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1.
The sexual agglutinin from the mating-type minus gametes ofChlamydomonas reinhardtii was purified by gel filtration and hydroxyapatite chromotography. The minus agglutinin was identified as a single glycopolypeptide termed Agg(-) of very high molecular weight by SDS-poly-acrylamide gel electrophoresis. It was also observed as a glycoprotein with agglutinin activity on non-SDS polyacrylamide gels. The native agglutinin appeared to be composed of a complex of Agg(-) subunits. It consisted of about 60% protein and 40% carbohydrate and the activity was diminished by a mild periodate oxidation. When the plus agglutinin was also purified and compared with the minus agglutinin, it was found that both agglutinins migrate in the same position by SDS-polyacrylamide gel electrophoresis, whereas their behaviors on gel filtration and hydroxyapatite chromatography are different.Abbreviations
mt
+/–
mating-tape plus or minus
- SDS
sodium dodecyl sulfate
- Ve
elution volume
- Vo
void volume
- kDa
kilodalton 相似文献
2.
Isolation and composition of the constitutive agglutinins from haploid Saccharomyces cerevisiae cells 总被引:4,自引:0,他引:4
P. C. Sijmons A. J. A. Nederbragt F. M. Klis H. Van Den Ende 《Archives of microbiology》1987,148(3):208-212
Sex-specific agglutinins from the cell surface of haploid cells of Saccharomyces cerevisiae (X2180, mta and mt) were purified and analysed. The constitutive agglutinin from mta cells was extractable with 3 mM dithiothreitol. It was shown to be a glycoprotein (3% mannose) with an apparent Mr of 43,000 based on gel filtration, but in SDS-PAGE it behaved as a much smaller molecule (Mr between 18,000 and 26,000). About one in three amino acids was a hydroxyamino acid. Its biological activity was resistant to boiling for 1 h, but sensitive to pronase. Intact mt cells retained their agglutinability in the presence of dithiothreitol but limited trypsinizing released a biologically active agglutinin fragment. It had an apparent Mr of 320,000 (gel filtration). When analysed by SDS-PAGE, a single diffuse band with an apparent Mr of 225,000 was observed. The protein was 94% (w/w) mannose with a trace of N-acetyl glucosamine. Its biological activity was almost completely lost after boiling for 1 h. Both agglutinins behaved as monovalent molecules and specifically inhibited the biological activity of both noninduced and pheromone-induced cells. Pheromone treatment of mta cells resulted in an apparent 32-fold increase in agglutinin activity at the cell surface, whereas pheromone treatment of mt cells only doubled the apparent agglutinin activity.Abbreviations mt
mating type
- DTT
dithiothreitol
- SDS-PAGE
sodium dodecyl sulphate polyacrylamide gel electrophoresis
- YPG
yeast-peptone-glucose
- PAS
periodic-acid-Schiff reagent 相似文献
3.
Marieke R. Samson Frans M. Klis Wieger L. Homan Piet van Egmond Alan Musgrave Herman van den Ende 《Planta》1987,170(3):314-321
Sexual interaction between gametes of opposite mating type (mt) of the unicellular green alga Chlamydomonas eugametos starts with agglutination of the cells via particular glycoproteins on the flagellar surface. Purification of these socalled agglutinins was achieved by a three-step procedure consisting of, successively, gel filtration, anion-exchange chromatography, and high-performance gel filtration. The amino-acid and sugar compositions of both agglutinins showed a high degree of similarity; the most prominent amino acids were hydroxyproline, serine and glycine, and the main sugars were arabinose and galactose. The carbohydrate portions represented about half of the molecular mass of both agglutinins. Using high-performance gel filtration, a calibration curve was constructed for high-molecular-mass compounds from which the Stokes' radius of the sexual agglutinins could be estimated. The mt
+ agglutinin had a Stokes' radius of 39 nm and a sedimentation coefficient of 9.3 S. From these data its molecular mass was estimated to be 1.2·106. The corresponding data for the mt
- agglutinin were 38 nm, 9.7 S and 1.3·106, respectively. The biological activity of both agglutinins was destroyed by mild periodate treatment. Treatment with specific glycosidases had a differential effect on the biological activity of the agglutinins. These observations indicate that carbohydrate side-chains are needed for biological activity and perhaps are responsible for the specifity of the sexual agglutinins. A comparison of both agglutinins is given and their possible structure is discussed in relation to their amino-acid and sugar compositions.Abbreviations HP
high performance
-
mt
mating type
- SDS
sodium dodecyl sulfate 相似文献
4.
