Interaction of proteins with Triton X-100-substituted sepharose 4B

Interaction of a number of arbitrarily chosen proteins with Triton X-100-substituted Sepharose 4B has been investigated. Of the proteins examined, bovine serum albumin, hemoglobin, glutamate dehydrogenase, and pepsin were found immobilized on the adsorbent. Binding of these proteins occurred irrespective of pH and NaCl concentration. Cytochrome c, used as a model protein, was totally immobilized only at low pH. Adsorption of glutamate dehydrogenase and pepsin took place with retention of their catalytic activities. Moreover, glutamate dehydrogenase used as a model allosteric enzyme, was found to retain its native properties upon binding to the adsorbent in the forms of suspension or column. Results are discussed in terms of specific interactions involving the hydrophobic region of Triton X-100 and the apolar patches or crevices present on the surface of protein molecules. Possible potential of the matrix as a method for preparation of biologically active immobilized proteins and its application in continuous operations are also discussed.     

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).     

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).     

Purification of an Acidic Structural Protein from rat Skin Using Triton X-100

An acidic protein was extracted with neutral-salt solutions from rat skin. When precipitated by dialysis against dilute acetic acid, the structural protein was separated from contaminating soluble collagens and other soluble proteins. The precipitate was dissolved in buffers containing 1% Triton X-100 and purified to apparent size and charge homogeneity by chromatography on a DEAE Bio-Gel A column. Triton X-100 was necessary for achieving nondestructive disaggregation of the protein which could be reversed by dialyzing out the detergent against methanol-dilute acetic acid.     

Interaction of beef liver lipase with mixed micelles of tripalmitin and Triton X-100

When concentrated dispersions of tripalmitin in Triton X-100 are added to reaction mixtures containing soluble beef liver lipase, the rate of hydrolysis of tripalmitin increases with incubation time. When the diluted substrate is aged at 37 degrees C for 3 hr before the addition of enzyme, the rate of hydrolysis is greater than the rate with freshly diluted dispersions and is constant for at least 2 hr. The reciprocal of the rate of hydrolysis is a complex function of the reciprocal of the substrate concentration when measured with freshly diluted substrate dispersions. A linear relationship between these reciprocals is obtained when measured with aged preparations of substrate. The rate and extent of increase of the velocity of hydrolysis of the aged substrate in relation to the velocity of hydrolysis of freshly diluted substrate are directly proportional to the substrate concentration and inversely proportional to the Triton X-100 concentration. The apparent V(max) of beef liver lipase for tripalmitin in diluted and aged dispersions is independent of the Triton X-100 concentration, while the apparent K(m) is inversely proportional to the Triton X-100 concentration. The apparent K(m) for tripalmitin complexes at zero Triton X-100 concentration was judged to be 7.5 x 10(-5) m. The molecular size of dispersion complexes does not change significantly as dispersions are aged. The spherical diameter of the complexes assessed by gel filtration techniques is in the order of 100 A.     

Type A and type B monoamine oxidase activities in rat brain and liver mitochondria: a comparison of their properties using the non-ionic detergent Triton X-100

1. The effects of the non-ionic detergent Triton X-100 on the heterogeneity of monoamine oxidase activities were studied and compared in synaptic (fractions SM and SM2) and non-synaptic (fraction M) brain mitochondria and liver mitochondria. 2. Triton X-100 inhibited type A and type B monoamine oxidase activities in all four mitochondrial fractions in a concentration-dependent manner. Liver mitochondrial enzymatic activities were much more sensitive to this inhibition than those of brain mitochondria. The activities in the SM fraction of synaptic brain mitochondria were the least susceptible. 3. In all four mitochondrial fractions, type A activities were more sensitive to inhibition than type B activities. 4. These results suggest that the membrane micro-environment around the enzyme molecules in situ may be important in the functional expression of the activity of the enzyme.     

gamma-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100.

Abstract— The treatment of cerebellar membranes of rat brain with a low concentration of Triton X-100 followed by sufficient washing results in an increase of the Na⁺-independent binding of [³H]GABA and a total loss of the Na ⁺-dependent binding of [³H]GABA. The Na⁺-independent binding of [³H]GABA was more abundant in membranes of cerebellum than in membranes of other rat brain regions and mainly localized in the synaptic membrane fraction of a cerebellar homogenate. In the Triton-treated membranes, the Na⁺-independent binding of [³H]GABA was a saturable process, which could be resolved into two components, a high and a low affinity component with dissociation constants of 4.5 and 30 nm , respectively. The neurophysiological agonists, muscimol, GABA, and imidazole acetic acid, and the antagonist, bicuculline, inhibited the high affinity Na⁺-independent binding of [³H]GABA by 50% at 0.003, 0.012, 0.3 and 10 μm respectively. These data suggest that the Na⁺-independent binding of [³H]GABA in the Triton-treated cerebellar membranes represents the synaptic receptors of GABA. It is emphasized that extensive washing of the membranes after a Triton treatment is necessary in order to detect the high affinity Na⁺-independent binding of [³H]GABA.     

