Protein chromatography on adsorbents with hydrophobic and ionic groups. Purification of human erythrocyte glycophorin.

Human erythrocyte glycophorin was purified rapidly by (a) chromatography of a Triton X-100 extract of erythrocyte 'ghosts' on N-(3-carboxypropionyl)aminodecyl-Sepharose in buffers containing Triton X-100 or sodium dodecyl sulphate, or (b) chromatography of whole 'ghosts', solubilized in sodium dodecyl sulphate, on dodecyl-Sepharose, in buffers containing sodium dodecyl sulphate. The products contained 85-95% glycophorin (electrophoretic band PAS-1) and the major contaminants were glycoproteins PAS-2 (possibly a subunit of glycophorin) and PAS-3.     

Covalent labelling of the lutropin binding site. Evidence for a single Mr 90000 sialoglycopolypeptide.

Membrane-associated sialoglycopolypeptides of rat ovaries were oxidized with NaIO4, reduced with NaB3H4 and solubilized with Triton X-100. The solubilized proteins carrying the 3H label were subjected to affinity chromatography on human choriogonadotropin coupled to agarose. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate followed by fluorography revealed a single component of apparent Mr 90000. This component was abolished when ovaries saturated with choriogonadotropin were used as starting material. The above result is identical to that obtained previously by conventional detection methods [ Metsikk ö & Rajaniemi (1982) Biochem. J. 208, 309-316] and indicates that the 3H-labelled lutropin/choriogonadotropin sialoglycopolypeptide was observed. The affinity-purified 3H-labelled protein co-eluted with the choriogonadotropin-binding activity solubilized with Triton X-100 from rat ovarian particles, showed a Stokes' radius of 6.2 nm and sedimented as a single band with a sedimentation coefficient of 5.1 S. The sedimentation coefficient of this 3H-labelled protein was not significantly altered when boiled in 1% sodium dodecyl sulphate, indicating that non-covalently associated subunits were not present. The 3H-labelled protein cosedimented with the choriogonadotropin-binding activity solubilized with Triton X-100 from rat ovary. When 125I-choriogonadotropin-receptor complex was covalently crosslinked with glutaraldehyde, an Mr 130000 component was produced as detected by sodium dodecyl sulphate gel electrophoresis. This component was extracted from the polyacrylamide gel and subjected to sucrose-density-gradient centrifugation in 0.1% Triton X-100. A single band sedimenting at the position of the 125I-choriogonadotropin-receptor complex solubilized from a prelabelled ovary was observed, exhibiting a sedimentation coefficient of 6.5S. These data suggest that the lutropin-binding site is a single sialoglycopolypeptide of Mr 90000, which binds one molecule of hormone resulting in an apparent Mr 130000 complex. The large Stokes' radius (6.2 nm) of the binding site is accounted for by bound detergent.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01.

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35,000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [¹⁴C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35 000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

The stimulus-secretion coupling of glucose-induced insulin release. Environmental influences on L-glutamine oxidation in pancreatic islets.

Rat ovarian luteinizing hormone/human choriogonadotropin binding sites were labelled with 125I-choriogonadotropin in vivo, and the resulting 125I-choriogonadotropin-receptor complexes were solubilized by Triton X-100 and purified by use of antibodies to choriogonadotropin immobilized to agarose. The purified 125I-choriogonadotropin-receptor complex was treated with glutaraldehyde to crosslink radiolabelled hormone to the receptor. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the crosslinked product revealed a labelled Mr 130 000 major band in addition to the hormone and its alpha-subunit, indicating that a single receptor component was linked to the hormone. Unoccupied binding sites for luteinizing hormone were also solubilized by Triton X-100 from pseudopregnant rat ovaries, and attached to choriogonadotropin-agarose. The agarose gel was washed, and eluted with 0.1 M-sodium acetate, pH 4. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the pH 4 eluate revealed an Mr 90 000 major band which was abolished when ovaries presaturated with choriogonadotropin were used as starting material. These observations suggest that the hormone-binding component of the luteinizing hormone receptor is a polypeptide of Mr 90 000. This polypeptide was isolated and labelled with Na 125I. The labelled polypeptide showed a single band on sucrose density gradient centrifugation and on gel filtration on agarose.     

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     

Detergents inhibit exocytosis in PC 12 cells: evidence for an effect on ion fluxes

Membrane events in exocytosis were studied by examining the effect of different detergents on the K+-stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X-100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 microM and 10 microM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4). The inhibitory effect of 30 microM Triton X-100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X-100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X-100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X-100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13-14 degrees C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished. In the presence of 30 microM Triton X-100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50-60%. These data suggest that the site of action of Triton X-100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.     

