Purification and some properties of UDP-N-acetylglucosamine 4-epimerase from Bacillus subtilis.
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Abstract:
UDP-N-Acetylglucosamine 4-epimerase (EC 5.1.3.7) was purified from a cell extract of Bacillus subtilis by protamine sulfate treatment, ammonium sulfate fractionation, and column chromatographies on DEAE-Sephadex, hydroxylapatite, and Sephadex G-150. The purified enzyme was homogeneous on disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 62, 000 by gel filtration. The enzyme had an optimal pH in the range of 7.0 to 9.0, and was active at 30-35°C in the absence of added NAD and more active at 40-45°C in the presence of NAD. The enzyme activity was highly stimulated by small amounts of NAD. The estimated Km values were 4.4mM for UDP-N-acetylglucosamine and 0.13mM for NAD. No UDP-glucose 4-epimerase activity was found in the purified enzyme. The enzyme was inhibited by high concentrations of glucosamine and inhibition by glucosamine was competitive with UDP-N-acetylglucosamine. The inhibition constant was 37.0mM. N-Acetylglucosamine did not inhibit the enzyme.Keywords:
Sephadex
N-Acetylglucosamine
Acetylglucosamine
Ammonium sulfate
N-Acetylglucosamine
Amino sugar
Acetylglucosamine
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N-Acetylglucosamine
Acetylglucosamine
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N-Acetylglucosamine
Carbohydrate Metabolism
Acetylglucosamine
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A reaction mixture was devised for the synthesis of N-acetylglucosamine-6-phosphate in the bovine parotid gland extract. The reaction products were identified qualitatively as the phosphate esters of glucosamine and acetylglucosamine. The presence of three enzymes in the parotid gland, hexokinase, acetate activating enzyme and glucosamine-6-phosphate N-acetylase has been suggested. Cysteine, a component of the earlier mixture, inhibited the production of N-acetylglucosamine-6-phosphate probably by inhibiting the acetylation of glucosamine-6-phosphate. Cysteine was found to inhibit the color reaction in the procedure of Morgan-Elson method for the detection of N-acetylglucosamine.
N-Acetylglucosamine
Hexokinase
Acetylglucosamine
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N-Acetylglucosamine
Acetylglucosamine
Neuraminic acid
N-Acetylneuraminic acid
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N-Acetylglucosamine
Acetylglucosamine
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The effect of N ‐unsubstitution of chitin and chitin oligosaccharides on their hydrolysis by lysozyme was studied. Lysozyme digests of chitin deacetylated at about 70% of its glucosamine residues were fractionated by gel chromatography, paper chromatography and paper electrophoresis, giving six oligosaccharides with N ‐unsubstituted glucosamine residues, C1–C6, together with the monomer to tetramer of N ‐acetylglucosamine. Oligosaccharides C1, C2 and C3, which accounted individually for about 4–7% of the total glucosamine residues in the isolated saccharides, were identified as GlcNAc‐GlcNAc‐GlcNAc‐GlcN, GlcNAc‐GlcN‐GlcNAc‐GlcNAc and GlcNAc‐GlcN‐GlcNAc‐GlcN, respectively. Oligosaccharide C4, present in a smaller amount, and oligosaccharide C5, present in a very small amount, were identified as GlcNAc‐GlcNAc‐GlcN and GlcN‐GlcNAc‐GlcNAc, respectively. Oligosaccharide C6, accounting for about 7% of the glucosamine residues recovered, was identified as GlcNAc‐GlcN. On further digestion with excess lysozyme, tetrasaccharide C1 yielded small amounts of N ‐acetylglucosamine and GlcNAc‐GlcNAc‐GlcN in addition to major products, (GlcNAc) 2 and GlcNAc‐GlcN. Tetrasaccharide C2 was hydrolyzed by lysozyme into GlcNAc‐GlcN and (GlcNAc) 2 ; tetrasaccharide C3 into GlcNAc‐GlcN alone; trisaccharide C4 into N ‐acetylglucosamine and GlcNAc‐GlcN. In contrast, trisaccharide C5 was shown to be completely resistant to lysozyme. The results of digestion of the isolated oligosaccharides with lysozyme, together with the structural feature of these saccharides, were accounted for by the importance of the N ‐acetyl groups of the N ‐acetylglucosamine residues located on subsites C and E in the enzyme · substrate complexes.
