Interaction of Triton X-100 with the pigment-protein complexes of photosynthetic membranes.

The interaction of the non-ionic detergent Triton X-100 with photosynthetic membrane components of Pisum sativum (pea) is described. The detergent affected both the wavelength and the intensity of the 77K fluorescence-emission peaks of both Photosystem I and Photosystem II preparations, in addition to the effects on whole thylakoids recently described by Murphy & Woodrow [(1984) Biochem. J. 224, 989-993]. Below its critical micellar concentration, Triton X-100 had no effect on 77K fluorescence emissions even after prolonged incubations of up to 30 min. Above the critical micellar concentration of about 0.16 mg X ml-1, Triton X-100 caused a dramatic increase in the intensity of the 680 nm emission. The intensity of the 680 nm fluorescence emission continued to increase as more Triton X-100 was added, until limiting concentrations of detergent were reached. These limiting concentrations were proportional to the amount of membrane present and generally occurred at Triton X-100/chlorophyll (w/w) ratios of 100-200:1. In all cases the detergent effect was seen within 10 min, and is often considerably faster, with longer detergent treatments causing no further effects. The data are discussed in terms of a three-stage mechanism for detergent solubilization of membrane components.     

Membrane-surfactant interactions The effect of triton X-100 on sarcoplasmic reticulum vesicles

The effect of Triton X-100 on purified sarcoplasmic reticulum vesicles has been studied by means of chemical, ultrastructural and enzymic techniques. At low detergent/membrane ratios (about 1 Triton X-100 per 60 phospholipid molecules) the only effect observed is an increase in vesicle permeability. Higher surfactant concentrations, up to a 1:1 detergent/phospholipid ratio, produce a large enhancement of ATPase activity. Membrane solubilization occurs as a critical phenomenon when the surfactant/phospholipid molar ratio reaches a value around 1.5:1, corresponding to 2 μmol Triton X-100/mg protein. At this point, the suspension turbidity drops, virtually all the protein and phospholipid is solubilized and every organized structure disappears. Simultaneously, a dramatic increase in the specific activity of the solubilized ATPase is observed. The sudden solubilization of almost all the bilayer components at a given detergent concentration is attributed to the relative simplicity of this membrane system. Solubilization takes place at the same surfactant/membrane ratio, at least between 0.5 and 4 mg membrane protein/ml. The non-solubilized residue seems to consist mainly of delipidized aggregated forms of ATPase.     

Membrane-surfactant interactions. The effect of Triton X-100 on sarcoplasmic reticulum vesicles

The effect of Triton X-100 on purified sarcoplasmic reticulum vesicles has been studied by means of chemical, ultrastructural and enzymic techniques. At low detergent/membrane ratios (about 1 Triton X-100 per 60 phospholipid molecules) the only effect observed is an increase in vesicle permeability. Higher surfactant concentrations, up to a 1:1 detergent/phospholipid ratio, produce a large enhancement of ATPase activity. Membrane solubilization occurs as a critical phenomenon when the surfactant/phospholipid molar ratio reaches a value around 1.5:1, corresponding to 2 mumol Triton X-100/mg protein. At this point, the suspension turbidity drops, virtually all the protein and phospholipid is solubilized and every organized structure disappears. Simultaneously, a dramatic increase in the specific activity of the solubilized ATPase is observed. The sudden solubilization of almost all the bilayer components at a given detergent concentration is attributed to the relative simplicity of this membrane system. Solubilization takes place at the same surfactant/membrane ratio, at least between 0.5 and 4 mg membrane protein/ml. The non-solubilized residue seems to consist mainly of delipidized aggregated forms of ATPase.     

Activation of dynein 1 adenosine triphosphatase by organic solvents and by Triton X-100