Isolation of the porcine ileal intrinsic factor receptor by sequential affinity chromatography

An extract containing solubilised receptor was passed through four columns containing Sepharose to which had been covalently coupled anti-cobalophilin IgG, vitamin B-12-intrinsic factor, vitamin B-12, and free intrinsic factor, respectively. Following a wash the receptor was eluted with EDTA, then residual Triton X-100 micelles and vitamin B-12-intrinsic factor were removed by Sephadex G-200 filtration. The receptor was purified 84 000-fold, sodium dodecyl sulphate electrophoresis indicated two subunits and gel filtration of its vitamin B-12-inttrinsic factor complex resolved it into two molecular species.     

Glycoproteins and glycosaminoglycans synthesized by human keratinocytes in culture. Their role in cell-substratum adhesion.

Glycoproteins and proteoglycans synthesized by human keratinocytes in medium containing D-[1-14C]glucosamine were extracted and analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Extraction of the labelled keratinocytes with 0.5% Triton X-100 removed most of the glycoconjugates and left the cytoskeleton and nuclear residue adherent to the substratum. In addition to the cytoskeletal proteins, there was a relatively simple profile of glycoproteins and glycosaminoglycans associated with this adherent cytoskeleton. These consisted of eight glycoproteins in the mol.wt. range 99000-232000, five proteins in the keratin region (mol.wt. 42000-61000), hyaluronic acid and a sulphated glycosaminoglycan. Surface labelling of the keratinocytes with galactose oxidase (with or without neuraminidase)/KB3H4 revealed that many of the glycoproteins were exposed on the cell surface. The importance of the glycoproteins and proteoglycans in attaching the keratinocytes to the substratum was examined by studying their expression after incubation in medium containing tunicamycin and their degradation after digestion with trypsin and hyaluronidase. These studies, together with an examination of the glycoconjugates released by sequential extraction with 0.5% Triton X-100 followed by 0.2% sodium dodecyl sulphate, revealed that the glycoprotein of mol.wt. 232000 has an important role in mediating the attachment of keratinocytes to the substratum.     

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.     

Electrophoretically monodisperse cytochrome c oxidases

A discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions has been used to demonstrate monodispersity of procaryotic and eucaryotic cytochrome c oxidase preparations. Alkaline treated bovine enzyme which contains nine subunits as analysed by subsequent discontinuous SDS-polyacrylamide gel electrophoresis is a monodisperse dimer in 0.1% Triton X-100 and a monomer in 0.1% dodecyl maltoside. The Mr-values corrected for bound detergent are 286,000 in Triton X-100 and 152,000 in dodecyl maltoside respectively. The two-subunit bacterial cytochrome c oxidase of Paracoccus denitrificans is proved to be a monomer with a corrected Mr of 76,000 in both nonionic detergents Triton X-100 and dodecyl maltoside.     

Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes

1. A fraction enriched in plasma membranes of human tumour KB cell line, a permissive cell for adenovirus type 5, was obtained. 2. Electrophoresis of the membranes in polyacrylamide gels with buffers containing sodium dodecyl sulphate showed that the membranes after reduction with 2-mercaptoethanol contained over 20 polypeptide species. Three polypeptides were glycosylated and had apparent mol.wts. of 92000, 72000 and 62000. 3. The glycoproteins and the specific receptors responsible for adenovirus adsorption to the membranes were readily extracted into solutions containing low concentrations of Triton X-100. Glycolipids and proteins were also made soluble. A membranous residue obtained after Triton X-100 extraction was enriched in several proteins that appeared to consist of polypeptides of lower molecular weight than the average of KB membrane polypeptides. 4. Sphingomyelin, cholesterol and triglycerides were similarly concentrated in the insoluble residue remaining after successive extractions of KB membranes with Triton X-100. Further, ceramide trihexoside was significantly less easily extracted from KB membranes than lactosyl ceramide. 5. The differences noted in the ease of extraction of membrane components are discussed. 6. The components of membranes made soluble by detergent extraction and containing the large part of the KB membrane glycoproteins were subjected to chromatography on Sepharose 6B and DEAE-cellulose and to isoelectric focusing in the presence of buffers containing Triton X-100. In general, the degree of separation into fractions enriched in individual glycoproteins was disappointing. Possible reasons for the poor fractionation of membrane components by chromatographic systems conveniently used for purification of proteins and glycoproteins of non-membranous origin are briefly discussed.     

Some observations on the choice of detergent for solubilization of the human erythrocyte membrane.

