Differential effects of detergents upon the soluble and particulate guanylate cyclases from rat lung.

The enzyme guanylate cyclase is present in both particulate and soluble form in rat lung homogenates. As previously reported, the soluble enzyme can be activated by preincubation in the presence of O₂. The inactive (nonactivated) soluble enzyme is also stimulated by nonionic detergents, in the order Tween 20 > Lubrol PX > Triton X-67 > Triton X-100. The activated enzyme, however, was inhibited by these detergents in the reverse order. Sodium deoxycholate and lysolecithin were potent inhibitors of both inactive and activated enzyme. The activity of the particulate enzyme was stimulated by Lubrol PX > Triton X-100 > Triton X-67 > Tween 20. At a low concentration of lysolecithin or deoxycholate the particulate activity was increased; however, when detergent/protein > 1, inhibition was seen. In the case of deoxycholate, the inhibition could be reversed if excess deoxycholate was removed either by chromatography or by forming mixed micelles with Lubrol PX; however, deoxycholate inhibition of the soluble enzyme was irreversible. The stimulation by detergents of the particulate enzyme was apparently the result of solubilization. The effects upon the activity of the soluble enzyme were interpreted in terms of a model which assumes two hydrophobic regions on the enzyme surface. The two regions differ in hydrophobicity with the more hydrophobic region only being exposed as a result of activation. Interaction of a nonionic detergent with the less hydrophobic region stimulates activity, while interaction with the more hydrophobic region results in inhibition.     

Binding of fibrinogen to platelet integrin αIIbβ3 in solution as monitored by tracer sedimentation equilibrium

Fibrinogen showed essentially no binding (K_D>1 mM ) to platelet αIIbβ3 integrin in solution in the presence of Triton or octylglucoside above critical micellar concentrations. Under these conditions the integrin was an αβ monomer. After removal of the detergent from the Triton containing buffer (25 mM Tris/HCl;, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, pH 7.4) the integrin formed aggregates with hexamers as the most prominent species, as demonstrated by analytical ultracentrifugation and electron microscopy. Tracer sedimentation equilibrium experiments indicate that fibrinogen binds to the integrin aggregates, but with a surprisingly large K_D (at least 3 μM ). This value is 10- to 100-fold higher than values determined by solid phase assays or with integrins reconstituted onto lipid bilayers.     

Stabilization of Crystalline Acid Carboxypeptidase from Penicillium janthinellum by Nonionic Surfactants,and Inhibition of Enzyme Activity by Anionic Compounds

The crystalline acid carboxypeptidase from Penicillium janthinellum IFO-8070 was stabilized by the addition of nonionic surfactants, such as Triton X-100, Brij 35, Span 40, and Tween 20. In the presence of these stabilizers, extremely diluted enzyme (0.3 μg/ml of 50 mm sodium acetate buffer, pH 3.7) was almost completely stable after 2 days incubation at 25°C. About 35% and 20% of the enzyme activities were activated by the addition of Triton X-100 and Brij 35, respectively. Triton X-100 completely retarded inactivation at freezing (?15°C). On the other hand, anionic surfactants of SLS and LBSA, and cationic surfactant of cetyltrimethylammonium bromide strongly inactivated the enzyme. The inhibition of the fatty acid series was roughly proportional to the molecular weight of the inhibitor. Di-, and Tri-carboxylic acids also inhibited the enzyme activity.     

SIALYLTRANSFERASES IN RAT BRAIN: INTRACELLULAR LOCALIZATION AND SOME MEMBRANE PROPERTIES

Abstract— Total rat cerebral homogenate, with nuclei removed, yielded sialyltransferase activity peaks that were distinct from the protein distribution profile in a continuous sucrose density gradient. Marker enzyme studies and electron microscopic examinations on the gradient fractions suggested that most of the sialyltransferase activities were not associated with the synaptosomes.
The sialyltransferases appeared to be localized in the smooth microsomal membranes and the Golgi complex derivatives. The sialyltransferase activities were stimulated by non-ionic detergent mixture, Triton CF-54/Tween 80 (2/1, w/w), the effect being much more pronounced with exogenous substrates. The stimulatory effect was dependent on detergent concentration. With 1 mg detergent mixture per mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; exogenous GM_1a, 700; exogenous DS-fetuin, 230. The action of the nonionic detergents appears to be on a hydrophobic segment of the enzyme molecule, bearing the active site, which is buried in the membrane lipid bilayer. This was substantiated by the partial trypsin resistance of the sialyltransferase activities and the abolition of that resistance when trypsiniza-tion was performed in the presence of nonionic detergents. Furthermore, the sialyltransferase activities were markedly inhibited by organic solvents; and these inhibitory effects were inversely proportional to the solvent dielectric constants.     

