Determination of Triton X-100 binding to membrane proteins by polyacrylamide gel electrophoresis

The molecular weight of proteins in protein-detergent complexes can be determined from ultracentrifugation experiments if the amount of bound detergent is known. A new sensitive method to measure the binding of the nonionic detergent Triton X-100 to proteins has been developed. For the membrane proteins studied, less than 50 mug of protein was required to achieve an accuracy of 10% in the determination of the detergent-protein weight ratio. The proteins were equilibrated with the detergent by electrophoresis into polyacrylamide gels containing radioactively labelled Triton X-100. The gels were then sliced and the amount of bound detergent calculated from the increase in radio-activity in the slices containing the protein zone. The amounts of protein were determined by amino acid analysis of identical protein zones cut from gels running parallel.     

Extraction of Triton X-100 and its determination in virus-inactivated human plasma by the solvent--detergent method

For inactivation of lipid-enveloped viruses during the production of fresh frozen and lyophilized human plasma, the solvent-detergent method was applied. In this process, the solvent tri-n-butyl phosphate is removed by extraction with castor oil. The removal of the non-ionic detergent Triton X-100 is performed by solid-phase extraction using reversed-phase supports. For this purpose, different polymer- and silica-based supports were tested. The highest capacity for Triton X-100 was achieved with C18 silica gels. These supports can bind more than 0.1 ml of Triton X-100 per ml of support. None of the proteins, e.g., clotting factors, bind to the support and therefore they pass through the column and their biological activity is hardly affected. The determination of detergent during the production process was also studied. The application of special columns allowing direct sample injection was introduced. This is a simple method for the rapid in-process determination of Triton X-100 in human plasma by reversed-phase chromatography under isocratic conditions. Using the method developed here, less than 1.0 ppm of Triton X-100 can be detected in less than 12 min without any sample pretreatment.     

Stabilisation and partial purification of Triton X-100 solubilised trihydroxycoprostanoyl-CoA synthetase from rat liver

The surface characteristics of sixteen "monobloc" titanium-6% aluminum-4% vanadium (Ti6A14V) femoral components (two of the 6-Ti-28 type and 14 of the 6-Ti-32 type) retrieved after periods of 78-131 months following loosening of the femoral component, as well as two unimplanted controls, were studied. The femoral heads were examined by a combination of noncontact light profilometry, scanning electron microscopy, and energy-dispersive X-ray analysis. No consistent correlations were found between classical surface roughness parameters (average, root mean square, peak-to-valley roughness, and radius of curvature) and any clinical parameter studied (patient gender, weight, and height; primary diagnosis; implantation time; or calculated force applied on the hip joint). This extensive quantitative topographic analysis suggests that wear mechanisms in vivo are complex and that wear of titanium alloy femoral heads is partly attributed to a combination of an imperfect nature of the surface before implantation, removal of the oxide layer causing abrasion of the alloy, subsequent deformation of the bearing surface including polishing, and, to a very small degree, patient parameters.     

5-Br-PADAP-TritonX-100光度法连续测定铜和锰

研究了非离子型表面活性剂TritonX 1 0 0存在下 ,用 5 Br PADAP光度法联合测定铜和锰的方法。结果表明 :在 pH 9 0的硼砂缓冲介质中 ,5 Br PADAP与铜 (锰 )生成紫红色络合物 ,λmax=575nm ,ε575=1 0 4× 1 0 5。铜量在 0～ 1 4μg/2 5mL(锰量在 0～ 1 0 μg/2 5mL)的范围内符合比尔定律。实测了钢样中铜和锰的含量 ,结果令人满意。     

Trace amounts of Triton X-100 modify the inhibitor sensitivity of the mitochondrial porin

Transport properties of mitochondrial porin were investigated on the basis of changes in the activity of hexokinase utilizing external ATP. Production of glucose 6-phosphate is inhibited by polyanion both in intact brain mitochondria and in contact point vesicles. Hexokinase activity is restored by solubilization of the enzyme by high ionic strength or 0.5-1% Triton X-100. In very low concentrations (0.001-0.005%) Triton does not mobilize hexokinase from its binding sites but it is able to release polyanion-inhibition completely. This finding provides an explanation for the discrepancy observed in the transport properties of porin when studied 'in situ' or in artificial lipid membranes.     