A new mannose-binding lectin was isolated from Sternbergia lutea bulbs by affinity chromatography on an α(1-2)mannobiose-Synsorb column and purified further by gel filtration. This lectin
(S. lutea agglutinin; SLA) appeared homogeneous by native-gel electrophoresis at pH 4.3, gel filtration chromatography on a Sephadex
G-75 column, and SDS-polyacrylamide gel electrophoresis, These data indicate that SLA is a dimeric protein (20 kDa) composed
of two identical subunits of 10 kDa which are linked by non-covalent interactions.
The carbohydrate binding specificity of the lectin was investigated by quantitative precipitation and hapten inhibition assays.
It is an α-D-mannose-specific lectin that interacts to form precipitates with various α-mannans, galactomannan and asialo-thyroglobulin,
but not with α-glucans and thyroglobulin. Of the monosaccharides tested only D-mannose was a hapten inhibitor of the SLA-asialothryroglobulin
precipitation system, whereas D-glucose, D-galactose and L-arabinose were not. The lectin appears to be highly specific for
terminal α(1-3)-mannooligosaccharides. The primary structure of SLA appears to be quite similar to that of the snow drop (Galanthus nivalis) bulb lectin which is a mannose-binding lectin from the same plant family Amaryllidaceae. The N-terminal 46 amino acid sequence
SLA showed 7% homology with that of GNA. Abbreviations: AAA, Allium ascalonicum agglutinin (shallot lectin); ASA, Allium sativum
agglutinin (garlic lectin); AUA, Allium ursinum agglutinin (ramsons lectin); DAP, 1,3-diaminopropane; GNA, Galanthus nivalis
agglutinin (snowdrop lectin); HHA, Hippeastrum hybr. agglutinin (amaryllis lectin); LOA, Listera ovata agglutinin (orchid
twayblade lectin); NPA, Narcissus pseudonarcissus agglutinin (daffodil lectin); PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered saline, SLA, Sternbergia lutea agglutinin; SDS, sodium dodecyl sulfate; Me, methyl; Bn, benzyl; PNP,
p-nitrophenyl.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
5.
An antitryptic activity has been identified in the flour of dry non-germinated seed of an African leguminous plant, the bambara pea (Voandzeia subterranea). This inhibitor has been purified by trichloracetic acid and ammonium sulphate precipitations followed by gel filtration on Sephadex G25, ion exchange chromatography and gel filtration on Sephadex G75. Antitryptic activity increased 50-fold. Its purity has been verified by electrophoresis on polyacrylamide gel and gel chromatography. Its MW is 13 200 in the denatured and reduced forms and 26 300 in the native form. It is resistant to thermal denaturation and appears to be in monomeric form when entirely denatured. 相似文献
6.
A new agglutinin from the Tulipa gesneriana bulbs 总被引:1,自引:0,他引:1
Two agglutinins with different agglutinating activity exist in Tulipa gesneriana bulbs. One is the T. gesneriana lectin which agglutinates yeasts as reported previously [Oda, Y. and Minami, K. (1986) Eur. J. Biochem. 159, 239-245]. The other agglutinin is a new one which agglutinates animal erythrocytes and was purified from the tulip bulbs using affinity chromatography on thyroglobulin-Sepharose 4B. The agglutinin agglutinated mouse and rat erythrocytes at a minimum concentration of 2 micrograms/ml and 30 micrograms/ml respectively, but did not agglutinate erythrocytes from other animals and yeasts even at a concentration of 1000 micrograms/ml. The agglutinin appeared homogeneous by disc gel electrophoresis at pH 4.3 and gel filtration. Its relative molecular mass was determined by gel filtration to be approximately 40,000. It was suggested that the agglutinin was composed of two different subunits of 26 kDa and 14 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis. Binding of radioiodinated agglutinin to mouse erythrocytes indicated that the presence of a high-affinity site with a dissociation constant of 2.00 X 10(-9) M. In inhibition experiments thyroglobulin glycopeptides were the most potent inhibitors; thyroglobulin was also a potent inhibitor. Orosomucoid and mucin showed weak inhibition. The other glycoproteins, glycopeptides and sugars examined showed no inhibition. 相似文献
7.