Utility of enzymes from <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> and <Emphasis Type="Italic">Prevotella ruminicola</Emphasis> as detergent additives

In this study, we investigated the application of cellulase and protease purified from rumen bacteria as detergent additives. Cellulase and protease were purified from the rumen cellulytic bacteria Fibrobacter succinogenes S85, and Prevotella ruminicola 23, respectively. An inhibitor test indicated that the purified protease belongs to the category of serine proteases and metalloproteases. Both the enzymes were effective at a high temperature (50 degrees C) and neutral pH (pH 7-8), but the protease activity increased with the increase in temperature and pH. The purified protease was treated with ten types of surfactants/detergents; it was found to retain over 60% of its activity in the presence of anionic and nonionic detergents. The cellulose plus protease combination was still effective after treatment with Triton X-100 and Tween 80, but the residual activity was low after treatment with Tween 20 than that after treatment with other nonionic detergents. Washing tests indicated that enzyme addition produced no significant improvement in the removal of grass stains, but individual enzyme addition in surfactants/detergents, especially in nonionic detergents, could improve the washing performance of the detergents by improving its ability to remove blood stains. This suggested that the surfactant/detergent class, enzyme properties, and the mixing ratio of ingredients should be considered simultaneously to enhance the washing performance.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Purification and reconstitution of the Ca2+-ATPase from plasma membrane of pig erythrocytes.

The Ca2+-ATPase from plasma membranes of pig erythrocytes was purified by mixed micelle gel chromatography (Wolf, H.U., Diekvoss, G., and Lichtner, R. (1977) Acta Biol. Med. Germ. 36, 847-858). The enzyme was activated at high concentrations of Tween 20 (10 mg/ml) or by appropriate mixtures of Triton X-100 and phospholipids. It was highly unstable in the absence of Ca2+ and activator protein. The Ca2+-ATPase was incorporated into liposomes by freeze-thaw sonication. After removal of non-ionic detergent by passage through a phenyl Sepharose 4B column, the reconstituted vesicles catalyzed a rapid ATP-dependent uptake of Ca2+. Modulator protein from brain substituted for the natural activator protein and stimulated Ca2+ uptake in reconstituted vesicles.     

Organization and structure of two mixed micellar phases of the sphingomyelin/Triton X-305 system

Properties of mixed dispersions of sphingomyelin and the nonionic detergent, Triton X-305, were investigated by analytical ultracentrifugation and by autocorrelation spectroscopy of scattered laser light. These properties were compared with those of the sphingomyelin/Triton X-100 mixed micellar system reported previously [S. Yedgar, Y. Barenholz, and V. G. Cooper (1974) Biochim. Biophys. Acta 363, 98-111]. The substitution of the 30-unit ethylene oxide chain of Triton X-305 for the 10-unit chain of the Triton X-100 resulted in the appearance of two micellar phases at all detergent/lipid mixture ratios studied, whereas only a single mixed micellar phase was observed using Triton X-100. Despite this difference, the properties of the mixed lipid/detergent micelles obtained using Triton X-100 have been verified in the following respects: The detergent aggregation numbers in the mixed micelles are quite constant over a wide range of detergent molar fractions, being about 70 and 400 for the lighter and heavier mixed micellar phases, respectively. The detergent aggregation numbers are larger in the mixed micelle than in the pure detergent micelle. Very large sphingomyelin aggregation numbers can be accommodated within the mixed micelles, apparently by the critical intervention of the detergent molecules to produce a stable micellar structure.     

Purification of 2,3-oxidosqualene cyclase from rat liver.

