Mechanism of inhibition of beta-hexosaminidase B from human liver by short- and medium-chain monocarboxylic acids

Short- and medium-chain monocarboxylic acids showed an inhibitory effect on enzymatic activity of beta-hexosaminidase B (Hex B) when 4-methylumbelliferyl-2-acetamido-2-deoxyglucopyranoside (MU-GlcNAc) was used as substrate: 1. Two groups were distinguished according to the chain length of the monocarboxylic acids: the first was only constituted by acetic acid (C2) whereas the second group exhibited a broader chain length specificity for medium-chain monocarboxylic acids (between C6 and C9). 2. Both groups were reversible competitive inhibitors (Km = 0.52 +/- 0.15 mM; KiC2 = 21.4 +/- 3.0 mM; KiC7 = 3.4 +/- 0.5 mM). Competition experiments between C2 and C7 (as representent of medium-chain monocarboxylic acids group) demonstrated that these inhibitors were bound to different subsites. 3. Competition experiments between C2 and 2-acetamido-2-deoxy-D-galactonolactone (GalNAcLone) (a competitive inhibitor of lysosomal hexosaminidases) demonstrated that these two inhibitors were mutually exclusive, i.e. they were probably bound at the same subsite. This feature and the structural analogy of C2 with the acetyl residue of GalNAcLone (and of the saccharidic part of the substrate) suggested that C2 bound to the substrate site where the N-acetyl residue of the beta-N-acetyl hexosaminide was positioned. 4. The inhibitory effect of medium-chain monocarboxylic acids (C6 and C9) was dependent on their physical state. Below the critical micellar concentration (CMC), detected by a dye spectral shift method, no significant inhibition was detected, but as extensively reported using C7, an obvious inhibitory effect occurred at concentrations higher than CMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeabilization of microorganisms by Triton X-100.

A simple permeabilization procedure has been developed which allows the reliable determination of enzyme activitiesin situ in a variety of different microorganisms. Permeabilization is obtained by freezing cell suspensions in the presence of a low concentration of the anionic detergent Triton X-100. After thawing, the cells can be used directly in the enzyme assays. The procedure has been optimized using the yeastSaccharomyces cerevisiae. Yeast cells are completely permeabilized by Triton X-100 concentrations of 0.05% (v/v), and permeabilization is independent of cell age and cell concentration. The treatment makes the cells freely diffusible for macromolecules up to molecular weights around 70,000. Cytoplasmic and mitochondrial amino acid biosynthetic enzymes as well as aminoacyl-tRNA synthetases could be readily measured in treated cells. The method has been successfully applied to the determination of enzyme activities in other fungi as well as in gram-positive and gramnegative bacteria.     

Triton X-100 inhibition of yeast plasma membrane associated NADH-dependent redox activities

Plasma membrane (PM) vesicles isolated from the yeast Saccharomyces cerevisiae (wild-type NCIM 3078, and a MG 21290 mutant pma 1-1) were used to monitor the effect of the detergents, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (Chaps) and Triton X-100, on (H+)-ATPase (E.C. 3.6.1.35), NADH oxidase and NADH-hexacynoferrate (III)[HCF (III)] oxidoreductase (E.C. 1.6.99.3) activities. The results obtained show that Triton X-100 inhibited both membrane bound and solubilized NADH-dependent redox activities. The nature of this inhibition as determined for NADH-HCF(III) oxidoreductase was non-competitive and the Ki values for wild and mutant enzymes were 1.2 x 10(-5) M and 8.0 x 10(-6) M, respectively. The findings are interpreted, in view of the established reports, that the active site architecture of PM bound NADH-dependent oxidoreductase in yeast is likely to be different than in other eukaryotes.     

