Enrichment of enzyme activity on deformylation of 1-NFK-lysozyme.
(1/395)
The formamide linkage of an inactive lysozyme derivative (1-NFK-lysozyme), formed by selective ozonization of tryptophan 62 in hen egg-white lysozyme [EC 3.2.1.17] was hydrolyzed with dilute acid faster in the frozen state at about --10 degrees than at 20 degrees. On hydrolysis of 1-NFK-lysozyme the low lytic activity increased to approximately 80% of that of native lysozyme. It is suggested that the binding ability associated with kynurenine 62 in the lysozyme derivative formed by this hydrolysis may be responsible for increase in enzymatic activity. (+info)
Preferential acid-catalyzed hydrolysis of the formamide linkage of N'-formylkynurenine in frozen solution.
(2/395)
Acid-catalyzed hydrolysis of the formamide linkage of N-acetyl-N'-formyl-L-kynurenineamide in frozen dilute hydrochloric acid solution followed first-order kinetics, yielding N-acetyl-L-kynurenineamide as the sole reaction product. The maximum rate of reaction in the frozen solution was found at around -7.5 degrees and approximated that of the reaction in liquid solution at 40 degrees. By freezing the dilute acid solution at -8 degrees the reaction was accelerated by 60 times compared with that in super-cooled liquid solution at the same temperature. (+info)
The kynurenine metabolic pathway in the eye: studies on 3-hydroxykynurenine, a putative cataractogenic compound.
(3/395)
The rabbit lens has an elevated content of 3-hydroxykynurenine (30HKYN) in spite of a very low activity of the enzymes leading to its synthesis. The iris/ciliary body, on the contrary, has very high activity of 30HKYN synthesizing enzymes but a content of 30HKYN lower than that of the lens. These observations suggest that 30HKYN is formed in the iris/ ciliary body, released into the aqueous humor and then taken up into the lens where it may be used for the synthesis of UV filtering products. An excessive accumulation of 30HKYN in the lens has been associated with cataract formation. We found that available selective inhibitors of kynurenine hydroxylase reduced 30HKYN synthesis in both the lens and the iris/ciliary body. (+info)
Identification of glutathionyl-3-hydroxykynurenine glucoside as a novel fluorophore associated with aging of the human lens.
(4/395)
A novel fluorophore was isolated from human lenses using high performance liquid chromatography (HPLC). The new fluorophore was well separated from 3-hydroxykynurenine glucoside (3-OHKG) and its deaminated isoform, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside, which are known UV filter compounds. The new compound exhibited UV absorbance maxima at 260 and 365 nm, was fluorescent (Ex(360 nm)/Em(500 nm)), and increased in concentration with age. Further analysis of the purified compound by microbore HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 676 Da. This mass corresponds to that of an adduct of GSH with a deaminated form of 3-OHKG. This adduct was synthesized using 3-OHKG and GSH as starting materials. The synthetic glutathionyl-3-hydroxykynurenine glucoside (GSH-3-OHKG) adduct had the same HPLC elution time, thin-layer chromatography R(F) value, UV absorbance maxima, fluorescence characteristics, and mass spectrum as the lens-derived fluorophore. Furthermore, the (1)H and (13)C NMR spectra of the synthetic adduct were entirely consistent with the proposed structure of GSH-3-OHKG. These data indicate that GSH-3-OHKG is present as a novel fluorophore in aged human lenses. The GSH-3-OHKG adduct was found to be less reactive with beta-glucosidase compared with 3-OHKG, and this could be due to a folded conformation of the adduct that was suggested by molecular modeling. (+info)
Growth inhibition of multiresistant enterococci by interferon-gamma-activated human uro-epithelial cells.
(5/395)
Nosocomial infections with enterococci are an increasing problem in modern medical practice due to the development of resistance to a wide range of antibiotics, including the glycopeptides vancomycin and teicoplanin. An increasing number of vancomycin-resistant enterococci (VRE) have been cultured from clinical specimens -- especially from patients undergoing immunosuppressive therapy -- and bacteraemia caused by these VRE, subsequent to colonisation of epithelial surfaces, is a significant cause of mortality in such patients. Recent evidence showed that the induction of indoleamine 2,3 dioxygenase (IDO) by interferon-gamma (IFN-gamma) inhibited growth of group B streptococci by depleting the essential amino acid L-tryptophan. This study describes the IFN-gamma-induced expression of IDO -- shown at a transcriptional level by Northern blot analysis, at translational level by Western blot and also at a functional level by L-tryptophan degradation to L-kynurenine -- in the uro-epithelial cell line RT4. The depletion of L-tryptophan resulted in growth inhibition of enterococci, and this was confirmed by abrogation of the inhibitory effect by re-supplementation with excess L-tryptophan. Multiresistant enterococci, including vancomycin-resistant strains resistant to all commercially available antibiotics, were inhibited by the IFN-gamma-induced expression of IDO and subsequent L-tryptophan degradation. This may be an important mechanism in the local restriction of colonisation of the urinary tract by endogenous enterococci and in inhibiting the spread of the bacteria beyond the epithelial barrier. (+info)
Human lens coloration and aging. Evidence for crystallin modification by the major ultraviolet filter, 3-hydroxy-kynurenine O-beta-D-glucoside.
