Effect of the nonionic detergent Triton X-100 on mitochondrial succinate-oxidizing enzymes

Specific activities of succinate:coenzyme Q reductase, ubiquinone:cytochrome c reductase, cytochrome oxidase, succinate:cytochrome c reductase, succinate oxidase, and ubiquinol oxidase have been measured in rat liver mitochondria in the presence of Triton X-100. The last three activities are much more sensitive to Triton X-100 than the first ones; the data suggest that the electron transport chain components cannot react with each other in the presence of the detergent. At least in the case of succinate:cytochrome c reductase, reconstitution of the detergent-treated membranes with externally added phospholipids reverses the inhibition produced by Triton X-100. These results support the idea that the respiratory chain components diffuse at random in the plane of the inner mitochondrial membrane; the main effect of the detergent would be to impair lateral diffusion by decreasing the area of lipid bilayer. When detergent-treated mitochondrial suspensions are centrifuged in order to separate the solubilized from the particulate material, only the first three enzyme activities mentioned above are found in the supernatants. After centrifugation, a latent ubiquinol:cytochrome c oxidase activity becomes apparent, whereas the same centrifugation process produces inhibition of cytochrome c oxidase in the presence of certain Triton X-100 concentrations. These effects could be due either to a selective solubilization of regulatory or catalytic subunits or to a conformational change of the enzyme-detergent complex.     

Characterization of mixed micelles of phospholipids of various classes and a synthetic,homogeneous analogue of the nonionic detergent triton X-100 containing nine oxyethylene groups

The synthesis and high-pressure liquid chromatographic purification of the homogeneous nonionic surfactant $\text{p-}\text{(1,1,3,3-tetramethylbutyl)phenoxynonaoxyethylene}$ glycol (OPE-9) in quantities suitable for membrane solubilization studies is reported. Micelles of OPE-9 and mixed micelles of OPE-9 with dimyristoyl and dipalmitoyl phosphatidylcholine as well as phosphatidylserine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin, and palmitic acid were characterized by column chromatography on 6% agarose. It was found that at 28°C OPE-9 micelles have a Stokes' radius of 32 Å, giving a molecular weight for a spherical micelle of about half that of micelles of the polydisperse nonionic surfactant Triton X-100 under the same conditions. The micelle size is temperature dependent: at 40°C the OPE-9 micelles have a Stokes' radius of 44 Å, giving a molecular weight for a spherical micelle of about twice that of the OPE-9 micelles at 28°C. The size of the mixed micelles varies linearly (as measured by $\text{K}{}_{\mathrm{<mtext>av</mtext>}}$) with the mole fraction of phospholipid. The mixed micelle size was found to be relatively independent of the absolute concentration of surfactant over a four-fold range if the mole fraction of phospholipid is kept constant. The usefulness of the OPE-9/phospholipid mixed micelle system for lipolytic enzyme substrates and membrane-related studies is considered.     

GM1-ganglioside-triton X-100 mixed micelles: Changes of micellar properties studied by laser-light scattering and enzymatic methods

The micellar properties of mixtures of GM1 ganglioside and the non-ionic amphiphile Triton X-100 in 25 mM Na phosphate-5 mM di Na EDTA buffer (pH = 7.0) were investigated by quasielastic light scattering in a wide range of Triton/GM1 molar ratios and in the temperature range 15–37°C. These measurements: (a) provided evidence for the formation of mixed micelles; (b) allowed the determination of such parameters as the molecular weight and the hydrodynamic radius of the mixed micelles; (c) showed the occurrence of statistical aggregates of micelles with increasing temperature and micelle concentration. Galactose oxidase was chosen for studying the relation between enzyme activity and micellar properties. The action of the enzyme on GM1 was found to be strongly dependent on the micellar structure. In particular: (a) galactose oxidase acted very poorly on homogeneous GM1 micelles, while affecting mixed GM1/Triton X-100 micelles; (b) at fixed GM1 concentration the oxidation rate increased by enhancing Triton X-100 concentration and followed a biphasic kinetics with a break at a certain Triton X-100 concentration; (c) the formation of statistical micelle aggregates was followed by inhibition of the enzyme activity.     