Triton X-100 inhibition of yeast plasma membrane associated NADH-dependent redox activities

Plasma membrane (PM) vesicles isolated from the yeast Saccharomyces cerevisiae (wild-type NCIM 3078, and a MG 21290 mutant pma 1-1) were used to monitor the effect of the detergents, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (Chaps) and Triton X-100, on H⁺-ATPase (E.C. 3.6.1.35), NADH oxidase and NADH- hexacynoferrate (III)[HCF (III)] oxidoreductase (E.C. 1.6.99.3) activities. The results obtained show that Triton X-100 inhibited both membrane bound and solubilized NADH-dependent redox activities. The nature of this inhibition as determined for NADH–HCF(III) oxidoreductase was non-competitive and the Ki values for wild and mutant enzymes were 1.2?×?10^?5?M and 8.0?×?10^?6?M, respectively. The findings are interpreted, in view of the established reports, that the active site architecture of PM bound NADH-dependent oxidoreductase in yeast is likely to be different than in other eukaryotes.     

Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine

A mixed micellar assay for protein kinase C was developed to investigate the specificity and stoichiometry of activation by phospholipids and diacylglycerols. Triton X-100 mixed micelles containing 8 mol % phosphatidylserine (PS) and 2.5 mol % sn-1,2-dioleoylglycerol (diC18:1) activated rat brain protein kinase C in the presence of Ca2+ to the same degree as sonicated PS/diC18:1 did in the standard assay. However, protein kinase C activity was more responsive to diC18:1 in the mixed micellar assay than the standard assay. At 8 mol % PS and 100 microM Ca2+, diC18:1 stimulated maximally at 1 mol %. At 2.5 mol % diC18:1 and 100 microM Ca2+, PS did not activate until 3 mol % and then did so cooperatively with maximal stimulation occurring at 6-8 mol %. Direct evidence for a Ca2+-, PS-, and diC18:1-dependent interaction of protein kinase C with mixed micelles was obtained by molecular sieve chromatography on Sephacryl S-200. These data permit inferences pertaining to the number of diC18:1 and PS molecules/micelle which are required for activation. For diC18:1, a single molecule may be sufficient but no more than 2 molecules are required. For PS, greater than 4 but less than 10 molecules are required. These data establish that a phospholipid bilayer is not required for protein kinase C activation and that activation of monomeric protein kinase C occurs.     

Biodegradation of octylphenol polyethoxylate surfactant Triton X-100 by selected microorganisms

Octylphenol polyethoxylate (OPEO(n)) surfactants are used in numerous commercial and industrial products. Large amounts of such surfactants and their various residual biodegradation by-products are ultimately released into the environment. OPEO(n) biodegradation was performed in this study using pure cultures of Pseudomonas species and strains under different environmental conditions. Environmental factors including the pH, nitrogen sources, and growth kinetics of the cells were investigated. The intermediates of Triton X-100 biotransformation were detected by high performance liquid chromatography-mass spectrophotograph (HPLC-MS). We found the highest specific growth rate (mu) was 0.56 h(-1) and this was achieved by strain E with an initial concentration of Triton X-100 of 5000 mg L(-1). A pH level of 7 was most favorable for cell growth for all five strains. The highest specific growth rate was achieved using (NH(4))(2)SO(4) as the sole nitrogen source for strain E. Strain A showed an enhancement of growth when between 0.2 and 1.4 mg L(-1) of H(2)O(2) was added. Detection of intermediates was possible after four days of transformation and the octylphenol triethoxylate (OPEO(3)) peak was predominant, while the high molecular weight peaks had all disappeared. The kinetic analysis demonstrated that the greatest maximum specific growth rate (mu(max)) and the greatest saturation constant (K(s)) of 0.83 h(-1) and 5.24 mg L(-1), respectively, were obtained for strain E in 5000 mg L(-1) Triton X-100. The higher K(i) revealed that strain A was resistant to higher Triton X-100 concentrations.     

Triton X-100 solubilized bone matrix-induced alkaline phosphatase

1. Solubilized and membrane-bound alkaline phosphatase showed Michaelis-Menten behavior in a wide range of different substrate concentrations. 2. Membrane-bound alkaline phosphatase has a molecular weight of 130,000 and its minimum active configuration comprises two identical subunits of about 65,000. 3. The two forms of the enzyme behave similarly with respect to NaCl, urea and guanidine HCl. 4. Catalytic groups have pK values of about 8.5 and 9.7 for both membrane-bound and solubilized enzyme.     

