An NTP-Binding Protein That Cross with a Ras-Specific Antibody in the Myceia of Neurospora crassa

A Ras-related NTP-binding protein was partially purified froma membrane fraction derived from the mycelia of Neurospora crassa.[-³²P]ATP and [-³²P]GTP were incubated with mem brane and solublefractions which were then irradiated with UV light to inducecrosslinking of tightly bound nucleotides. After SDS-polyacrylamidegel electrophoresis, blotting onto a nitrocellulose filter andautoradiography it was apparent that most of the proteins thatbound [-³²P]-GTP also bound [-³²P]ATP. Pretreatment of the membranefraction with Ras-specific antibody effectively blocked thebinding of [-³²P]ATP and [-³²P]GTP to several ATP-GTP-bindingproteins. The band of a protein with a molecular weight of 26kDa on the SDS-polyacrylamide gel cross-reacted strongly withthe Ras-specific antibody. The protein was extracted from thegel and further purified by repeated gel electrophoresis. Thepurified protein bound [-³²P]ATP, [-³²P]-GTP, [-³²P]CTP and[-³²P]UTP at 1.6x10^– M and was autophosphorylated in thepresence of [-³²P]ATP and [-³²P]GTP at 1.7x10 M. Pretreatmentof the protein with Ras-specific antibody partially blockedthe autophosphorylation in the presence of these nucleotides.The binding of [-³²P]ATP to the NTP-binding protein was blockedby addition of ATP at 10^–4–10^–3 M. ATP ata concentration of 10^–4 M prevented the binding of [-³²P]to a greater extent than did GTP at the same concentration.Binding of [-³²P]CTP and [-³²P]UTP to the protein was also observed. (Received October 7, 1991; Accepted July 14, 1992)     

Glycosidases from Cotyledons of Pisum sativum L.

-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 4.0–4.5. 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.     

Mycobacterial arabinan biosynthesis: the use of synthetic arabinoside acceptors in the development of an arabinosyl transfer assay

Information on the biosynthesis of the D-arabinans of the cellwall of Mycobacterium tuberculosis is rapidly emerging, withthe promise of new targets for drug development against tuberculosis.Accordingly, arabinosyl transferase assays were developed utilizingsynthesized [1–¹⁴C]-β-D-arabinofuranosyl-1-monophosphoryldecaprenolas donor and a variety of O- and S-alkyl arabinosides as acceptors.These were: -D-Araf-(15)--D-Araf-O- and -S-alkyl di-arabinosidesand -D-Araf-(15)--D-Araf-(15)--D-Araf-O- and -S-alkyl triarabinosides.Whereas the O- and S-alkyl monosaccharide acceptors were inactive,the O- and S-alkyl disaccharide and the O- and S-alkyl trisaccharideacceptors (<C12) possessed considerable acceptor activity,and the trisaccharide acceptors were more potent than the correspondingdisaccharides. The O-alkyl disaccharide acceptors with a C8alkyl chain were more active than those containing the C6 orC10 analogs. Chemical analysis of the enzymatically synthesizedproducts of the reactions demonstrated that β-D-arabinofuranosyl-1-monophosphoryldecaprenolwas an effective donor for two of the three potential arabinosyltransferases: β-D-arabinofuranosyl-1-monophosphoryldecaprenol:arabinan (15) arabinosyl transferase and β-D-arabinofuranosyl-1-monophosphoryl-decaprenol:arabinan β(12) arabinosyl transferase. The β(12) arabinosyltransferase activity was more in evidence in the presence ofthe O-alkyl disaccharide acceptor, whereas both transferaseswere about equivalent in the presence of the S-alkyl trisaccharideacceptor. The tuberculosis drug, ethambutol, a known mycobacterialarabinosyl transferase inhibitor, was inactive within thesearabinosyl transferase/acceptor based assay systems, supportingother evidence that a third activity, responsible for the formationof 13 linkage, is the drug target. acceptor arabinan biosynthesis glycosyltrans-ferase assay mycobacteria     