The presence of low concentrations of methanol or isopropyl alcohol (2-5%, v/v) in the assay medium stabilizes the latency of dynein 1 from sea urchin sperm flagella, with about a 50% decrease in ATPase level compared to that in the absence of solvent. Somewhat higher concentrations (10-20%, v/v) of these solvents in the assay give a 5-10-fold activation of ATPase activity. Dioxane, formamide, and dimethylformamide, on the other hand, always activate the ATPase activity, with a 5-10-fold increase observed at about 15% (v/v). The activation of latent ATPase activity by solvents is reversible for short exposures, especially in the presence of ATP and at low temperature, but the activation becomes irreversible upon more prolonged exposure. The rate constant for irreversible activation by 16% methanol at 21 degrees C is 0.08 min-1, compared to rates of 0.44 and 0.02 min-1 for activation by 0.05% Triton X-100 at 21 and 0 degree C, respectively. The slowness of this reversible activation induced by methanol and by Triton X-100 suggests that it is the result of large-scale conformational changes in the structure of the dynein. However, the activation by methanol occurs without the dissociation of the alpha and beta subunits of dynein that is observed with Triton X-100. The presence of 1 mM MgATP, or of 100 microM MgATP and 10 microM vanadate substantially protects latent dynein from activation by 0.05% Triton X-100.     

Interactions between sarcoplasmic reticulum calcium adenosintriphosphatase and nonionic detergents

The interaction of Triton X-100 and other nonionic detergents with a delipidated preparation of the Ca2+ ATPase from sarcoplasmic reticulum has been studied. Binding of radiolabeled Triton X-100 was determined by column chromatography at 6 degrees C, and two classes of binding sites were observed. Below the critical micelle concentration (cmc), binding of Triton occurred at 35-40 equivalent sites on the delipidated ATPase with a binding constant of 2.7 X 10(4) M-1. Near the cmc cooperative binding of an additional 70 molecules of the detergent was observed. The binding of monomeric Triton X-100 below the cmc was associated with a parallel activation of over half of the ATPase activity, and the remainder of the activity was recovered after the detergent concentration was increased to the cmc. The ability to reactivate ATPase activity was more dependent on the polar poly(oxyethylene) portion of nonionic detergents than on the hydrocarbon portion. Generalizing for all amphiphiles, these results suggest that there are discrete binding sites on the Ca2+ ATPase for phospholipid molecules in the native membrane and that the polar head groups of phospholipids interact more strongly with the protein than the hydrophobic acyl chains. Perturbations in micelle structure were observed for several nonionic detergents by measurement of cis-parinaric acid fluorescence and differential scanning calorimetry, and discontinuities in Arrhenius plots occurred at the transition temperature of the detergent used for reactivation of ATPase activity. It is concluded that both the physiol state of teh micelle and the intrinsic behavior of the ATPase polypeptide affect the temperature dependence of ATPase activity.     

Purification and preliminary characterization of mitochondrial complex I (NADH: ubiquinone reductase) from broad bean (Vicia faba L.).

NADH:ubiquinone reductase (EC 1.6.19.3), or complex I, was isolated from broad bean (Vicia faba L.) mitochondria. Osmotic shock and sequential treatment with 0.2% (v/v) Triton X-100 and 0.5% (w/v) [3-cholamidopropyl)dimethylammonio]-1-propanesulfate (CHAPS) removed all other NADH dehydrogenase activities. Complex I was solubilized in the presence of 4% Triton X-100 and then purified by sucrose-gradient centrifugation in the presence of the same detergent. The second purification step was hydroxylapatite chromatography. Substitution of CHAPS for Triton X-100 helped remove contaminants such as ATPase. The high molecular mass complex is composed of at least 26 subunits with molecular masses ranging from 6000 to 75,000 kD. The purified complex I reduced ferricyanide and ubiquinone analogs but not cytochrome c. NADPH could not substitute for NADH as an electron donor. The KM for NADH was 20 microM at the optimum pH of 8.0. The NH2-terminal sequence of several subunits was determined, revealing the ambiguous nature of the 42-kD subunit.     

The Two Km's for ATP of Corn-Root H+-ATPase and the Use of Glucose-6-Phosphate and Hexokinase as an ATP-Regenerating System

Plasma membrane vesicles derived from corn (Zea mays L.) roots retain a membrane-bound H+-ATPase that is able to form a H+ gradient across the vesicle membranes. The activity of this ATPase is enhanced 2- to 3-fold when Triton X-100 or lysophosphatidylcholine is added to the medium at a protein:detergent ratio of 2:1 (w/w). In the absence of detergent, the ATPase exhibits only one Km for ATP (0.1-0.2 mM), which is the same as for the pumping of H+. After the addition of either Triton X-100 or lysophosphatidylcholine, two Km's for ATP are detected, one in the range of 1 to 3 [mu]M and a second in the range of 0.1 to 0.2 mM. The Vmax of the second Km for ATP increases as the temperature of the assay medium is raised from 15[deg]C to 38[deg]C. The Arrhenius plot reveals a single break at 30[deg]C, both in the absence and in the presence of detergents. In the presence of Triton X-100 the H+-ATPase catalyzes the cleavage of glucose-6-phosphate when both hexokinase and ADP are included in the assay medium. There is no measurable cleavage when the apparent affinity for ATP of the H+-ATPase is not enhanced by Triton X-100 or when 1 mM glucose is included in the assay medium. These data indicate that when the high-affinity Km for ATP is unmasked with the use of detergent, the ATPase can use glucose-6-phosphate and hexokinase as an ATP-regenerating system.     

