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1.
Cathepsin B (EC 3.4.22.1) was purified from buffalo liver. The enzyme activity against-benzoyl-dl-arginine-naphthylamme (BANA) was substantially reduced by heat (above 37C) and by nondenaturing concentrations of urea (3 M) and guanidine hydrochloride (1 M). Cathepsin B was significantly activated by 1.5 mM EDTA alone. The activation of the enzyme was further enhanced in the presence of thiol compounds, e.g., cysteine thioglycolic acid, 2,3-dimercapto-1-propenol, and dithioerythritol (DTE). The minimum concentration of the thiol compound required for optimal activation of cathepsin B was found to be lowest (0.2 mM) for DTE. The BANA hydrolyzing activity of cathepsin B was substantially reduced by Cu2+ (20–200M) and Ca2+ (30–250 mM) as well as by thiol blocking reagents, e.g., iodoacetate, 5,5-dithiobis(2-nitro-benzoic acid) (DTNB), andp-hydroxymercuribenzoate (pHMB). The enzyme activity was completely abolished when the molar ratio of the reagent: cathepsin B was close to 1. The number of free sulfhydryl groups in cathepsin B was determined to be 2 by titration against DTNB and pHMB. Modification of one free thiol group of cathepsin B resulted in complete loss of BANA hydrolyzing activity. 相似文献
2.
B. Sylvén 《Histochemistry and cell biology》1968,15(2):150-159
Summary Since it was recently observed that purified cathepsin B will hydrolyse the chromogenic substrate LNA the obvious possibility of demonstrating the activity of this enzyme in tissue sections was explored. Chemical and histochemical experiments suggested that all aminopeptidase activity could be excluded by choosing a sufficiently acid pH for incubation, whereby the selective demonstration of cathepsin B activity was possible. This conclusion was further supported by different attempts to influence the histochemical reaction by preincubation with suitable activating and/or inhibiting reagents. This more specific procedure was used on sections from liver, kidney and granulomas rich in active macrophages. The suggested cathepsin B activity was demonstrated in autophagic vacuoles, and in other lysosome-like enlarged vacuoles or granules, appearing as a consequence of cellular injury as well as in actively phagocytosing macrophages.The following Abbreviations have been used
AM Pase(s)
aminopeptidase(s) in general
-
NA
naphthylamides in general. — Substrates
-
LA
Leucine amide
-
GPA
Gly-Phe-amide
-
LNA
L-leucyl--naphthylamide
-
Arg-NA
L-arginyl--naphthylamide
-
BANA
Benzoyl-dl-arginine--naphthylamide 相似文献
3.
H. G. Aghajanyan A. M. Arzumanyan A. A. Arutunyan T. N. Akopyan 《Neurochemical research》1988,13(8):721-727
Two cystatins were purified from tissue extract of bovine brain by alkaline treatment, acetone fractionation, gel chromatography on Sephadex G-75, and affinity chromatography on S-carboxymethyl-papain-Sepharose. One of the inhibitors had a relatively high molecular mass, 25 kDa (HMM-cystatin) with pI 4.7, and the other, 11 kDa (LMM-cystatin) with pI 5.23. Both inhibitors showed considerable stability at pH 2 and 80°C. The cystatins inhibited papain, ficin, and cathepsins B and H, but not trypsin, chymotrypsin, thermolysin, nagarse, and cathepsin D. Ki values for the complexes of papain and the inhibitors were estimated to be 2.8×10–10 M for HMM-cystatin and 1.3×10–9 M for LMM-cystatin. Both purified cystatins prevented degradation of substance P by soluble fraction and lysosomal extract obtained from synaptosomes, but did not suppress the cleavage of the peptide by synaptosomal plasma membranes.Abbreviations HMM-cystatin
high molecular mass inhibitor
- LMM-cystatin
low molecular mass inhibitor
- SP
substance P
- SPM
synaptosomal plasma membranes
- p-CMB
4-chloromercuribenzoic acid
- BK
bradykinin
- Bz-Arg-Nap
N-benzoyl-dl-arginine--naphthylamide
- Arg-Nap
dl-arginine--naphthylamide
- P-Pxy-Hb
hemoglobin initially coupled with pyridoxal-5-phosphate 相似文献
4.