Xyloglucan and r{beta}-D-Glucan in Cell Walls of Rice Seedlings

Cell walls of 4-day old rice seedlings were extracted successivelywith ammonium oxalate-oxalic acid, 4% KOH and 24% KOH. A rß-D-glucanpreparation and a xyloglucan preparation were isolated fromthe 4% KOH extract and 24% KOH extract, respectively. Methylationanalysis and enzymic degradation studies of the polysaccharidesshowed that the former was built up predominantly of repeating-oligosaccharideunits of 3-O-rß-cellobiosyl-D-glucose and 3-O-rß-cellotriosyl-D-glucosein a molar ratio of 2.6 : 1.0, and the latter was of repeating-oligosaccharideunits of -D-xylosyl-(16)-rß-D-glucosyl-(14)-[-D-xylosyl-(16)]-rß-D-glucosyl-(14)-D-glucose,-D-xylosyl-(16)-rß-D-glucosyl-(14)-D-glucose and cellobiose. ¹ Present address: Department of Botany, Iowa State University,Ames, Iowa 50011, U.S.A. (Received August 29, 1981; Accepted January 12, 1982)     

Occurrence of a Novel Galactopinitol and its Changes with Other Non-Reducing Sugars during Development of Leucaena leucocephala Seeds

A new cyclitol which is abudant in the late developmental stagesof leucaena (Leucaena leucocephala (Lam.) de Wit) seeds wasidentified by HPLC, NMR, and GC-MS as O--D-galactopyranosyl-(1 1)-3-O-methyl-D-chiro-inositol, a new galactopinitol. Thisgalactopinitol was initially detected midway through seed developmentand increased to 10.2 mg (g DW)^-1, but decreased in mature seedsto its about a half. Stachyose content increased greatly andremained the most abundant of the soluble sugars in mature seeds(25.6 mg (g DW)^-1). Artifical drying at 73% relative humidityof 70 DPA immature seeds induced the accumulation of raffinose,stachyose, galactopinitol and galactinol, but the total amountsof these sugars were only about half of those found in matureseeds. Seed germination decreased following an initial increaseafter 8 d artitifical drying to a moisture content of 24%, andthis dehydration damage probably is because of underdevelopmentof seed tissue. Galactopinitol changes in a similar fashionto the oligosaccharides during the late developmental stageand dehydration experiment, implying that galactopinitol mayplay a role in desiccation tolerance of leucaena seeds. ¹Contribution no. 79 of Taiwan Forestry Research Institute.     

A novel alpha1,2-fucosyltransferase (CE2FT-2) in Caenorhabditis elegans generates H-type 3 glycan structures

The 1,2-fucosyltransferase family (1,2FT) is the largest familyof glycosyltransferases in the genome of the free-living nematodeCaenorhabditis elegans, and early evidence suggests that eachmember may have a unique activity. Here we describe a C. elegansgene (designated CE2FT-2) encoding an 1,2FT that has the potentialto generate the sequence Fuc1-2Galβ1-3GalNAc-R, which isthe H-type 3 blood group structure. The CE2FT-2 cDNA encodesa putative transmembrane protein that shows 42% amino acid identityto a previously cloned C. elegans 1,2FT (termed CE2FT-1), buthas a very low identity (16–20%) to 1,2FT sequences inhumans, rabbits, and mice. A recombinant form of CE2FT-2 expressedin human 293T cells has a high 1,2FT activity toward Galβ1-3GalNAc-O-pNP,but unexpectedly, the enzyme is inactive toward the acceptorGalβ-O-phenyl. Thus, CE2FT-2 differs from all other 1,2FTspreviously described from animals that all utilize Galβ-O-phenyl.CE2FT-2 is expressed at all stages of worm development, butremarkably, promoter analysis of the CE2FT-2 gene using greenfluorescent protein reporter constructs indicates that the CE2FT-2is expressed exclusively in pharyngeal cells of the worm fromembryo to an adult stage. Because pharyngeal cells are knownto secrete their glycoconjugates to the nematode surface, theseresults may indicate that products of CE2FT-2 contribute tointeractions of the nematode with its environment or are usedas ligands for bacterial attachment. These findings, along withthose on other 1,2FTs in C. elegans, suggest that each 1,2FTin this organism may have a unique acceptor specificity, expressionpattern, and biological function.     