Solubilization of the human erythrocyte membrane by seven detergents is described. Components released into the supernatant or retained in the residue were identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two non-ionic detergents exhibiting little u.v. absorption were more efficient than u.v.-absorbing Triton X-100. Evidence is presented of an interchange between protein PAS 1 and protein PAS 2.     

Effects of different surfactants on motility and DNA integrity of brown trout (Salmo trutta fario) and common carp (Cyprinus carpio) spermatozoa

In this study we evaluated effects of surfactants on motility parameters and DNA integrity of spermatozoa of freshwater teleost fish. Common carp (Cyprinus carpio) and brown trout (Salmo trutta fario) spermatozoa were exposed to either sodium dodecyl sulphate (SDS, anionic surfactant) or octoxynol 9 ( Triton X-100, nonionic surfactant). Both surfactants added at activation caused a decrease in sperm motility characteristics measured by computer-assisted sperm analysis (CASA). Intraspecific differences in speed and trajectory of movement were detected. Triton X-100 and SDS when added to non activated sperm were also effective in the decrease of sperm motility and caused an increase of DNA fragmentation. Our results suggest that not only sperm motility apparatus but also DNA are targets for surfactant action. Therefore any exposure of spermatozoa to surfactants, in aquaculture conditions or natural environment, would have a negative impact on fish reproduction.     

A comparison of ornithine aminotransferase from human and rat sources

Ornithine aminotransferase was purified from human liver, rat liver and rat kidney. Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a subunit molecular weight of 45,000 in all three cases. Estimations of the native molecular weights of ornithine aminotransferase were determined by Sephadex G-200 chromatography in the presence and absence of 0.1% (w/v) Triton X-100. Human and rat enzymes were tetrameric in the presence of detergent but the rat subunits aggregated further in its absence. Characterisation of ornithine aminotransferase from the two rat sources indicated that they were the same protein. The human and rat enzymes were similar but not identical.     

The molecular size of N-methylglutamate dehydrogenase of Pseudomonas aminovorans.

N-Methylglutamate dehydrogenase, purified to a specific activity of 0.29 unit/mg of protein, gave one band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a molecular weight of 130 000. Enzyme-Triton complexes were found to have a partial specific volume of 0.73 cm3/g, suggesting that the protein binds less than 0.1 g of Triton/g of protein. A molecular weight for the intact enzyme in the presence of 1% (w/v) Triton X-100 of 550 000 suggested that the enzyme may be a tetramer.     

A simple and rapid procedure for removal of triton X-100 from protein solution

A simple and rapid procedure for removal of Triton X-100 from protein solutions is described. The procedure is based on the extraction of Triton X-100 with chloroform from the protein solution. By the addition of sodium dodecyl sulfate before the extraction, the procedure was improved effectively and the sample thus prepared was used directly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method can be used for the removal of other nonionic detergents and for samples of larger size.     

Radioimmunoassay of the 148-kilodalton cartilage protein. Distribution of the protein among bovine tissues.

A sensitive and specific radioimmunoassay for the 148 kDa cartilage protein (previously referred to as 'cartilage matrix protein') was developed. The protein is insoluble in conventional buffers and was therefore dissolved in sodium dodecyl sulphate. Excess sodium dodecyl sulphate was bound in mixed micelles with Triton X-100 before assay. Antibodies raised against the 148 kDa cartilage protein did not react with other cartilage macromolecules. Neither did they react with guanidinium chloride extracts of a number of non-cartilaginous tissues. The protein, then, is unique for cartilage. It was demonstrated in tracheal, nasal-septum, xiphisternal, auricular and epiphysial cartilage. Surprisingly, the protein was detected neither in extracts of articular cartilage nor in extracts of the anulus fibrosus or the nucleus pulposus of the intervertebral disc.     

Cell surface proteins of encapsulated Streptococcus cremoris: identification and immunochemical characterization

Cell surface proteins of two slime-forming, encapsulated Streptococcus cremoris strains, MLS96 and T₅ from the fermented milk product viili, were extracted with the non-ionic detergent Triton X-100. The isolated protein antigens were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immuno-blotting with antisera produced against whole Strep. cremoris cells. When protein profiles of these strains were compared, seven prominent polypeptides were found common to both and were recognized by both antisera. Five of these polypeptides with molecular weights of 70000, 54000, 50000, 47000 and 40000 were identified as cell wall components. The remaining two polypeptides with molecular weights of 42000 and 26000 are being studied further in connection with slime formation for which modified Triton X-100 extraction provides a suitable method for isolation of the surface-associated antigens of lactic streptococci.