A coupled enzyme assay for GlcNAc1: UDP-galactose galactosyltransferase has been developed that allows this enzyme to be assayed spectrophotometrically and in nondenaturing polyacrylamide gels. Utilizing three, intermediate enzymes, galactosyltransferase activity has been coupled to the production of NADH with a stoichiometry of 2 mol of NADH produced for each mol of galactose transferred to GlcNAc. The enzyme reactions coupled to the production of UDP by galactosyltransferase can be summarized as follows: The activities of partly purified bovine milk galactosyltransferase and galactosyltransferase in dialyzed fetal calf serum have been determined spectrophotometrically by measuring NADH production at 340 nm. The reaction is dependent on N-acetylglucosamine, UDP-galactose, and Mn2+. For both enzyme sources, activities calculated from NADH production are similar to those determined from assays that use radioactive sugar nucleotide substrates. Both galactosyltransferase activities have been localized on 7.5% nondenaturing polyacrylamide gels after electrophoresis by incubating the gel with an agarose indicator gel containing the coupled enzyme system. Enzyme activity is marked by NADH fluorescence, which is dependent on the presence of N-acetylglucosamine in the indicator gel. The intensity of fluorescence increases with increasing galactosyltransferase activity applied to the gel. 相似文献
A soluble galactosyltransferase was purified 22,000-fold from bovine cornea. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-acetyl-d-glucosamine, α- and β-glucosaminides, bovine cornea and nasal septum agalactokeratan, and to glycoproteins containing terminal nonreducing N-acetylglucosaminyl units. When N-acetyl-d-glucosamine served as acceptor, the product formed by the cornea transferase contained galactose glycosidically linked to carbon atom 4 of N-acetyl-d-glucosamine; the same glycosidic linkage was found in [14C]keratan preparations isolated from reaction mixtures where keratan containing terminal nonreducing N-acetylglucosaminyl units served as acceptor. The cornea enzyme exhibited a markedly lower Km with keratan than with N-acetyl-d-glucosamine. The physical and kinetic properties of the cornea galactosyltransferase and of the milk A-protein (A-protein + α-lactalbumin = lactose synthase), including modulations of acceptor specificity by α-lactalbumin, were compared. The results of these studies strongly suggest that the two glycosyltransferases are similar, if not identical. Efforts to demonstrate the presence of other soluble galactosyltransferases in cornea were unsuccessful; no change in the ratios of products formed with several acceptors was observed at any stage of purification. It is suggested that in bovine tissues a single galactosyltransferase participates in the synthesis of both high and low molecular weight galactosides including the assembly of the repeating disaccharide [O-β-galactopyranosyl-(1 → 4)-N-acetylglucosamine] of cornea keratan sulfate. 相似文献
Two galactosyltransferase activities (1 and 2) were measured in the pancreas, liver and gut of the developing rat embryo. 1. N-Acetylglucosamine:Galactosyltransferase. UDP [14C]galactose + N-acetylglucosamine → [14C]galactosyl-β-(1 → 4)-N-acetylglucosamine + UDP. 2. N-Acetylgalactosamine-protein:Galactosyltransferase. UDP [14C]galactose + N-acetylgalactosamine-protein → [14C]galactosyl-β-(1 → 3)-N-acetylgalactosamine-protein + UDP. Galactosyltransferases 1 and 2 increased in the pancreas, about 10- and 40-fold in specific activity, respectively, from 11 to 12 days in utero to birth. During this period the activities of both transferases in the liver were somewhat variable, but showed no definite trend. A drop in the level of galactosyltransferase 1 in the pancreas occurred at birth or shortly thereafter. The “Golgimarker” enzyme for liver, galactosyltransferase 1, may be absent or present at low levels in adult rat pancreas.Zymogen granule membrane preparations apparently are devoid of these galactosyltransferase activities. Bromodeoxyuridine, which inhibits the development of the synthetic capability of the specific exocrine proteins, had essentially no effect on the normal accretion of the galactosyltransferase activities in organ cultures of pancreatic rudiments from 13-day rat embryos. 相似文献