S. Hayashi K. Matsuzaki Y. Takasaki H. Ueno K. Imada 《World journal of microbiology & biotechnology》1992,8(3):276-279
-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The Km value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate. 相似文献
5.
T. Vanha-Perttula V. K. Hopsu V. Sonninen G. G. Glenner 《Histochemistry and cell biology》1965,5(2):170-181
Summary Hog kidney homogenate was fractionated initially following the steps presented by De La Haba
et al. (1959) for the purification of cathepsin C from beef spleen. The preparation was further fractionated by gel filtration using Sephadex G 200, starch gel and immunoelectrophoresis. The following enzymes were identified in the fractions obtained:Cathepsin C, which liberated ammonia from glycyl-phenylalanine amide and naphthylamine from glycyl-phenylalanyl--naphthylamide, pH optimum at 5.0, was activated by cysteine and inhibited by sulfhydryl reagents.An aminopeptidase, which liberated first of all glycine from glycyl-phenylalanine amide and glycyl-phenylalanyl--naphtylainide and after that ammonia and naphthylamine, respectively, hydrolysed numerous amino acid naphthylamides, pH optimum at 7.0–7.5, was activated by Co++ and inhibited by EDTA.A peptidase, which liberated glycine from glycyl-phenylalanine amide and naphthylamide, did not hydrolyse amino acid naphthylamides, maximally active at neutral pH, was inhibited by EDTA.Several esterases, two in gel filtration, 5–6 in starch gel and immunoelectrophoresis, hydrolysing 5-bromoindoxyl acetate. The activities were sensitive to E 600.Both the studies on the characteristics of these activities as well as starch gel and immunoelectrophoretic studies support the view that none of the esterase activities is identical with cathepsin C. Cathepsin C, on the other hand, does not hydrolyse significantly 5-bromoindoxyl acetate and, consequently, this substrate can not be used to demonstrate cathepsin C histochemically.Glycyl-phenylalanyl--naphthylamide is recommended as a new sensitive chromogenic substrate for cathepsin C in biochemical studies in which the role of the aminopeptidase (s) can be adequately excluded but cannot be used in the histochemical demonstration of this enzyme. 相似文献
6.
The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed. 相似文献
7.
Josef Gabelsberger Wolfgang Liebl Karl-Heinz Schleifer 《Applied microbiology and biotechnology》1993,40(1):44-52
A -glucosidase of the hyperthermophilic bacterium Thermotoga maritima has been purified from a recombinant Escherichia coli clone expressing the corresponding gene. The enzyme was found to be a dimer with an apparent molecular mass of approximately 95 kDa as determined by size exclusion chromatography. It was composed of two apparently identical subunits of about 47 kDa (determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). The enzyme had a bbroadsubstrate specificity and attacked -glucoside, -galactoside, -fucoside, and, to a very small extent, also -xyloside substrates. -Glycosidic bonds were not hydrolysed. Kinetic measurement of the hydrolysis of o-nitrophenyl--d-glucopyranoside (oNPGlc) and o-nitrophenyl--d-galactopyranoside (oNPGal) in the concentration ranges 0.05–20 mm and 0.1–10 mm, respectively, at 75°C resulted in non-linear Lineweaver-Burk and Eadie-Hofstee 3lots whereas cellobiose and lactose did not induce this type of effect. Lactose caused substrate inhibition above 350 mm. The enzyme was optimally active at about pH 6.1. The T. maritima -glucosidase represents the most thermostable -glucosidase described to date. In 50 mm sodium phosphate buffer, pH 6.2, at an enzyme concentration of 50 g/ml, the pure enzyme without additives retained more than 60% of its initial activity after a 6-h incubation at 95°C.
