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1.
An enzyme-linked immunosorbent assay (ELISA)-based glycosyltransferase assay has been used to measure UDP-Gal:N-acetylglucosamine beta-1,4-galactosyl-transferase (EC 2.4.1.38) activity in detergent extracts of chinese hamster ovary (CHO) cells. LEC11 cells (a mutant of the CHO cell line, Pro -5), which are known to express a complex array of carbohydrate structures, were used to develop the assay for use with whole cell extracts. A detergent-solubilized preparation of the enzyme from whole cells was used to convert the substrate, lactotriglycosylceramide, to the product, neolactotetraglycosylceramide. The monoclonal antibody, 1B2, which specifically binds to the Gal beta 1-4GlcNAc epitope, was used in an ELISA to identify and quantify the product. The enzyme activity in the preparations was found to be similar to that obtained by conventional radioactive assay methods. The beta-galactosyltransferase found in LEC11 cell detergent extracts exhibited an absolute requirement for the nucleotide sugar and MnCl2. The activity of the enzyme was also strictly dependent on the presence of exogenous glycolipid acceptor. When Triton X-114 was used to solubilize the LEC11 beta-galactosyltransferase, activity was found in both the hydrophilic and the hydrophobic phases, suggesting the presence of two forms of the enzyme. The ELISA-based assay was used to compare beta-1,4-galactosyltransferase activity in detergent extracts of four CHO cell lines: Pro-5, Lec1, LEC11, and LEC12 and in detergent-solubilized microsomes from human leukemia cells. The results from this study demonstrate the utility of the ELISA-based assay for measuring glycosyltransferase activity in detergent-solubilized whole cells and microsome preparations. 相似文献
2.
The development of an enzyme-linked immunosorbent assay (ELISA) for uridine 5'-diphospho-N-acetyl-glucosamine: alpha mannoside beta 1----6 N-acetylglucosaminyltransferase (GnT-V) is reported. The assay quantitates the enzymatic conversion of the specific synthetic GnT-V acceptor GlcNAc beta 1----2Man alpha 1----6Man beta-R (5) to the product GlcNAc beta 1----2[GlcNAc-beta 1----6]Man alpha 1----6Man beta-R (6) when these oligosaccharide structures were covalently attached to bovine serum albumin which was then coated on microtiter wells. Conversion of 5 to 6 was detected using a polyclonal antiserum raised against the product 6 and from which antibodies cross-reacting with acceptor 5 had been removed by affinity adsorption. GnT-V activity detected by ELISA was linearly proportional to both enzyme concentration and time under appropriate experimental conditions where 50-300 fmol of product was formed per microtiter well. GnT-V activity could be measured by ELISA in Triton X-100 extracts of hamster kidney acetone powder and in human serum. The twofold increase in GnT-V activity which is known to accompany Rous sarcoma virus transformation of baby hamster kidney cells could also be quantitated using the ELISA. 相似文献
3.
The detergent solubility properties of a malarial (Plasmodium knowlesi) variant antigen expressed on the surface of infected erythrocytes 总被引:2,自引:0,他引:2
Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase-catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X-100, or the zwitterion 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). After addition of Triton X-100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pk1(A+), which has 125I-variant antigens of Mr 210,000 and 190,000, and with clone Pk1(B+)1+, which has variant antigens of Mr 200,000-205,000. SDS yielded maximal levels of immunoprecipitated 125I-variant antigens. Variant-specific immunoprecipitation was detected in some experiments with Triton X-100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X-100-insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X-100-soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I-variant antigens either have a structure that renders them intrinsically insoluble in Triton X-100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents. 相似文献
4.