Dimorphism is an ability of certain fungi related to its adaptation to the environment and provides a selective advantage under stress conditions and is associated to the development of human diseases. Hyphae inducing- and inhibitory-effect of farnesol on hyphae formation by the dimorphic yeast Yarrowia lipolytica was evaluated through digital image analysis. The agitation speed of the culture was the most effective hyphae inducer in comparison to bovine calf serum and N-acetylglucosamine. In low agitation system, bovine calf serum was more effective for hyphae formation inducing 57 % of hyphae transition. Farnesol inhibited hyphae formation even in low concentration (300 μM) and this effect increased with increasing concentrations. In the presence of N-acetylglucosamine, this effect was more evident in comparison to the presence of bovine calf serum, which might have protected the cells from farnesol. Digital image analysis was an important tool to evaluate this phenomenon. 相似文献
1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37°C for 1h released about 60% of the membrane-bound UDP-galactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with α-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn2+ that could not be replaced by Ca2+, Mg2+, Zn2+ or Co2+, and was active over a wide pH range (6–8) with a pH optimum of 6.5. The apparent Km for UDP-galactose was 10.8μm. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000–70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2–5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-ε-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the ε-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction. 相似文献
We immobilized human milk galactosyltransferase covalently to CNBr- and tresylchloride-activated Sepharose. The enzyme was also immobilized non-covalently to Concanavalin A-Sepharose and to monoclonal anti-galactosyltransferase antibodies which were boundvia their Fc-fragment to Protein G-Sepharose. With the covalent methods, up to 72% of the enzyme could be bound to the carrier, but more than 90% of the specific activity was lost. In contrast, non-covalent immobilization yielded only about 50% immobilization efficiency, but 21% and 25% of specific activity, respectively, could be recovered. The stability of immobilized galactosyltransferase as compared to native enzyme was considerably increased: at room temperature, 55% of initial immobilized activity was lost after 65 hours compared to 95% of loss of soluble enzyme activity. Immobilized galactosyltransferase was then used for continuous galactosylation of the glycoproteins ovalbumin, endo H-treated yeast invertase and bovine serum albumin-N-acetylglucosamine in a slurry reactor. 55%, 35% and 25%, respectively, of all acceptor sites on these glycoproteins could be galactosylated by this method. 相似文献
A glucosidase preparation with activity toward certain glucose-containing oligosaccharides was partially purified from calf liver membranes by Triton X-100 solubilization and DEAE-cellulose and hydroxylapatite chromatography. The enzyme preparation hydrolyzed the glucose residues from (glucose)1,(mannose)9(N-acetylglucosamine)1, and (glucose)2(mannose) 9(N-acetylglucosamine)1 but was totally inactive toward (glucose)3(mannose)9(N-acetylglucosamine) 1. In contrast, crude membrane preparations of the calf liver were active toward all three substrates. The partially purified enzyme had a pH optimum of 6.7 and was very unstable in the absence of added 20% glycerol. The rate of glucose release from the one-and two-glucose-containing oligosaccharides was significantly decreased when four or five of the mannose residues were first removed from the substrate. The release of glucose from (glucose)1(mannose)9(N-acetylglucosamine)1 was inhibited by p-nitrophenyl-α-d-glucoside much more effectively than by p-nitrophenyl-β-d-glucoside, suggesting that this glucose residue may be linked α to the mannose residue. We conclude that during oligosaccharide processing at least two different glucosidases are involved in glucose removal. 相似文献
O-Glycosidic analogs of N-acetylglucosamine are good substrates for galactosyltransferase, and as the O-substituted group becomes more hydrophobic, the apparent Km decreases as much as 2000-fold. l-leucine, leucine-amide, norleucine, valine, ?-amino-n-caproic acid and tyrosine-agaroses all retain galactosyltransferase in the presence of 1.25 m ammonium sulfate. The enzyme is eluted quantitatively with a five- to tenfold purification by a decreasing linear gradient of ammonium sulfate. Galactosyltransferase was not specifically bound on any of a series of ω-aminoalkylagaroses tested. A simple and highly efficient procedure for the isolation of galactosyltransferase from bovine skim milk was developed and consisted of a 40–60% ammonium sulfate precipitation of the enzyme from skim milk followed by chromatography on (1) norleucine-Sepharose, (2) UDP-hexanolamine-Sepharose, and (3) α-lactalbumin-Sepharose. 相似文献
