scholarly journals The solubilization of a glucuronyltransferase involved in pea (Pisum sativum var. Alaska) glucuronoxylan synthesis

1989 ◽  
Vol 264 (3) ◽  
pp. 643-649 ◽  
Author(s):  
K W Waldron ◽  
E A H Baydoun ◽  
C T Brett
Keyword(s):  
Pisum Sativum ◽  
Cholic Acid ◽  
Gel Filtration ◽  
Partial Separation ◽  
Ionic Detergent ◽  
Solubilized Enzyme ◽  
Triton X

A glucuronyltransferase involved in glucuronoxylan biosynthesis was obtained from the epicotyls of 1-week-old etiolated pea (Pisum sativum var. Alaska) seedlings and was solubilized in Triton X-100, a non-ionic detergent. The enzyme was inactivated by SDS and inhibited by Derriphat 160 and cholic acid. The enzyme was active in the presence of NN-dimethyldodecylanium-N-oxide, but was not solubilized by it. The stimulatory effect of UDP-D-xylose on the particulate and solubilized enzymes was the same, but the optimum Mn2+ concentration was lower for the solubilized enzyme, and the product formed by the solubilized enzyme has altered structure and solubility properties. Gel filtration of the solubilized enzyme on Sepharose CL-6B permitted partial separation of the stimulatory effect of UDP-D-xylose from the activity in the absence of UDP-D-xylose. The solubilized enzyme was more stable than the particulate enzyme and could be stored for 2 weeks at -20 degrees C without loss of activity.

scholarly journals Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

1984 ◽  
Vol 32 (7) ◽  
pp. 690-696 ◽  
Author(s):  
J Fischer ◽  
G Uhlenbruck ◽  
P J Klein ◽  
M Vierbuchen ◽  
R Fischer
Keyword(s):  
Molecular Weight ◽  
Gastric Mucosa ◽  
High Molecular Weight ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Gastric Cancer Cells ◽  
Tissue Sections ◽  
Gradient Gel Electrophoresis ◽  
Triton X

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

scholarly journals Effects of detergent on the sulphation of chondroitin by cell-free preparations from chick-embryo epiphyseal cartilage

1992 ◽  
Vol 285 (2) ◽  
pp. 577-583 ◽  
Author(s):  
G Sugumaran ◽  
J E Silbert
Keyword(s):  
Chick Embryo ◽  
Supernatant Fraction ◽  
Random Pattern ◽  
Type I ◽  
Type Ii ◽  
Enzyme Preparations ◽  
Enzyme Substrate ◽  
Ionic Detergent ◽  
The Individual ◽  
Triton X

The effects of the non-ionic detergent Triton X-100 on 6-sulphation of two species of endogenous nascent proteochondroitin by a chick-embryo cartilage microsomal system was examined. Sulphation of the larger (Type I) species with adenosine 3′-phosphate 5′-phosphosulphate was slightly diminished when Triton X-100 was present, whereas sulphation of the smaller (Type II) species was slightly enhanced. An ordered rather than random pattern of sulphation was obtained for the smaller proteoglycan, but with a considerably lower degree of sulphation than that of the larger proteochondroitin. These differences were consistent with other differences between these two species as described previously. Sulphation of exogenous [14C]chondroitin and exogenous proteo[3H]chondroitin by the microsomal system with Triton X-100 present produced ordered rather than random sulphation patterns. When a 100,000 g supernatant fraction was utilized for sulphation of [14C]chondroitin or proteo[3H]chondroitin, Triton X-100 was not needed, and ordered sulphation was still obtained. When hexasaccharide was used, sulphation of multiple N-acetylgalactosamine residues of the individual hexasaccharides resulted. This was relatively independent of Triton X-100 or the concentration of the hexasaccharide acceptors. With soluble enzyme, sulphation of multiple N-acetylgalactosamine residues on the individual hexasaccharide molecules was even greater, so that tri-sulphated products were found. This suggests that ordered rather than random sulphation of chondroitin with these enzyme preparations is due to enzyme-substrate interaction rather than to membrane organization.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

scholarly journals Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114

1991 ◽  
Vol 280 (3) ◽  
pp. 745-751 ◽  
Author(s):  
N M Hooper ◽  
A Bashir
Keyword(s):  
Phase Separation ◽  
Membrane Proteins ◽  
Aqueous Phase ◽  
Hydrophobic Membrane ◽  
Rich Phase ◽  
Glycosyl Phosphatidylinositol ◽  
Ionic Detergent ◽  
Membrane Spanning ◽  
Triton X ◽  
Insoluble Pellet