A rapid and simple purification of milligram amounts of 2,3-oxidosqualene cyclase, an integral membrane enzyme that catalyzes the cyclization of squalene epoxide to lanosterol, is reported. Several nonionic detergents (Triton X-100, Tween 80, Emulphogene, and lauryl maltoside) were evaluated for solubilization of oxidosqualene cyclase from rat liver microsomes. At a detergent concentration of 5 mg/ml, lauryl maltoside was approximately 10 times more effective than Emulphogene in the solubilization of oxidosqualene cyclase; Triton X-100 and Tween 80 were less effective than Emulphogene as judged by the relative specific activities of the solubilized enzyme. Treatment of microsomes with lauryl maltoside resulted in a selective solubilization of the cyclase with concomitant activation of the enzyme. The solubilized enzyme was purified to homogeneity by fast protein liquid chromatography. The purified enzyme consists of a single subunit that has an apparent molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme obeys saturation kinetics and the apparent Km of (2,3)-oxidosqualene is 15 microM; the apparent kcat/Km is 200 M-1.min-1. An improved assay of the enzyme that utilizes high performance liquid chromatography methods is also described.     

Solubilization and partial purification of the rabbit parotid Na/K/Cl-dependent bumetanide binding site

Summary We demonstrate that the high affinity bumetanide binding site of the rabbit parotid acinar cell can be extracted from a basolateral membrane fraction using relatively low concentrations (0.07%, wt/vol; 1 mg membrane protein/ml) of the nonionic detergent Triton X-100. This extracted site cannot be sedimented by ultracentrifugation at 100,000 ×g × 1 hr. Bumetanide binding to this site retains the ionic characteristics of bumetanide binding to native membranes but shows a fivefold increase in binding affinity (K d=0.57±0.15 m vs.K d=3.3±0.7 m for native membranes). Inactivation of the extracted bumetanide binding site observed at detergent/protein ratios>1 can be prevented or (partially) reversed by the addition of exogenous lipid (0.2% soybean phosphatidylcholine). When the 0.07% Triton extract is fractionated by sucrose density gradient centrifugation in 0.24% Triton X-100, 0.2% exogenous lipid and 200mm salt, the high affinity bumetanide binding site sediments as a single band withS _20,w =8.8±0.8 S. This corresponds to a molecular weight 200 kDa for the bumetanide binding protein-detergent-lipid complex and represents a sevenfold purification of this site relative to the starting membrane fraction. In contrast to previous attempts to purify Na/K/Cl cotransport proteins and their associated bumetanide binding sites, the present method avoids harsh detergent treatment as well as direct covalent modification (inactivation) of the transporter itself. As a consequence, one can follow the still active protein through a series of extraction and purification steps by directly monitoring its bumetanide binding properties.     

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.     

Renaturation and ligand blotting of the major subunit of the rat asialoglycoprotein receptor after denaturing polyacrylamide gel electrophoresis

Rat hepatic asialoglycoprotein receptors (ASGP-Rs) bind terminalclustered galactosyl or N-acetylgalactosaminyl residues withhigh affinity. The affinity-purified ASGP-R consists of threesubunits designated RHL1, RHL2, and RHL3. The ligand-bindingactivity of individual subunits was investigated by ligand blotting,after separation of subunits by SDS-PAGE under nonreducing conditions,electrotransfer to nitrocellulose, and incubation with ¹²⁵I-asialo-orosomucoid(ASOR). No ligand-binding to any subunits could be detectedwhen proteins such as BSA, casein, gelatin, or fat-free drymilk were used as blocking agents. However, subsequent incubationof BSA-blocked nitrocellulose blots with some nonionic detergentsresulted in renaturation of RHL1. ¹²⁵I-ASOR-binding to RHL2or RHL3 was weaker and could be detected only after longer exposure.Similarly, direct use of detergents such as Tween 20, NonidetP-40, or Triton X-100 as blocking agents also preserved theASOR-binding activity of RHL1. Ionic detergents tested did notshow any ability to renature the ligand-binding activity ofRHL subunits. Among nonionic detergents tested, Tween 20, Tween85, Lubrol PX, Nonidet P-40, and Triton X-100 were more effectivethan Tween 40, Tween 65, Tween 80, or Brij 35, whereas SPAN,digito-nin, or octyl-glucoside showed no effect. Weak ¹²⁵I-ASORbinding to RHL2 or RHL3 could be detected only when the Tweenseries or Lubrol PX were used. Incubation of blots with dithiothreitolcaused a dose-dependent loss of binding activity. The carbohydraterecognition domain (CRD) of RHL1, isolated after subtilisindigestion of ASGP-R bound to ASOR-Sepharose, retained ligand-bindingactivity as assessed by its binding to ASOR-Sepharose and byligand blotting. ¹²⁵I-ASOR binding to electroblotted CRD afterSDS-PAGE was also dependent on the presence of nonionic detergents.We conclude that restoration of ligand-binding activity of RHL1after SDS-PAGE by some nonionic detergents is not dependenton the presence of the cytoplasmic, transmembrane, or stalkdomains of this subunit. asialoglycoprotein receptor Ligand blotting detergent renaturation RHL1     

