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1.
Activities of - and ß-glucosidase, - and ß-galactosidase,-mannosidase, ß-1,3-glucanase, acid and neutral invertaseswere detected in the cytoplasmic fraction as well as in cellwalls isolated from callus cultures of cotton. Activity of ß-mannosidase,however, could not be detected in the cell walls. Transfer ofcallus to a fresh medium did not immediately influence the activitiesof -glucosidase and ß-galactosidase but increasedsignificantly ß-glucosidase, -mannosidase, acid andneutral invertases. Addition of cycloheximide (1 and 100 mgl1) further stimulated acid and neutral invertases butnot other enzymes tested. Sodium chloride (NaCl) was effectivein extracting a-glucosidase, ß-glucosidase, ß-galactosidase,acid and neutral invertases. EDTA extracted most of the -galactosidase,-mannosidase, ß-1,3-glucanase and some -glucosidase.But, NaCl and EDTA could not extract some of the - and ß-glucosidasesand also acid and neutral invertases as evidenced from the residualand extra cellular activity. Studies with whole cells as a sourceof enzyme revealed that some of these enzymes were associatedwith the cell surface. Callus, glycosidases, glucanase, growth, Gossypium hirsutum 相似文献
2.
-Mannosidase, ß-N-acetylglucosaminidase, - and ß-galactosidaseand ß-glucosidase were partially purified from leavesof Pisum sativum by ammonium sulphate fractionation and columnchromatography on DEAE-Sephadex A-50 and hydroxylapatite. Atleast two molecular forms of each enzyme were resolved by thesetechniques except for ß-glucosidase of which onlyone form was resolved. Except for one form of -galactosidase,all of the glycosidases thus purified were completely boundby Sepharose-linked Concanavalin A. The binding was stronglyinhibited by cr-methyl-D-mannoside and no binding to Sepharose-6-Boccurred indicating that these glycosidases contain mannose-richoligosaccharides. The glycoprotein nature of -mannosidase, ß-galactosidaseand ß-glucosidase was further demonstrated by chromatographyon phenylboronate agarose columns. The differences in the concentrationof cr-methyl-D-mannoside and sorbitol required to elute thevarious glycosidases from Sepharose-linked Con A and phenylboronateagarose, respectively, suggested that these enzymes are glycosylatedto various degrees or that structural variation in their carbohydratemoieties occur. This is the first demonstration that glycosylationof several glycosidases present in a single plant species isapparently a generalized feature of these enzymes. Key words: Pisum sativum, Glycosidase, Glycoprotein 相似文献
3.
A monoclonal antibody (46-12-C12) for use in a solid-phase enzyme-linkedimmunosorbent assay (ELISA) specific for an anionic peroxidase(APRX) from peanut (Arachis hypogaea L.) suspension cell mediumwas developed. The McAb (IgG1) had a high affinity (2.77 ? 1011)and specificity for APRX, and showed only weak interaction witha-amylase and virtually no reactivity with other enzymes, suchas MCPRX (peanut), minor CPRX (peanut), peroxidase (horseradish),RuBP case (spinach), -glucosidase (rice), ß-glucosidase(almonds), acid phosphatase (potato), catalase (bovine liver)and glucose-6-phosphatase (yeast). Sample dilution curves werefound to parallel the standard curve. The detection limit was0.002 ? 1012 mol APRX. The absorbance was linear at concentrationsbetween 0.00424 ? 1012 mol APRX. Three hundredsamples could be analysed per day by one person, with a semi-automaticperformance. Using this assay, levels of APRX have been determinedin a number of biological extracts of different origin. Key words: Peanut, anionic peroxidase, monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) 相似文献
4.
Distribution of Glycosidases and Acid Invertase Activities in Relation to Elongation Growth in Pearl Millet Internode 总被引:1,自引:0,他引:1
CHANDA S. V.; JOSHI A. K.; KRISHNAN P. N.; SINGH Y. D. 《Journal of experimental botany》1986,37(9):1406-1415
The mean cell length along a differentiating internode and alliedchanges in the activities of ß-glucosidase, - andß-galactosidase. -mannosidase and acid invertase,together with the contents of reducing and non-reducing sugars,were examined in pearl millet (Pennisetum americanum L. Leekecv. BJ-104). The specific activities of cytoplasmic -mannosidase,wall ß-glucosidase, and cytoplasmic and wall acidinvertase showed close relationships with the rate of cell elongation.The linear regressions of the rate of cell elongation, and thespecific activities of wall ß-glucosidase and cytoplasmicand wall invertase showed significant positive correlations(P<0·05), whereas cytoplasmic -mannosidase was negativelycorrelated (P<0·01). The results are discussed in the light of cell wall looseningand the provision of carbon substrates for cell elongation. Key words: Glycosidases, acid invertase, sugars, cell elongation, Pennisetum americanum L., Leeke 相似文献
5.