Effect of triton X-100 on compactin production from<Emphasis Type="Italic">Penicillium citrinum</Emphasis>

Glucose alone was found to be the most effective carbon source for producing compactin. An initial glucose concentration of 40 g/L gave the highest compactin concentration of 250 mg/L. Among the various nitrogen sources, when 5 g/L of pharmamedia and soybean meal as the sole nitrogen source were used, respectively, the compactin concentration was higher than 250 mg/L. Especially, in the case of the mixture of 6 g/L of pharmamedia and 8 g/L of soybean meal, the compactin concentration was 400 mg/L. To select the best surfactant for effective compactin production, various surfactants were investigated. When Triton X-100 was used, the maximum compactin concentration was 445 mg/L. With the initial concentration ranging from 1.5 to 2.0 g/L, the compactin concentration was the highest at 465–450 g/L. The cell concentration was similar to that of the control without the addition of Triton X-100. On the other hand, when the above 4.0 g/L of Triton X-100 were used, the cell concentration decreased. Using the based results the continuous fed-batch cultures by adding the Triton X-100 were carried out for 10 days in an air-lift bioreactor. When 1.5 g/L of Triton X-100 was added to the culture broth at 0, 4, and 8 days of culture, respectively, the compactin production was increased with the increase of culture time. The maximum compactin concentration after 10 days of culture was 1,200 mg/L, which was about 2.0-fold higher than that of the control without the addition of Triton X-100.     

Role of phospholipid and protein-protein associations in activation and stabilization of soluble Ca2+-ATPase of sarcoplasmic reticulum

The effect of increasing concentrations of the nonionic detergent Triton X-100 on catalytic activity, stability, phospholipid content, and aggregational state of solubilized Ca2+ ion activated adenosinetriphosphatase (Ca2+-ATPase) of sarcoplasmic reticulum has been investigated. Increasing concentrations of Triton X-100 in the range 0.2-0.6% (w/v) inhibited ATP hydrolysis and p-nitrophenyl phosphate hydrolysis in parallel to the extent of 50% and 95%, respectively. Inactivation of p-nitrophenyl phosphate hydrolysis by preincubation in excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 25 degrees C was monophasic and first order at all concentrations of Triton X-100. The rate constant for inactivation increased sharply in the range 0.1-0.6% Triton X-100. At higher concentrations, the increase was less marked. Protein-protein associations of the solubilized ATPase were assessed by glutaraldehyde cross-linking and by ultracentrifugation in sucrose gradients. Both methods indicated a decrease in these associations in the 0.1-0.5% range. Cross-linking studies established that above 0.5% Triton X-100 the enzyme is greater than 90% monomeric. The amount of phospholipid associated with the ATPase, recovered from sucrose gradients, decreased from about 50 mol of phospholipid/mol of ATPase at 0.1% Triton X-100 to about 3 mol of phospholipid/mol of ATPase at 0.5% and higher concentrations. Monomeric ATPase and aggregated ATPase isolated from equilibrium mixtures of these components had similar phospholipid/protein ratios. The results indicated that with increasing Triton X-100 concentrations, inhibition of catalysis, destabilization, loss of protein-protein associations, and loss of phospholipid occur concurrently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of human erythrocyte cytoskeletal ATPase

