Effect of triton X-100 on the activity and solubilization of rat liver mitochondrial phosphatidylserine decarboxylase

It was shown that, among ionic and nonionic detergents tested, only Triton X-100 was able to stimulate the activity of rat liver phosphatidylserine decarboxylase, whereas other detergents were without effect or were inhibitory. The solubilization procedure of phosphatidylserine decarboxylase from mitochondrial membranes with Triton X-100 was elaborated. The dependence of the solubilized decarboxylase on the Triton X-100 to phosphatidylserine ratio and the inhibitory effect of Triton X-100 at its molar ratio to phospholipid higher than 5.6 was observed. No divalent cation requirement and no dependence of the ionic strength for the solubilized enzyme were observed. Kinetic parameters were determined.     

Characterization of triton X-100-solubilized prostaglandin E binding protein of rat liver plasma membranes.

Rat liver plasma membranes bind prostaglandins E1 and E2 (PGE) with high affinity and specificity. We have solubilized plasma membranes, prelabeled with radioactive PGE1, in water solutions of Triton X-100. We sedimented this material into sucrose density gradient containing H2O and D2O. From numerical integration of the sedimentation equation, taking explicitly into account the density and viscosity gradients present during the centrifugation, we have determined a value of s20,w = 5.6 to 5.7 X 10(-13) s and a partial specific volume, v = 0.80 to 0.81 cm3/g, for the PGE binding protein-Triton X-100 composed of 60% (w/w) protein and 40% (w/w) detergent. Gel filtration in water solutions of Triton X-100 gives a Stokes radius of 53 A for the complex. These data imply a molecular weight of 105,000 for the detergent-free binding protein and a frictional ratio of 1.3 for the complex. If the detergent is bound to the protein in a monolayer, about 40% of the PGE binding protein's surface would be covered with detergent. The procedures used in the analysis of the sedimentation behavior of the PGE binding protein-detergent complex, when coupled with a gel filtration measurement of the Stokes radius, allow valid determination of the size, shape, and extent of detergent binding of a wide variety of membrane proteins, even when they are present as minor components of complex mixtures.     

Lipid exchange between mixed micelles of phospholipid and triton X-100

If phospholipase catalyzed hydrolysis of phospholipid dissolved in a detergent mixed micelle is limited to the phospholipid carried by a single micelle, then hydrolysis ceases upon exhaustion of that pool. However, if the rate of phospholipid exchange between micelles exceeds the catalytic rate then all of the phospholipid is available for hydrolysis. To determine phospholipid availability we studied the exchange of 1,2-dioleoyl-sn-glycero-3-phosphocholine between mixed micelles of phospholipid and non-ionic Triton detergents by both stopped-flow fluorescence-recovery and nuclear magnetic resonance-relaxation techniques. Stopped-flow analysis was performed by combining mixed micelles of Triton and phospholipid with mixed micelles that contained the fluorescent phospholipid 1-palmitoyl-2-(12-[{7-nitro-2-1, 3-benzoxadiazo-4-yl}amino]dodecanoyl)-sn-glycero-3-phosphocholine (P-2-NBD-PC). The concentration dependence of fluorescence recovery suggested a second-order exchange mechanism that was saturable. The true second-order rate constant depends on the specific mechanism for exchange, which was not determined in this study, but the rate constant will be on the order of 106 to 107 M-1s-1. Incorporation of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine into micelles increased the rate of proton relaxation and gave a limiting relaxation time of 1.3 ms. The results demonstrate that phospholipid exchange was rapid and that the phospholipid content of a single micelle did not limit the rate of phospholipid hydrolysis by phospholipases.     

The effect of triton X-100 on bovine brain synaptic membranes

Bovine brain synaptic membranes which were frozen and then extensively washed showed low affinity [³H]muscimol binding. These membranes contained GABA and calmodulin, apparently tightly bound within the membrane fraction. Membranes which were additionally treated with the detergent Triton X-100 showed high affinity [³H]muscimol binding. These membranes did not appear to contain GABA or calmodulin. Transmission electron microscopy studies demonstrated that the washed membrane fraction contained many synaptosomal and vesicular structures. Triton treatment led to the extensive rupture of these structures. These studies explain the well-reported findings of tightly bound GABA and calmodulin in brain membrane fractions, as being due to the entrapment of these compounds inside sealed membrane-bound structures which are still present after a freezethaw and extensive wash treatment, their complete removal requiring Triton-treatment to rupture the vesicles.     

Nitroxide spin labeled analogs of the non-ionic detergent triton X-100

Two nitroxide spin labeled analogs of the non-ionic detergent, Triton X-100, have been synthesized. Electron spin resonance spectra of these compounds in solution, in egg lecithin multilayers, and in aqueous dispersion of dimyristoylphosphatidylcholine vesicles are described.     

Solubilization of human erythrocyte membrane glycoproteins by triton X-100.

1. The enzymic removal of sialic acid residues from the glycoproteins of the human erythrocyte decreases the solubilization of membrane glycoprotein by Triton X-100. 2. The solubilization of asialoglycoprotein by Triton X-100 may be restored by the addition of borate. 3. Use of this non-ionic detergent in the presence of borate, as a general procedure for the mild solubilization of membrane glycoproteins deficient in sialic acid residues, is discussed.     

