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S Ferguson-Miller

Publications and source records attributed to S Ferguson-Miller.

At least 55 records · Page 3Linked to original sources
PubMed1990

Respiratory control and ADP:O coupling ratios of isolated chick heart mitochondria.

Heart mitochondria isolated from 14- to 21-day-old chicks are highly coupled and often have respiratory control ratio (RCR) values exceeding 100. This paper presents data from a study of some of the properties of these mitochondria. The studies show that: (a) The ADP:O ratios and the state 4 rates of respiration are highly dependent upon the concentration of mitochondria at which these parameters are measured. (b) The mitochondrial isolate is contaminated with at least two divalent cation-stimulated ATPase, of which one is the F1F0-ATPase of broken mitochondria. (c) The oligomycin-sensitive component of state 4 respiration is completely inhibited by ethylene glycol bis(beta-amino-ethylether) N,N'-tetraacetic acid (EGTA). This inhibition is biphasic and attributable to the differential affinity of EGTA for Ca(II) and Mg(II). (d) Ca(II) and Mg(II) stimulate state 4 respiration, thereby depressing RCR values. These cations also decrease ADP:O ratios from greater than or equal to 3.25 to 3.0 for some NAD-linked substrates. (e) Uncoupled (i.e., oligomycin-insensitive) state 4 respiration can be abolished by treating the mitochondria with Nagarse and by preincubating mitochondria with exogenous substrate. (f) The ADP:O ratios obtained when these heart mitochondria oxidize pyruvate/malate, alpha-ketoglutarate, and beta-hydroxybutyrate are fractional and significantly greater than 3.0.

3-Hydroxybutyric Acid↗
PubMed1989

Synthesis, location, and lateral mobility of fluorescently labeled ubiquinone 10 in mitochondrial and artificial membranes.

To explore the influence of the long isoprene chain of ubiquinone 10 (UQ) on the mobility of the molecule in a phospholipid bilayer, we have synthesized a fluorescent derivative of the head-group moiety of UQ and measured its lateral diffusion in inner membranes of giant mitochondria and in large unilamellar vesicles. The diffusion coefficients, determined by the technique of fluorescence redistribution after photobleaching, were 3.1 X 10(-9) cm2 s-1 in mitochondria and 1.1 X 10(-8) cm2 s-1 in vesicles. Similar diffusion rates were observed for fluorescently labeled phosphatidylethanolamine (PE) with the same moiety attached to its head group (4-nitro-2,1,3-benzooxadiazole: NBD). Fluorescence emission studies carried out in organic solvents of different dielectric constants, and in vesicles and mitochondrial membranes, indicate that NBDUQ is located in a more hydrophobic environment than NBDPE or the starting material IANBD (4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitro-2,1,3- benzoxadiazole). Fluorescence quenching studies carried out with CuSO4, a water-soluble quenching agent, also indicate that NBDUQ is located deeper in the membrane than NBDPE. These results suggest that ubiquinone and PE are oriented differently in a membrane, even though their diffusion rates are similar. Conclusions regarding whether or not diffusion of UQ is a rate-limiting step in electron transfer must await a more detailed knowledge of the structural organization and properties of the electron transfer components.

4-Chloro-7-nitrobenzofurazan↗
PubMed1989

Independent control of respiration in cytochrome c oxidase vesicles by pH and electrical gradients.

The effects of altering the pH and electrical components of the membrane potential on the visible spectra and oxygen consumption rates of cytochrome oxidase vesicles were examined during steady-state respiration using cytochrome c as the substrate. Heme a was found to be 30-55% reduced in the presence of a membrane potential, becoming more reduced when the electrical gradient (delta psi) was abolished by valinomycin and more oxidized when the pH gradient (delta pH) was abolished by nigericin, with little increase (1.2-1.8-fold) in the rates of oxygen consumption in either case. When both gradients were eliminated, heme a reduction was close to initial levels, and activity was stimulated up to 8-fold. The magnitude of the changes in heme a reduction levels upon elimination of a gradient component was shown to be positively correlated with the magnitude of the respiratory control ratio of the vesicle preparation. Kinetic analysis of the dependence of oxidase activity on cytochrome c concentration indicated that changes in the Michaelis constant of the enzyme for its substrate are not a major factor in regulation by either delta pH or delta psi. These results suggest a dual mechanism for respiratory control in cytochrome oxidase vesicles under steady-state conditions, in which the electrical gradient predominantly affects electron transfer from cytochrome c to heme a, possibly by altering the reduction potential of heme a, while the pH gradient affects electron transfer from heme a (CuA) to heme a3 (CuB), possibly by a conformationally mediated change in the reduction potential of heme a3 or in the kinetics of the electron-transfer process.