Treatment of kidney microvillar membranes with the non-ionic detergent Triton X-114 at 0 degrees C, followed by low-speed centrifugation, generated a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and a detergent-depleted or aqueous phase. Those ectoenzymes with a covalently attached glycosyl-phosphatidylinositol (G-PI) membrane anchor were recovered predominantly (greater than 73%) in the detergent-insoluble pellet. In contrast, those ectoenzymes anchored by a single membrane-spanning polypeptide were recovered predominantly (greater than 62%) in the detergent-rich phase. Removal of the hydrophobic membrane-anchoring domain from either class of ectoenzyme resulted in the proteins being recovered predominantly (greater than 70%) in the aqueous phase. This technique was also applied to other membrane types, including pig and human erythrocyte ghosts, where, in both cases, the G-PI-anchored acetylcholinesterase partitioned predominantly (greater than 69%) into the detergent-insoluble pellet. When the microvillar membranes were subjected only to differential solubilization with Triton X-114 at 0 degrees C, the G-PI-anchored ectoenzymes were recovered predominantly (greater than 63%) in the detergent-insoluble pellet, whereas the transmembrane-polypeptide-anchored ectoenzymes were recovered predominantly (greater than 95%) in the detergent-solubilized supernatant. Thus differential solubilization and temperature-induced phase separation in Triton X-114 distinguished between G-PI-anchored membrane proteins, transmembrane-polypeptide-anchored proteins and soluble, hydrophilic proteins. This technique may be more useful and reliable than susceptibility to release by phospholipases as a means of identifying a G-PI anchor on an unpurified membrane protein.

scholarly journals Acetylcholinesterase aus Rindererythrozyten. Reinigung und Eigenschaften des mit und ohne Triton X-100 solubilisierten Enzyms / Acetylcholinesterase from Bovine Erythrocytes. Purification and Properties of the En­zyme Solubilized in the Presence and the Absence of Triton X-100

1979 ◽  
Vol 34 (9-10) ◽  
pp. 721-725 ◽  
Author(s):  
Heinz Großmann ◽  
Manfred Liefländer
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Multiple Forms ◽  
Terminal Amino Acid ◽  
Enzyme Preparations ◽  
Purification And Properties ◽  
Solubilized Enzyme ◽  
Terminal Amino ◽  
Triton X ◽  
Bovine Erythrocytes

Abstract Acetylcholinesterase was released from bovine erythrocytes by Triton X-100 treatment and pu­rified by twofold affinity chromatography. The detergentfree enzyme was obtained with a specific activity of 4130 U /mg (303 000-fold purification) and a 25% yield. Alternatively, the commercial available crude enzyme was purified. The latter preparation has an uniform molecular weight (Mr 175 000). The Triton-solubilized enzyme, however, can be resolved after removal of the detergent in eight multiple forms (Mr 175 000 and multiple values), in the presence of Triton there exists only one form (Mr 338 000). The amino acid composition of the two enzyme preparations differs significantly. No differences were observed with respect to other properties: SDS gel electrophore­sis revealed two protein bands (Mr 166 000 and 86 000) with both preparations. The enzyme is a glycoprotein with a pI value of 4.3 and contains strongly bound phosphatidylethanolamine. The N-terminal amino acid has been found to be Glu (or Gin).

scholarly journals Gel Filtration Chromatographic Study on the Micelle Formation of Triton X-100

1989 ◽  
Vol 62 (6) ◽  
pp. 1725-1730 ◽  
Author(s):  
Noriaki Funasaki ◽  
Sakae Hada ◽  
Saburo Neya
Keyword(s):  
Gel Filtration ◽  
Micelle Formation ◽  
Chromatographic Study ◽  
Triton X

Dual effect of non-ionic detergent Triton X-100 on insulin amyloid formation

2019 ◽  
Vol 173 ◽  
pp. 709-718 ◽  
Author(s):  
Katarina Siposova ◽  
Erik Sedlak ◽  
Tibor Kozar ◽  
Michal Nemergut ◽  
Andrey Musatov
Keyword(s):  
Amyloid Formation ◽  
Dual Effect ◽  
Ionic Detergent ◽  
Triton X

scholarly journals Differential effects of non-ionic detergents on microsomal and sarcolemmal adenylate cyclase in cardiac muscle

1978 ◽  
Vol 175 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Prakash V. Sulakhe ◽  
Njanoor Narayanan
Keyword(s):  
Enzyme Activity ◽  
Sarcoplasmic Reticulum ◽  
Adenylate Cyclase ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Protein Ratio ◽  
Guinea Pig Heart ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Ionic Detergent ◽  
Triton X