Unusual degradation of alpha -beta complexes in Xenopus oocytes by beta -subunits of Xenopus gastric H-K-ATPase

The catalytic -subunit of oligomeric P-type ATPases such asNa-K-ATPase and H-K-ATPase requires association with a -subunit after synthesis in the endoplasmic reticulum (ER) to become stably expressed and functionally active. In this study, we have expressed the-subunit of Xenopus gastricH-K-ATPase (HK) in Xenopus oocytes together with -subunits of H-K-ATPase (HK) or Na-K-ATPase (NK) and have followed the biosynthesis, assembly, and cell surface expression of functional pumps. Immunoprecipitations ofXenopus HK from metabolicallylabeled oocytes show that it is well expressed and, when synthesizedwithout -subunits, can leave the ER and become fully glycosylated.Xenopus HK can associate with both coexpressed HK and NK, but the - complexes formed aredegraded rapidly in or close to the ER and do not produce functionalpumps at the cell surface as assessed by⁸⁶Rb uptake. A possibleexplanation of these results is thatXenopus HK may contain atissue-specific signal that is important in the formation or correcttargeting of functional - complexes in the stomach but thatcannot be recognized in Xenopusoocytes and in consequence leads to cellular degradation of the -complexes in this experimental system.

     

The Effects of Salinity upon Galactosyl-Glycerol Content and Concentration of the Marine Red Alga Porphyra purpurea (Roth) C.Ag.

Changes in the major alcohol-soluble, low molecular weight carbohydratesof P. purpurea, O--D-galactopyranosyl-(1-2)-glycerol (‘floridoside’)and O--D-galactopyranosyl-(1-1)-glycerol (‘isofloridoside’),have been examined in response to salinity variation. ‘Floridoside’is shown to vary in absolute amount, increasing in hypersalineand decreasing in hyposaline media. ‘Isofloridoside’content per cell does not change in a similar manner. Responsesare almost identical under light or dark conditions, ‘floridoside’changes being complete within 24 h. Decreasing the externalwater potential using ionic and non-ionic solutes has the sameeffect upon galactosyl-glycerol content. The amount of ‘floridoside’synthesized, and degraded under hypersaline and hyposaline conditionsrespectively is shown to be insufficient to restore cell volumeto its original value. It is therefore suggested that the primaryfunction of ‘floridoside’ increases in concentratedsea-waters is that of a compatible solute, serving to protectthe cell during periods when the external salt content is increaseddramatically.     

Effect of TNF-alpha on SMIT mRNA levels and myo-inositol accumulation in cultured endothelial cells

Previously we have shown that hyperosmolarity increasesNa⁺-myo-inositolcotransporter (SMIT) activity and mRNA levels in cultured endothelialcells. Because hyperosmolarity and cytokines, such as tumor necrosisfactor- (TNF-), activate similar signal transduction pathways, weexamined the effect of TNF- on SMIT mRNA levels andmyo-inositol accumulation. In contrastto the effect of hyperosmolarity, TNF- caused a time- andconcentration-dependent decrease in SMIT mRNA levels andmyo-inositol accumulation. The effectof TNF- on myo-inositolaccumulation was found in large-vessel endothelial cells (derived fromthe aorta and pulmonary artery) and cerebral microvessel endothelialcells. In bovine aorta and bovine pulmonary artery endothelial cells,TNF- activated nuclear factor (NF)-B. TNF- also increasedceramide levels, and C₂-ceramidemimicked the effect of TNF- on SMIT mRNA levels andmyo-inositol accumulation in bovineaorta endothelial cells. Pyrrolidinedithiocarbamate, genistein, and7-amino-1-chloro-3-tosylamido-2-hepatanone, compounds that can inhibitNF-B activation, partially prevented the TNF--induced decrease inmyo-inositol accumulation. The effectof TNF- on myo-inositolaccumulation was also partially prevented by the protein kinase Cinhibitor calphostin C but not by staurosporine. These studiesdemonstrate that TNF- causes a decrease in SMIT mRNA levels andmyo-inositol accumulation in culturedendothelial cells, which may be related to the activation of NF-B.