Function of Lysosomes and Lysosomal Enzymes in the Senescing Corolla of the Morning Glory (Ipomoea purpurea) 总被引:2,自引:0,他引:2
The rapid senescence of the Ipomoea corolla is characterizedby the breakdown of protein and nucleic acids. At the onsetof wilting the activities of deoxyribonuclease (DNase), ribonuclease(RNase), and ß-glucosidase are increased dramatically,while other hydrolytic activities such as the actions of protease,aminopeptidase, -glucosidase, phosphatase, esterase, and -amylaseare only slightly changed. Isolated corolla discs show a course of senescence similar tothat of the intact organ. When floating on solutions of cycloheximidethe activities of DNase, RNase, and ß-glucosidasedo not increase. Actinomycin D inhibits the increase in RNaseactivity. It is concluded that protein synthesis is a prerequisitefor the changes in these enzyme activities in the senescingcorolla. The function of the lysosomal compartment in the process ofsenescence is illustrated by electron micrographs showing theautophagic activity of vacuoles. The last phase of senescenceis characterized by the breakdown of the tonoplast and completedigestion of the cytoplasmic constituents in the autolysingcells. 相似文献
6.
Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid 总被引:2,自引:1,他引:1
Duffin Kevin L; Lange Gary W.; Welply Joseph K.; Florman Richard; O'Brien Paul J.; Dell Anne; Reason Andrew J.; Morris Howard R.; Fliesler Steven J. 《Glycobiology》1993,3(4):365-380
The N-linked oligosaccharides of frog (Rana pipiens) rhodopsinwere analysed by sequential exoglycosidase digestion and gelfiltration chromatography, following reductive tritiation. Inaddition, selected tryptic glycopeptides obtained from frogretinal rod outer segment membranes were examined by electrospraymass spectrometry (ES-MS), fast atom bombardment mass spectrometry(FAB-MS), amino acid sequence and composition analysis, andcarbohydrate composition analysis. The amino acid sequence datademonstrated that the glycopeptides were derived from rhodopsinand confirmed the presence of twoN-glycosylation sites, at residuesAsn2 and Asn15. The predominant glycan (60% of total) had thestructure GlcNAcß12Man13(Man16)Manß14GlcNAcß14GlcNAc-(Asn),with the remaining structures containing 13 additionalhexose residues, as reported previously for bovine rhodopsin.Unlike bovine rhodopsin, however, a sizable fraction of thetotal giycans of frog rhodopsin also contained sialic acid (NeuAc),with the sialylated oligosaccharides being present exclusivelyat the Asn2 site. FAB-MS analysis of oligosaccharides releasedfrom the Asn2 site gave, among other signals, an abundant quasimolecularion corresponding to a glycan of composition NeuAc1Hex6HexNAc3(where Hex is hexose and HexNAc is N-acetylhexosamine), consistentwith a hybrid structure. The potential biological implicationsof these results are discussed in the context of rod outer segmentmembrane renewal. glycoforms oligosaccharide structure rhodopsin 相似文献
7.
Developing cotton fibre was analysed from 12 days post anthesis(DPA) till maturity for the activity of wall degrading enzymes,ß-galactosidase, ß-glucosidase, -mannosidaseand ß-1,3-glucanase. Each enzyme was estimated inthree different fractions namely cytoplasmic, ionically wall-boundand covalently wall-bound. There was a significant correlationbetween ß-galactosidase and ß-glucosidaseactivities in the covalently bound fraction, and the rate offibre elongation. Similarly, covalently bound ß-1,3-glucanaseactivity showed an increasing trend up to 18 DPA, i.e. aboutthe time when maximum rate of fibre elongation was achieved. The results presented here suggest that covalently wall-boundglycosidases may have an importafit role in cell wall loosening.Earlier reports providing evidence against the involvement ofthese enzymes in elongation growth in intact system, may perhapsbe due to scant attention paid to the subcellular distributionof these enzymes. Gossypium hirsutum, cotton fibre, glucanase, glycosidase, wall-loosening 相似文献
8.