Diradylglycerols alter fatty acid inhibition of monoacylglycerol acyltransferase activity in Triton X-100 mixed micelles

The activity of hepatic monoacylglycerol acyltransferase (MGAT) (EC 2.3.1.22), a developmentally expressed microsomal enzyme, is inhibited by long-chain fatty acids, and stimulated by its product 1, 2-diacyl-sn-glycerol. Because the quantities of fatty acids and diacylglycerols are likely to vary in membranes during different physiological conditions and could thereby alter MGAT activity, we examined their combined effects on MGAT in Triton X-100/phospholipid mixed micelles. MGAT's product, 1,2-diC18:1-sn-glycerol, which is also normally a cooperative activator of the activity, reversed the 50% inhibition caused by 10 mol % oleic acid. The presence of oleic acid also allowed low concentrations (<10 mol %) of 1, 2-diC18:1-sn-glycerol to stimulate MGAT activity without the lag that is observed in the absence of fatty acid. At 12.6 mol %, 1, 2-monoC18:1-sn-glycerol ether, which alone has no effect on MGAT activity, became an activator in the presence of 10 mol % oleic acid. Kinetic studies revealed that in the presence of 15 mol % oleic acid, 1,2-diC18:1-sn-glycerol ether increased the apparent Vmax by 3. 8-fold while minimally altering the apparent Km for palmitoyl-CoA. Other neutral lipids including tri-C18:1-glycerol, ceramide, and cholesterol oleate did not stimulate MGAT in either the presence or the absence of fatty acid. Assay conditions altered MGAT's apparent relative preferences for potential monoradylglycerol substrates. The presence of phospholipids and of MGAT's 1,2-diacyl-sn-glycerol product increased the enzyme's apparent preference for its 2-monoacyl-sn-glycerol substrate by selectively increasing the apparent Vmax 2.7-fold only when 2-monoC18:1-sn-glycerol was the substrate. Thus, in addition to previously reported regulation of MGAT by phospholipids and intracellular lipid second messengers, these studies lend additional support to the hypothesis that changes in other membrane-associated lipids, such as long-chain fatty acids and diradylglycerols, may also profoundly alter the activity of MGAT.     

Triton X-100 as a specific inhibitor of the mammalian NADH-ubiquinone oxidoreductase (Complex I)

Triton X-100 inhibits the NADH oxidase and rotenone-sensitive NADH-Q1 reductase activities of bovine heart submitochondrial particles (SMP) with an apparent Ki of 1x10-5 M (pH 8.0, 25 degrees C). The NADH-hexammineruthenium reductase, succinate oxidase, and the respiratory control ratio with succinate as the substrate in tightly coupled SMP are not affected at the inhibitor concentrations below 0.15 mM. The succinate-supported aerobic reverse electron transfer is less sensitive to the inhibitor (Ki=5x10-5 M) than NADH oxidase. Similar to rotenone, limited concentrations of Triton X-100 increase the steady-state level of NAD+ reduction when the nucleotide is added to tightly coupled SMP oxidizing succinate aerobically. Also similar to rotenone, Triton X-100 partially protects Complex I against the thermally induced deactivation and partially activates the thermally deactivated enzyme. The rate of the NADH oxidase inhibition by rotenone is drastically decreased in the presence of Triton X-100 which indicates a competition between these two inhibitors for a common specific binding site. In contrast to rotenone, the inhibitory effect of Triton X-100 is instantly reversed upon dilution of the reaction mixture. The NADH-Q1 reductase activity of SMP is inhibited non-competitively by added Q1 whereas a simple competition between Q1 and the inhibitor is seen for isolated Complex I. The results obtained show that Triton X-100 is a specific inhibitor of the ubiquinone reduction by Complex I and are in accord with our previous findings which suggest that different reaction pathways operate in the forward and reverse electron transfer at this segment of the mammalian respiratory chain.     

Isoelectric focusing of alkaline phosphatase isoenzymes in polyacrylamide gels. Use of Triton X-100 and improved staining technic

The examination of alkaline phosphatase isoenzymes by means of isoelectric focusing in polyacrylamide gel rods using the apparatus and focusing method of Righetti and Drysdale is discussed. A simultaneous coupling procedure using alpha-naphthyl phosphate and fast blue salt R in 2-amino-2-methyl-1,3-propanediol buffer, pH 9.68, containing MgCl2 and ZnSO4 proved sensitive for developing the enzyme bands. Also discussed are the effects seen with the incorporation of Triton X100 into the gel and sample mixtures. Enzyme, which remained at the top of the gel without using this detergent, entered the gel easily with the addition of Triton X-100 into the application solution. Incorporation of Triton into the gel matrix resulted in some enzyme band patterns that showed distinct differences from gels containing no Triton.     

Opposing roles of the Staphylococcus aureus virulence regulators, Agr and Sar, in Triton X-100- and penicillin-induced autolysis

The regulation of murein hydrolases is a critical aspect of peptidoglycan growth and metabolism. In the present study, we demonstrate that mutations within the Staphylococcus aureus virulence factor regulatory genes, agr and sar, affect autolysis, resulting in decreased and increased autolysis rates, respectively. Zymographic analyses of these mutant strains suggest that agr and sar exert their effects on autolysis, in part, by modulating murein hydrolase expression and/or activity.     

