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PubMedDate not supplied

Allergenicity testing of supermethrin, phenoxyacetic acid and DNCB using in vivo and in vitro modifications of the local lymph node assays, maximization and epicutaneous testing.

The purpose of this study was to compare two methods of testing for allergenicity: in vivo and in vitro modifications of local lymph node assays (LLNA) in mice and the maximization and epicutaneous skin tests in guinea pigs as per the Organization for Economic Cooperation and Development (1981). Two pesticides-the synthetic pyrethroid insecticide supermethrin (SM) and the herbicide phenoxyacetic acid (PAA)-were evaluated using this testing battery. 1-Chloro-2,4-dinitrobenzene (DNCB) was selected as a reference allergen for the local lymph node assay. In vitro modification of LLNA proliferative response per standard cell count in lymphocyte cultures derived from treated Balb/c mice did not differ from control mice. Results of the in vivo modification showed that treatment with 50% PAA and 50% SM resulted in a lower proliferation response of lymphocytes in lymph nodes compared with control animals. The vigour of the proliferative response varied more in in vivo modification of LLNA. Stimulation indices were <3, so PAA and SM did not indicate classification as allergens. Lymphocyte proliferation in 1% DNCB-activated lymph nodes was approximately fivefold higher than in those derived from control mice. Proliferation response in vitro calculated as stimulation index was higher in DNCB-treated mice than those observed in vivo, but differences were not dramatic. Auricular lymph node weight and cellularity in mice treated with PAA and SM were similar to controls. The DNCB stimulation index for lymph node cellularity was 5.5. Lymph node weight was three times higher in comparison with controls. In the maximization test in guinea pigs SM and PAA acid resulted in 40% and 50% of animals demonstrating sensitization, respectively. Epicutaneous administration resulted in weaker reaction. Both SM and PAA are mildly strong sensitizers by this battery.

Acetates↗
PubMed2003

Surface-modified poly(lactide-co-glycolide) nanospheres for targeted bone imaging with enhanced labeling and delivery of radioisotope.

Surface-modified nanospheres can be utilized for targeting drugs and diagnostic agents to the bone and bone marrow while extending their circulation time in the blood stream. The surface modification of poly(lactide-co-glycolide) (PLGA) nanospheres by radioisotope carrying poly(ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) triblock copolymers (Poloxamer 407) has been assessed by in vitro characterization and in vivo biodistribution studies after intravenous administration of the nanospheres to the mouse. A hydroxyphenylpropionic acid, a ligand for (125)I and (131)I labeling, was conjugated to the hydroxyl group of the Poloxamer 407 by using dicyclohexyl carbodiimide. The ligand-conjugated Poloxamer 407 was adsorbed onto the surface of PLGA nanospheres. Surface coating was confirmed by measuring both size distribution and the surface charge of the nanospheres. Besides, (125)I-labeling efficiency, radiolabeling stability, whole body imaging, and biodistribution of the radioisotope-labeled nanospheres were examined. Ligand-labeled, surface-modified PLGA nanospheres were in 100-nm size ranges, which may be adequate for long-circulation and further bone imaging. (125)I-labeling efficiency was >90% and was more stable at human serum for 24 h. A noticeable decrease in liver or spleen uptake was obtained by the surface-modified nanospheres. (125)I-labeled nanospheres showed higher blood maintenance and bone uptake compared with stannous colloid with the same size distribution. Therefore, a fully biodegradable, radioisotope-carrying, surface-modified nanosphere system has been developed as a promising tool for targeting bone and bone marrows.

Animals↗
PubMed2006

Enhancing surface free energy and hydrophilicity through chemical modification of microstructured titanium implant surfaces.