1. About 4 and 23% of the homogenate adenylate cyclase activity was recovered in the microsomal and sarcolemmal fractions isolated from guinea-pig heart ventricles. 2. Cardiac microsomal adenylate cyclase activity [basal as well as p[NH]ppG (guanyl-5′-yl imidodiphosphate)- and NaF-stimulated] was increased over 2-fold in the presence of Lubrol-PX (0.01–0.1%). 3. The sarcolemmal enzyme, however, showed concentration-dependent inhibition caused by the detergent under all assay conditions, except when p[NH]ppG was included in the assay. In the latter case, the detergent (0.01–0.02%) caused a modest increase (30–45%) in enzyme activity. 4. Another non-ionic detergent, Triton X-100, also stimulated the microsomal cyclase and inhibited the sarcolemmal enzyme. 5. With either membrane fraction, Lubrol-PX solubilized the enzyme when the detergent/membrane protein ratio was 2.5 (μmol of detergent/mg of protein). 6. The findings with homogenate and a washed particulate fraction resembled those obtained with sarcolemma, and those with isolated sarcoplasmic reticulum resembled those with microsomal preparations. 7. p[NH]ppG, and to some extent NaF, protected the detergent-induced inactivation of the enzyme observed at higher detergent concentrations (0.5% Lubrol-PX and 0.05–0.5% Triton X-100). 8. In the absence of detergents, p[NH]ppG increased the basal enzyme activity about 2-fold in microsomal fractions, but did not appreciably stimulate the sarcolemmal enzyme. Isoproterenol, on the other hand, increased the sarcolemmal enzyme activity (>2-fold) in the presence of p[NH]ppG and caused only moderate stimulation (31%) of the microsomal enzyme under these conditions. 9. These findings support the view that, although the bulk of adenylate cyclase resides in heart sarcolemma (plasma membrane), the microsomal activity cannot be accounted for solely by contamination of the microsomal fraction with sarcolemma, as has been suggested by others [Besch, Jones & Watanabe (1976) Circ. Res.39, 586–595; Engelhard, Plut & Storm (1976) Biochim. Biophys. Acta451, 48–61]. Further, the results of this study show that cardiac sarcoplasmic-reticulum membranes possess this enzyme.

Reaktionen der Hydrogenase aus Rhodopseudomonas capsulata im partikelgebundenen und gelösten Zustand

1969 ◽  
Vol 24 (5) ◽  
pp. 603-612 ◽  
Author(s):  
J.-H. Klemme
Keyword(s):  
Cytochrome C ◽  
Ammonium Sulfate ◽  
Deae Cellulose ◽  
Reaction Rates ◽  
Rhodopseudomonas Capsulata ◽  
Hydrogenase Activity ◽  
Original Activity ◽  
Dispersing Agent ◽  
Solubilized Enzyme ◽  
Triton X

In cell free extracts of Rps. capsulata obtained by exposure of cells to ultrasonic oscillation, about 90% of the hydrogenase is associated with the particulate chromatophore fraction. The particulate enzyme reacts with methylene blue (MB), menadione, phenazonium methosulfate (PMS), dichlorophenolindophenol (DCPIP), cytochrome c, p-benzoquinone (BQ), ferricyanide and O2,, but does not react with benzylviologen (BV), pyridinnucleotides and flavinnucleotides. Treatment of chromatophores with sodiumlaurylsulfate inactivates the hydrogenase reaction with PMS, DCPIP, BQ and ferricyanide. The MB-linked or menadione-linked hydrogenase is not destroyed by the detergent. The hydrogenase reaction with BV is increased more than 20-fold after incubation of the chromatophores with the lipid-dispersing agent. Treatment of chromatophores with acetone and petroleum ether almost completely inactivates the hydrogenase reaction with PMS and BQ. The reaction rate of the DCPIP-linked and the ferricyanide-linked hydrogenase is somewhat decreased, whereas the MB-linked, the menadione-linked and the BV-linked hydrogenase reactions still exhibit about 100% of the original activity. By extraction of the acetone-treated chromatophores with glycine-NaOH-buffer (pH 9), about 10 — 15% of the particulate hydrogenase is solubilized. The enzyme was 9-fold purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose. The purified enzyme contains no cytochrome. The relative reaction rates of the solubilized enzyme with different electron acceptors are similar to the corresponding reaction rates of the acetonetreated chromatophores. Extraction of chromatophores with n-butanol results in the solubilization of 5 — 10% of the particulate enzyme. By extraction of acetone-treated chromatophores with 0,5% Triton X-100, 40% of the particulate hydrogenase is solubilized. The fractionation of the extract with ammonium sulfate results in the isolation of a cytochrome c-containing particle which exhibits a 3-fold increased hydrogenase activity.

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