Identification of an Antigenic Determinant on Anionic Peanut Peroxidase by Monoclonal Antibodies 总被引:4,自引:0,他引:4
Two monoclonal antibodies (4612-C12 and 236-C12)raised against anionic peanut peroxidase were found to haveindependent epitope sites. These topographic sites were foundto be located within a tryptic glycopeptide (Atgp) from theanionic isozyme by both indirect and non-competive ELISA andWestern blotting. The Atgp has a Mr equal to 11 000 of which 70% is carbohydrateand the peptide is probably highly hydrophobic as determinedby its high RF (0.83) value and the amino acid composition.McAb 236-C12 recognized a contiguous epitope which encompassedalso the sole N-linked oligosaccharide on the anionic isozyme.That the monoclonal antibody also recognized the oligosaccharideon the -amylase, ß-glucosidase, acid phosphatase,and horse-radish peroxidase may be related to similarities insugars. Sugar removal from the Atgp or from the cross-reactivepeptide of enzymes caused loss of antibody affinity. The monoclonal antibody 4612-C12 recognized specificallya conformational epitope near the region of the cysteine, tryptophaneand methionine residue on Atgp. Digestion of the anionic isozymeby trypsin resulted in a 40-fold loss of affinity with thismonoclonal antibody. Moreover, treatment of the Atgp with performicacid or trifluoromethane sulphonic acid caused a loss of affinitybetween the treated Atgp and this monoclonal antibody. Key words: Monoclonal antibodies, peanut, anionic peroxidase, glycopeptide, trypsin digest 相似文献
9.
The major isoenzymes of -mannosidase (EC 3.2.1.24
[EC]
) and ß-galactosidase(ECf 3.2.1.23
[EC]
) have been separated from cotyledons of gardenpea, Pisum sativum L. (Vicieae), chick pea, Cicer arietinumL. (Cicereae), and cowpea, Vigna unguiculata (L.) Walp. (Phaseoleae).Some of their properties have been determined, including pHoptima, Km values for p-nitrophenyl glycosidc substrates, andthe effects of several inhibitors. Swainsonine, an indolizidinealkaloid, was the most effective inhibitor of mannosidase 1,with I30 values of 5.6 x 108 M (cowpea), 1x 107M (chick pea) and 2.9 x 197 M (pea). The most effectiveinhibitor of ß-galactosidase 2 from all sources wasD-galactonic acid-1,4-lactonwe (-lactone), with Ki values rangingbetween 3.0 and 3.9x 103 M. An inhibitor of the E. coliß-galactosidose, p-aminophenyl thio-ß-D-galactopyranoside,did not inhibit any of the legume ß-galctosidases;rather it enhanced the activites of the enzymes from chick peaand cowpea cotyledons. Etiolated hull and seed tissues frompea pods developing in darkness contained similar acid glycosidaseactivities to normal green tissues, thus the chloroplast isan unlikely location for ß-galactosidase 2. The majorß-galactosidasesdetected with an indigogenic substrate (5-bromo-4-chloro-3-indoxyl-ß-D-galactopyranoside)following gel electrophoresis of extracts from pea hull, seedcoats and cotyledons appeared to be different from ß-galactosidase2. Acid glycosidase, cotyledon, isoenzyme, -lactone, legume, swainsonine 相似文献
10.
The role of glycosidases activity in the regulation of pollentube extension in Amaryllis vittata during in vitro germinationwas investigated. No significant change in the enzyme activities(-glucosidase, -galactosidase, rß-glucosidase andrß-galactosidase) at different stages of tube growthwas found. No increase in patent rß-glucosidase activityassayed directly in a suspension of intact germinating pollenwas observed. The results are discussed in the light of thedifferential role of wall-bound glycosidases in cells showingoverall surface growth and tip growth i.e., pollen tubes. (Received February 4, 1981; Accepted June 5, 1981) 相似文献
11.