Human erythrocyte cytoskeletal ATPase was extracted with 0.2 mM ATP (pH 8.0) from Triton X-100 treated ghosts. The ATPase fraction contained mainly spectrin, actin, and band 4.1. When the ATPase fraction was applied to a Sepharose 4B column, 90% of the ATPase activity was recovered in a spectrin, actin, and band 4.1 complex fraction and none was detected in the spectrin fraction. A specific activity of the complex ATPase was 60-120 nmol/(mg protein X h). No ATPase activity was detected in the presence of EDTA. The presence of magnesium in the incubation medium was essential for the ATPase activity. The activity was activated by KCl and was almost completely inhibited by 10(-5) M free calcium in the presence of 0.2 mM MgCl2. The Ki for Ca2+ was 7 X 10(-7) M. Phalloidin and DNase 1, which affect actin, inhibited this K,Mg-ATPase activity by 95%, but cytochalasin B did not inhibit it. N-Ethylmaleimide activated the ATPase 1.6-fold. The order of affinity for nucleotides was ATP greater than ITP greater than CTP, ADP, AMP-PNP, GTP. A specific ATPase activity of purified actin was 50 nmol/(mg X h). These results suggest that the cytoskeletal ATPase is actin ATPase and the actin ATPase is activated by spectrin and band 4.1.     

Adenosine triphosphatase of rat liver mitochondria: detergent solubilization of an oligomycin- and dicyclohexylcarbodiimide-sensitive form of the enzyme.

The hydrolytic activity of the ATPase bound to purified inner membrane vesicles of rat liver mitochondria can be increased threefold by washing extensively with a high ionic strength phosphate buffer. The specific ATPase activities of such phosphate-washed membranes are the highest reported to date for a mitochondrial membrane preparation (21-24 mumol of ATP hydrolyzed min-1 mg-1 in bicarbonate buffer at 37 degrees C). Deoxycholate (0.1 mg/mg of protein) extracts from these membranes a soluble, cold-stable ATPase complex which exhibits a specific activity under optimal assay conditions of 12 mumol of ATP hydrolyzed min-1 mg-1. This complex is not sedimented by centrifugation at 201000 g for 90 min, and readily passes through a 250-A Millipore filter. The ATPase activity of the soluble complex is inhibited 95% by 2.4 muM oligomycin. In addition, inhibitions of 60% or better are obtained in the presence of 1-8 muM dicyclohexylcarbodiimide, p-chloromercuribenzoate, venturicidin, and aurovertin. While a similar complex may be extracted with Triton X-100 this preparation is always lower in both specific activity and in inhibitor sensitivities than the complex extracted with deoxycholate. Detergents of the Tween and Brij series and other detergents of the Triton series are also much less effective than deoxycholate in solubilizing the oligomycin-sensitive. ATPase complex of rat liver. It is concluded that deoxycholate is superior to other detergents as an extractant of the oligomycin-sensitive ATPase complex of rat liver mitochondria, and that the complex extracted with deoxycholate possesses a closer similarity to the membrane-associated ATPase than does the complex extracted with Triton X-100. These studies document the first report of a detergent-solubilized, oligomycin-sensitive ATPase preparation from rat liver mitochondria.     

The inhibitory effects of polyoxyethylene detergents on human erythrocyte acetylcholinesterase and Ca2+ + Mg2+ ATPase

The activities of acetylcholinesterase and Ca2+ + Mg2+ ATPase were measured following treatment of human erythrocyte membranes with nonsolubilizing and solubilizing concentrations of Triton X-100. A concentration of 0.1% (v/v) Triton X-100 caused a significant inhibition of both enzymes. The inhibition appears to be caused by perturbations in the membrane induced by Triton X-100 incorporation. No acetylcholinesterase activity and little Ca2+ + Mg2+ ATPase activity were detected in the supernatant at 0.05% Triton X-100 although this same detergent concentration induced changes in the turbidity of the membrane suspension. Also, no inhibition of soluble acetylcholinesterase was observed over the entire detergent concentration range. The inhibition of these enzymes at 0.1% Triton X-100 was present over an eightfold range of membrane protein in the assay indicating an independence of the protein/detergent ratio. The losses in activities of these two enzymes could be prevented by either including phosphatidylserine in the Triton X-100 suspension or using Brij 96 which has the same polyoxyethylene polar head group but an oleyl hydrophobic tail instead of the p-tert-octylphenol group of Triton X-100. The results are discussed in regard to the differential recovery of enzyme activities over the entire detergent concentration range.     

Purification and chemical modification of a phosphatidylinositol kinase from sheep brain.