Thirion J Wattiaux R Jadot M 《Biology of the cell / under the auspices of the European Cell Biology Organization》2003,95(2):99-105
We found a dipeptidyl aminopeptidase activity in the parasitic protozoan Giardia lamblia with properties similar to the lysosomal cathepsin C of rat-liver lysosomes. Subcellular fractionation of this parasite indicated that the cathepsin C activity is located in organelles not distinguishable from the ones containing acid phosphatase, a known marker enzyme of Giardia lysosome-like peripheral vesicles. Contrary to the rat lysosomal enzyme, Giardia cathepsin C behaved like a membrane protein. Moreover, the enzyme was not solubilized by Triton X-100 or Triton X-100/SDS at 0 degrees C but could be substantially solubilized by octylglucoside, Triton X-100 at 37 degrees C or by a pretreatment with the cholesterol complexing agent beta-cyclodextrin before the Triton/SDS treatment carried out at 0 degrees C. These observations suggest that binding/anchorage of this enzyme to membranes occurs in cholesterol-rich microdomains. 相似文献
5.
The hydrolytic activity of the ATPase bound to purified inner membrane vesicles of rat liver mitochondria can be increased threefold by washing extensively with a high ionic strength phosphate buffer. The specific ATPase activities of such phosphate-washed membranes are the highest reported to date for a mitochondrial membrane preparation (21-24 mumol of ATP hydrolyzed min-1 mg-1 in bicarbonate buffer at 37 degrees C). Deoxycholate (0.1 mg/mg of protein) extracts from these membranes a soluble, cold-stable ATPase complex which exhibits a specific activity under optimal assay conditions of 12 mumol of ATP hydrolyzed min-1 mg-1. This complex is not sedimented by centrifugation at 201000 g for 90 min, and readily passes through a 250-A Millipore filter. The ATPase activity of the soluble complex is inhibited 95% by 2.4 muM oligomycin. In addition, inhibitions of 60% or better are obtained in the presence of 1-8 muM dicyclohexylcarbodiimide, p-chloromercuribenzoate, venturicidin, and aurovertin. While a similar complex may be extracted with Triton X-100 this preparation is always lower in both specific activity and in inhibitor sensitivities than the complex extracted with deoxycholate. Detergents of the Tween and Brij series and other detergents of the Triton series are also much less effective than deoxycholate in solubilizing the oligomycin-sensitive. ATPase complex of rat liver. It is concluded that deoxycholate is superior to other detergents as an extractant of the oligomycin-sensitive ATPase complex of rat liver mitochondria, and that the complex extracted with deoxycholate possesses a closer similarity to the membrane-associated ATPase than does the complex extracted with Triton X-100. These studies document the first report of a detergent-solubilized, oligomycin-sensitive ATPase preparation from rat liver mitochondria. 相似文献
6.
Immunochemical Analysis of Triton X-100-Insoluble Residues from Micrococcus lysodeikticus Membranes
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Triton X-100-insoluble residues from Micrococcus lysodeikticus membranes were analyzed by crossed immunoelectrophoresis after dispersal of the residues in sodium dodecyl sulfate (SDS). Conditions which produce no obvious distortion of the immunoprecipitate profile and which allow qualitative and quantitative analyses of the antigens present in the extracts are described. Two main antigens were detected; these were identified as succinate dehydrogenase (EC 1.3.99.1) and adenosine triphosphatase (EC 3.6.1.3). As determined by peak area estimations, the maximal release of succinate dehydrogenase and of adenosine triphosphatase from Triton X-100-insoluble membrane residues occurred at protein/SDS ratios of about 4.3:1 (0.2% SDS) and 6.8:1 (0.13% SDS), respectively. A comparison of enzyme activities of SDS extracts with those of untreated, control Triton X-100-insoluble membrane residues indicated that both the succinate dehydrogenase and the adenosine triphosphatase antigens were released with a full (or enhanced) catalytic potential at or below concentrations of SDS required to effect maximal solubilization of the enzyme in question. Evidence is also presented to suggest that the more acidic of the two components detected by crossed immunoelectrophoresis for the heterogeneous adenosine triphosphatase antigen is more sensitive to SDS than is the other. Both succinate dehydrogenase and adenosine triphosphatase lost catalytic activity and were denatured at protein/SDS ratios lower than 3.4:1. 相似文献
7.