     

Glucose Induced H+ Influx and Transient Currents in Excised Roots: particularly those of Zea mays

The application of D-glucose to solutions bathing excised maize,wheat, pea and bean roots causes a rapid depolarization of theelectrical potentials between the cut tops of the roots andthe bathing solutions. Similar effects are observed for theplasma membrane potentials of maize lateral roots. A flow cell apparatus was used to demonstrate qualitative andquantitative relations between glucose induced H⁺ influx andthe transient decrease in current through the root. The currentchanges appear to be due entirely to H⁺ fluxes. Current andH⁺ fluxes are strongly influenced by external pH, the optimumpH for glucose induced current change being about 4.0. A similarpH optimum was found for 3-O-methyl-D-glucopyranoside but 1-O-methyl--D-glucopyranosidedid not significantly affect the trans-root potential at anypH, suggesting a significant role for the anomeric hydroxylgroup of glucose. Compounds which depolarize the trans-root potential also inhibitthe glucose induced depolarization. Surface -SH groups are probablynot involved in the glucose/H⁺ cotransport. Eadie-Hofstee plots relating the depolarization of trans-rootpotential to the concentrations of D-glucose or 3-O-methyl-D-glucopyranosidehave shown that K_m values increase with increasing monosaccharideconcentration and are very similar to reported values of 3-O-methyl-D-glucopyranosideuptake in maize root segments. K_m values for a similar rangeof D-glucose concentrations do not vary significantly with pHor with membrane depolarization due to a 10-fold increase ofKCl concentration. However, V_max is lowered by an increase inexternal pH or a decrease in trans-root potential. It appearsthat both proton and electrical gradients can affect glucoseinduced H⁺ influx. The auxin herbicide, 2, 4-dichlorophenoxyethanoic acid (0.01mM) stimulates the glucose induced depolarizations in a mannerconsistent with an increase in cytoplasmic pH. This is discussedin relation to the reported action of indole-3-acetic acid andfusicoccin on maize root tissue.     

Hearing and glycoconjugates: localization of Ley, Lex and sialosyl-Lex in guinea pig cochlea, particularly at the tectorial membrane and sensory epithelia of the organ of Corti

Immunohistological examination of guinea pig cochleas was performedusing a panel of 25 monoclonal antibodies directed to variouslacto-, ganglio- and globo-series carbohydrate epitopes as wellas mucin-type epitopes. Lacto-series structures were found tobe localized at specific sites of the tectorial membrane (TM)and Corti's organ, i.e. 13 fucosyl type 2 chain (Le^x) at Kimura'smembrane, marginal band and covering net of TM; 12, 13 difucosyltype 2 chain (Le^y) at covering net; and sialosyl-Le^x and sialosyl-iat Kimura's membrane and sensory epithelia, particularly sensorytips of hair cells of Corti's organ. In striking contrast, ganglio-seriesstructures (GM3, GD3, GD2, 9-O-Ac-GD3) were detected at spiralganglion cells, neuronal fibres and stria vascularis, but werecompletely absent from Corti's organ and most of the TM. Otherepitope structures defined by various antibodies were not detectableat any location. The functional roles of lacto-series carbohydrateepitopes expressed at TM and Corti's organ remain unknown. However,the expression of Le^y (but not other structures) in associationwith developmental deficiency of TM induced by 6-N-propyI-2-thio-uracilin rats suggests that Le^y plays some role in normal TM development.The presence of Le^x at Kimura's membrane and sialosyl-Le^x athair cell sensory tips of Corti's organ suggests the intriguingpossibility that these fucosylated/sialosylated carbohydratestructures play some role in interactions (either attractiveor repulsive) of these inner ear components, which have beenimplicated in the physiology of hearing, i.e. the conversionof sound waves to nerve impulses. cochlea Corti's organ glycoconjugate sialosyl fucosyl type 2 chain tectorial membrane     

Part of the Lysyl Residues in Wheat {alpha}-Amylase is Methylated as N-{varepsilon}-Trimethyl Lysine

Amino acid analyses of wheat -amylase purified from germinatingseeds by affinity chromatography showed a high content of amodified lysyl residue. The modified residue was identifiedas N--trimethyl lysine. The presence of trimethyl lysine in-amylase is discussed in terms of isozymes. ¹ Present address: National Institutes of Health, Bldg. 10,Rm. 9B-15, Bethesda, MD 20205, U.S.A. (Received August 20, 1981; Accepted March 19, 1982)     

Two different glycosyltransferase defects that result in GalNAc{alpha}-O-peptide (Tn) expression