A membrane-bound phosphatidylinositol (PtdIns) kinase has been purified approximately 9500-fold to apparent homogeneity from sheep brains. The purification procedure involves: solubilisation of the membrane fraction with Triton X-100, ammonium sulphate fractionation and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 1149 nmol.min-1.mg-1. The molecular mass of the enzyme was estimated to be 55 kDa by SDS/PAGE and 150 +/- 10 kDa by HPLC gel filtration in the presence of Triton X-100. Kinetic measurements have shown that the apparent Km value of PtdIns kinase for the utilisation of PtdIns is 22 microM and for ATP 67 microM. Mg2+ was the most effective divalent cation activator of PtdIns kinase, with maximal enzymatic activity reached at a concentration of 10 mM Mg2+. In addition to adenosine and ADP, the 2'(3')-O-(2,4,6-trinitrophenyl) derivative of ATP was found to be a strong competitive inhibitor of the enzyme, with a Ki of 32 microM. Enzymatic activity was found to be stimulated by Triton X-100 but inhibited by deoxycholate.     

Triton X—100加KI对燕麦根细胞质膜ATP酶的溶解

在酸性条件下,1％ Triton X—100加 0.25mol/L KI能有效地溶解燕麦根细胞质膜ATP酶。溶解的ATP酶水解ATP的最适pH在6.5左右,酶活性受到Na_3VO_4和DES的强烈抑制,而不受Na_2MoO_4和NaN_3的抑制。溶解的酶液经透析后,K~ —ATP酶活性占Mg~(2 ),KCl—ATP酶活性的85％。     

Purification of a putative K+-ATPase from Streptococcus faecalis

We have purified a novel membrane ATPase from Streptococcus faecalis by the following procedure: extraction of membranes with Triton X-100 followed by fractionation of the extract by successive DEAE-cellulose chromatography, hydroxylapatite chromatography and Cm-Sepharose chromatography. The overall yield was 5%. The purified ATPase appears to consist of a single polypeptide component of Mr = 78,000. The Triton-solubilized purified enzyme has a specific activity of approximately 50 mumol of ATP hydrolyzed per min per mg, is dependent on phospholipids for activity, and is strongly inhibited by vanadate (I50 = 3 microM). Maximal ATPase activity is displayed at pH 7.3. Mg2+-ATP, for which the enzyme has a Km of 60 microM, is the best substrate. The ATPase forms an acylphosphate intermediate that can also be detected in native membranes as the major acylphosphate component. The purified ATPase, when reconstituted into soybean phospholipid vesicles, exhibits coupling, e.g. the ATPase activity can be stimulated at least 8-fold by valinomycin in the presence of potassium. Based on these observations we conclude that the enzyme we have purified is an ion-motive ATPase, most likely a K+-ATPase.     

Glycosyl-phosphatidylinositol anchored acetylcholinesterase as substrate for phosphatidylinositol-specific phospholipase C from Bacillus cereus.

We investigated the enzymatic properties of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus towards glycosyl-phosphatidylinositol anchored acetylcholinesterase (AChE) from bovine erythrocytes and Torpedo electric organ as substrate. The conversion of membrane from AChE to soluble AChE by PI-PLC depended on the presence of a detergent and of phosphatidylcholine. In presence of mixed micelles containing Triton X-100 (0.05%) and phosphatidylcholine (0.5 mg/ml) the rate of AChE conversion was about 3 times higher than in presence of Triton X-100 alone. Furthermore, inhibition of PI-PLC occurring at Triton X-100 concentrations higher than 0.01% could be prevented by addition of phosphatidylcholine. Ca2+, Mg2+ and sodium chloride had no effect on PI-PLC activity in presence of phosphatidylcholine and Triton X-100, whereas in presence of Triton X-100 alone sodium chloride largely increased the rate of AChE conversion. Determination of kinetic parameters with three different substrates gave Km-values of 7 microM, 17 microM and 2 mM and Vmax-values of 0.095 microM.min-1, 0.325 microM.min-1 and 56 microM.min-1 for Torpedo AChE, bovine erythrocyte AChE and phosphatidylinositol, respectively. The low Km-values for both forms of AChE indicated that PI-PLC not only recognized the phosphatidylinositol moiety of the anchor but also other components thereof.     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

Characterization of the Na+-stimulated ATPase of Propionigenium modestum as an enzyme of the F1F0 type