Roughness-induced hydrophobicity, well-known from natural plant surfaces and intensively studied toward superhydrophobic surfaces, has currently been identified on microstructured titanium implant surfaces. Studies indicate that microstructuring by sandblasting and acid etching (SLA) enhances the osteogenic properties of titanium. The undesired initial hydrophobicity, however, presumably decelerates primary interactions with the aqueous biosystem. To improve the initial wettability and to retain SLA microstructure, a novel surface modification was tested. This modification differs from SLA by its preparation after acid etching, which was done under protective gas conditions following liquid instead of dry storage. We hypothesized that this modification should have increased wettability due to the prevention of contaminations that occurs during air contact. The main outcome of dynamic wettability measurements was that the novel modification shows increased surface free energy (SFE) and increased hydrophilicity with initial water contact angles of 0 degrees compared to 139.9 degrees for SLA. This hydrophilization was kept even after any drying. Reduced hydrocarbon contaminations were identified to play a possible role in altered surface thermodynamics. Such surfaces aim to retain the hydrophilicity and natural high surface energy of the Ti dioxide surface until surgical implants' insertion and are compared in this in vitro study with structural surface variants of titanium to compare roughness and chemically induced wettability.

Acids↗
PubMed2006

Inhibition of phospholipase C-beta1-mediated signaling by O-GlcNAc modification.

Here we report inhibition of phospholipase C-beta1 (PLC-beta1)-mediated signaling by post-translational glycosylation with beta-N-acetylglucosamine (O-GlcNAc modification). In C2C12 myoblasts, isoform-specific knock-down experiments using siRNA showed that activation of bradykinin (BK) receptor led to stimulation of PLC-beta1 and subsequent intracellular Ca2+ mobilization. In C2C12 myotubes, O-GlcNAc modification of PLC-beta1 was markedly enhanced in response to treatment with glucosamine (GlcNH2), an inhibitor of O-GlcNAase (PUGNAc) and hyperglycemia. This was associated with more than 50% inhibition of intracellular production of IP3 and Ca2+ mobilization in response to BK. Since the abundance of PLC-beta1 remained unchanged, these data suggest that O-GlcNAc modification of PLC-beta1 led to inhibition of its activity. Moreover, glucose uptake stimulated by BK was significantly blunted by treatment with PUGNAc. These data support the notion that O-GlcNAc modification negatively modulates the activity of PLC-beta1.

Acetylglucosamine↗
PubMed2005

Effect of the covalent modification of horseradish peroxidase with poly(ethylene glycol) on the activity and stability upon encapsulation in polyester microspheres.

Encapsulation of proteins in polyester microspheres by coacervation methods frequently causes protein inactivation and aggregation. Furthermore, an often-substantial amount of the encapsulated proteins is released within the first 24 h from the microspheres. To overcome these problems poly(ethylene glycol) (PEG) was employed as excipient and protein-modifying agent. The model protein horseradish peroxidase (HRP) was chemically modified or co-lyophilized with PEG of differing molecular weights, namely PEG(5000), PEG(20000), and PEG(40000). The lyophilized preparations were encapsulated in poly(D,L-lactide-co-glycolic) acid (PLGA) microspheres by a coacervation method. Covalent modification of HRP with PEG increased the encapsulation efficiency (EE) from 83% to about 100% while PEG when used as an excipient reduced the EE. Encapsulation caused aggregation of ca. 5% of non-modified HRP and the residual specific activity was only 57%. Covalent modification with PEG reduced HRP aggregation to less than 1% and improved its residual activity to more than 95%. When PEG was used as excipient similar results were found with respect to a reduction in encapsulation-induced aggregation, but no more than 80% of residual activity was obtained even for the best formulation after encapsulation. It was also found that covalent modification of HRP with PEG substantially reduced the unwanted initial "burst" release observed during the initial 24 h of in vitro release from about 70% to 23%. Furthermore, HRP activity and stability were also improved during in vitro release for HRP-PEG conjugates. The data show that covalent modification of proteins with PEG might be useful to improve protein stability during coacervation encapsulation and subsequent release as well as to increase EE and reduce the burst release.

Chemistry, Pharmaceutical↗
PubMed2002

Thermal and chemical modification of dentin by 9-11-microm CO2 laser pulses of 5-100-micros duration.