Correspondence to: W. Liebl 相似文献
8.
Tadashi II Masayuki Kubota Satoshi Okuda Takashi Hirano Mamoru Ohashi 《Glycoconjugate journal》1995,12(2):162-172
Negative-ion fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from heparin and heparan sulfate. The positional isomers of the sulfate group of monosulfated disaccharides were distinguished from each other by negative-ion fast atom bombardment tandem mass spectra, which provide an easy way of identifying the positional isomers. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of di- and trisulfated disaccharides.Abbreviations FABMS
fast atom bombardment mass spectrometry
- CID
collision induced dissociation
- MIKE
mass analysed ion kinetic energy
- MS/MS
mass spectrometry/mass spectrometry
- HPLC
high performance liquid chromatography
- UA
d-gluco-4-enepyranosyluronic acid
- CS
chondroitin sulfate
- DS
dermatan sulfate
- HA
hyaluronan
- Hep
heparin
- HS
heparan sulfate
- UA(14) GlcNAc
2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose
- UA(14)GlcNAc6S
2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose
- UA2S(14)GlcNAc
2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose
- UA2S(14)GlcNAc6S
2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose
- UA(14)GlcN6S
2-amino-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose
- UA2S(14)GlcN
2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose
- UA2S(14)GlcN6S
2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose
- UA(14)GlcNS
2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose
- UA(14)GlcNS6S
2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose
- UA2S(14)GlcNS
2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose
- UA2S(14)GlcNS6S
2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose
- UA(13)GalNAc
2-acetamido-2-deoxy-3-O-(-d-Gluco-4-enepyranosyluronic acid)-d-galatose
- UA(13)GalNAc4S
2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose
- UA(13)GalNAc6S
2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose
- UA2S(13)GalNAc
2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose
- UA2S(13)GalNAc4S
2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose
- UA2S(13)GalNAc6S
2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose
- UA(13)GalNAcDiS
2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose
- UA(13)GlcNAc
2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose. 相似文献
9.
Delphi Chatterjee James T Douglas Sang-Nae Cho Thomas H Rea Robert H Gelber Gerald O Aspinall Patrick J Brennan 《Glycoconjugate journal》1985,2(3-4):187-208
A stratagem for the synthesis ofneoglycoproteins suitable for the selective serodiagnosis of leprosy is described in which synthetic 3,6-di-O-methyl--d-glucopyranose, the epitope of phenolic glycolipid I fromMycobacterium leprae, was used. Condensation of 8-methoxycarbonyloctanol with the acetobromo derivative of 3,6-di-O-methylglucose gave 8-methoxycarbonyloctyl 2,4-di-O-acetyl-3,6-di-O-methyl--d-glucopyranoside in 65% yield, and with absolute stereospecificity for the anomer. The deacylated product was converted to the crystalline hydrazide and coupled to bovine gamma globulin, bovine serum albumin and poly-d-lysinevia intermediate acyl azide formation to produce the 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranosyl polypeptides. Theneoglycoproteins were highly sensitive in ELISA and emulated the specificity of the native glycolipid in analysis of sera from patients throughout the spectrum of leprosy and from different geographical regions. The 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranoside-bovine serum albumin was also synthesized and shown to have about one-half the activity of the -linkedneoglycoprotein. A different synthetic approach produced the 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhamnopyranoside-bovine serum albumin which was also highly sensitive and specific for the serodiagnosis of leprosy. The presence of the second sugar unit, similar to that in the native glycolipid but for the absence ofO-methyl groups, seemed to provide a probe with greater felicity for the serological detection of tuberculoid leprosy.Thus, the results indicate that highly sensitive and specific antigen probes for the serodiagnosis of leprosy can be constructed based only on the terminal one or two sugars of phenolic glycolipid I, and the synthetic approach leads to the formation of haptens with absolute stereospecificity.Nomenclature BGG
bovine gamma globulin
- PGL-I
phenolic glycolipid I
- PDL
poly-d-lysine
- PBS
phophate-buffered saline
- 3,6-Me2-Glc-Link-BSA
8-carbonyloctyl 3,6-di-O-methyl-glucopyranoside-bovine senalbumin
- 3,6-Me2-Glc-Rha-Link-BSA
8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhan pyranoside-BSA 相似文献
10.