Proton nuclear magnetic resonance studies of the manganese (II) binding site of concanavalin A. Effect of saccharides on the solvent relaxation rates

The longitudinal relaxation rate ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$) of water protons was studied in solutions of Mn(II)-concanavalin A at a number of frequencies. These relaxation rates were lowered in the presence of a variety of saccharides which have affinities for concanavalin A which range over two orders of magnitude. A good correlation was found in which saccharides which bind tightly have the greatest effect and saccharides which bind weakly or not at all have little effect on the $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ values. The temperature dependence of the proton relaxation rates showed that the lowering of these rates in the presence of saccharides was most likely due to a change in the exchange rate of solvent interacting with protein-bound Mn(II), $\text{1}\text{T}{}_{\mathrm{<mtext>m</mtext>}}$.An analysis of the temperature and frequency dependence of the $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$ (transverse) solvent proton relaxation rates resulted in evaluation of a number of parameters for solvent water molecules interacting in the first coordination sphere of Mn(II) bound to concanavalin A. The ratio of the number of water molecules (q) to the Mn(II)-proton distance (r) obtained from a computer fit of the data over a limited temperature range is in accord with the findings of Koenig et al. ((1973) Proc. Nat. Acad. Sci.70, 475) and Meirovitch and Kalb ((1973) Biochim. Biophys. Acta303, 258). However, our studies of $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$ of water over a more extensive temperature range are best fit with the following conclusions: at low temperatures (<20 °C), the data are consistent with an outer-sphere relaxation process. At higher temperatures (> 30 °C), the water molecule in the inner coordination sphere of the bound Mn(II) begins exchanging more rapidly and contributes to the relaxation processes ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$). The relaxation time of protons in the inner coordination shell, T_1M, contributes over the entire temperature range and produces a frequency dependence in the relaxivity data from 6 to 100 MH_z since the contributions to the correlation times are in the range 10^?9-10^?8 sec.     

Phosphatidylserine decarboxylase: analysis of its action towards unsaturated and saturated phosphatidylserine and the effect of Triton X-100 on activity.

The membrane-bound enzyme phosphatidylserine decarboxylase (Tetrahymena pyriformis) was found to have activity both in a crude, particulate form and when it is in a soluble form in the presence of the nonionic surfactant Triton X-100. This surfactant has routinely been included in the assay of phosphatidylserine decarboxylases from all sources; its effect on the activity of the Tetrahymena enzyme has now been characterized and a detailed consideration of the functioning of this surfactant in the assay of this membrane-bound enzyme is presented. The activity of the enzyme towards natural phosphatidylserine is found to be greater than towards saturated phosphatidylserine, both with and without Triton present; this finding is considered in terms of the effect of the thermotropic phase transition of the saturated material on the physical state of the phospholipid, rather than simply in terms of the specificity of the enzyme for phosphatidylserine containing unsaturated fatty acid groups. At high molar ratios of Triton to phospholipid, the activity of the enzyme is dramatically decreased. The decreased activity of the enzyme toward unsaturated Phosphatidylserine is considered in terms of a surface dilution model and the greatly diminished activity towards the saturated analogue is suggested to be the result of lipid phase separation.     

Analysis of phospholipase C (Bacillus cereus) action toward mixed micelles of phospholipid and surfactant.