BACKGROUND AND OBJECTIVES: Previous studies have shown that dentin can be thermally modified by pulsed CO(2) laser irradiation to form a more highly mineralized tissue. The implications are important for the potential laser modification or removal of dentinal and root caries and the transformation of dentin to a more acid resistant mineralized tissue. STUDY DESIGN/MATERIALS AND METHODS: Time resolved radiometry measurements with TEA CO(2) laser pulses were used to determine the magnitude of the absorption coefficients of dentin at the highly absorbed CO(2) laser wavelengths and to measure the temperature excursions during lambda = 9.3, 9.6, 10.3, and 10.6 microm laser irradiation at irradiation intensities of 0.1-8 J/cm(2) per pulse. In addition, photoacoustic and transient reflectance measurements were used to monitor the loss of water and organics and to detect the thresholds for surface modification and tissue ablation. RESULTS: The absorption coefficients were measured to be 5,000; 6,500; 1,200; and 800 cm(-1) at lambda = 9.3, 9.6, 10.3, and 10.6 microm, respectively. The surface temperatures of dentin were markedly higher than those measured on enamel for similar irradiation intensities due to the lower reflectance losses of dentin and the lower thermal diffusivity of dentin at the respective wavelengths. Hence, lower fluences are required for the thermal decomposition of dentin. Ablation typically occurred with the first few laser-pulses during multiple pulse irradiation and eventually ceased after modification of dentin to a more highly mineralized enamel-like tissue. The debris ejected during the initial laser pulses shielded the surface by as much as 60% at the low fluences employed in this study. Optical and electron microscopy and IR spectroscopy indicated that incident laser pulses with incident fluence as low as 0.5 J/cm(2) at 9.3 and 9.6 microm wavelengths with a duration of 5-8-micros were sufficient to induce chemical and morphological changes in dentin. CONCLUSIONS: In this study, the laser parameters for the efficient thermal modification of dentin with minimum heat deposition at CO(2) laser wavelengths were firmly established.

Dental Caries↗
PubMed2005

Dynamics of temperature dependent modifications of blood in the near-infrared.

BACKGROUND AND OBJECTIVES: Laser irradiation induces blood coagulation by heating of blood components. It is a complex phenomenon which encompasses a variety of processes, such as cell shape modification, cell membrane rupture, protein denaturation, aggregation, and finally blood gelation. An in vitro study was performed to investigate heating temperatures leading to transformation of blood and to have a better understanding of the dynamics of temperature dependent modifications of blood in the near-infrared. STUDY DESIGN/MATERIALS AND METHODS: Slow heating of whole blood and hemolized blood was performed using a specific optical chamber. Eight hundred and five and 940 nm light transmission of blood was measured as a function of time during heating at various temperatures (60-75 degrees C). RESULTS: During heating of whole blood, three phases were clearly identified. For hemolized blood, only phase 3 was present. For whole blood, the duration of each phase was correlated to blood temperature. A temporary increase of transmission was observed during heating with a maximum at 65 degrees C. The analysis of the dynamics of temperature dependent modifications of blood are consistent with cell shape modification, denaturation, and aggregation of blood, resulting in the formation of an aqueous gel-matrix. CONCLUSIONS: "Slow" vessel heating which is now proposed as the optimal mechanism for permanent vessel closure should be reconsidered in terms of our results. In that particular case, the optical coefficients of blood could be different from those expected. In case of a large blood vessel, this could be an advantage since a "temporary" higher transmission of light could lead to a more homogeneous heating of the blood vessel. In case of a small blood vessel, this phenomenon could lead to a collateral damage of the skin.

Blood↗
PubMedDate not supplied

Monitoring 2-D gel-induced modifications of proteins by MALDI-TOF mass spectrometry.

In addition to more than 200 endogenously produced post-translational modifications, a detailed analysis of 2-D gel-separated proteins must also consider other modifications that a protein can experience during various steps of its separation. This review describes the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to investigate some of these modifications, which can originate during sample preparation and/or during the separation phase. The analyses described were mostly conducted at pH 9-9.5, and yielded reliable information on stable adduct formation that involved protein-bound amino acids and a number of gel components, including acrylamide derivatives, gel cross-linkers, and Immobiline chemicals. The -SH group of Cys was found to be the prime target of such adducts; however, longer reaction times revealed the involvement of the epsilon-NH2 of Lys. The same analysis revealed that the failure to achieve full reduction/alkylation prior to any electrophoretic step could result in protein-protein interaction, which could lead to a number of spurious spots in the final 2-D map. The implications of these modifications on the MS analysis in particular and on proteome research in general are discussed.

Alkylation↗
PubMedDate not supplied

Graphic-aided study of metabolic modifications of plasma in cancer using proton magnetic resonance spectroscopy.