The effects of the membrane perturbing reagents linoleic acid and benzyl alcohol on the activities of four rat liver Golgi membrane enzymes, N-acetylglucosaminyl-, N-acetylgalactosaminyl-, galactosyl-, and sialytransferases and several soluble glycosyltransferases, bovine milk galactosyl- and N-acetylglucosaminyltransferases and porcine submaxillary N-acetylgalactosaminyltransferases have been studied. In rat liver Golgi membranes, linoleic acid inhibited the activities of N-acetylgalactosaminyl- and galactosyltransferases by 50% or greater, sialyltransferase by 10–15%, and N-acetylglucosaminyltransferase not at all. The isolated bovine milk N-acetylglucosaminyltransferase and porcine submaxillary N-acetylgalactosylaminyltranferase were not inhibited but bovine milk galactosyltransferase was inhibited by 95% or greater. The inhibition by linoleic acid on Golgi membrane galactosyltransferase appears to be a direct effect of the reagent on the enzyme. Incorporation of bovine milk galactosyltransferase into liposomes formed from saturated phospholipids, DMPC, DPPC, and DSPC (dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine) prevented inhibition of the enzyme activity suggesting that the lipid formed a barrier which did not allow linoleic acid access to the enzyme. The water soluble benzyl alcohol was more effective in inhibiting enzymes of the isolated rat liver Golgi complex. All four glycosyltransferases were inhibited, the N-acetylglucosaminyl- and N-acetylgalactosaminyltransferases by more than 95%. A higher concentration of benzyl alcohol was necessary to inhibit the galactosyltransferases than was required for the other Golgi enzymes. Benzyl alcohol also inhibited the isolated bovine milk N-acetylglucosaminyl- and galactosyltransferases 90% to 95%, respectively, but did not affect the isolated porcine submaxillary gland N-acetylgalactosaminyltransferase. Benzyl alcohol did not inhibit the milk galactosyltransferase incorporated into DMPC or DPPC liposomes but showed a complex effect on the activity of the enzyme incorporated into DSPC vesicles, a stimulation of activity at low concentrations followed by an inhibition. A lipid environment consisting of saturated lipids appears to present a barrier to inhibiting substances such as linoleic acid and benzyl alcohol, or lipid may stabilize the active conformation of the enzyme. The different effects of these reagents on four transferases of the Golgi complex suggest that the lipid environment around these enzymes may be different for each transferase. 相似文献
Summary We have characterized the effects of serum andN-acetylglucosamine in a glucose-deprived condition on the glycosylation of antibody light chains, as well as the resulting
biological properties of those antibodies. We have chosen for our investigation the human hybridoma lines producing monoclonal
antibodies reactive to lung adenocarcinoma. Each antibody possess aN-glycosylated carbohydrate chain in the hypervariable region of the light chains. When the cell lines were grown in the absence
of glucose, variant light chains with varying molecular masses were found to be secreted. Analysis of these light chains produced
in a glucose-deprived condition revealed that the changed molecular-mass of the variant light chains is due to different glycosylation.
Addition ofN-acetylglucosamine or fetal calf serum to the glucose-free medium led to the creation of other light chains that exhibit increased
antigen binding activity. 相似文献
1.1. The effect of fetal calf serum as an additive for reconstituted freeze-dried bovine and porcine blood for feeding Glossina palpalis palpalis was determined.
2.2. Reproductive performance of tsetse flies fed reconstituted freeze-dried bovine and porcine blood supplemented with fetal calf serum was higher than that of flies fed only freeze-dried blood.
3.3. Sonicated freeze-dried porcine blood supplemented with fetal calf serum resulted in a reproductive performance equivalent to that of flies fed on non-sonicated freeze-dried porcine blood and fetal calf serum.
4.4. A 50:50 mixture of freeze-dried porcine and freeze-dried bovine blood reconstituted and sonieated resulted in a reproductive performance equivalent to the highest ever attained for in vitro-reared G.p. palpalis.