Annerose und Fritz Anders 《Molecular & general genetics : MGG》1963,94(1):1-18
Zusammenfassung Beim wildenPlatypoecilus maculatus aus Mexiko, der im männlichen Geschlecht heterogamet ist (Gordon 1946), ist neben dem gonosomalen noch ein autosomales System von Geschlechtsrealisatoren am Werke (vgl.Öktay 1959). Es vermag in extremen Konstellationen, die zufalls- oder selektionsbedingt sein können, denXY-Mechanismus epistatisch zu überlagern, so da\XY-Individuen zu determiniert werden können.XY- sind voll vital und fertil und ergeben in Paarungen mitXY- nebenXX- undXY- voll vitale und fertileXY-.Auf Grund der vorliegenden Kreuzungsergebnisse und der Befunde anderer Autoren wird angenommen, daß der normalerweise rein gonosomale Geschlechtsbestimmungsmodus vonPlatypoecilus maculatus aus dem primitiveren autosomal polyfaktoriellen, der unter anderem bei einem nahen Verwandten dieser Art,Xiphophorus helleri, vorkommt (Kosswig 1931 ff.), hervorgegangen ist.Mit 1 Textabbildung 相似文献
11.
T. Lebret 《Journal of Ornithology》1955,96(1):43-49
Summary 1. Aerial chases of a female duck by a number of (Reihen) include three different forms of chasings: a) chases in connection with swimming courtship (Gesellschaftsspiel); b) territorial chases performed by a which is driving away a of a pair that had entered the territory of the former; c) chases of a in brooding mood by a number of intending to copulate.2. Aerial chases of type a) are part of the courtship, they are courtship flights. They are preceded and often followed by swimming courtship and they are characterized by the birds uttering their courtship calls. They may be observed from late August to the beginning of May.3. Chases of type c) are characterized by the female's call and gesture of repulsion. It only occurs when the female is laying or brooding. The periods of chases of type a) and c) overlap, which may explain the controversy betweenGeyr vonSchweppenburg andChristoleit.4. The Red-crested Pochard- has a special gliding performance during its courtship flight. 相似文献
12.
Teruo Yoshino Minobu Sadamitsu Megumi Minagawa Gerd Reuter Roland Schauer 《Glycoconjugate journal》1988,5(4):377-384
The preparation of benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside, which is a key intermediate for chemical synthesis of oligosaccharide components of glycosphingolipids, was achieved by an improved method. The 3-O-p-methoxybenzyl and 3-O-methyl derivatives were prepared from benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside through stannylation. By using benzyl -d-lactoside as starting material, benzyl 3-O-methyl-, 3-O-benzyl- and 3-O-p-methoxybenzyl--d-lactoside were regioselectively synthesized using the same procedure. 相似文献
13.