Ho Wong J Ng TB 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,828(1-2):130-135
A homotetrameric agglutinin with a molecular mass of 130 kDa was isolated from seeds of the haricot bean. The agglutinin was isolated using a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and gel filtration by fast protein liquid chromatography on Superdex 200. Haricot bean agglutinin was adsorbed on DEAE-cellulose and Affi-gel blue gel. The hemagglutinating activity of the agglutinin was stable up to 40 degrees C. It underwent a 40% decline when the temperature was raised to 50 degrees C and a complete loss when the temperature was further increased to 80 degrees C. The hemagglutinating activity exhibited a time-dependent loss in activity when the agglutinin was incubated at 100 degrees C for different durations. No activity was discernible when the agglutinin was left at 100 degrees C for 1 min. The activity also underwent a decline in the presence of 500 mM FeCl(3) and CaCl(2). Haricot bean agglutinin manifested a weaker mitogenic activity than concanavalin A toward mouse splenocytes. It exhibited antiproliferative activity toward the tumor cell lines M1 [leukemia], HepG2 [hepatoma] and L1210 [leukemia] cells. 相似文献
8.
Summary The protein composition of the flagellar membrane of C. eugametos mt
–gametes was analyzed using SDS-polyacrylamide gel electrophoresis. The association of the proteins with the membrane was assessed by differential extraction and an assay for glycosylation. Particular attention was paid to integral membrane proteins that could be associated with the mt
– agglutinin, the membrane-bound sexual receptor by which the mt
– gamete binds to its mt
+ partner. This agglutinin is a peripheral membrane glycoprotein and must be bound to the flagellar surface by an integral membrane anchor protein that connects the agglutinin with the cell's interior. Immunoaffinity chromatography was performed using Mab 66.3, a monoclonal antibody specific for the mt
– agglutinin, in order to isolate protein complexes consisting of agglutinin molecules and associated components. Only one integral membrane glycoprotein (Mr = 125 kDa) was isolated that has an association with the agglutinin. It did not bind Mab 66.3, but did bind the lectin wheat germ agglutinin. This was an expected property of the membrane anchor protein because previous research (Kooijman et al. 1989) has shown that cross-linking a WGA-binding glycoprotein by this lectin induces sexual responses that are similar to those induced by agglutinin-agglutinin interactions during mating. We conclude that the 125-kDa glycoprotein is the membrane anchor for the agglutinin.Abbreviations
BSA
Bovine serum albumin
-
CBB
Coomassie Brilliant Blue
-
CHAPS
3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate
-
GTC
guanidine thiocyanate
-
mt
–/mt
+
mating type minus/plus
-
PAS
periodic acid Schiff
-
PBS
phosphate buffered saline
-
SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
-
TBS
TRIS-buffered saline
-
WGA
wheat germ agglutinin 相似文献
9.
N.J. Brewin D.H. Northcote 《Biochimica et Biophysica Acta (BBA)/General Subjects》1973,320(1):104-122
Cyclic AMP phosphodiesterase has been partially purified from soybean liquid suspension culture. It has a Km of 6.7·10?5 M and an optimum pH of 4.2. Its activity is not affected by inorganic cations. Methyl xanthines and papaverine are not inhibitory. It is inhibited by inorganic phosphate and dibutyryl cyclic AMP. At least 35% of the total phosphodiesterase activity of the cell is associated with the plasmalemmaThe enzyme can be resolved into two fractions by DEAE-cellulose chromatography. These have apparently identical properties and each is associated with acid phosphatase activity. It is suggested that these two fractions may represent different aggregation states of a multienzyme complex.A non-dialysable inhibitor of the enzyme has been isolated by gel filtration on Sephadex G-75. It is heat stable and its association with phosphodiesterase is reversible. 相似文献
10.
The effect of the plant lectins concanavalin A and wheat germ agglutinin on the membrane-bound Mg+2-dependent ATPase of an adrenergic clone of mouse neuroblastoma was examines. When cell membranes were treated with concanavalin A or wheat germ agglutinin, a dose-related increase in ATPase-specific activity was observed. Maximal stimulation was greater with wheat germ agglutinin than with concanavalin A; half-maximal and maximal stimulation occurred at similar lectin concentrations. Concanavalin A-dependent stimulation was blocked by α-methylmannoside but not by N-acetylglucosammine. Conversely, stimulation with wheat germ agglutinin was prevented by N-acetylglucosamine but not by α-methylmannoside. The combined effects of concanavalin A and wheat germ agglutinin were greater than the individual effects of either, but were not additive. The results suggest that these lectins interact specifically with membrane glycoproteins or glycolipids, resulting in enhancement of Mg+2-dependent ATPase activity. 相似文献
11.