The action of phospholipase C (Bacillus cereus) toward mixed micelles of phosphatidylcholine and the nonionic surfactant Triton X-100 is analyzed according to the “surfaceas-cofactor” kinetic scheme recently proposed for characterizing the action of lipolytic enzymes [Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem.250, 9013–9020]. According to this scheme, the enzyme first associates with the surface or mixed micelles, where the dissociation constant is K_s^A. The enzyme, now part of the mixed micelle surface, then binds the substrate phospholipid molecule in its active site and this binding is related to the Michaelis constant, K_m^B. The surface, or mixed micelles in this scheme, behaves kinetically as a cofactor in that, under initial rate conditions, the surface properties of the mixed micelles are virtually unchanged after catalysis. For phospholipase C with egg phosphatidylcholine as substrate, it was found that at pH 6.4 (the pH optimum for the enzyme) and 40 °C, V is about 2 × 10³ μmol min^?1 (mg of protein)^?1. K_s^A is about 2 mm and K_m^B is 1 to 2 × 10^?10 mol cm^?2. The kinetic constants for phospholipase C are compared with those previously reported for phospholipase A₂ and the membrane-bound enzyme phosphatidylserine decarboxylase determined under similar conditions.     

Lowry determination of protein in the presence of Triton X-100.

The Lowry procedure has been modified for use in the presence of Triton X-100 (TX-100) by the addition of 10% sodium dodecyl sulfate. The method is applicable to samples containing 40–120 μg protein.     

Reduction and subsequent oxidation of a cytochrome b of human neutrophils after stimulation with phorbol myristate acetate.

The level of nerve growth factor-like immunoreactivity in the lower limb muscles, the site where the dystrophic mice are effected, of both normal and dystrophic mice was studied by solid-phase radioimmunoassay. Nerve growth factor-like immunoreactivity levels of the homozygous dystrophic mice were about two times lower than those of the heterozygous dystrophic mice at 10–11 weeks and 7–8 weeks of age. The levels in 4–5 week old homozygous mice were too low for detection and remarkable differences between the homozygous and the heterozygous mice were observed at this age. These differences in the level of nerve growth factor-like immunoreactivity suggest that the factor may have some relation to the disease.     

Isoenzymes of an acyltransferase from rabbit mammary gland: Solubilization of the micelle-specific species with Triton X-100

The micelle-specific palmityl-CoA: monopalmityl-sn-glycerol 3-phosphate palmityltransferase isoenzyme from lactating rabbit mammary gland microsomes is selectively solubilized in Triton X-100 but not Tween 80. Both detergents inactivate the monomer-specific isoenzyme. The possibility that, $\text{in}$$\text{vivo}$, physiological surfactants regulate the relative activities of these two isoenzymes is discussed.     

Microviscosity of the lipid domains of normal and hypercholesterolemic very low density lipoprotein.

Very low density lipoprotein (VLDL) has been isolated from normal (n) and dietary-induced hypercholesterolemic (hc) rabbits. Incorporation of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene into the lipid domains of both n VLDL and hc VLDL allowed assessment of the fluidity characteristics of these particles, utilizing fluorescence polarization techniques. Over the temperature range of 5° – 45°, the lipid region of n VLDL consists of an invariant phase, characterized by a microviscosity, η, at 30° of 0.6 ± 0.2 poise and a fusion activation energy, ΔE, of 7.6 ± 1.5 kcal/mole. The lipid region of hc VLDL, over the same temperature range, also is invariant and is characterized by a value of η at 30° of 4.6 ± 0.3 poise, and a ΔE of 7.8 ± 1.5 kcal/mole. Thus, large differences in the fluidit of the lipid in n VLDL and hc VLDL are evident, most probably due to the greatly increased content of cholesterol esters in hc VLDL, compared to n VLDL.     