N-nitro-l-arginine (NG-nitro-l-arginine) is a potent nitric oxide synthase inhibitor which crosses the blood brain barrier and does not undergo extensive metabolism in vivo. In this study, effect of chronic pretreatment of N-nitro-l-arginine (75 mg/kg, i.p., twice daily for 7 days) on the harmaline- (100 mg/kg, s.c.), picrotoxin- (4 mg/kg, s.c.), pentylenetetrazole- (50 mg/kg, i.p.), andl-glutamic acid- (400 g/10 l/mouse, i.c.v.) induced increase in cerebellar cGMP was assessed. All the four drugs produced significant increase in cerebellar cGMP in vehicle pretreated control animals. Cerebellar cGMP increase induced by harmaline, picrotoxin, andl-glutamic acid was attentuated in N-nitro-l-arginine pretreated animals. These results indicate that in vivo cerebellar cGMP levels are increased by the prototype excitatory amino acid receptor agonist,l-glutamic acid and also by the drugs which augment the excitatory amino acid transmission. Furthermore, parenteral chronic administration of N-nitro-l-arginine blocks NO synthase in the brain and hence cerebellar cGMP response in chronic N-nitro-l-arginine treated animals could be used as a tool to assess the physiological functions of nitric oxide in vivo.Part of this work was presented at the Experimental Biology 93 FASEB Meeting at New Orleans, March 1993. 相似文献
14.
V. Varga R. Janáky K. -M. Marnela J. Gulyás P. Kontro S. S. Oja 《Neurochemical research》1989,14(12):1223-1227
A number ofD-glutamyl andL-aspartyl dipeptides, glutathione, -D-glutamylglycine and -D-glutamyltaurine, were tested for their efficacy to displace ligands specific for different subtypes of excitatory amino acid receptors from rat brain synaptic membranes. In general, theL enanthiomorphs of -glutamyl peptides were more potent displacers than -D-glutamylglycine and-taurine but the latter were more specific for the quisqualate type of receptors. -L-glutamyl-L-glutamate was the most effective dipeptide in displacing the binding of glutamate, 2-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) and 2-amino-5-phosphonoheptanoate (APH), whereas -L-glutamyl-L-aspartate was the most effective in the binding of kainate. Both oxidized and reduced glutathione were inhibitory, being most potent in the binding of AMPA. -L-Glutamylaminomethylsulphonate was most effective in the binding of APH. The most potent -L-glutamyl peptides (glutathione, -L-glutamyl-L-glutamate,-L-aspartate, and-glycine) may act as endogenous modulators of excitatory aminoacidergic neurotransmission. 相似文献
15.
Willy R. Kinzy David M. Belenky Frank G. Loontiens Nathan Sharon 《Glycoconjugate journal》1992,9(5):225-227
The synthesis of the methyl - and -N-dansyl-d-galactosaminides is described using methyl ,-2-azido-2-deoxy-d-galactopyranoside as starting material. This was reduced to the corresponding methyl ,-2-amino-2-deoxy-d-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl ,-N-dansyl-d-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl -N-dansyl-d-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with theN-acetyl-d-galactosamine specific lectin fromErythrina corallodendron.Abbreviations ECorL
Erythrina corallodendron lectin
- MeGalNDns
methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside
- MeGalNDns
methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside
Dedicated to Hilde De Boeck (1958–1991). 相似文献
16.
Piotr W. D. Scisłowski 《Molecular and cellular biochemistry》1977,18(2-3):93-99
Summary -Glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: acceptor oxidoreductase, EC 1.1.99.5) activity in mitochondria isolated from rat skeletal muscle has been studied. The pH optimum of the enzyme activity was about 7.4 and the apparent Km value for DL--glycerophosphate was approxinately 1.6mm. The activity of this enzyme was found to be inhibited by DL-glyceraldehyde-3-phosphate, phosphoenolpyruvate and 3-phosphoglycerate in a competitive manner: the apparent Ki values at pH 7.4 being 0.3mm, 1.5mm and 4.0mm respectively. The enzyme was found to be more sensitive to phosphoenolpyruvate at pH 7.0 than 7.6.The activity of -glycerophosphate dehydrogenase in rat skeletal muscle was also inhibited by palmitoyl-CoA and stearoyl-CoA in a competitive manner. The Ki values being about 9.0 m for both metabolites. This inhibition was partly reversed by Ca2+ and Mg2+ ions. Palmitoylcarnitine also exerted inhibitory effect on -glycerophosphate dehydrogenase activity but palmitate, carnitine and CoA added alone was without effect. It is proposed that the activity of -glycerophosphate dehydrogenase in rat skeletal muscle mitochondria may be controlled by changes of the cytosolic levels of some glycolytic intermediates and long-chain acyl-CoA esters. These results are discussed with respect to the regulation of -glycerophosphate shuttle activity in skeletal muscle. 相似文献
17.