Proton high-resolution MRS of human plasma allows the rapid detection, on the same spectrum, of many compounds originating from different metabolic pathways. In this paper, we illustrate the modifications of the plasma metabolic profiles recorded by proton NMR spectroscopy in different classes of cancers. These modifications can be easily monitored with graphic aids such as 'star plots' which define for each type of cancer a particular pattern describing the most altered metabolic pathways. By using 'star plots' three types of metabolic patterns have been distinguished: (i) the 'inflammatory' pattern characterized by an increase of glycosylated moieties of glycoproteins; (ii) a 'lipid modified' pattern, characterized by various modifications occurring mainly in the lipid moieties detected by MRS; and (iii) a pattern which is often observed in sarcomas and mainly characterized by an alteration in the N-acetyl glucosamine/N-acetyl neuraminic acid ratio. This study demonstrates the ability of proton MRS of plasma to rapidly detect the occurrence of metabolic modifications brought about by cancer evolution or therapy.

Acetylglucosamine↗
PubMed2003

Tissue-specific p53 responses to ionizing radiation and their genetic modification: the key to tissue-specific tumour susceptibility?

Although little is understood of the underlying mechanisms, there are tissue-specific responses to tumourigenic and therapeutic agents and these responses are influenced by genetic factors. Ionizing radiation is an important tumourigenic and therapeutic agent for which there is substantial evidence for such tissue-dependent and genotype-dependent responses. Because the p53 tumour suppressor protein is a major determinant of cellular responses to radiation, the present study has investigated whether modification of the p53 pathway contributes to tissue-dependent and genotype-dependent responses using inbred strains of mice. Comparison of responses in haemopoietic and epithelial cells in irradiated C57BL/6 and DBA/2 mice revealed significant differences in p53 and apoptotic responses in different cell types and in different cells of the same type, reflecting the complexity of damage responses operating in the whole organism. The data suggest that p53-mediated up-regulation of Bax is a major determinant of apoptosis in the spleen, but not in the intestine, whereas p53-mediated induction of p21(waf1) plays an anti-apoptotic role in the spleen, but not in the intestine. It is also shown that p53 stabilization and differential transactivational activities towards Bax or p21(waf1) are influenced by genetic factors that act in a tissue-specific manner. Analysis of ATM, a potential mediator of differential p53 activation, indicates that this key regulator of radiation responses is preferentially induced in epithelial cells, but is unlikely to account for genetic modification of p53 or apoptotic responses in the mouse strains studied. Polymorphisms in the p53 or DNA-PKcs genes are also unlikely to account for the genetic modifications that are reported here. There are numerous further potential modifiers of the p53 pathway, but analysis of backcross and inter-cross mice demonstrates that genes responsible for the complex modification of these in vivo responses can be identified by linkage analysis. This approach has the potential to reveal new or unexpected interactions involving the p53 pathway that determine both short-term and long-term effects of radiation exposure and the basis of tissue-specific responses and tumour susceptibility.

Animals↗
PubMed2005

Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol.

While phosphorylation and O-GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post-translational modifications have been difficult to analyze in proteomic studies. We describe advances in beta-elimination / Michael addition-based approaches which allow for mass spectrometry-based identification and comparative quantification of O-phosphate or O-GlcNAc-modified peptides, as well as cysteine-containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope-coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O-phosphorylation and O-GlcNAc modification sites. Specificity of O-phosphate vs. O-GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O-GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post-translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large-scale quantitative analysis of both protein expression and serine / threonine post-translational modifications.

Animals↗
PubMed1992

Inactivation and covalent modification of CTP synthetase by thiourea dioxide.

Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.

Binding Sites↗
PubMed1993

Analysis and modification of thiols in the hemolymph juvenile hormone binding protein of Manduca sexta.