Khraltsova LS Sablina MA Melikhova TD Joziasse DH Kaltner H Gabius HJ Bovin NV 《Analytical biochemistry》2000,280(2):250-257
UDP-Gal:Galbeta1-4GlcNAc alpha1,3-galactosyltransferase (alpha3GalT) is responsible for the synthesis of carbohydrate xenoantigen Galalpha1-3Galbeta1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of alpha3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Galbeta1-4GlcNAc (acceptor) are incubated with alpha3GalT in the presence of "cold" UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin. The standard curve for correct quantification of alpha3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl-donor UDP-Gal. In accordance with the known specificity of alpha3GalT, no enzymatic conversion of Le(x) into GalalphaLe(x) was observed using this assay. Human alphaGal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive. 相似文献
8.
Korekane H Taguchi T Sakamoto Y Honke K Dohmae N Salminen H Toivonen S Helin J Takio K Renkonen O Taniguchi N 《Glycobiology》2003,13(5):387-400
A rat intestinal beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified to apparent homogeneity by successive column chromatographic procedures using an assay wherein pyridylaminated lacto- N-triose II (GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcbeta1-3'LacNAc into GlcNAcbeta1-3'(GlcNAcbeta1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcbeta1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galbeta1-3GalNAcalpha1-O-paranitrophenyl (pNP) and GlcNAcbeta1-3GalNAcalpha1-O-pNP, into Galbeta1-3(GlcNAcbeta1-6) GalNAcalpha1-O-pNP (C2GnT activity) and GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-pNP (C4GnT activity), respectively. Based on the partial amino acid sequences of the purified protein, the cDNA encoding this enzyme was cloned. The COS-1 cells transiently transfected with this cDNA had high dI/C2/C4GnT activities in a ratio of 0.34:1.00:0.90, compared with non- or mock-transfected cells. The primary structure shows a significant homology with human and viral mucin-type core 2 beta1-6GnTs (C2GnT-Ms), indicating that this enzyme is the rat ortholog of human and viral C2GnT-Ms. This is the first identification and purification of this enzyme as a major carrier of dIGnT activity in the small intestine. This rat ortholog should mostly be responsible for making distal I-branch structures on poly-N-acetyllactosamine sequences in this tissue, as well as making mucin core 2 and core 4 structures, given that it also has high C2/C4GnT activities. 相似文献
9.
Rajesh Naz 《Molecular reproduction and development》1998,51(2):210-217
The presence and possible role of c-Abl proto-oncoprotein was investigated in human sperm cell. The c-Abl monoclonal antibody (mAb), against the protein tyrosine kinase domain of v-Abl protein, reacted specifically with the acrosomal region of methanol-fixed capacitated and non-capacitated human sperm cell in the indirect immunofluorescence technique. The c-Abl mAb predominantly recognized two protein bands of 145 kD and 95 kD in detergent-solubilized (Triton X-100 and NP-40) sperm and testes preparations in the Western blot procedure. The 95 kD protein band reacted stronger than the 145 kD band and was the only band detected in the lithium diiodosalicylate (LIS)–solubilized sperm preparation, and even in the Triton X-100/NP-40 extracts of sperm of some men. In the in vitro kinase assay using the Triton X-100–solubilized capacitated sperm preparation, the 95 kD protein was autophosphorylated at the tyrosine residues, which was inhibited in the presence of c-Abl mAb. The tyrosine phosphorylation of sperm proteins, especially of the 95 kD protein, has been shown to have a vital role in human sperm function, namely, the sperm capacitation/acrosomal exocytosis and binding to zona pellucida of oocyte. These findings suggest that the c-Abl or c-Abl-like proteins are present in mature sperm cells that are tyrosine autophosphorylated and may have a role in human sperm cell function. Mol. Reprod. Dev. 51:210–217, 1998. © 1998 Wiley-Liss, Inc. 相似文献
10.