(6/395)
The human lens becomes increasingly yellow with age and thereby reduces our perception of blue light. This coloration is associated with lens proteins (crystallins), but its molecular basis was unknown. Here we show that the coloration occurs because of the interaction of crystallins with a UV filter compound, 3-hydroxykynurenine glucoside (3-OHKG). Crystallin modification results from deamination of the 3-OHKG amino acid side chain, yielding an unsaturated ketone that is susceptible to nucleophilic attack by cysteine, histidine, and lysine residues. This novel protein modification contributes to age-related lens coloration and may play a role in human nuclear cataractogenesis. (+info)
Human placental indoleamine 2,3-dioxygenase: cellular localization and characterization of an enzyme preventing fetal rejection.
(7/395)
In order to test the hypothesis (Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, Mellor, Science 281 (1998) 1191-1193) that localized placental tryptophan catabolism prevents immune rejection of the mammalian fetus, the cellular localization and characteristics of human placental indoleamine 2,3-dioxygenase (EC 1.13.11.42) were studied. The localization of indoleamine 2, 3-dioxygenase activity was determined quantitatively using cell fractionation by differential and discontinuous sucrose gradient centrifugation. Enzyme activity was looked for in isolated brush border microvillous plasma membranes of placental syncytiotrophoblast. We found that this membrane preparation (which showed a 32.4-fold purification from the starting homogenate with reference to the activity of a membrane marker enzyme, alkaline phosphatase (EC 3.1.3.1)) was strongly negatively enriched with indoleamine 2,3-dioxygenase (which showed a one twenty-fifth decrease in its specific activity). Placental indoleamine 2, 3-dioxygenase is thus not expressed in the maternal facing brush border membrane of syncytiotrophoblast. 1-Methyl-DL-tryptophan which was used by Munn et al. as a key experimental tool for inhibiting indoleamine 2,3-dioxygenase in the murine model showed a competitive inhibition of human placental indoleamine 2,3-dioxygenase with L-tryptophan. The hypothesis, based on experiments performed in mouse, may therefore be applicable to avoidance of immune rejection of the fetus in human pregnancy. (+info)
UV filter compounds in human lenses: the origin of 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside.
(8/395)
PURPOSE: To investigate UV filter synthesis in the human lens, in particular the biosynthetic origin of the second most abundant UV filter compound, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside. METHODS: Human lenses were analyzed by high-performance liquid chromatography (HPLC) after separate incubation with 3H-tryptophan (3H-Trp), beta-benzoylacrylic acid, D,L-alpha-amino-beta-benzoylpropionic acid, or D,L-3-hydroxykynurenine O-beta-D-glucoside. The effect of pH on the model compound D,L-alpha-amino-beta-benzoylpropionic acid and D,L-3-hydroxykynurenine O-beta-D-glucoside was also investigated. RESULTS: UV filters were not detected in fetal lenses, despite a 5-month postnatal lens displaying measurable levels of UV filters. In adults no radiolabel was incorporated into 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside after 3H-Trp incubations. Beta-benzoylacrylic acid was readily reduced in lenses. D,L-alpha-amino-beta-benzoylpropionic acid and D,L-3-hydroxykynurenine O-beta-D-glucoside slowly deaminated at physiological pH and were converted to beta-benzoylpropionic acid and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside, respectively, after lens incubations. CONCLUSIONS: UV filter biosynthesis appears to be activated at or near birth. Compounds containing the kynurenine side chain slowly deaminate, and in the lens, the newly formed double bond is rapidly reduced. These findings suggest that 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside is derived from L-3-hydroxykynurenine O-beta-D-glucoside through this deamination-reduction process. The slowness of the deamination presumably accounts for the absence of incorporation of radiolabel from 3H-Trp into 4(2-amino-3-hydroxyphenyl)4-oxobutanoic acid O-beta-D-glucoside. (+info)