The soft-rot fungus Trichoderma reesei forms -xylosidase (EC 3.2.1.37) activity during cultivation on xylan and xylose, but not on glucose. When mycelia precultivated on glycerol were washed and transferred to fresh medium without a carbon and nitrogen source, -xylosidase formation was induced by xylan, xylobiose and xylose. A supply of 4 mm xylose and a pH of 2.5 provided optimal conditions for induction. -Xylosidase accounted for the major portion of total extracellular protein under these conditions, and could be purified to physical homogeneity by a single anion exchange chromatography step. A recombinant strain of T. reesei that carries multiple copies of the homologous xylanase II-encoding gene has a six-fold increased xylanase activity, but forms comparable -xylosidase activities. This shows that the rate of xylan hydrolysis has no effect on the induction of -xylosidase. Methyl--d-xyloside inhibited -xylosidase competitively and was a weak -xylosidase inducer. The induction by xylobiose and xylan was strongly enhanced by the simultaneous addition of methyl--d-xylosidese and xylan or xylobiose. The results suggest that a slow supply of xylose is a trigger for -xylosidase induction. 相似文献
18.
Philip Kyprianou Alan Betteridge Alastair S R Donald Winifred M Watkins 《Glycoconjugate journal》1990,7(6):573-588
The -2-l-fucosyltransferase in human plasma has been freed from -3-l-fucosyltransferase activity and purified approximately 200,000-fold by a series of steps involving ammonium sulphate precipitation, hydrophobic chromatography on Phenyl Sepharose 4B and affinity chromatography first on GDP-adipate-Sepharose and then on GDP-hexanolamine-Sepharose. The purified -2-l-fucosyltransferase had a Mr on gel filtration HPLC of 158,000 and showed optimal activity in the pH range 6.5–7.0. The enzyme transferred fucose equally well to Type 1 (Gal1-3GlcNAc) and Type 2 (Gal1-4GlcNAc) substrates but Type 3 (Gal1-3GalNAc) structures were less efficient acceptors. Competition experiments indicated that a single enzyme species in the purified preparation was responsible for reactivity with the Type 1 and Type 2 structures. Thus the differences in conformation between the Type 1 and Type 2 disaccharides do not appear to influence the capacities of their terminal non-reducing -d-galactosyl residues to function as acceptor substrates for the -2-l-fucosyltransferase expressed by the blood groupH gene in haemopoietic tissue. 相似文献
19.
Gabriele Möller Folkert Reck Hans Paulsen Kanwal J. Kaur Mohan Sarkar Harry Schachter Inka Brockhausen 《Glycoconjugate journal》1992,9(4):180-190
UDP-GlcNAc: Man3R 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M
r 42,000). TheV
max for the pure enzyme with [Man6(Man3)Man6](Man3)Man4GlcNAc4GlcNAc-Asn as substrate was 4.6 µmol min–1 mg–1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds anN-acetylglucosamine (GlcNAc) residue in 1–2 linkage to the Man3Man-terminus of the substrate. Several derivatives of Man6(Man3)Man-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the -linked mannose of Man6(Man3)Man-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the 3-linked mannose (Man) of the substrate increases theK
M 20-fold. Modifications on the 6-linked mannose or on the core structure affect mainly theK
M and to a lesser degree theV