Formation and characterization of mixed micelles of the nonionic surfactant Triton X-100 with egg, dipalmitoyl, and dimyristoyl phosphatidylcholines

Mixed micelles of the nonionic surfactant Triton X-100 and egg phosphatidylcholine were isolated by column chromatography on 6% agarose and by centrifugation at 35,000g. It was found that egg phosphatidylcholine bilayers are able to incorporate Triton X-100 at molar ratios of Triton to phospholipid below about 1:1, whereas above a molar ratio of about 2:1 Triton/phospholipid all of the phospholipid is converted into mixed micelles. Mixed micelles at a molar ratio of about 10:1 Triton/phospholipid were found to be in the same size range as pure micelles of Triton X-100. The formation of mixed micelles with dipalmitoyl phosphatidylcholine at room temperature, when the phospholipid is below its thermotropic phase transition, is shown to require relatively high concentrations of Triton X-100. The point at which dimyristoyl phosphatidylcholine bilayers are converted to mixed micelles was found to be less clear cut than with egg phosphatidylcholine, but above a molar ratio of about 2:1 Triton/phospholipid, all of this phospholipid is also in mixed micelles. The relevance of these results to the solubilization of membrane-bound proteins with Triton X-100 and the action of phospholipase A₂, which hydrolyzes phosphatidylcholine when it is in mixed micelles with Triton X-100, is discussed.     

A thermodynamic description of the self-association of flavin mononucleotide.

Systematic heat of dilution studies of the self-association of flavin mononucleotide (FMN) have been conducted as a function of ionic strength (0.05 – 2.0 m) and pH (5–9) in aqueous solution. The data are adequately described by the expression $\text{Q}{}_{\mathrm{T}}\text{= ΔH ? (}\text{ΔH}\text{K}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{Q}{}_{\mathrm{T}}\text{c}{}_{\mathrm{T}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for an isodesmic self-association. Q_T is the molar heat of dilution, ΔH and K are the derived enthalpy and equilibrium constants for the process FMN + (FMN)_i?1 ? (FMN)_i, and c_T is the concentration of FMN expressed in monomer units. Typical values derived for the various thermodynamic parameters at 25 °C are ΔG = ?3.56 kcal mol^?1, ΔH = ?3.72 kcal mol^?1, and ΔS = ?0.54 cal (mol · deg)^?1. These data, plus nuclear magnetic resonance evidence (Yagi, K., Ohishi, N., Takai, A., Kawano, K., and Kyogoku, Y., 1976, Biochemistry15, 2877–2880) argue in favor of an open-ended association of flavin molecules. The signs of the various thermodynamic parameters suggest that both hydrophobic and surface energy forces contribute significantly to the association, while the lack of any significant ionic strength dependence indicates the lack of any ionic centers in the association.     

Motility of the N-terminal tail of phosphorylase b as revealed by crosslinking.

There are lysyl-ε-NH₂ groups within about 3.5 Å distance across the intersubunit contact area of rabbit muscle phosphorylase $\text{b}$, as shown by cross-linking with malonic diimidate. These include the lysines of N-terminal region as revealed by limited tryptic digestion, but the contribution of the tail lysines to overall formation of covalent dimers is small. The fine structure of dimer band on dodecylsulfate-gelelectrophoretograms of crosslinked phosphorylases suggests that the tail retains its freedom in the phosphorylase $\text{b}$-AMP complex. Amidination induces the dissociation of phosphorylase $\text{b}$ dimer, which is slow relative to crosslinking.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

Molecular organization in bacterial cell membranes: IV. Isolation by preparative electrophoresis in sodium dodecylsulphate and properties of the two major polypeptide groups of a “soluble” fraction from Streptomyces albus membranes