Perstraction of intracellular pigments by submerged cultivation of <Emphasis Type="Italic">Monascus</Emphasis> in nonionic surfactant micelle aqueous solution

“Milking processing” describes the cultivation of microalgae in a water-organic solvent two-phase system that consists of simultaneous fermentation and secretion of intracellular product. It is usually limited by the conflict between the biocompatibility of the organic solvent to the microorganisms and the ability of the organic solvent to secret intracellular product into its extracellular broth. In the present work, submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle aqueous solution for pigment production is exploited, in which the fungus Monascus remains actively growing. Permeabilization of intracellular pigments across the cell membrane and extraction of the pigments to the nonionic surfactant micelles of its fermentation broth occur simultaneously. “Milking” the intracellular pigments in the submerged cultivation of Monascus is a perstraction process. The perstractive fermentation of intracellular pigments has the advantage of submerged cultivation by secretion of the intracellular pigments to its extracellular broth and the benefit of extractive microbial fermentation by solubilizing the pigments into nonionic surfactant micelles. It is shown as the marked increase of the extracellular pigment concentration by the submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle solution.     

The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes

The efficiency of several nonionic detergents and a homologous series of zwitterionic detergents for the extraction of acetylcholinesterase (EC 3.1.1.7) from bovine erythrocyte membranes was examined. Of the nonionic detergents examined, the polyoxyethylene-based Tweens were the least effective solubilizing agents. Within this series, increasing the length of the saturated fatty acid chain progressively decreased the efficiency of enzyme recovery, while unsaturation in the side chain reversed this trend. In the Lubrol detergents, where the chain length of the alcohol group is variable, an increase in the length of the polyoxyethylene glycol group decreased the recovery of acetylcholinesterase in the solubilized state, without affecting the efficiency of extraction of total erythrocyte protein. As with the other nonionic detergents examined, Triton X-100 and octyl beta-D-glucoside were maximally effective in solubilizing acetylcholinesterase activity at concentrations greater than their respective critical micelle concentrations. In the sulfobetaine (N-alkyldimethylaminopropane sulphonate) zwitterionic detergent series, the longer alkyl chain zwittergents Z 316 and Z 314 were more efficient than the shorter chain length members of the series (Z 310 and Z 312). In contrast to the higher chain length compounds, short chain analogs were maximally effective at or below their critical micelle concentrations. After purification by ion-exchange chromatography and affinity chromatography, the enzyme extracted with the various detergents gave sedimentation coefficients between 6.8S and 7.6S, consistent with a dimeric structure. Acetylcholinesterase could also be efficiently released by 0.2 mM EDTA or 0.5 M NaCl from bovine erythrocyte membranes previously depleted of 70-80% of the membrane lipids by butanol. Nonlinear Arrhenius plots of enzyme activity were found whether acetylcholinesterase was solubilized with Tween 20, Lubrol PX, or Triton X-100. The present work confirms that bovine erythrocyte acetylcholinesterase requires detergents to solubilize it from membranes and that its activity depends on the structure of the amphiphiles used to solubilize the enzyme.     

Quantitative determination by derivative spectrophotometry of Triton X-100 in solubilized preparations of membrane proteins

The amount of the nonionic detergent Triton X-100 in protein preparations cannot be determined spectrophotometrically from the normal spectrum because the spectral bands of the detergent and proteins overlap, but it was found that the detergent could be determined accurately by 4th-derivative spectrophotometry. The intensity of the derivative absorbance difference between the positive and negative peaks at about 277.5 and 281 nm, respectively, increased linearly with increase in the detergent concentration at more than its critical micelle concentration (about 0.03%). Proteins had little effect on these spectral bands of the detergent. This method is very simple and accurate for determination of the concentration of Triton X-100 in solubilized preparations of membrane proteins.     

Optimization of detergents for the assay of cathepsins B, L, S, and K

The differential effects of representative, commonly available ionic (SDS), nonionic (Brij 35, Tween 20, and Triton X-100), and zwitterionic (Chaps) detergents on the catalytic activity and properties of human cathepsins B, L, S, and K were examined. The presence of detergents in the assay buffer affected the activity of cathepsins to variable extents; Chaps enhanced the activity of all the enzymes while SDS was most detrimental. Tween 20 lowered cathepsin S activity, while it slightly enhanced that of all other cathepsins studied. The presence of detergents in the activation buffer was clearly beneficial to both cathepsins L and K, possibly by favoring the release of the enzyme from the walls of the incubation vessel. Overall, the results indicate that Chaps is the optimal detergent for use with this family of enzymes.     