Hemolymph-induced in vitro modifications of the hemolymph juvenile hormone binding protein from the tobacco hornworm, Manduca sexta, were analyzed by polyacrylamide gel electrophoresis, Western blots, and equilibrium dialysis. Upon hemolymph melanization, a complex reaction involving polymerization of phenolic compounds, multiple forms of the protein were detected. Under melanizing conditions, one slower- and several faster-migrating proteins were observed. When the hemolymph was treated with 100 mM catechol, a melanin precursor, two well-defined forms of the binding protein appeared. When the protein was incubated with excess catechol (> 100 mM) a single faster-migrating form appeared. Both fast and slow forms bound juvenile hormone I with similar efficiency. These results were duplicated using the thiol modifying reagents p-chloromercurobenzoate and N-ethylmaleimide, suggesting modification of cysteine residues. Using differential alkylation, it was determined that hemolymph juvenile hormone binding protein contained two cystine and two cysteine residues. One of the cysteines is exposed and readily accessible for modification. Since modification of the exposed free thiol did not alter binding, it presumably resides outside the hormone-binding domain. The analyses support the observation that there is a single form of the hemolymph juvenile hormone-binding protein in our strain of M. sexta.

Alkylation↗
PubMed1993

Identification of Ser143 as the site of modification in the active site of histidine ammonia-lyase.

Histidine ammonia-lyase (histidase) from Pseudomonas putida was irreversibly inactivated by L-cysteine at pH 10.5 in the presence of oxygen. Inactivation was accompanied by the formation of a new uv-absorbing species centered around 340 nm. L-[35S]cysteine labeling experiments revealed that 4 mol of L-cysteine was bound per mole of enzyme tetramer upon complete modification. However, the radiolabel was dissociated from the protein under denaturing conditions without loss of the 340-nm absorbance. Prior inactivation of histidase by cyanide, borohydride, or bisulfite precluded the formation of the 340-nm species in subsequent L-cysteine modification experiments. This suggests a common target site for modification of histidase by all of these reagents. Based on its strong absorbance at 340 nm an octapeptide was isolated from L-cysteine-inactivated histidase following trypsin and staphylococcal V8 protease digestion. Electrospray MS/MS revealed that this peptide (Gly138-SerValGlyAlaSerGlyAsp145) contained an unidentified modification of mass 184 Da located on Ser143. This peptide and the serine residue are conserved in all histidases and phenylalanine ammonia-lyases for which the amino acid sequence is available. Ser143 represents the binding site for an electrophilic cofactor required for histidase activity.

Amino Acid Sequence↗
PubMed1996

4-Hydroxy-2-nonenal cytotoxicity in renal proximal tubular cells: protein modification and redox alteration.

4-Hydroxy-2-nonenal (HNE), one of the major products of membrane lipid peroxidation, has been shown recently to be present in a form covalently attached to proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate (Toyokuni, S., et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2616-2620; Uchida, K., et al. (1995) Arch. Biochem. Biophys. 317, 405-411). In the present study, the mechanism of HNE cytotoxicity was studied using the renal tubular epithelial cells (LLC-PK1), focusing on the protein modification and alteration of cellular redox status induced by HNE. Upon treatment with HNE for 2 h, the LLC-PK1 cells were found to be resistant to the low concentration (10 microM) of HNE, while HNE at higher concentrations (> or = 50 microM) mediated cell death. The cytotoxicity of HNE appeared to be correlated with the HNE modification of cellular proteins. Among a number of proteins modified by HNE, a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was detected as one of the major targets of HNE in the cells. On the other hand, exposure of LLC-PK1 cells to HNE resulted in rapid reduction of cellular glutathione (GSH) levels, suggesting that HNE influenced primarily the redox status of the cells. Depletion of GSH with buthionine sulfoximine, a potent suppressor of GSH biosynthesis, before HNE treatment caused the cells to be sensitive to HNE cytotoxicity and to HNE modification of cellular proteins, whereas the increase in intracellular GSH levels by treatment with N-acetylcysteine before HNE treatment resulted in a dose-dependent inhibition of HNE-mediated protein modification. These results suggest that intracellular GSH is a determinant on cellular resistance against the HNE-mediated cytotoxicity.

Acetylcysteine↗
PubMed1996

Oxidative modification of nicotinamide nucleotide transhydrogenase in submitochondrial particles: effect of endogenous ubiquinol.