S Jayasree 《Journal of invertebrate pathology》2001,77(4):237-242
A natural agglutinin in the hemolymph of the marine prawn Penaeus indicus was isolated by gel filtration chromatography, purified using polyacrylamide gel electrophoresis, and characterized. Prawn agglutinin has a native molecular mass of 181 kDa and consists of two monomeric units (97 and 84 kDa), maintains some agglutinating activity over a wide pH range (7-9), and is inactivated at 85 degrees C. The agglutinin was denatured upon mixing with trichloroacetic acid, phenol, chloroform, and 45% ammonium sulfate. It was also sensitive to trypsin digestion. The results indicate that prawn agglutinin is proteinaceous in nature, with agglutinating, hemolytic, and antibacterial properties against marine bacteria and erythrocytes with carbohydrate binding sites. 相似文献
12.
Yamaguchi T 《Archives of microbiology》2004,181(2):106-111
Streptococcus intermedius strain 1208-1 cells were aggregated in the presence of saliva. The saliva agglutinin was purified by centrifugation, filtration, and gel filtration. SDS-PAGE analyses indicated that the purified agglutinin consisted of two high-molecular-mass proteins. Aggregation was dependent on calcium over pH 5.5, with 1 mM being the most effective concentration. Boiling inactivated purified agglutinin. S. intermedius strain 3 and Streptococcus mutans strain 1 were aggregated in the purified agglutinin. After adsorption with strain 1208-1 cells, the saliva sample did not exhibit any aggregation activity, and the agglutinin bands were no longer visible by SDS-PAGE. Adherence analyses demonstrated that the purified agglutinin immobilized on the surfaces of polystyrene wells, actinomyces cells, and apatite beads accounted for the binding of streptococcus cells. Agglutinin also effectively inhibited adherence to apatite beads coated with native saliva. 相似文献
13.
《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1996,1305(3):172-180
We describe the partial purification and characterization of two different types of homologous DNA pairing activity from rat testis nuclear extracts. The activities are separated from each other by single-stranded DNA-cellulose affinity chromatography. One activity requires single-stranded DNA ends and promotes the homologous pairing of single-stranded DNA fragments with double-stranded circular DNA and has an apparent molecular mass of 100 kDa as determined by gel filtration chromatography. This pairing activity does not require the addition of exogenous ATP and is strongly Mg2+-dependent. The second pairing activity promotes strand-transfer between single-stranded circular DNA and homologous double-stranded DNA fragments and has an apparent molecular mass of 30 kDa as determined by gel filtration chromatography. This pairing activity also does not require ATP but, in contrast to the former, is Mg2+-independent. 相似文献
14.
Field-grown wheat (Triticum aestivum L.) has been used as a developmental system to study the appearance of wheat-germ agglutinin during grain maturation. The lectin appears at the mid-grain growth period (30–34 days post-anthesis) and continues to be synthesised throughout the late stages of maturation and desiccation. An acidic endopeptidase activity, inhibited by pepstatin-phenanthroline is present in extracts of embryo and endosperm throughout maturation. After in-vivo labelling of immature embryos with [35S]methionine for 3 h and extraction in the presence of proteinase inhibitors, immunoprecipitates with anti-wheat-germ agglutinin were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, and found to contain three 35S-labelled polypeptides of Mr 46000, 18000 and 13000. Comparison of two-dimensional tryptic maps of 125I-labelled peptides indicate the three polypeptides are closely related.Abbreviations dpa
days post-anthesis
- PBS
phosphate-buffered saline
- RIA
radioimmunoassay
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- WGA
wheat-germ agglutinin 相似文献
15.
Xylanase of low molecular weight (K II) was isolated from the fungus Aspergillus niger IBT-90 cultivated in medium with wheat bran. K II was purified by precipitation with ammonium sulphate (20–80% saturation)
and gel filtration on Biogel P-10. This enzyme is most active in hydrolysis of birchwood xylan at 50°C and pH 5.5. Xylanase
K II has an ability to degrade 1,4-β-bonds and to debranch substrates. It degrades not only xylans but also cellulose, an
important factor for its application in bakery. Ag+, Fe3+ and NBS are strong inhibitors of the enzyme. DTT and Na+ activate xylanase K II by 24 and 13%, respectively. Enzyme K II used as additive to flour improves dough properties, increases
the volume of wheat–rye and whole meal bread, and increases the porosity of crumb and the moisture of the final product, consequently
extending the shelf life of bread. 相似文献
16.