The role of lipid phase structure in the interaction of lactate dehydrogenase with phosphatidylserine. Activity studies

Lactate dehydrogenase is one of the enzymes of the glycolytic path. It has been shown to be able to bind in vitro to cellular membranes. The presence of anionic phospholipids induces changes in the catalytic properties of the enzyme similar to those found when the enzyme is bound to natural membranes. In this study, a nonionic detergent (Tween 20), at concentrations not affecting the catalytic activity of LDH, was used to study the role of the lipid supra-molecular structure in the interaction between pig skeletal muscle lactate dehydrogenase and phosphatidylserine. Tween 20 changes the equilibrium of concentrations between the lipid supra-molecular forms. The detergent at the used concentration values did not alter the activity of the enzyme when it was used on its own, but did diminish the level of inhibition induced by the studied phospholipid. The obtained results showed that the interaction is reversible and that the bilayer structure of the lipid is essential for the inhibition.     

Biochemical and Electron Microscopic Studies of the Streptomyces reticuli Cellulase (Avicelase) in Its Mycelium-Associated and Extracellular Forms

Streptomyces reticuli is able to grow efficiently with crystalline cellulose (Avicel) as the sole carbon source. Cultivation in the presence of the nonionic detergent Tween 80 at a concentration of 0.1% led to a 10-fold increase in extracellular cellulolytic activity. Under these conditions, one single 82-kDa cellulase (Avicelase) capable of degrading crystalline and soluble cellulose as well as cellodextrins and p-nitrophenylcellobioside was purified to apparent homogeneity by a procedure which consisted of two consecutive anion-exchange chromatographies followed by chromatofocusing. Aggregation, which was a major problem during protein purification, could be avoided by including Triton X-100 at a concentration of 0.1% in every chromatographic step. The Avicelase was identified in extracellular and mycelium-associated forms, the latter of which could be released efficiently by nonionic detergents. In addition, a 42-kDa truncated form retaining cellulolytic activity was identified which had been generated from the 82-kDa enzyme by a protease. Antibodies raised against the mycelium-associated Avicelase reacted with the 42-kDa derivative and the extracellular form. The mycelial association of the enzyme was confirmed by immunofluorescence and immunoelectron microscopies.     

Toxicity of phenanthrene dissolved in nonionic surfactant solutions to Pseudomonas putida P2

The fraction in which direct contact occurs between micellar-phase phenanthrene and the bacterial cell surface was estimated by measuring the toxicity of nonionic surfactant (Tween 80 and Triton X-100) solutions to the phenanthrene-degrading bacterium, Pseudomonas putida P2. Cell viability of completely dissolved phenanthrene decreased by 30% at concentrations greater than 0.3 mg L(-1), which is equal to approximately one third of its solubility. Both nonionic surfactants had no effect on cell viability up to 5 g L(-1). Cell viability increased with increasing surfactant concentration at a fixed phenanthrene concentration, due to the decreased concentration of aqueous-pseudophase phenanthrene and the reduced fraction of direct contact. The fraction of direct contact was c. 20% or more below 3 g L(-1) of Triton X-100. The fraction of direct contact for Tween 80 was estimated to be lower than Triton X-100.     

The adjuvant effect of selenium nanoparticles,Triton X-114 detergent micelles,and lecithin liposomes for <Emphasis Type="Italic">Escherichia coli</Emphasis> antigens

We studied the adjuvant properties of micelles from nonionogenic detergents, liposome, and selenium nanoparticles containing extracellular and intracellular vaccine antigens of a weakly virulent α-hemolytic Escherichia coli B-5 strain used for the immunization of experimental animals. Triton X-100 was used as a nonionogenic detergent for micelle preparation. The liposomes were obtained on the basis of lecithin from a chicken egg and E. coli B-5 membrane lipids. Native lipoproteins of E. coli B-5 cells and peptides for the proteolytic hydrolysis of toxin-containing culture liquid were used as antigens for micelles and liposomes. The obtained data suggested that micelles, liposomes, and selenium nanoparticles can be used for immunization with cellular and extracellular E. coli antigens.