Extraction of 11 beta-hydroxysteroid dehydrogenase from rat liver microsomes by detergents 总被引:1,自引:0,他引:1
In these studies our goal was to solubilize the microsomal enzyme, 11 beta-hydroxysteroid dehydrogenase (11-HSD) as the first step in its purification. Enzyme was extracted from rat liver microsomes with representative detergents (Zwittergents, Tritons, modified sterols). Oxidation-reduction (O-R) ratios of extracts varied with detergent used and ranged from 0.18 (CHAPS) to 3.8 (Zwittergent 3-14) relative to a ratio of 1.7 in intact microsomes. All detergents solubilized 11-HSD using lack of sedimentation during high speed centrifugation as criterion. With Triton DF-18 and Triton X-100, optimum extraction of 11-HSD occurred in the detergent-protein ratio range of 0.1 to 0.2 O-R ratios decreased with increased Triton X-100, but were constant as Triton DF-18 was varied. The pH optimum of enzyme extraction was 9 at a detergent-protein ratio of 0.05 and 7.5-8.0 at a ratio of 0.2. Sodium chloride increased enzyme extraction by detergents; in the absence of detergent, salt extracted protein, but not enzyme. In aqueous solution at 0 degrees C or -15 degrees C, microsomal 11-oxidation activity rose within 24 h, then decreased; reductase activity consistently decreased. Oxidation and reduction activities were inversely related in the microsomal bound enzyme. No relationship between these activities appeared in detergent-solubilized enzymes. Possible mechanisms to account for the unexpected behavior of this enzyme are discussed. 相似文献
11.
Bumgarner GW Zampell JC Nagarajan S Poloso NJ Dorn AS D'Souza MJ Selvaraj P 《Journal of biochemical and biophysical methods》2005,64(2):99-109
A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 degrees C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 degrees C. The combination of saponin/Triton X-100 at 4 degrees C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 degrees C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl beta-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54). 相似文献
12.
Steven R. Coats Warren J. Pledger Midori Awazu Thomas O. Daniel 《Journal of cellular physiology》1996,168(2):412-423
Current models of platelet-derived growth factor (PDGF) β receptor itinerary are based upon the properties of receptors recovered from nonionic detergent-solubilized cellular extracts. Comparing several commonly used cell extraction procedures, we have determined that up to 50% of immunoreactive PDGF β receptors reside in a Triton X-100 insoluble pool in a wide distribution of cultured cell lines, including Balb/c-3T3, NIH 3T3, and Swiss fibroblasts, primary murine and human fibroblasts, and primary human glial cells. Many properties of Triton insoluble receptors are distinct from the well-characterized PDGF β receptors, including (1) delayed arrival of newly synthesized receptors into the Triton insoluble fraction, (2) prolonged half-life in the presence of PDGF, (3) increased abundance with increasing cell density, (4) inaccessibility to modification by extracellular compartment enzymes, (5) cofractionation with cytoskeletal proteins, and (6) a higher basal tyrosine phosphorylation state. PDGF stimulates accumulation of tyrosine phosphorylated PDGF β receptors in the Triton X-100 insoluble fraction. Cell surface PDGF β receptors modified by enzymatic desialylation redistribute to the insoluble fraction. These findings distinguish the itinerary of a large subpopulation of PDGF β receptors from those characterized previously. Receptors in this fraction represent a long-lived tyrosine phosphorylated population that may effect responses for extended periods following ligand activation. © 1996 Wiley-Liss, Inc. 相似文献
13.