Miller Arnold L.; Norton Valerie; Robertson Robin; Jenks Michael; Yeh Richard Y.; Wright Darin 《Glycobiology》1993,3(4):313-318
We previously reported that I-cell disease lymphoblasts maintainnormal or near-normal intracellular levels of lysosomal enzymes,even though N-acetylglucosamine-1-phosphotransferase activityis severely depressed or absent (Little et al., Biochem. J.,248, 151159, 1987). The present study, employing subcellularfractionation on colloidal silica gradients, indicates thatboth light and heavy lysosomes isolated from I-cell diseaseand pseudo-Hurler polydystrophy lymphoblasts possess normalspecific activity levels of N-acetyl-ß-D-hexosaminidase,-D-mannosidase and ß-D-glucuronidase. These currentfindings are in contrast to those of cultured fibroblasts fromthe same patients, where decreased intralysosomal enzyme activitiesare found. Column chromatography on Ricinus communis revealedthat N-acetyl-ß-D-hexosaminidase in both heavy andlight I-cell disease lysosomal fractions from lymphoblasts possessesan increased number of accessible galactose residues (3050%)as compared to the enzyme from the corresponding normal controls.Endo-ß-N-acetylglucos-aminidase H treatment of N-acetyl-ß-D-hexosaminidasefrom the I-cell lysosomal fractions suggests that the majorityof newly synthesized high-mannose-type oligosaccharide chainsare modified to complex-type carbohydrates prior to being transportedto lysosomes. This result from lymphoblasts differs from previousfindings with fibroblasts, where N-acetyl-ß-D-hexosaminidasefrom I-cell disease and pseudo-Hurler polydystrophy lysosomesexhibited properties associated with predominantly high-mannose-typeoligosaccharide chains. The current results imply that differentcell types may modify the carbohydrate side chains of lysosomalenzymes in a differential manner, and that selected cell typesmay also employ mechanisms other than the mannose-6-phosphatepathway for targeting lysosomal enzymes to lysosomes. I-cell disease lymphoblasts lysosomes mannose-6-phosphate oligosaccharide chains pseudo-Hurler polydystrophy 相似文献
12.
ELPIDINA E. N.; DUNAEVSKY Y. E.; BELOZERSKY M. A. 《Journal of experimental botany》1990,41(8):969-977
Protein bodies were isolated from cotyledons of dry buckwheatseeds by homogenization in acetone with subsequent purificationin a 1.26 g cm3 to 1.53 g cm3 linear density gradientof a mixture of acetone with CCI4. The purified fraction ofprotein bodies with globoids (PB I) had a buoyant density of1.481.51 g cm3 and was intact according to microscopicdata. Localization of hydrolytic enzymes and proteinase inhibitorsin the PB I fraction and in the fraction of the cytoplasm andmembrane material (CMM) was studied. It was shown that acidhydrolytic enzymes, such as aspartic proteinase, carboxypeptidase,acid phosphatase, -D-mannosidase and N-acetyl-ß-glucosaminidase,as well as chymotrypsin and trypsin inhibitors were predominantlylocalized in the PB I. BAPAase and SH-activated caseinase activitieswere equally distributed between the PB I and CMM fraction.The activities of leucine aminopeptidase and SH-independentcaseinase were noticeably predominant in the CMM fraction. Key words: Buckwheat, subcellular fractionation, protein bodies, hydrolases 相似文献
13.
Carbonera Daniela; Rovati Laura; Guano Fulvio; Balestrazzi Alma 《Journal of experimental botany》1995,46(3):347-354
Topoisomerase II was partially purified from Daucus carota cellsby a procedure including ammonium sulphate fractionation, ion-exchange,and affinity chromatography steps. The type II enzyme, identifiedfor its ability to unknot knotted P4 DNA and decatenate Trypanosomacruzi kDNA, requires ATP and Mg2+ for activity. The unknottingactivity was sensitive to an inhibitor of the mammalian typeII enzyme, the drug VP16 (IC50 32 mmol m3), whereas inhibitorsof DNA gyrase showed a limited effect on activity. The SDS-PAGEanalysis of the dsDNA cellulose fraction revealed the presenceof four polypeptides of apparent molecular masses of 72, 71,34, and 33 kDa among which only a polypeptide of about 70 kDacrossreacted with antibodies against yeast topoisomerase II.Immunoprecipitation experiments with monoclonal antibodies tothe and ß isoforms of the human enzyme confirmedthe recognition of a polypeptide of 70 kDa. The sedimentationcoefficient (S) of the topoisomerase II in the phosphocellulosefraction, calculated by analytical glycerol gradient, was 6.1corresponding to a molecular mass of about 123 kDa. Resultssuggest the presence in carrot of a protein of molecular massof 70 kDa having the typical properties of an eukaryotic topoisomeraseII and carrying epitopes recognized by MoAbs to both human and ß enzymes. The 70 kDa polypeptide might then representthe monomer of a homodimer enzyme of 123 kDa. Key words: Daucus carota, topoisomerase II, immunoprecipitation 相似文献
14.