Tetrasaccharide
Trisaccharide
N-Acetylglucosamine
Oligosaccharide
Acetylglucosamine
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ABSTRACT The effect of glucosamine and N-acetylglucosamine on aggregation and energy metabolism was investigated over an 8-h period in cells dissociated by 0·25 % (w/v) trypsin from the skeletal muscle of 9-day-old chick embryos. At 8 h, 0·023 M glucosamine and N-acetylglucosamine inhibited the aggregation of cells suspended in Eagle’s minimal essential medium by 18·9 % and 16·4% respectively, as judged on a basis of aggregate size. Glucosamine and N-acetylglucosamine reduced the cellular ATP level by a mean of 36 % and 27 % respectively; values reflected in the 32% and 19% loss of total adenine nucleotides caused by these sugars. The adenine nucleotide balance was also changed from a mean control ATP/AMP ratio of I3·7 to 8·75 by N-acetylglucosamine and to 8·0 by glucosamine. Intracellular lactate/pyruvate ratios were similarly disrupted in cells incubated with 0·023 M hexosamine. Although the hourly values fluctuated, it was seen that the amount of lactic acid relative to pyruvic acid, considered as an average for the 8-h period, was raised from 6:1 in controls to 8:1 in N-acetylglucosamine-treated and to 10:1 in glucosamine-treated cell preparations. Compared to controls at 8 h, glucosamine enhanced the production of lactate into the suspension medium by 99%. The N-acetyl analogue caused cells to produce more lactic acid than did controls for 4–5 h only, for by 8 h 25 % less of this metabolite was assayed in the culture medium. The incorporation of D-[U-14C]glucose into glycogen paralleled the results of extracellular lactic acid assays. N-acetylglucosamine inhibited the incorporation by 30 % at 4 h, although by 6 h, and for the remainder of the experimental period, there was more 14C-labelled glycogen in these cells than in controls. By contrast, glucosamine inhibited the incorporation of radioactive glucose into glycogen by 42% at 4 h and, unlike N-acetylglucosamine, consistently thereafter. Glucosamine also enhanced cellular oxygen uptake throughout the experimental period, to the extent of 59 % at 8 h. The oxygen uptake of N-acetylglucosamine-treated cells was similar to controls until about the 5th hour, when there was a subsequent inhibition which had accumulated to 13 % by the end of the experiment. The release of 14COs by cells was inhibited by glucosamine. This hexosamine depressed production by 19% at 12 h whereas N-acetylglucosamine inhibited this evolution by 9 % at this time. The metabolic effects of these hexosamines on chick muscle cells in vitro are mainly attributed to a central alteration of the adenine nucleotide balance although certain other documented effects of glucosamine are considered to be involved. An inhibition of cell aggregation by glucosamine and N-acetylglucosamine is discussed in terms of a depressed cellular metabolic economy.
N-Acetylglucosamine
Acetylglucosamine
Amino sugar
Pyruvic acid
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Acetylglucosamine
N-Acetylglucosamine
Galactosamine
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Nitrous acid, which specifically depolymerises polymers containing hexosamines with a primary amino group, was used to analyse the hexosamine‐containing polymers in the hyphal wall of Mucor mucedo. N ‐Acetylglucosamine was found to occur in three polymeric fractions. One fraction which was solubilished by HNO 2 treatment contained N ‐acetylglucosamine interspersed with glucosamine; no homopolymer of glucosamine (chitosan) was detected. Another fraction became HNO 2 ‐soluble after treatment with pronase or alkali; this points to the occurrence of a heteropolymer containing N ‐acetylglucosamine and glucosamine in which some of the glucosamine residues are linked to peptides via their amino groups. The residue remaining after pronase and HNO 2 treatment appeared to consist of a homopolymer of N ‐acetylglucosamine (chitin).
Pronase
N-Acetylglucosamine
Muramic acid
Hexosamines
Residue (chemistry)
Acetylglucosamine
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