The activity of acetyl-CoA carboxylase, measured in various ways, was studied in 15000g extracts of rat liver hepatocytes and compared with the rate of fatty acid synthesis in intact hepatocytes incubated with insulin or glucagon. Hepatocyte extracts were prepared by disruption of cells with a Dounce homogenizer or by solubilization with 1.5% (v/v) Triton X-100. Sucrose-density-gradient centrifugation demonstrated that the sedimentation coefficient of acetyl-CoA carboxylase from cell extracts was 30-35S, regardless of the conditions of incubation or disruption of hepatocytes. Solubilization of cells with 1.5% Triton X-100 yielded twice as much enzyme activity (measured by [14C]bicarbonate fixation) in the sucrose-gradient fractions as did cell disruption by the Dounce homogenizer. Analysis by high-performance liquid chromatography of acetyl-CoA carboxylase reaction mixtures showed that [14C]malonyl-CoA accounted for 10-60% of the total acid-stable radioactivity, depending on the method for disrupting hepatocytes and on the preincubation of the 15000g extract, with or without citrate, before assay. Under conditions in which incubation of cells with insulin or glucagon caused an activation or inhibition, respectively, of acetyl-CoA carboxylase, only 25% of the acid-stable radioactivity was [14C]malonyl-CoA and enzyme activity was only 13% (control), 16% (insulin), and 57% (glucagon) of the rate of fatty acid synthesis. Under conditions when up to 60% of the acid-stable radioactivity was [14C]malonyl-CoA and acetyl-CoA carboxylase activity was comparable with the rate of fatty acid synthesis, there was no effect of insulin or glucagon on enzyme activity. 相似文献
14.
The present method of quantitating soluble asialoglycoprotein (galactosyl) receptor activity relies on the selective precipitation of receptor-ligand complexes to allow separation from free ligand. To provide an alternative to selective precipitation procedures, a simple and rapid method to assay for detergent-solubilized galactosyl receptor activity has been developed which uses permeabilized, fixed cells as a source of immobilized solid-phase receptors. Isolated rat hepatocytes were treated with digitonin to make available the internal as well as the external receptors. The permeable cells were also treated with glutaraldehyde to prevent further protein loss during subsequent exposure to detergents such as Triton X-100. The permeable/fixed cells, which retained about 70% of their total 125I-asialo-orosomucoid (125I-ASOR)-binding activity, with 89% specific binding, were insoluble even in 0.5% Triton X-100 and were easily pelleted. The permeable/fixed cells can be prepared in advance and stored frozen for months. A detergent extract of receptor is mixed with a constant amount of both 125I-ASOR and permeable/fixed cells. Soluble active receptors compete with immobilized receptors on the treated cell for binding of the 125I-ASOR. The assay is reproducible, linear over a broad range of soluble receptor concentration, and can quantitate receptor activity from as few as 10(5) hepatocytes. A modified purification procedure for the rat hepatic galactosyl receptor using this competition assay is also described. 相似文献
15.
R R Russell 《Analytical biochemistry》1979,97(1):173-175
Triton X-100 was used to overcome the inhibition by sodium dodecyl sulfate (SDS) of fructosyltransferase from Streptococcus mutants. This procedure allowed the detection of enzyme activity in a tube assay and also after SDS-gel electrophoresis. 相似文献
16.
Lubov' S. Khraltsova Marina A. Sablina Tatyana D. Melikhova David H. Joziasse Herbert Kaltner Hans-J. Gabius Nicolai V. Bovin 《Analytical biochemistry》2000,280(2):250
UDP-Gal:Galβ1-4GlcNAc α1,3-galactosyltransferase (α3GalT) is responsible for the synthesis of carbohydrate xenoantigen Galα1-3Galβ1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of α3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Galβ1-4GlcNAc (acceptor) are incubated with α3GalT in the presence of “cold” UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin. The standard curve for correct quantification of α3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl-donor UDP-Gal. In accordance with the known specificity of α3GalT, no enzymatic conversion of Lex into GalαLex was observed using this assay. Human αGal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive. 相似文献
17.