Extraction of Enzymes from Cell Walls of Sugar Beet Cells Grown in Suspension Culture 总被引:1,自引:0,他引:1
Various glycosidases were extracted from cell walls of sugarbeet cells grown in suspension culture using three successiveprocedures employing saline, EDTA, and commercial cellulase.Saline was effective for extracting acid invertase, ß-galactosidase,and ß-glucosidase. EDTA extracted most of the -galactosidaseand some of the ß-glucosidase. It was most effectivebetween 27?C and 40?C. Commercial cellulase could extract mostof the -mannosidase in the cell wall when used at 27?C for 96h. These three procedures could not extract some of the acidinvertase and ß-glucosidase. The results suggest thatthe cell wall glycosidases are associated with different polysaccharides. These extraction procedures were also applied to the cell wallsof intact tissues, such as cotyledons, hypocotyls plus roots,and mature roots of sugar beets. EDTA as well as saline wasquite effective for extracting bound enzymes from the cell wallof intact tissue, which indicated that extraction with EDTAis useful for liberating bound enzymes from plant cell walls. (Received October 19, 1987; Accepted March 16, 1988) 相似文献
15.
Changes in the Activities of Various Glycosidases during Carrot Cell Elongation in a 2,4-D-Free Medium 总被引:1,自引:0,他引:1
Masuda Hiroshi; Ozeki Yoshihiro; Amino Shin-ichi; Komamine Atsushi 《Plant & cell physiology》1985,26(6):995-1001
Changes in the activities of some glycosidases were studiedin carrot suspension cultures with and without 2,4-D. Remarkablecell elongation occurs in a medium without 2,4-D, while fewcells elongate in a medium containing it. Glycosidases werefractionated into soluble, ionically wall-bound, tightly wall-boundand extracellular enzymes. The optimum pHs of all the ionicallybound glycosidases were in an acidic range, 4.45.0. The activities of the ionically and tightly bound ß-xylosidasesand ß-galactosidases were higher in elongating thanin non-elongating cells. Furthermore, the activities of theseenzymes increased with cell elongation during culture, suggestingthat they may play important roles in cell elongation. Higheractivities of soluble and cell wall-bound ß-glucosidaseand -mannosidase were found in non-elongating rather than inelongating cells. The activities of all soluble glycosidasesexcept ß-xylosidase were also higher in non-elongatingcells. Only ß-xylosidase and ß-galactosidaseactivities were detectable in the medium of the elongation culture.
1 Present address: Department of Agricultural Chemistry, ObihiroUniversity of Agriculture and Veterinary Medicine, Obihiro,Hokkaido 080, Japan. 相似文献
16.
Various oligosaccharides were studied with regard to their effecton the in vitro growth of Camellia japonica pollen tube. Sucrose,raffinose, melezitose, cellobiose, turanose and isomaltose,especially the first four, promoted pollen tube growth, whilemaltotriose, trehalose, gentiobiose, palatinose, melibiose,lactose and lactulose had little effect. Maltose strongly inhibitednot only the tube growth on sugar-free medium but also sugar-stimulatedgrowth, except in the case of sucrose stimulation. Glycosidaseactivities toward the growth-stimulating oligosaccharides weredetected in the extract of sucrose-grown pollen, but the activitiesof -glucosidase and -galactosidase were much lower than thoseof ß-fructosidase and ß-glucosidase. Maltosesuppressed the increase in UDP-glucose level of the glucose-grownpollen but not that of the sucrose-grown one. These resultssuggest that maltose acts, directly or indirectly, somewherein the pathway from glucose to UDP-glucose via glucose-1-phosphate,but does not interfere with the direct conversion of sucroseto UDP-glucose. (Received December 1, 1984; Accepted May 24, 1985) 相似文献
17.