This study shows for the first time that different glycosyltransferasedefects in the biosynthesis of O-linked oligosaccharides giverise to the same GalNAc-O-Ser/Thr determinant on Tn erythrocytesand colorectal carcinoma cells. The O-linked oligosaccharidesisolated from the glycophorins of Tn erythrocytes containedpredominantly -Nacetylgalactosamine-O-Ser/Thr (Tn antigen) andsialyl-Tn. A marked reduction in normal sialylated oligosaccharideswas also observed. Monoclonal antibody BRIC 111 raised againstTn erythrocytes reacted with both Tn erythrocytes and colorectalcarcinoma tissues. Weak staining was detected in the supranucleararea and at the surface membranes in normal colorectal cells,but was absent from goblet cell vesicles. An increase in supranuclearstaining over controls was found in tumour tissue and in themajority of resection margin specimens. The highest levels ofstaining were present in transitional mucosa, adjacent to thetumours where goblet vesicles were also positive. Glycosylationdefects in the same patients were further studied by determinationof the activity of glycosyltransferases in mucosal tissue fromcontrol and cancer patients. The reduction in or loss of ß1-3 N-acetylglucosaminyl transferase activity to GalNAc-peptidein asialo-ovine submaxillary gland glycoprotein was detectedby direct assay and by isolation of the oligosaccharides fromthe incubation products. No differences in N-acetylglucosaminyl-,galactosyl- or sialyl-transfer to Galß1-3GaINAc inantifreeze glycoprotein or in sialyl transferase to asialo-ovinesubmaxillary gland glycoprotein were detected. Our study showsthat the GalNAc-O-Ser/Thr determinant on Tn erythrocytes andin colorectal carcinoma results from different glycosyltransferasedefects in separate biosynthetic pathways for haematopoieticand epithelial tissues. -N-Acetylgalactosamine-O-Ser Thr colon cancer erythrocyte O-glycosylation glycosyltransfer Tn     

Uptake and Accumulation of {alpha}-Naphthalene Acetic Acid by Cell Suspensions of Galium mollugo L.

Fidgeon, C. and Wilson, G. 1988. Uptake and accumulation ofa-naphthalene acetic acid by cell suspensions of Galium mollugoL.—J. exp. Bot. 39: 241-249. Galium mollugo cell suspensions require -NAA for continued growthand cell division. The kinetics of -NAA uptake from the medium(B₅) by Galium cells was assessed using 1-¹⁴C -NAA in a standardratio of cells to medium (0.25 g: 2.5 cm³). It was found thatthe uptake of -NAA was rapid, over 90% being taken up within4 h. Cells which had accumulated -NAA for 4 h or more did notrelease it back into the medium. It was found that Galium cellsaccumulated -NAA against a significant concentration gradient;suggesting the participation of an active component in the uptakemechanism. The effect of free-space and surface adsorption onthe uptake of -NAA was determined by means of a repeated washtechnique. These two factors were found to be of importanceonly during the first hour of uptake. Neither dead cells norplasmolysed cells absorbed -NAA. It is clear that, in the normal growth cycle, Galium cells cantake up the available -NAA within 3 or 4 h of inoculation andthat this can stimulate a cell division response of 3-4 generationsover the subsequent 14 d. Key words: Galium, cell suspension, -naphthalene acetic acid     

srb: a Bacillus subtilis Gene Encoding a Homologue of the {alpha}-Subunit of the Mammalian Signal Recognition Particle Receptor

We cloned a Bacillus subtilis gene (srb) encoding a homologueof the mammalian signal recognition particle receptor -subunit(SR). The gene is 987 bp in length and encodes a 329-amino acidprotein. The deduced amino acid sequence of the protein shared26.6, 36.2 and 49.7% identity with those of mammalian SR, archaebacterialDP and Escherichia coli FtsY, respectively. The protein containsthree conserved GTP-binding elements like the other three SRPreceptor proteins, though the N-terminal portion of the putativeB. subtilis protein was shorter than the others. Secondary structureprediction showed that an amphipathic -helix is positioned inthe N-terminal region. A defect in srb inhibited cell growthand protein translocation.     