Limited proteolysis was used to probe and compare the conformation of the rat lung vasoactive intestinal peptide (VIP) receptor in membrane-bound and detergent-solubilized states. It had been shown previously that the activity of the detergent-solubilized VIP receptor is sensitive to the nature of the detergent used for extraction (Patthi, S., Simerson S. and Velicelebi, G. (1988) J. Biol. Chem., 263, 19363-19369). Receptors that were extracted from the membrane using digitonin retained the ability to bind 125I-VIP, while those solubilized in Triton X-100 displayed little or no detectable activity. In order to correlate the differences observed in the activity of the receptor with its folded state, membrane-bound and detergent-solubilized receptors were covalently labeled with 125I-VIP and subjected to limited proteolysis using trypsin, chymotrypsin or carboxypeptidase Y. Digitonin-solubilized receptors most closely resembled the membrane-bound protein in terms of protease sensitivity and proteolytic cleavage products. By contrast, receptors solubilized in Triton X-100 displayed increased sensitivity to proteases and produced distinctly different proteolytic patterns. Thus, the differences observed in the activities of receptors solubilized in digitonin and those solubilized in Triton X-100 could be correlated with detectable differences in the conformation of the protein in each respective detergent solution. These results suggest that digitonin provides an environment that is more compatible with the native folded state of the receptor, similar to its conformation in the membrane. 相似文献
18.
Yuan X Ren F Zeng G Zhong H Fu H Liu J Xu X 《Applied microbiology and biotechnology》2007,76(5):1189-1198
The adsorption behavior of five surfactants, cetyltrimethylammonium bromide (CTAB), Triton X-100, Tween 80, sodium dodecyl
sulfate (SDS), and rhamnolipid, on a Pseudomonas aeruginosa strain and the effect of temperature and ionic strength (IS) on the adsorption were studied. The change of cell surface lypohydrophilic
property caused by surfactant adsorption was also investigated. The results showed that the adsorption kinetics of the surfactants
on the cell followed the second-order law. CTAB adsorption was the fastest one under the experimental conditions, and it took
longest for SDS adsorption to equilibrate because of electric repulsion. The adsorption of Triton X-100 and Tween 80 was characterized
by short equilibration time, and rhamnolipid adsorption reached equilibrium in about 90 min. The adsorption isotherms of all
the surfactants on the bacterium fitted Freundlich equation well, but the adsorption capacity and mode were variations for
the surfactants as indicated by k and n parameters in the equations. The adsorption mode for all the surfactants except SDS is probably hydrophilic interaction because
the adsorption totally turned the cell surface to be more hydrophobic. Neither the temperature nor the IS had significant
effect on CTAB adsorption, but higher IS significantly enhanced SDS adsorption and modestly strengthened adsorption of Triton
X-100, Tween 80, and rhamnolipid. Higher temperature strengthened adsorption of SDS but weakened the adsorption of Triton
X-100, Tween 80, and rhamnolipid. 相似文献
19.
The Galbeta1-->4(SO(3)(-)-->6)GlcNAc moiety is present in various N-linked and O-linked glycans including keratan sulfate and 6-sulfosialyl-Lewis X, an L-selectin ligand. We previously found beta1,4-galactosyltransferase (beta4GalT) activity in human colonic mucosa, which prefers GlcNAc 6-O-sulfate (6SGN) as an acceptor to non-substituted GlcNAc (Seko, A., Hara-Kuge, S., Nagata, K., Yonezawa, S., and Yamashita, K. (1998) FEBS Lett. 440, 307-310). To identify the gene for this enzyme, we purified the enzyme from porcine colonic mucosa. The purified enzyme had the characteristic requirement of basic lipids for catalytic activity. Analysis of the partial amino acid sequence of the enzyme revealed that the purified beta4GalT has a similar sequence to human beta4GalT-IV. To confirm this result, we prepared cDNA for each of the seven beta4GalTs cloned to date and examined substrate specificities using the membrane fractions derived from beta4GalT-transfected COS-7 cells. When using several N-linked and O-linked glycans with or without 6SGN residues as acceptor substrates, only beta4GalT-IV efficiently recognized 6SGN, keratan sulfate-related oligosaccharides, and Galbeta1-->3(SO(3)(-)-->6GlcNAcbeta1-->6) GalNAcalpha1-O-pNP, a precursor for 6-sulfosialyl-Lewis X. These results suggested that beta4GalT-IV is a 6SGN-specific beta4GalT and may be involved in the biosynthesis of various glycoproteins carrying a 6-O-sulfated N-acetyllactosamine moiety. 相似文献
20.
The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20–30 kdalton range.The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60–80 kdalton region and an increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed. 相似文献