Studies on Triton X-100 detergent micelles.

Triton X-100 micelle formation at 25 degrees C was studied by use of sedimentation equilibrium and fluorescence spectroscopic techniques. The apparent molecular weight of the major Triton X-100 micelle was found to be 81250, indicating a micelle number of 125. A micelle number of 121 was obtained with fluorescence titration experiments, which showed one molecule of 1-anilino-8-naphthalene sulfonate binding per micelle with an apparent association constant of 0.9 x 10(5) M. The fluorescent titration experiments also indicated the presence of another TX-100 binding species of variable size.     

Structural changes induced by Triton X-100 on sonicated phosphatidylcholine liposomes

Solubilization of sonicated unilamellar vesicles by Triton X-100 is a complex process. Solubilization starts at low detergent concentrations, as compared to the case of large vesicles, and is accompanied by the simultaneous rapid formation of large multilamellar liposomes. Measurements of lipid and detergent distribution indicate that, at a 1:1 lipid:detergent mole ratio, about one-third of the lipid, with most of the detergent, is solubilized in the form of mixed micelles. The remaining two-thirds are in the form of multilamellar liposomes, virtually free of detergent. Higher detergent concentrations also bring about the solubilization of these liposomes.     

Triton X-100 in Giemsa Staining of Blood Parasites

The effects of a surface active agent, Triton X-100, were studied in the routine Giemsa staining of seven blood parasites: Plasmodium vivax, Trypanosoma cruzi, T. lewisi, Leishmania donovani, Toxoplasma gondii, and microfilariae of Dirofilaria immitis and of Wuchereria bancrofti. Concentrations of Triton X-100 ranging from 0.01% to 0.5% were used in staining (a) both thick and thin blood films of all organisms except L. donovani, (b) tissue smears of L. donovani, and (c) tissue and peritoneal fluid smears of T. gondii. In general, the addition of the detergent to the Giemsa solution resulted in cleaner preparations and better stained organisms. Morphological details were more distinct, thus facilitating microscopical detection and identification of species. The most beneficial concentration of Triton X-100 was found to be 0.1%. Since it has a hemolytic effect on erythrocytes, concentrations above 0.01% cannot be used in staining thin blood films. It is suggested also that the use of Triton-Giemsa may help prevent transfer of some of these organisms from one slide to another during mass staining procedures as it has been demonstrated to do with malaria parasites (Brooke and Donaldson, 1950).     

The interaction of phosphatidylcholine bilayers with Triton X-100

The interaction of multilamellar phosphatidylcholine vesicles with the non-ionic detergent Triton X-100 has been studied under equilibrium conditions, specially in the sub-lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X-100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X-100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration. The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier-transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H-NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier-transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel-to-fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P-NMR and 2H-NMR spectra, and a new low-temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X-100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one-component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.     

Triton X-100 in Giemsa Staining of Blood Parasites

The effects of a surface active agent, Triton X-100, were studied in the routine Giemsa staining of seven blood parasites: Plasmodium vivax, Trypanosoma cruzi, T. lewisi, Leishmania donovani, Toxoplasma gondii, and microfilariae of Dirofilaria immitis and of Wuchereria bancrofti. Concentrations of Triton X-100 ranging from 0.01% to 0.5% were used in staining (a) both thick and thin blood films of all organisms except L. donovani, (b) tissue smears of L. donovani, and (c) tissue and peritoneal fluid smears of T. gondii. In general, the addition of the detergent to the Giemsa solution resulted in cleaner preparations and better stained organisms. Morphological details were more distinct, thus facilitating microscopical detection and identification of species. The most beneficial concentration of Triton X-100 was found to be 0.1%. Since it has a hemolytic effect on erythrocytes, concentrations above 0.01% cannot be used in staining thin blood films. It is suggested also that the use of Triton-Giemsa may help prevent transfer of some of these organisms from one slide to another during mass staining procedures as it has been demonstrated to do with malaria parasites (Brooke and Donaldson, 1950).