Animals↗
PubMed1988

Effect of subunit III removal on control of cytochrome c oxidase activity by pH.

Studies were undertaken to assess the postulated involvement of subunit III in the proton-linked functions of cytochrome c oxidase. The effect of pH on the steady-state kinetic [corrected] parameters of subunit III containing and subunit III depleted cytochrome oxidase was determined by using beef heart and rat liver enzymes reconstituted into phospholipid vesicles. The TNmax and Km values for the III-containing enzyme increase with decreasing pH in a manner quantitatively similar to that reported by Thornstrom et al. [(1984) Chem. Scr. 24, 230-235], giving three apparent pKa values of less than 5.0, 6.2, and 7.8. The maximal activities of the subunit III depleted enzymes (beef heart and rat liver) show a similar dependence on pH, but the Km values are consistently higher than those of the III-containing enzyme, an effect that is accentuated at low pH. The pH dependence of TNmax/Km for both forms of the enzyme (+/- subunit III) indicates that protonation of a group with an apparent pKa of 5.7 lowers the affinity for substrate (cytochrome c) independently of a continued increase in maximal velocity. N,N'-Dicyclohexylcarbodiimide (DCCD) decreases the pH responsiveness of the electron-transfer activity to the same extent in both III-containing and III-depleted enzymes, indicating that this effect is mediated by a peptide other than subunit III. Control of intramolecular electron transfer by a transmembrane pH gradient (or alkaline intravesicular pH) is shown to occur in cytochrome oxidase vesicles with cytochrome c as the electron donor, in agreement with results of Moroney et al. [(1984) Biochemistry 23, 4991-4997] using hexaammineruthenium(II) as the reductant.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Yeast and horse liver alcohol dehydrogenases: potential problems in target size analysis and evidence for a monomer active unit.

Yeast and horse alcohol dehydrogenases are commonly used as standards for radiation inactivation analysis of proteins, usually assuming that the minimal functional unit corresponds to the physical size in solution, a tetramer (Mr = 148,000) and a dimer (Mr = 80,000), respectively. Results described in this paper demonstrate that molecular weight overestimates may be obtained for the yeast protein as a result of its unusual sensitivity to secondary radiation products. Irradiation in the presence of sulfhydryl reagents results in a smaller functional size estimate (67,000 +/- 3000) than that obtained in their absence (128,000 +/- 5000), indicating that some sulfhydryl groups in the enzyme may be particularly susceptible to attack by radiolytic species. Analysis of the horse liver enzyme reveals that although it has structural and functional similarities to the yeast protein, it is not as prone to secondary radiation damage and gives a minimal functional size estimate (33,000 +/- 1000) that most closely corresponds to a monomer. Quantitation of disappearance of the protein from a sodium dodecyl sulfate gel as a function of radiation dose also gives a target size (48,000 +/- 3000) in reasonable agreement with the monomer molecular weight. These results indicate that the individual subunits of horse liver alcohol dehydrogenase have independent catalytic capacity and imply that the same may be true for the yeast enzyme.

Alcohol Dehydrogenase↗
PubMed1986

Methionine-80-sulfoxide cytochrome c: preparation, purification and electron-transfer capabilities.

In order to explore the electron-transferring properties of methionine-80-sulfoxide cytochrome c, the pure, chromatographically homogeneous methionine-80-sulfoxide cytochrome c was previously published procedure (Ivanetich, K.M., Bradshaw, J.J. and Kaminsky, L.S. (1976) Biochemistry 15, 1144-1153) was found to produce a mixture of products. In the pure derivative, visible spectroscopy indicates that the 695 nm band indicative of the Met-80-Fe coordination is missing, amino acid analysis indicates that only one methionine is modified to the sulfoxide, and the E0' is found to be 240 mV vs. N.H.E. For succinate cytochrome c reductase activity, the Km for modified cytochrome was about one-ninth that of the native protein, while the maximum turnover number of the reductase with the modified protein was only about 54% of that with native protein. In contrast, the activity with cytochrome oxidase measured polarographically using ascorbate and TMPD under two different buffer/pH conditions, gave Km values that were very similar for both the native and modified cytochromes c, but the maximum turnover numbers of the oxidase with the modified protein were less than 40% of native in either buffer. It is concluded that the Met-80-sulfoxide cytochrome c in the reduced form is able to maintain substantially its heme crevice structure and thus maintain Km values similar to those of native protein. However, the low maximum turnover numbers for oxidase activity with the modified protein in the reduced state indicate that electron transfer itself has been significantly decreased, probably because the parity of acid/base and electrostatic interactions of Met-80 sulfur with the Fe in the two redox states has been disrupted.