Cellular lysis of Streptococcus faecalis induced with triton X-100.

Lysis of exponential-phase cultures of Streptococcus faecalis ATCC 9790 was induced by exposure to both anionic (sodium dodecyl sulfate) and nonionic (Triton X-100) surfactants. Lysis in response to sodium dodecyl sulfate was effective only over a limited range of concentrations, whereas Triton X-100-induced lysis occurred over a broad range of surfactant concentrations. The data presented indicate that the bacteriolytic response of growing cells to Triton X-100: (i) was related to the ratio of surfactant to cells and not the surfactant concentration per se; (ii) required the expression of the cellular autolytic enzyme system; and (iii) was most likely due to an effect of the surfactant on components of the autolytic system that are associated with the cytoplasmic membrane. The possibility that Triton X-100 may induce cellular lysis by releasing a lipid inhibitor of the cellular autolytic enzyme is discussed.     

Electrophoresis and electrofocusing of rhodopsin solubilized by triton X-100]

Detergent-rhodopsin micells (component I) were separated from other fast and slow migrating protein components under electrophoresis of triton X-100 solubilized bovine rod outer segments (ROS). Treatment of ROS by alum caused a complete disappearance of non-rhodopsin proteins and the appearance of slow migrating band (component II). Preliminary bleaching of dark extracts did not affect the migration rate of the component I. The addition of urea to solubilizing mixture caused the increase of component I content and the diffusity components I and II bands. The rate of electrophoretic migration and the content of components I and II sharply decreased together with the appearance of fast migrating pink-brown band after the addition of 2-mercaptoethanol. The extracts from alum-treated ROS were separated into 15-20 protein bands under acrylamide gel isoelectric focusing. Such protein heterogeneity probably depended on the ability of triton X-100 to form micells with different isoelectric points during the interaction with ampholines in the electric field. These micells, having different isoelectric points, are shown to contain one and the same protein--opsin.     

A procedure for the estimation of microgram quantities of triton X-100

A spectrophotometric procedure is reported for the assay of microgram amounts of the nonionic detergent Triton X-100. The method is based on the reaction of ammonium cobaltothiocyanate with the poly (ethylene oxide) groups of Triton X-100 to form a blue precipitate. The latter is extracted into ethylene dichloride and assayed spectrophotometrically. Capable of assaying as little as 40 μg of Triton X-100, the procedure is applicable in the presence of proteins provided a small easily determined correction is applied. High ionic strength (up to 2 m NaCl tested) did not interfere with the method.     

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.     

Reactivation of progessive motility in hamster sperm modified by triton X-100

Epididymal hamster sperm, denuded of their plasma membrane with Triton X-100 and washed free of cytosol, were found to become progressively motile in defined media containing ATP¹ and salts. The chemical requirements for motility were investigated and several experimental observations were made that were relevant to the mechanism and control of sperm motility.     

Turbidimetric method for quantitative determination of triton X-100 with silicotungstic acid

The Formation of Triton X-100-silicotungstic acid complex was studied. Quantitative turbidimetric determination of the detergent based on this process was suggested. This method allows to determining the complex formation at any wavelength in the range from 350 (epsilon 350 = 15,600 cm-1 M-1) to 600 nm (epsilon 600 = = 9090 cm-1 M-1). The calibration curve for Triton X-100 recorded at 350 nm is linear in the concentration range of 0 to 30 micrograms/ml. A sigmoid calibration curve was observed at longer wavelengths. A linear fragment of the calibration curve recorded at 600 nm was found at a concentration of Triton X-100 of about 5 micrograms/ml. The complex nature of calibration curves can be explained by heterogeneity of the complex dispersion.     

Cytoskeletal structures inEuglena gracilis after triton X-100 extraction and dry cleaving

Summary A three-dimensional network of structural filaments was visible with common electron microscopes in the cytoplasm ofEuglena gracilis green cells extracted with buffers containing the nonionic detergent Triton X-100. A similar filamentous web was detected at the periphery of critical point dried cells cleaved on grids by means of an adhesive tape. SDS-polyacrylamide gel electrophoresis of the detergent-resistent cytoskeleton showed that actin or actin-like proteins of molecular weight in the range of 43–45 K are not among the components having a structural role inEuglena. The significance of these findings was discussed in relation to the capability of the alga to change the cell shape.The study was supported by grants from Consiglio Nazionale delle Ricerche (CNR) and Ministero della Pubblica Istruzione of Italy.     

Ammonium molybdate-stannous chloride determination of orthophosphate in the presence of triton X-100

A colorimetric method for the determination of orthophosphate in the presence of Triton X-100 and the extent of their mutual interference is presented. Effects of albumin and trichloroacetic acid on the reaction are also examined. The method is based on the very sensitive reaction developed for determination of orthophosphate by complex formation with ammonium molybdate followed by reduction with stannous chloride. The method allows determination of 0.005 μmol of orthophosphate in the presence of up to 0.5% Triton X-100 and as little as 0.3% ($\text{v}\text{v}$) Triton X-100 in the absence of phosphate.