Calf brain membranes have previously been shown to enzymatically transfer N-acetyl[14C]glucosamine from UDP-N-acetyl[14C]glucosamine into N-acetyl[14C]glucosami-nylpyrophosphoryldolichol, N,N′-diacetyl[14C]chitobiosylpyrophosphoryldolichol and a minor labeled product with the chemical and chromatographic properties of a [14C]trisaccharide lipid (Waechter, C. J., and Harford, J. B. (1977) Arch. Biochem. Biophys.181, 185–198). This paper demonstrates that incubating calf brain membranes containing endogenous, prelabeled N-acetyl[14C]glucosaminyl lipids with unlabeled GDP-mannose enhances the formation of the [14C]trisaccharide lipid. The intact [14C]trisaccharide lipid behaves like a dolichol-bound trisaccharide, in which the glycosyl group is linked via a pyrophosphate bridge, when chromatographed on SG-81 paper or DEAE-cellulose. Mild acid treatment releases a water-soluble product that comigrates with authentic β-Man-(1→4)-β-GlcNAc(1→4)-GlcNAc. The free [14C]trisaccharide is converted to N,N′-diacetyl[14C]chitobiose by incubation with a highly purified β-mannosidase. These findings indicate that the trisaccharide lipid formed by calf brain membranes is β-mannosyl-N,N′-diacetylchito-biosylpyrophosphoryldolichol. The two glycosyltransferases responsible for the enzymatic conversion of the N-acetylglucosaminyl lipid to the trisaccharide lipid have been studied using exogenous, purified [14C]glycolipid substrates. Calf brain membranes enzymatically transfer N-acetylglucosamine from UDP-N-acetylglucosamine to exogenous N-acetyl[14C] glucosaminylpyrophosphoryldolichol to form [14C]disaccharide lipid. The biosynthesis of [14C]disaccharide lipid is stimulated by unlabeled UDP-N-acetylglucosamine under conditions that inhibit N-acetylglucosaminylpyrophosphoryldolichol synthesis. Unlike the formation of N-acetylglucosaminylpyrophosphoryldolichol the enzymatic addition of the second N-acetylglucosamine residue is not inhibited by tunicamycin. Exogenous purified [14C] disaccharide lipid is enzymatically mannosylated by calf brain membranes to form the [14C] trisaccharide lipid. The formation of the [14C]trisaccharide lipid from exogenous [14C] disaccharide lipid is stimulated by unlabeled GDP-mannose and Mg2+, and inhibited by EDTA. Exogenous dolichyl monophosphate is also inhibitory. These results strongly suggest that the calf brain mannosyltransferase involved in the synthesis of the trisaccharide lipid requires a divalent cation and utilizes GDP-mannose, not mannosylphosphoryldolichol, as the direct mannosyl donor. 相似文献
The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically. 相似文献
A wide variety of medically important biofilm forming bacteria produce similar polysaccharide intracellular adhesins (PIAs). The PIA structures consist of partially de-N-acetylated β-(1→6)-N-acetylglucosamine polymers. These exopolysaccharides are key components of the bacterial biofilm matrix. Here, we describe the efficient synthesis of PIA oligosaccharides using an acid reversion reaction of N-acetylglucosamine in HF·pyridine. The PIA oligosaccharides produced by this reaction can be purified to homogeneity by size exclusion chromatography. Chemistry was developed to conjugate the PIA oligosaccharides to bovine serum albumin using a new heterobifunctional linker containing a thiol and an N-methylhydroxylamine functional group. These glycoconjugates may serve as useful precursors for the development of defined conjugate vaccines against PIA producing bacterial strains. 相似文献
An assay for fructokinase activity is described that permits an accurate estimation of specific fructokinase in crude tissue preparations without interference of hexokinase activity. It utilizes two properties of hexokinases which differentiate hexokinase from fructokinase: (1) hexokinase activity is more labile to [H+] than is fructokinase, and (2) hexokinase activity is markedly inhibited by N-acetylglucosamine while fructokinase activity is relatively unaffected. 相似文献
