt. Higher values of tau below Tt indicate the almost complete hydrophobic environment. The decay curves of r(t) were separated into two phases: an initial fast decreasing phase of the order of one nanosecond and a second almost constant phase. This indicates that the orientational motion of DPH in the hydrocarbon region is described by a wobbling diffusion restricted by a certain anisotropic potential. The results were analyzed on the model that the wobbling diffusion is confined in a cone with a uniform diffusion constant. Though temperature dependence of the cone angle was sigmoidal, that of the wobbling diffusion constant was like the exponential function. The change in the cone angle at Tt was sharper than that in the wobbling diffusion constant at Tt. Estimated values of the viscosity in the cone were an order of magnitude smaller than the values of "microviscosity" which were estimated from the steady-state emission anisotropy without considering the restrictions on the rotational motion.

Benzene Derivatives↗
PubMed1977

A theory of fluorescence polarization decay in membranes.

Decay of fluorescence polarization after an impulsive excitation is correlated with wobbling motion of fluorescent molecules in membranes. The motion is characterized by two parameters, a "wobbling diffusion constant" and a "degree of orientational constraint" both of which can be determined directly from experimentally obtained decay. Detailed discussion, including theoretically calculated time-courses of polarization decay, is given for several types of molecules embedded in lipid bilayers; these types cover a large part of fluorescent probes available at present. The theory is useful for the analysis of fluorescence polarization decay in any system where the orientation of fluorophore is restricted by the surrounding structure.

Liposomes↗