The present paper describes the sensitivity of the mitochondrial nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) to oxidative modification, and the effects of endogenous ubiquinol on this modification. A comparison is made between the effects of treatment with ADP-Fe3+ and ascorbate and with peroxynitrite, using kinetic, electrophoretic, and immunological analyses, together with lipid peroxidation measurements. The transhydrogenase was inactivated by both types of oxidative modification, but apparently through different mechanisms. Ubiquinol protected the enzyme against inactivation only when the modification was caused by ADP-Fe3+ and ascorbate treatment. Kinetic measurements revealed a threefold increase of the Km value of the enzyme for NADPH after exposure to ADP-Fe3+ and ascorbate, and a twofold increase of the Km values for both NADH and NADPH after exposure to peroxynitrite. NAD(H) exerted a protection against trans-hydrogenase inactivation when added to the preincubation in the case of peroxynitrite, but neither NAD(H) or NADP(H) protected in the case of ADP-Fe3+ and ascorbate. Using immunoblotting it was shown that the enzyme became both aggregated and fragmented, although to different extents, depending on the oxidative system used. Again, ubiquinol prevented these effects only in the case of ADP-Fe3+ and ascorbate treatment. Furthermore, there occurred a striking decrease in the 66-kDa trypsin fragment after exposure of the enzyme to ADP-Fe3+ and ascorbate, and of the 48-kDa trypsin fragment after exposure to peroxynitrite. It is concluded that the mitochondrial nicotinamide nucleotide transhydrogenase is sensitive to oxidative stress and that the mechanism underlying this can vary according to the challenge to which the enzyme is exposed. Endogenous ubiquinol may play a role in protecting the enzyme against agents perturbing the lipid phase of the membrane.

Adenosine Diphosphate↗
PubMed1997

Mapping the mechanism-based modification sites in L-aspartase from Escherichia coli.

Inactivation of the enzyme L-aspartase from Escherichia coli by the substrate analog aspartate beta-semialdehyde has previously been shown to occur by the mechanism-based conversion to the corresponding product aldehyde, followed by covalent modification of cysteine-273 (F. Giorgianni et al. (1995) Biochemistry 34, 3529). Inactivation by the product analog, fumaric acid aldehyde (FAA), has now been examined directly by adding a reduction step to the modification protocol in order to stabilize the resulting enzyme-FAA derivative(s). HPLC and mass spectrometric analyses of proteolytic digests of inactivated L-aspartase have confirmed the modification at cysteine-273, and have also identified an additional modified peptide. The inactivation at this additional site involves a crosslink between cysteine-140 and an adjacent lysine. Site-directed mutagenesis studies have shown that cysteine-140 is a very reactive and accessible nucleophile that is not, however, directly involved in enzyme activity. The adjacent lysine-139 that is modified does appear to play a role in substrate binding. A double mutant in which both of the reactive cysteines have been replaced is almost completely insensitive to modification by these substrate and product analogs.

Aspartate Ammonia-Lyase↗
PubMed1999

Oxidative modification of apolipoprotein E in human very-low-density lipoprotein and its inhibition by glycosaminoglycans.

The mechanism of metal ion-catalyzed oxidative modification of apolipoprotein E (apoE) in human very-low-density lipoprotein (VLDL) and its inhibition by glycosaminoglycan (GAG) was investigated in vitro. The VLDL oxidation catalyzed by Cu2+ led to the lipid peroxidation, the formation of aggregates, and covalent modification of apoE. The modified apoE lost heparin-binding activity. These results suggest that the lipid peroxidation of VLDL and modification of apoE cause impairment of lipid uptake by cells and deposit the oxidized lipids in the tissues. The lipid peroxidation and oxidative modification of apoE in VLDL mediated by Cu2+ and an aqueous radical generator were suppressed by GAG, heparan sulfate, heparin, and chondroitin sulfate A, even though GAGs demonstrated no ability to scavenge alpha,alpha-diphenyl-beta-picrylhydrazyl radical. There were no relationships between inhibitory activity of GAGs in the VLDL oxidation and their number of sulfate groups which possess chelating activity of metal ion. Therefore, it can be considered that the inhibition of VLDL oxidation by GAGs is possibly due to the interaction between GAG and VLDL which bring about the steric hindrance, interference with the reaction between VLDL particle and the reactive oxygen species. These studies suggest that GAGs preserve the biological functions of apoE from oxidative stress.

Adult↗