β-环糊精和Triton X-100存在下邻菲啰啉-四碘荧光素分光光度法测定水中微量锌

研究了在β-环糊精(β-CD)和Triton X-100存在下,Zn(Ⅱ)与邻菲啰啉和四碘荧光素的显色反应,建立了光度法测定水中微量锌的新方法.结果表明络合物的最大吸收波长为570nm,表观摩尔吸光系数为1.75×105L·mol-1·cm-1,25 mL溶液中锌质量在0～20 μg范围内服从比尔定律.方法已用于管网水中微量锌的测定.     

Simultaneous detection of X- and Y-bearing human sperm by double fluorescence in situ hybridization

Double fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in decondensed human sperm nuclei. Biotinylated X chromosome specific (TRX) and digoxigenin-labeled Y chromosome specific (HRY) probes were simultaneously hybridized to sperm preparations from 12 normal healthy donors. After the hybridization, the probes were detected immunocytochemically, using two different and independent affinity systems. Ninety-six percent of the 12,636 sperm showed fluorescent labeling, of which 47.4% were haploid X and 46.8% were haploid Y. A frequency of 0.46% of XX-bearing sperm (0.28% disomic, 0.18% diploid) and 0.38% YY-bearing sperm (0.21% disomic, 0.17% diploid) was found. The overall proportions of X- and Y-bearing sperm in the ejaculates were 47.9% and 47.2%, respectively, which was not significantly different from the expected 50:50 ratio. In addition 0.21% of cells appeared to be haploid XY-bearing sperm, 0.62% were diploid XY-bearing cells, and 0.05% of cells were considered to be tetraploid cells. The application of double FISH to human sperm using X-chromosome and Y-chromosome probes has allowed a more accurate assessment of the sex chromosal complements in sperm than single FISH method and quinacrine staining for Y-bodies.     

Triton X-100微乳液介质中锡催化KBrO3氧化5-NO2-PADAP褪色反应动力学光度法测定痕量锡

基于在弱酸性微乳液介质中,加热温度为80℃时,痕量锡(Ⅳ)催化KBrO3氧化2-(5-硝基-2-吡啶偶氮)-5-二乙氨基酚(5-NO2-PADAP)的褪色反应,建立了测定痕量锡(Ⅳ)的动力学光度法。于450 nm波长处测定5-NO2-PADAP褪色溶液的吸光度降低值ΔA,ΔA与锡的质量浓度在0.053~1.0μg/L范围呈线性关系,方法检出限为0.016μg/L。结合巯基葡聚糖凝胶分离富集,方法用于环境水样中痕量锡(Ⅳ)的测定,结果与AAS测定值相符,相对标准偏差为2.2%~3.8%,加标回收率在102%~104%间;方法用于地质样品中痕量锡(Ⅳ)的测定,结果与认定值相符。     

Human beta-glucuronidase. I. Recognition and uptake by animal fibroblasts suggests animal models for enzyme replacement studies

As part of an effort to develop an animal model for studies of uptake of human beta-glucuronidase, fibroblasts were established from primary explants of connective tissue from nine different animal species, and examined for their ability to take up human platelet beta-glucuronidase. Endogenous fibroblast beta-glucuronidase was inactivated by heating extracts to 65 degrees for 30 min. Human beta-glucuronidase was stable to this treatment. Uptake of human beta-glucuronidase by animal fibroblasts was measured as heat-stable beta-glucuronidase present in fibroblasts after exposure to partially purified human platelet beta-glucuronidase for 48 hr. Althought all animal fibroblasts examined exhibited some uptake capacity for human beta-glucuronidase, the uptake capacity of different animal fibroblasts varied over a 10-fold range. The uptake capacity of bovine fibroblasts was at least 80% that of human fibroblasts. Rat and hamster fibroblasts showed about half the uptake capacity of human fibroblasts. The rat fibroblasts resembled the human fibroblasts in the kinetics of uptake of hihg uptake (platelet) enzyme, poor uptake of human placental enzyme, and lack of appreciable turnover of enzyme taken up over 4 days. Heating extracts of rat organs containing added human beta-glucuronidase at 65 degrees selectively inactivated rat enzyme.     

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 degrees 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.     

在Triton Χ-100存在下利用火焰原子吸收法测定硅铁合金中的镍

本文报导了表面活性剂Triton Χ—100对火焰原子吸收法测定镍的增敏作用。结果表明TritonΧ—100不但能起到增敏作用,且能抑制干扰离子的干扰。该法测定硅铁合金中镍的线性范围为0.2～0.4μg/ml。检出限为0.04μg/ml。适合大批量样品测定。