-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH4)2SO4, and chromatography on ConcanavalinA1-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.04.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful. 相似文献
18.
Purification and characterization of avian {beta}1,4 galactosyltransferase: comparison with the mammalian enzyme 总被引:4,自引:1,他引:3
Hathaway Helen J.; Runyan Raymond B.; Khounlo Sayadeth; Shur Barry D. 《Glycobiology》1991,1(2):211-221
Avian ß1,4 galactosyltransferase (GalTase) was purifiedfrom chicken serum, partially characterized, and compared tomammalian GalTase using antibody cross-reactivity, North-ernblot hybridization and amino acid sequence analysis. The enzymewas purified to apparent homogeneity by lactalbumin(LA)-agaroseaffinity chromatography followed by preparative SDS-polyacrylamidegel electrophoresis, and identified as two proteins of apparentmolecular masses of 39 and 46 kD. Chicken serum GalTase hada Km for UDPGal of 42 µM, for GlcNAc of 10 mM and hadoptimal activity in the presence of 1020 mM MnCl2 Substrateand linkage specificity analyses indicated that the purifiedenzyme behaves as a traditional Gal ß1,4 GlcNAc:GalTase,since: (i) the avian ß1,4 GalTase bound to -LA; (ii)terminal GlcNAc residues served as good acceptors for chickenserum GalTase; (iii) the enzyme was inhibited by high concentrationsof GlcNAc; (iv) the galactosylated product was sensitive toß1,4-specific ß-galactosidase. Finally,the disaccharide reaction product comigrated with authenticß1,4 N-acetyllactosamine standard. No other GalTaseactivities were detectable using a battery of defined glycosidesubstrates. Polyclonal antibodies raised against the two gel-purifiedGalTase proteins showed reactivity with avian GalTase by ELISAand immunoprecipitation assays. The antibodies also inhibitedGalTase activity toward both high mol. wt and monosaccharideacceptor substrates. Despite similar kinetics and substratespecificity, the avian and mammalian GalTases showed littleoverall structural similarity, since polyclonal anti-avian GalTaseIgG failed to react with mammalian GalTase purified from bovinemilk, and conversely anti-bovine milk GalTase IgG did not reactwith the avian enzyme. Furthermore, in Northern blot analysis,no hybridization was detected when chicken embryo liver poly(A)+RNA was probed with a mouse GalTase cDNA, even under conditionsof reduced stringency. Amino acid sequence analysis identifiedthree of five tryptic peptides that are homologous to the mammaliansequence within a putative substrate binding domain and thecarboxy terminal domain of the enzyme. Their overall structuraldisparity leads us to believe that regions of homology betweenthe avian and mammalian GalTases may represent active sitesof the enzyme. avian ß1,4 galactosyltransferase homology mammalian purification 相似文献
19.