max, e.g., substitutions of the Man6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower theK
M, whereas various other substitutions at the 3-position increase theK
M slightly. Man6(Man3)4-O-methyl-Man4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.Abbreviations BSA
Bovine serum albumin
- Bn
benzyl
- Fuc, F
l-fucose
- Gal, G
d-galactose
- GalNAc, GA
N-acetyl-d-galactosamine
- Glc
d-glucose
- GlcNAc, Gn
N-acetyl-d-glucosamine
- HPLC
high performance liquid chromatography
- Man, M
d-mannose
- mco
8-methoxycarbonyl-octyl, (CH2)8 COOOCH3
- Me
methyl
- MES
2-(N-morpholino)ethanesulfonate
- NMR
nuclear magnetic resonance
- PMSF
phenylmethylsulfonylfluoride
- pnp
p-nitrophenyl
- SDS
sodium dodecyl sulfate
- T
transferase
- Tal
d-talose
- Xyl
d-xylose;
- {0, 2 + F}
Man6 (GlcNAc2Man3) Man4GlcNAc4 (Fuc6) GlcNAc
- {2, 2}
GlcNAc2Man6 (GlcNAc2Man3) Man4GlcNAc4GlcNAc; M5-glycopeptide, Man6 (Man3) Man6 (Man3) Man4 GlcNAc4GlcNAc-Asn
Enzymes: GlcNAc-transferase I, EC 2.4.1.101; GlcNAc-transferase II, EC 2.4.1.143; GlcNAc-transferase III, EC 2.4.1.144; GlcNAc-transferase IV, EC 2.4.1.145; GlcNAc-transferase V, UDP-GlcNAc: GlcNAc2 Man6-R (GlcNAc to Man) 6-GlcNAc-transferase; GlcNAc-transferase VI, UDP-GlcNAc: GlcNAc6(GlcNAc2) Man6-R (GlcNAc to Man) 4-GlcNAc-transferase; Core 1 3-Gal-transferase, EC 2.4.1.122; 4-Gal-transferase, EC 2.4.1.38; 3-Gal-transferase, UDP-Gal: GlcNAc-R 3-Gal-transferase; blood group i 3-GlcNAc-transferase, EC 2.4.1.149; blood group I 6-GlcNAc-transferase, UDP-GlcNAc: GlcNAc3Gal-R (GlcNAc to Gal) 6-GlcNAc-transferase. 相似文献
20.
Filamentous fungi are capable of secreting relatively large amounts of heterologous recombinant proteins. Recombinant human glycoproteins expressed in this system, however, carry only carbohydrates of the oligomannose type limiting their potential use in humans. One approach to the problem is genetic engineering of the fungal host to permit production of complex and hybrid N-glycans. UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (GnT I) is essential for the conversion of oligomannose to hybrid and complex N-glycans in higher eukaryotic cells. Since GnT I is not produced by fungi, we have introduced into the genome ofAspergillus nidulans the gene encoding full-length rabbit GnT I and demonstrated the expression of GnT I enzyme activity at levels appreciably higher than occurs in most mammalian tissues. All the GnT I activity in theAspergillus transformants remains intracellular suggesting that the rabbit trans-membrane sequence may be capable of targeting GnT I to the fungal Golgi apparatus.Abbreviations CM
complete medium
- Gal-T
UDP-Gal:GlcNAc -1,4-galactosyltransferase (EC 2.4.1.38/90)
- GnT I
UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101)
- HPLC
high performance liquid chromatography
- M3-octyl
Man1-6[Man1-3]Man-octyl
- PAGE
polyacrylamide gel electrophoresis
- MES
2-(N-morpholino)ethane sulfonate
- PCR
polymerase chain reaction
- PEG
polyethylene glycol
- PMSF
phenyl methyl sulfonyl fluoride
- SDS
sodium dodecyl sulfate
- SSC (1×)
0.15m NaCl/0.015m sodium citrate (pH 7.0)
- STC
1.2m sorbitol, 100mm Tris-HCl, pH 7.4, and 10mm CaCl2
- STET
0.1m NaCl, 10mm Tris-HCl, pH 8.0, 1mm EDTA, pH 8.0, 5% Triton-X-100
Deceased. This paper is dedicated to the memory of Lorne S. Reid. 相似文献