To investigate the physical and kinetic properties of sperm carnitine acetyltransferase, the enzyme was purified from bovine spermatozoa and heart muscle. Carnitine acetyltransferase was purified 580-fold from ejaculated bovine spermatozoa to a specific activity of 85 units/mg protein (95% homogeneity). Sperm carnitine acetyltransferase was characterized as a single polypeptide of Mr 62,000 and pI 8.2. Heart carnitine acetyltransferase was purified 650-fold by the same procedure to a final specific activity of 71 units/mg protein. The kinetic properties of purified bovine sperm carnitine acetyltransferase were consistent with the proposed function of this enzyme in acetylcarnitine pool formation. Product inhibition by either acetyl-l-carnitine or CoASH was not sufficient to predict significant in vivo inhibition of acetyl transfer. At high concentrations of l-carnitine, bovine sperm and heart carnitine acetyltransferases were most active with propionyl- and butyryl-CoA substrates, although octanoyl-, iso-butyryl-, and iso-valeryl-CoA were acceptable substrates. Binding of one substrate was enhanced by the presence of the second substrate. Carnitine analogs that have significance in reproduction, such as phosphorylcholine and taurine, did not inhibit carnitine acetyltransferase. Bovine sperm and heart carnitine acetyltransferases were indistinguishable on the basis of purification behavior, pI, pH optima, kinetic properties, acyl-CoA specificity, and sensitivity to sulfhydryl reagents and divalent cations; thus there was no indication that bovine sperm carnitine acetyltransferase is a sperm-specific isozyme. 相似文献
The chitinolytic properties of Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus, are reported. E. americana was shown to produce chitinolytic activity in the absence of chitin and in the presence of glucose and N-acetylglucosamine, indicating constitutive synthesis by these strains. A single 33-kDa protein with chitinolytic activity was purified to homogeneity from culture filtrates, by hydrophobic interaction chromatography using a phenyl-group substituted matrix. This enzyme, by virtue of differential activity against chromogenic chitooligosaccharides and against dye-labelled soluble carboxymethylated chitin (CM-chitin-RBV), was demonstrated to be an endochitinase. Our data suggest this 33-kDa chitinase appeared to be the only chitinolytic enzyme produced by E. americana, strains of which do not grow using chitin as a carbon source. The significance of these findings in the context of mushroom disease is discussed. 相似文献
Arthrobacter protophormiae produced a high level of extracellular endo-β-N-acetylglucosaminidase when cells were grown in a medium containing ovalbumin. The enzyme was induced by the glycopeptide fraction of ovalbumin prepared by pronase digestion. Production of the enzyme was also induced by glycoproteins such as yeast invertase and bovine ribonuclease B but not by monosaccharides such as mannose, N-acetylglucosamine, and galactose. The enzyme was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis and has an apparent molecular weight of about 80,000. The enzyme showed a broad optimum pH in the range of pH 5.0 to 11.0. The enzyme hydrolyzed all heterogeneous ovalbumin glycopeptides, although the hydrolysis rates for hybrid type glycopeptides were very low. The substrate specificity of A. protophormiae endo-β-N-acetylglucosaminidase was very similar to that of Endo-CII from Clostridium perfringens. Therefore, the enzyme induction by A. protophormiae seems to have a close relation to the substrate specificity of the enzyme. 相似文献
A convenient means was devised for the purification of milligram quantities of a soluble form of the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF II receptor). The receptor was purified to near homogeneity from bovine serum by affinity chromatography on agarose-pentamannosephosphate in the absence of detergent. Approximately 2.5 mg of receptor were obtained from 500 ml of fetal calf serum. The concentration of receptor in serum decreased sharply with development. Fetal calf serum Man-6-P/IGF II receptor was immunologically similar to detergent-solubilized, membrane-bound Man-6-P/IGF II receptor from bovine liver. N-Terminal sequence analysis revealed that the purified serum receptor, but not the solubilized, membrane-associated receptor, contains stoichiometric amounts of bound IGF II. The results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography studies suggest that the fetal calf serum receptor (in contrast to the solubilized, membrane-bound bovine testis receptor) does not aggregate. The affinity of the fetal calf serum receptor for bovine testis beta-galactosidase approximated one-half that observed for solubilized, membrane-bound bovine testis receptor. 相似文献