Enzymatic 3-O-sulfation of terminal ß-Gal residueswas investigated by screening sulfotransferase activity presentin 37 human tissue specimens toward the following synthesizedacceptor moieties: Galß1,3GalNAc-O-Al, Galß1,4GlcNAcß-O-Al,Galß1,3GlcNAcß-O-Al, and mucin-type Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-O-Bnstructures containing a C-3 methyl substituent on either Gal.Two distinct types of Gal: 3-O-sulfotransferases were revealed.One (Group A) was specific for the Galß1, 3GalNAc-linkage and the other (Group B) was directed toward the Galß1,4GlcNAcbranch ß1,6 linked to the blood group T hapten. Enzymeactivities found in breast tissues were unique in showing astrict specificity for the T-hapten. Galß-O-allylor benzyl did not serve as acceptors for Group A but were veryactive with Group B. An exainination of activity present insix human sera revealed a specificity of the serum enzyme towardß1,3 linked Gal, particularly, the T-hapten withoutß1,6 branching. Group A was highly active toward T-haptenlacrylamidecopolymer, anti-freeze glycoprotein, and fetuin O-glycosidicasialo glycopeptide; less active toward fetuin triantennaryasialo glycopeptide; and least active toward bovine IgG diantennaryglycopeptide. Group B was moderately and highly active, respectively,with the latter two glycopeptides noted and least active withthe first two. Competition experiments performed with Galß1,3GaLNAc-O-Aland Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-O-Bnhaving a C-3 substituent (methyl or sulfate) on either Gal reinforcedearlier findings on the specificity characteristics of GroupA and Group B. Group A displayed a wider range of optimal activity(pH 6.07.4), whereas Group B possessed a peak of activityat pH 7.2. Mg2+ stimulated Group A 55% and Group B 150%, whereasMn+2 stimulated Group B 130% but inhibited Group A 75%. Ca2+stimulated Group B 100% but inhibited Group A 35%. Group A andGroup B enzymes appeared to be of the same molecular size (<100,000Da) as observed by Sephacryl S-100 HR column chromatography.The following effects upon Gal: 3-O- sulfotransferase activitiesby fucose, sulfate, and other substituents on the carbohydratechains were noted. (1) A methyl or GlcNAc substituent on C-6of GalNAc diminished the ability of Galß1,3GalNAc-O-Alto act as an acceptor for Group A. (2) An 1,3-fucosyl residueon the ß1,6 branch in the mucin core structure didnot affect the activity of Group A toward Gal linked ß1,3to GalNAc-. (3) Lewis x and Lewis a terminals did not serveas acceptors for either Group A or B enzymes. (4) Eliminationof Group B activity on Gal in the ß1,6 branch owingto the presence of a 3-fucosyl or 6-sulfo group on GlcNAc didnot hinder any action toward Gal linked ß1,3 to GalNAc.(5) Group A activity on Gal linked ß1,3 to GalNAcremained imaffected by 3'-sulfation of the ß1,6 branch.The reverse was true for Group B. (6) The acceptor activityof the T-hapten was increased somewhat upon C-6 sulfation ofGalNAc, whereas, C-6 slalylation resulted in an 85% loss ofactivity. (7) A novel finding was that Galß1,4GlcNAcß-O-Aland Galß1,3GlcNAcß-O-M, upon C-6 sulfationof the GlcNAc moiety, became 100% inactive and 5- to 7-foldactive, respectively, in their ability to serve as acceptorsfor Group B. human tissues glycoprotein galactose:sulfotransferase specificities kinetic properties 相似文献
20.
We previously showed that human melanoma, CHO and other cellscan convert ß-xylosides into structural analogs ofganglioside GM3. We have investigated several potential acceptorsincluding a series of n-alkyl-ß-D-glucosides (n =69). All were labeled with 3H-galactose when incubatedwith human melanoma cells. Octyl-ß-D-glucoside (GlcßOctyl)was the best acceptor, whereas neither octyl--D-glucoside norN-octanoyl-methylglucamine (MEGA 8) were labeled. Analysis ofthe products by a combination of chromatographic methods andspecific enzyme digestions showed that the acceptors first receiveda single Galß1,4 residue followed by an 2,3 linkedsialic acid. Synthesis of these products did not affect cellviability, adherence, protein biosynthesis, or incorporationof radio-labeled precursors into glycoprotein, glycolipid orproteoglycans. To determine which ß1,4 galactosyltransferase synthesized Galß1,4GlcßOctyl,we analyzed similar incubations using CHO cells and a mutantCHO line (CHO 761) which lacks GAG-core specific ß1,4galactosyltransferase. The mutant cells showed the same levelof incorporation as the control, eliminating this enzyme asa candidate. Thermal inactivation kinetics using melanoma cellmicrosomes and rat liver Golgi to galactosylate GlcßOctylshowed the same half-life as UDP-Gal:GlcNAc ß1,4 galactosyltransferase,whereas LacCer synthase was inactivated at a much faster rate.We show that GlcßOctyl is a substrate for purifiedbovine milk UDP-Gal:GlcNAc ß1,4 galactosyltransferaseFurthermore, the galactosylation of GlcßOctyl by CHOcell microsomes can be competitively inhibited by GlcNAc orGlcNAcßMU . These results indicate that UDP-Gal:GlcNAcß1,4 galactosyltransferase is the enzyme used forthe synthesis of the alkyl lactosides when cells or rat liverGolgi are incubated with alkyl ß glucosides. alkylglucosides galactosyltransferase glycolipid artificial acceptors 相似文献