Hydroxyproline-rich bacterial agglutinin from potato : extraction, purification, and characterization 总被引:26,自引:11,他引:15
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A protein, extracted from Katahdin potato (Solanum tuberosum L. cv `Katahdin') tubers and purified by ion exchange chromatography and gel filtration, agglutinates avirulent strains of the bacterial wilt pathogen, Pseudomonas solanacearum, but only weakly agglutinates virulent strains. The agglutinin has very low hemagglutinating activity (in contrast to potato lectin) and is a glycoprotein containing about 61% carbohydrate. The carbohydrate moiety contains 91% (weight%) arabinose, 5% galactose, 3% glucose, and 1% glucosamine. The protein portion is rich in hydroxyproline (42%), lysine (16%), serine (9%), and proline (9%). The entire agglutinin has a molecular weight of 91,000 ± 5,000 and is very basic (pI > 11). Shape estimations based on the concentration dependence of the sedimentation coefficient, the high viscosity ([η] = 92.7), the frictional coefficient (f/fo = 2.15), and axial ratio (a/b = 25) indicate that the agglutinin is a prolate ellipsoid. 相似文献
17.
H. Khyami-Horani 《World journal of microbiology & biotechnology》1996,12(5):525-529
A newly isolated Bacillus species, which grew optimally at 30°C and pH 10, produced a carboxymethylcellulase in a medium containing 10 g CM-cellulose/l. The enzyme, when partially purified by gel filtration, had a mass of about 29 kDa as determined by both SDS-PAGE and gel filtration chromatography. It was optimally active at pH 9.5 and 40°C, and was stable from pH 7 to 11 at 4°C for 24 h. The enzyme was stimulated by Ca2+ (1mm) but was completely inhibited by Hg2+ (1mm). Neither EDTA nor EGTA (10mm) affected the activity.The author is with the Department of Biological Sciences, University of Jordan. PO Box 2686, Amman 11181, Jordan 相似文献
18.
《Biochemical Engineering Journal》2008,41(3):445-451
Lipase production (8.02 ± 0.24 U/ml) by the yeast Aureobasidium pullulans HN2.3 isolated from sea saltern was carried by using time-dependent induction strategy. The lipase in the supernatant of the yeast cell culture was purified to homogeneity with a 3.4-fold increase in specific lipase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography and anion-exchange chromatography. According to the data on SDS polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 63.5 kDa. The optimal pH and temperature of the purified enzyme were 8.5 and 35 °C, respectively. The enzyme was greatly inhibited by Hg2+, Fe2+ and Zn2+. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride, not inhibited by ethylene diamine tetraacetic acid (EDTA), but weakly inhibited by iodoacetic acid. It was found that the purified lipase had the highest hydrolytic activity towards peanut oil. 相似文献
19.
The effect of disulfide-reducing agent dithiothreitol (DTT) on the plus and minus agglutinins ofChlamydomonas reinhardtii gametes was studied. Live gametes of mating-type plus (mt
+) lost their flagellar agglutinability by DTT treatment without any loss of cell motility and concurrently released into the medium agglutinin in an inactive form. DTT treated cells also lost completely their cell body-agglutinin. By contrast, the mating-type minus (mt
-) gametes neither lost their agglutinability nor released agglutinin into the medium by DTT even at very high concentrations. In vitro experiments showed that plus agglutinin in solution is just as sensitive as that in vivo to DTT, whereas minus agglutinin is totally insensitive, and the sulfhydryl-oxidizing agent diamide restores the plus agglutinin activity immediately and completely. Isolated flagella from themt
+ gametes were also inactivated by DTT, but they retained the inactivated agglutinin on the surfaces. The results indicate that plus agglutinin, but not minus agglutinin, possesses disulfide bonds which are essential for the recognition/adhesion activity.Abbreviations
mt
+/-
mating-type plus or minus
- DTT
dithiothreitol 相似文献
20.
Yoshimi Kawade 《Microbiology and immunology》1973,17(2):129-140
Interferon was produced in high yields in mouse L cell cultures infected with Newcastle disease virus, with a specific activity of the order of 106 units per mg protein. It was partially purified by zinc acetate precipitation, carboxymethyl Sephadex chromatography, Sephadex gel filtration and pressure dialysis. On electrophoresis in polyacrylamide gel, it consisted of a fast-moving sharp component and a slow, broadly distributed component(s). The highest specific activity of the former component so far obtained was 8 × 107 units per mg protein, numerically the highest value ever reported for interferon. It was considered likely, however, that the protein components in the purified samples, revealed by staining of the electrophoresis gel, still represented mostly impurities. Gel filtration experiment indicated some heterogeneity of interferon in molecular weight but the major component was estimated to be 30 000 in molecular weight. Interferon activity could be maintained without added preservatives for prolonged periods, provided that the protein concentration of the sample itself was high. One interferon unit as defined in this paper was found to correspond to 0.3 international unit. 相似文献