Animals↗
PubMed1985

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.

Animals↗
PubMed1984

The functional and physical form of mammalian cytochrome c oxidase determined by gel filtration, radiation inactivation, and sedimentation equilibrium analysis.

When solubilized in laurylmaltoside, cytochrome oxidases from beef heart and rat liver mitochondria exist as monodisperse populations that are stable, highly active, and have apparent molecular weights of 300,000 to 350,000, as measured by gel filtration. To determine whether these are monomeric (2 heme A, 2 Cu) or dimeric forms of the enzyme, we performed radiation inactivation and sedimentation equilibrium analyses. From radiation inactivation experiments under two different sets of conditions, we obtained estimates for the functional molecular weight of beef heart cytochrome oxidase of 114,000 and 99,000, much less than a dimer and significantly smaller than a 200,000 molecular weight monomer containing one copy of each of the 12 subunits normally present in the complex. The same functional size is obtained for a rat liver oxidase preparation depleted of subunit III. The physical molecular weight of cytochrome oxidase was determined by sedimentation equilibrium measurements in solvents of different densities using mixtures of H2O and D218O. Estimates of Mr = 194,000 +/- 9,000 for the beef heart oxidase and Mr = 152,000 +/- 6,000 for the rat liver enzyme were obtained, consistent with the size predicted for monomers of their subunit composition. From these results we conclude that mammalian cytochrome oxidases from beef heart and rat liver exist in laurylmaltoside as monomers capable of high rates of electron transfer and normal substrate binding. Further, these functions appear to be associated with a subset of the peptides present in the monomer, mainly composed of subunits I and II.

Animals↗
PubMed1983

Lipid and subunit III depleted cytochrome c oxidase purified by horse cytochrome c affinity chromatography in lauryl maltoside.

Cytochrome oxidase is purified from rat liver and beef heart by affinity chromatography on a matrix of horse cytochrome c-Sepharose 4B. The success of this procedure, which employs a matrix previously found ineffective with beef or yeast oxidase, is attributed to thorough dispersion of the enzyme with nonionic detergent and a low density of cross-linking between the lysine residues of cytochrome c and the cyanogen bromide activated Sepharose. Beef heart oxidase is purified in one step from mitochondrial membranes solubilized with lauryl maltoside, yielding an enzyme of purity comparable to that obtained on a yeast cytochrome c matrix [Azzi, A., Bill, K., & Broger, C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2447-2450]. Rat liver oxidase is prepared by hydroxyapatite and horse cytochrome c affinity chromatography in lauryl maltoside, yielding enzyme of high purity (12.5-13.5 nmol of heme a/mg of protein), high activity (TN = 270-400 s-1), and very low lipid content (1 mol of DPG and 1 mol of PI per mol of aa3). The activity of the enzyme is characterized by two kinetic phases, and electron transfer can be stimulated to maximal rates as high as 650 s-1 when supplemented with asolectin vesicles. The rat liver oxidase purified by this method does not contain the polypeptide designated as subunit III. Comparisons of the kinetic behavior of the enzyme in intact membranes, solubilized membranes, and the purified delipidated form reveal complex changes in kinetic parameters accompanying the changes in state and assay conditions, but do not support previous suggestions that subunit III is a critical factor in the binding of cytochrome c at the high-affinity site on oxidase or that cardiolipin is essential for the low-affinity interaction of cytochrome c. The purified rat liver oxidase retains the ability to exhibit respiratory control when reconstituted into phospholipid vesicles, providing definitive evidence that subunit III is not solely responsible for the ability of cytochrome oxidase to produce or respond to a membrane potential or proton gradient.

Animals↗
PubMed1982

The active form of cytochrome c oxidase: effects of detergent, the intact membrane, and radiation inactivation.

Cytochrome oxidase is a multisubunit, intrinsic membrane protein with a complex function that includes oxidation of cytochrome c, reduction of oxygen and generation of a membrane potential. To clarify the relationship of its normal function to protein and membrane structure, we have examined the kinetic behavior of rat liver cytochrome oxidase in the intact inner mitochondrial membrane and in detergent solubilized states. Dissolution of rat liver mitochondrial membranes alters the kinetic parameters of the oxidase in a manner dependent in part on the dispersing agent, and characterized by a large increase in maximal activity which is not attributable to exposure of more oxidase or diminished affinity for cytochrome c. The most profound effect of solubilization of the membrane is seen on the low affinity reaction of cytochrome c, suggesting that the electron transfer pathway from this site to oxygen is sensitive to alterations in hydrophobic interactions within the oxidase. Purified rat liver and beef heart oxidase exists predominantly in a monodisperse, 300 kilodalton form in laurylmaltoside (Rosevear et al., 1980). However, a smaller, 130 kd species that exhibits high turnover rates equal to the 300 kd form is detected in some beef heart preparations, implying that the dimer may not be essential for high activity. Radiation inactivation studies on purified oxidase reveal a molecular weight for the functional unit of approximately 70 kd. It is concluded that less than a complete set of subunits may be sufficient for both normal binding of cytochrome c and rapid electron transfer to oxygen.