Hydrodynamic properties of porcine bestrophin-1 in Triton X-100

Bestrophin-1 (Best-1) is an integral membrane protein, defects in which cause Best vitelliform macular dystrophy. Best-1 is proposed to function as a Cl⁻ channel and/or a regulator of Ca⁺⁺ channels. A tetrameric (or pentameric) stoichiometry has been reported for recombinant best-1. Using a combination of gel exclusion chromatography and velocity sedimentation we examined the quaternary structure of native best-1 and found that it migrates as a single species with a Stokes radius of 7.3 nm, sedimentation coefficient (S_20,w) of 4.9, and partial specific volume (ν) of 0.80 ml/g. The mass of the protein-detergent complex is calculated to be 206 kDa, with the protein component estimated to be ∼138 kDa. Given a monomeric mass of 68 kDa, we conclude that native best-1 solubilized with Triton X-100 is a homodimer. The differences between this observation and a prior report were examined by comparing recombinant best-1 with tissue derived best-1 using gel exclusion chromatography. Much of the recombinant best-1 eluted in the column void (V_o) fraction, unlike that extracted from RPE cells. We conclude that the minimal functional unit of best-1 is dimeric. This stoichiometry differs from that previously measured for recombinant best-1, suggesting that further studies are necessary to determine the stoichiometry of functional best-1 in RPE membranes.     

The activity of Triton X-100 soluble chlorophyllase in liposomes

Chlorophyllase (chlorophyll-chlorophyllidohydrolase, EC 3.1.1.14) was isolated and purified from Phaseolus vulgaris L. chloroplasts and etioplasts dissolved in 1% Triton X-100 and 10% glycerol. A 100 and 40-fold purification, respectively, was achieved. Enzyme preparations from both sources had similar affinities for chlorophyll a when assayed in a Triton X-100 medium. When electrophoresed in sodium dodecyl sulphate polyacrylamide gels the major band in both preparations migrated as a peptide of 30,000 daltons. Chlorophyll containing liposomes were also used as a substrate for chlorophyllase. The rate of hydrolysis did not follow Michaelis-Menten kinetics. When chlorophyllide a or methyl chlorophyllide a was incorporated in the liposomes, then in the presence of phytol dissolved in methanol, methylchlorophyllide a and chlorophyll a were shown to be synthesized. Apparently the purified enzyme in the presence of lipids, is endowed with both synthetic and hydrolytic activity.Abbreviations DEAE diethylaminoethyl - MeOH methanol - SDS sodium dodecyl sulphate     

Hydrodynamic properties of porcine bestrophin-1 in Triton X-100

Bestrophin-1 (Best-1) is an integral membrane protein, defects in which cause Best vitelliform macular dystrophy. Best-1 is proposed to function as a Cl- channel and/or a regulator of Ca++ channels. A tetrameric (or pentameric) stoichiometry has been reported for recombinant best-1. Using a combination of gel exclusion chromatography and velocity sedimentation we examined the quaternary structure of native best-1 and found that it migrates as a single species with a Stokes radius of 7.3 nm, sedimentation coefficient (S20,w) of 4.9, and partial specific volume (nu) of 0.80 ml/g. The mass of the protein-detergent complex is calculated to be 206 kDa, with the protein component estimated to be approximately 138 kDa. Given a monomeric mass of 68 kDa, we conclude that native best-1 solubilized with Triton X-100 is a homodimer. The differences between this observation and a prior report were examined by comparing recombinant best-1 with tissue derived best-1 using gel exclusion chromatography. Much of the recombinant best-1 eluted in the column void (Vo) fraction, unlike that extracted from RPE cells. We conclude that the minimal functional unit of best-1 is dimeric. This stoichiometry differs from that previously measured for recombinant best-1, suggesting that further studies are necessary to determine the stoichiometry of functional best-1 in RPE membranes.     

Triton X-100 as a channel-forming substance in artificial lipid bilayer membranes

The nonionic detergent Triton X-100, introduced into artificial membranes of various lipid compositions, induced current fluctuations that may correspond to the formation of channels across the bilayer. Independently of the lipid used, these fluctuations vary in amplitude between 1 and 4·10^?10, $\text{Ω}{}^{\mathrm{?1}}$, show a strong dependence on the applied voltage, and are selective for cations in the sequence Rb⁺ > K⁺ > Na⁺ > Li⁺. These results are discussed in relation to the chemical structure of the Triton X-100 molecule.     

Purification of an acidic structural protein from rat skin using Triton X-100.

An acidic protein was extracted with neutral-salt solutions from rat skin. When precipitated by dialysis against dilute acetic acid, the structural protein was separated from contaminating soluble collagens and other soluble proteins. The precipitate was dissolved in buffers containing 1% Triton X-100 and purified to apparent size and charge homogeneity by chromatography on a DEAE Bio-Gel A column. Triton X-100 was necessary for achieving nondestructive disaggregation of the protein which could be reversed by dialyzing out the detergent against methanol-dilute acetic acid.     

The cyclic AMP-dependent initiation of DNA synthesis by T51B rat liver epithelioid cells.

The brief rise in the cellular cyclic AMP content which occurs late in the prereplicative phases of rat hepatocytes in vivo and T51B rat liver epitheloid cells in vitro seems to be necessary for the initiation of DNA synthesis. Thus, the extracellular calcium-deprivation in T51B rat liver cells in culture which induces a late G-1 block is rapidly reversible (cells surge into S phase within one hour) either by creating a cyclic AMP surge by the addition of calcium or 3-isobutyl-1-methyl xanthine (a cyclic 3',5'-nucleotide phosphodiesterase inhibitor) or by the exogenous addition of low concentrations of cyclic AMP itself (i.e., 10(-8)-10(-5) M). On the other hand, prevention of the calcium-induced cyclic AMP surge by imidazole (a cyclic 3',5'-nucleotide phosphodiesterase activator) blocked the initiation of DNA synthesis by the calcium-deprived T51B cells.