TNF-alpha is a mitogen in skeletal muscle

Emerging evidence suggests that tumor necrosis factor (TNF)- plays a role in muscle repair. To determine whether TNF- modulates satellite cell proliferation, the current study evaluated TNF- effects on DNA synthesis in primary myoblasts and on satellite cell activation in adult mouse muscle. Exposure to recombinant TNF- increased total DNA content in rat primary myoblasts dose-dependently over a 24-h period and increased the number of primary myoblasts incorporating 5-bromo-2'-deoxyuridine (BrdU) during a 30-min pulse labeling. Systemic injection of TNF- stimulated BrdU incorporation by satellite cells in muscles of adult mice, whereas no BrdU was incorporated by satellite cells in control mice. TNF- stimulated serum response factor (SRF) binding to the serum response element (SRE) present in the c-fos gene promoter and stimulated reporter gene expression controlled by the same element. Our data suggest that TNF- activates satellite cells to enter the cell cycle and accelerates G₁-to-S phase transition, and these actions may involve activation of early response genes via SRF. cytokine; cell cycle; satellite cells; serum response factor; c-fos     

Sugar-Controlled Ca2+ Uptake and {alpha}-Amylase Secretion in Cultured Cells of Rice (Oryza sativa L.)

Sugar starvation-induced synthesis and extracellular liberationof -amylase molecules in suspension-cultured cells of rice (Oryzasativa L.) required Ca²⁺, although the level of translatable-amylase mRNA was not affected in the presence of Ca²⁺. Sugardepletion markedly stimulated Ca²⁺ uptake by rice cells andsucrose supplementation reduced it. Immunohistochemical andelectron probe microanalyzer studies indicated an apparent resemblancebetween the distribution pattern of Ca²⁺ and that of -amylasemolecules induced in the sugar-depleted cells. Ca²⁺ uptake wasreduced by sucrose, maltose, fructose, and glucose similarlyat more than 5 mM, but was unaffected by mannitol (88 mM), 6-deoxy-D-glucose(10 mM), and 3-O-methyl-D-glucose (10 mM). Furthermore, an effectiveCa²⁺ channel blocker, La³⁺ significantly inhibited the Ca²⁺uptake and the synthesis and extracellular liberation of -amylasemolecules in the absence of sucrose, while a general P-typeATPase inhibitor, vanadate greatly stimulated both in the presenceof sucrose. We concluded that, by controlling the Ca²⁺ uptake,metabolic sugars regulate the protein synthesis and posttranslationalsecretory processes of -amylase molecules in rice cells. ⁴ Invited research fellow of the Japan Society for the Promotionof Science. Present address: Plant Physiology Department, WarsawAgricultural University, Rakowiecka Str. 26/30 02-528 Warsaw,Poland.     

Treatment with the {alpha}-glucosidase 1 inhibitor 6-O-butanoyl castanospermine reduces the detection of LFA-1 (CD18/CD11a) by monoclonal antibodies

The antiviral clinical candidate 6-O-butanoyl castano-spermine(MDL 28,574), an -glucosidase 1 inhibitor, was examined forits effect on elementary parameters of immune function. It didnot affect the mitogenic response of uninfected human mononuclearleukocytes or the detection of a range of cell surface markers,with the exception of the integrin LFA-1 (CD18/CD11a), whichwas reduced, after cell growth in vitro. The detection of LFA-1was also reduced on both human and murine cells after oral administrationof the compound to xenochimaeric or normal mice, respectively.Altered LFA-1 expression or function may contribute to reducedcell adhesion and the observed reduction in the in vitro allogeneicresponse by uninfected cells, as well as the previously describedprevention of cell conjugate and HTV-induced syncytium formation. adhesion 6-O-butanoyl castanospermine CD18 CD11a LFA-1     

Overexpression and purification of non-glycosylated recombinant endo-{beta}-N-acetylglucosaminidase F3

The gene for endo-ß-N-acetylglucosaininldase F₃ wascloned into the high-expression vector pMAL c-2, and expressedin Escherichia coli as a fusion protein. A key step in the purificationemployed Poros II (HS) chromatography, which greatly facilitatedisolation of the enzyme from crude intracellular lysates. Theunfused enzyme was recovered following digestion with FactorX_a, and was isolated in a homogeneous form. The enzyme is non-glycosylatedand fully active, and is a very useful analytical tool for investigatingthe structure of asparagine-linked glycans, especially thosewith core-substituted 1,6 fucosyl residues. deglycosylation Endo F₃ endoglycosidase MBP-Endo F₃