Animals↗
PubMed1982

Lateral mobility of cytochrome c on intact mitochondrial membranes as determined by fluorescence redistribution after photobleaching.

Lateral mobility of an active fluorescent derivative of cytochrome c on the membranes of giant mitochondria was measured by fluorescence redistribution after photobleaching. A diffusion coefficient of 1.6 X 10(-10) cm2/sec was determined for the labeled cytochrome c on inner mitochondrial membranes under conditions where succinate oxidase activity was demonstrated. This relatively low rate of diffusion, together with results of other investigators, is explained in terms of a model involving a dynamic equilibrium between freely diffusing and associated forms of electron-transfer components.

Animals↗
PubMed1981

An effective electron donor to cytochrome oxidase. Purification, identification, and kinetic characterization of a contaminant of ruthenium red, hexaamineruthenium II/III.

When the calcium-transport inhibitor, ruthenium red, is chromatographed on a cation exchange resin, it yields a number of colored fractions and a colorless component that absorbs in the ultraviolet. The electron transfer activity previously ascribed to ruthenium red (Schwerzmann, K., Gazzotti, P., and Carafoli, E. (1976) Biochem. Biophys. Res. Commun. 69, 812) fractionates exclusively with the UV-absorbing material. On the basis of spectral, physical, and activity studies, we have identified this compound as Ru(NH3)62+/3+. It is shown that Ru(NH3)62+/3+ is an efficient electron donor directly to cytochrome oxidase, without mediation by cytochrome c. The steady state kinetics of electron transfer from Ru(NH3)62+ to purified oxidase resembles that of cytochrome c, showing a biphasic pattern but higher apparent Km values (Km1 = 8 microM, Km2 = 88 microM). Under conditions that favor tight binding to the oxidase, cytochrome c acts as a competitive inhibitor of Ru(NH3)62+, indicating that the two electron donors interact with cytochrome oxidase at the same site(s). The efficiency of Ru(NH3)62+ as an electron mediator to cytochrome aa3 and the similarity of its kinetic behavior to that of cytochrome c, make it a potentially valuable tool for investigating the mechanism of energy conservation in the terminal segment of the mitochondrial respiratory chain.

Animals↗
PubMed1980

Alkyl glycoside detergents: a simpler synthesis and their effects on kinetic and physical properties of cytochrome c oxidase.

Octyl glucoside is an effective, nonionic, solubilizing agent for membrane proteins with the advantage of ease of removal by dialysis. In order to study the detergent-sensitive activity of cytochrome c oxidase, we chose this detergent because of its simple structure and the possibility of synthesizing analogues to test the structural dependence of the detergent specificity. A procedure was therefore developed that facilitates large-scale preparation of octyl glucoside and related alkyl glycosides, improving on previous methods by eliminating crystallization steps and employing a one-step purification of the final product on Dowex 1. This new purification procedure is particularly important for achieving the level of purity required to obtain the disaccharide, longer alkyl chain detergents in soluble form. Of the alkyl glycosides prepared (octyl beta-D-glucopyranoside, octyl beta-D-lactopyranoside, dodecyl beta-D-lactopyranoside, dodecyl beta-D-cellobiopyranoside, and dodecyl beta-D-maltopyranoside), lauryl (dodecyl) maltoside was found to be the most successful as an activator of purified beef and Neurospora cytochrome oxidase, giving two- to tenfold higher activities than octyl glucoside and other commercially available detergents, Tween-20 and Triton X-100. Kinetic studies using two different steady-state assay systems indicate that the activity changes are not the result of altered binding of the substrate but rather reflect a detergent effect on the state of association of the enzyme (as a monomer, dimer, or polymer) as well as on its intrinsic activity. By gel filtration procedures, lauryl maltoside and octyl glucoside were found to exist as monodisperse populations of micelles of 50 000 and 8000 daltons, respectively. The small uniform micelles and chemically well-defined structures of lauryl maltoside and octyl glucoside make them superior to other nonionic detergents for the study of membrane proteins in general and cytochrome oxidase in particular, since its activity in lauryl maltoside most closely approaches that of the physiological state.

Animals↗