The ATP-hydrolyzing activity of Propionigenium modestum was extracted from the membranes with Triton X-100 or by incubation with EDTA at low ionic strength. The ATPase in the Triton extract was highly sensitive to dicyclohexylcarbodiimide but not to vanadate. These properties are characteristic for enzymes of the F1 F0 type. The ATPase was specifically activated by Na+ ions yielding a 15-fold increase in catalytic activity at 5 mM Na+ concentration. The additional presence of 1% Triton X-100 caused a further 1.5-fold activation. In the absence of Na+ Triton stimulated the ATPase about 13-fold. The Triton-stimulated ATPase was further activated about 1.5-2-fold by Na+ addition. The ATPase extracted by the low-ionic-strength treatment was purified to homogeneity by fractionation with poly(ethylene glycol) and gel chromatography. The enzyme had the characteristic F1-ATPase subunit structure with Mr values of 58,000 (alpha), 56,000 (beta), 37,600 (gamma), 22,700 (delta), and 14,000 (epsilon). The F1-ATPase was not stimulated by Na+ ions. The membrane-bound ATPase was reconstituted from the purified F1 part and F1-depleted membranes, thus further indicating an F1 F0 structure for the ATPase of P. modestum. Upon reconstitution the ATPase recovered its stimulation by Na+ ions, suggesting that the binding site for Na+ is localized on the membrane-bound F0 part of the enzyme complex.     

The reaction of horseradish peroxidase with hydroperoxides derived from Triton X-100

All of the commercially available Triton X-100 examined gave Compound I upon reaction with horseradish peroxidase, followed by its gradual transition into Compound II. Titration of horseradish peroxidase with Triton X-100 to form Compound I indicated that 1% (v/v) aqueous solutions of the detergent contained 0.4 to 3.2 microM equivalent peroxide but iodometric titration revealed 1.1 to 5.0 microM peroxide, suggesting the occurrence of different types of peroxides, reactive and unreactive with the peroxidase. The rate constant for Compound I formation was 1.5 X 10(7) M-1 S-1 at pH 7.4 at 25 degrees C, and for conversion into Compound II apparent first-order rate constants were 5.2 X 10(-3) to 1.7 X 10(-2) S-1. These results indicate that the Triton peroxides are as highly reactive as hydrogen peroxide. The amount of Triton peroxides increased as aqueous solutions of the detergent were allowed to stand, but the peroxides were destroyed by treatment with sodium borohydride. Although freshly prepared aqueous solutions of sodium cholate, sodium dodecyl sulfate, Tween 20 (polyoxyethylene sorbitan monolaurate), and Emasol 1130 (an equivalent of Tween 20) did not contain any detectable amount of peroxide, aged solutions of sodium dodecyl sulfate and Emasol 1130 contained peroxides. These observations suggest the need for appropriate precautions when biologically active substances vulnerable to attack by peroxides are incubated with Triton X-100 either for their solubilization from biomembranes or for other processing.     

Inhibitory effects of detergents on rat CYP1A1-dependent monooxygenase: comparison of mixed and fused systems consisting of rat CYP 1A1 and yeast NADPH-P450 reductase

Inhibitory effects of detergents Triton X-100 and Chaps on 7-ethoxycoumarin O-deethylation activity were examined in the recombinant microsomes containing both rat CYP1A1 and yeast NADPH-P450 reductase (the mixed system) and their fused enzyme (the fused system). Triton X-100 showed competitive inhibition in both mixed and fused systems with K(i) values of 24.6 and 21.5 microM, respectively. These results strongly suggest that Triton X-100 binds to the substrate-binding pocket of CYP1A1. These K(i) values are far below the critical micelle concentration of Triton X-100 (240 microM). Western blot analysis revealed no disruption of the microsomal membrane by Triton X-100 in the presence of 0-77 microM Triton X-100. On the other hand, Chaps gave distinct inhibitory effects to the mixed and fused systems. In the fused system, a mixed-type inhibition was observed with K(i) and K(i)' values of 1.2 and 5.4 mM of Chaps, respectively. However, in the mixed system, multiple inhibition modes by Chaps were observed. Western blot analysis revealed that the solubilized fused enzyme by Chaps preserved the activity whereas the solubilized CYP1A1 and NADPH-P450 reductase reductase showed no activity in the mixed system. Thus, the comparison of the mixed and fused systems appears quite useful to elucidate inhibition mechanism of detergents.