Two polypeptide fractions have been purified from a “soluble” fraction of n-butanol-extracted Streptomyces albus membranes by preparative electrophoresis in sodium dodecylsulphate. They accounted for approx. 80% of the protein of the fraction (i.e. 20% of the total membrane protein). The ultraviolet spectrum of Group 1 (relative mobility 1.0) revealed the presence of nucleotide material, while that of Group 3 (relative mobility 0.65±0.05) showed the presence of a possibly aggregated protein-like material. About 100 and 30% of the protein contents (Lowry method) of Groups 3 and 1, respectively, were recovered as amino acid residues. These results confirm the protein nature of both fractions and suggest an overestimation of the protein value in Group 1. Both polypeptide groups can be classified as “extrinsic” membrane proteins on the basis of their similar amino acid composition (Vanderkooi, G. and Capaldi, R. A. (1972) Ann. N.Y. Acad. Sci. 195, 135–138). Three N-terminal amino acids were found in each fraction: one common (alanine), methionine, leucine or isoleucine (Group 3) and glutamic acid, lysine (Group 1). The sedimentation coefficients calculated were 2.46 S for Group 3 and 1.54 S for Group 1. Analysis of the isolated groups by gel electrophoresis under non-dissociating conditions or with Triton X-100, gave aggregate-like patterns.Sodium dodecylsulphate electrophoresis revealed an anomalous staining behaviour of Group 3 depending upon the dissociating conditions. The whole “soluble” fraction bound 0.40 mg dodecylsulphate /mg protein (0.55 mg detergent/mg protein corrected for overestimation). After dialysis, the fraction retained 10% of the bound dodecylsulphate. Circular dichroism of the isolated groups after exhaustive dialysis showed similar spectra, although of lower dichroism, to those obtained by other authors on soluble enzymes treated with sodium dodecylsulphate. Strong acid conditions were required to change the CD spectra of the polypeptides.     

Vacuum ultraviolet circular dichroism spectrum of beta-turn in solution.

The vacuum ultraviolet circular dichroism spectrum of an isolated 4 → 1 hydrogen bonded β-turn is reported. The observed spectrum of N-acetyl-Pro-Gly-Leu-OH at ? 40°C in trifluoroethanol is in good agreement with the theoretically calculated CD spectrum of the β-turn conformation. This spectrum, particularly the presence of a strong negative band around 180 nm and a large ratio $\text{[θ]}{}_{201}\text{[θ]}{}_{225}$, can be taken as a characteristic feature of the isolated β-turn conformation. These CD spectral features can thus be used to distinguish the β-turn conformation from the β-structure in solution.     

The effect of in vivo treatment with EHDP and/or 1,25-DHCC on calcium uptake and release in isolated kidney mitochondria

Previous work on calcium transport (uptake and release) in isolated mitochondria, in vitro, has shown that addition of EHDP to the medium does not influence calcium uptake, but does delay calcium release. In vivo treatment of normal chicks with high doses of EHDP (10 mg P/kg body weight/day) has now also been found not to affect the in vitro calcium uptake in isolated chick kidney mitochondria, but to delay the subsequent release as compared with controls. The effect is not due to the decrease in 1,25-DHCC, since chronic administration of this metabolite did not correct the delay. In fact 1,25-DHCC in itself had a delaying effect on accumulated calcium release.     

Fluorescence polarization studies of human and rhesus A-I apolipoproteins and their complexes with phosphatidylcholine.

Human and rhesus A-I apolipoproteins, covalently labelled with dansyl chloride, were used in fluorescence polarization studies of: 1) the monomeric structure of the free proteins in solution; 2) the interaction of the apolipoproteins with sonicated egg phosphatidylcholine; and 3) the size of the saturated complexes of protein with phospholipid. The results indicate that both monomeric apolipoproteins have relatively rigid, yet asymmetrical structures, with Stokes radii of 24.2 ± 0.5 Å, in neutral aqueous solutions. Axial ratios are of the order of $\text{6}\text{1}$ or $\text{4}\text{1}$ for hydrated, prolate or oblate ellipsoids, respectively. A molar excess of about 200 phosphatidylcholine molecules are required to saturate each apolipoprotein. At saturation, the complexes with both proteins have Stokes radii of 40.6 ± 1.7 Å. Since the radius of phosphatidylcholine vesicles is around 125 Å, we conclude that the complexes are relatively small structures derived from disruption of the lipid vesicles, rather than from adsorption of the proteins on intact vesicles.