Two ultramicro-scale methods for plasma uric acid analysis with uricase.
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Ascorbic Acid↗
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Conformational stability of bovine alpha-crystallin. Evidence for a destabilizing effect of ascorbate.
Short-term incubation of bovine alpha-crystallin with ascorbate alters the protein conformational stability. The denaturation curves with urea and guanidinium-chloride show different patterns, suggesting a deviation from a two-state mechanism owing to the presence of one or more intermediates in the unfolding of ascorbate-modified alpha-crystallin. Furthermore, the latter protein profiles are shifted to lower denaturant concentrations indicating a destabilizing action of ascorbate, which is capable of facilitating protein dissociation into subunits as demonstrated by gel filtration with 1.5 M-urea. The decrease in conformational stability cannot be ascribed to any major structural alteration, but rather to localized changes in the protein molecule. In fact, no difference between native and ascorbate-treated alpha-crystallin can be detected by amino acid analysis but perturbation of the tryptophan and tyrosine environment is indicated by alterations in intrinsic fluorescence. Furthermore, turbidity and light-scattering measurements suggest an involvement of the lysine side chains, since aggregability patterns with acetylsalicylic acid are significantly altered. The ascorbate-destabilizing effect on the conformational stability of alpha-crystallin, probably exerted through oxidative modification of amino acid residues and/or the formation of covalent adducts, provokes unfavourable steric interactions between residues along the polypeptide chains, thus favouring aggregation and insolubilization of crystallins which can lead to cataract formation, as also demonstrated by proteolytic digestion patterns which show a lower rate of degradation of the ascorbate-modified alpha-crystallin.
Acrylamide↗
Immunological monitoring of Fenton fragmentation of fibrinogen.
Fibrinogen is transformed into insoluble "neofibe" by reaction with up to 100 microM Cu(II) and 1.5 mM ascorbate. The soluble peptides which are released during the reaction can be monitored by amino acid analysis and by measuring released keto-carbonyl (with DNPH). Immunologic characterization of the soluble peptides, with antibodies directed against fibrino-peptide A (FPA) clearly show the release of this epitope, optimally at 50 microM Cu(II). Anti-FPB gives no evidence of that epitope. However, N-terminal amino acid analyses reveals the presence of 3 peptides terminating in ALA (alpha chain FPA), GLU (beta chain FPB) and SER/ASP (unknown). The release of fibrinopeptides is interpreted within the context of a general mechanism for OH'-induced peptide chain cleavage via intermediate Schiff-base hydrolysis.
Enzyme-Linked Immunosorbent Assay↗
Amidation of joining peptide, a major pro-ACTH/endorphin-derived product peptide.
Based on sequence data, rat and mouse pro-adrenocorticotropin (ACTH)/endorphin could give rise to joining peptide, a short acidic peptide that could terminate with a glutamic acid alpha-amide. Rat and mouse pituitary cells were found to cleave the pro-ACTH/endorphin precursor at an -Arg-Arg- site to produce primarily joining peptide-sized material. The amounts of joining peptide were approximately equimolar to the other major pro-ACTH/endorphin-derived products. Using antisera specific for the COOH-terminal modifications of joining peptide and three analytical approaches which separate amidated from glycine-extended forms of joining peptide, it was found that most of the joining peptide in murine anterior and intermediate pituitary was amidated. Identification of the amidated and glycine-extended forms of joining peptide was confirmed by amino acid analysis of the purified molecules. When anterior pituitary corticotrope tumor cells were grown in culture medium lacking ascorbate, there was no detectable ascorbate in the cells; nevertheless, a significant fraction of the joining peptide produced was alpha-amidated, indicating that production of alpha-amidated product was not totally dependent on ascorbate. The amidation state of the joining peptide produced by mouse corticotrope tumor cells was responsive to added ascorbate. Cells grown in medium containing ascorbic acid at the levels found in plasma concentrated the ascorbate to the levels normally found in pituitary tissue, and nearly all of the joining peptide produced was alpha-amidated. The amidation state of secreted joining peptide mirrored the amidation state of the joining peptide in the cells.
Amides↗
Ascorbic acid-induced crosslinking of lens proteins: evidence supporting a Maillard reaction.
The incubation of calf lens extracts with 20 mM ascorbic acid under sterile conditions for 8 weeks caused extensive protein crosslinking, which was not observed with either 20 mM sorbitol or 20 mM glucose. While no precipitation was observed, ascorbic acid did induce the formation of high-molecular-weight protein aggregates as determined by Agarose A-5m chromatography. Proteins modified by ascorbic acid bound strongly to a boronate affinity column, however, crosslinked proteins were present mainly in the unbound fraction. These observations suggest that the cis-diol groups of ascorbic acid were present in the primary adduct, but were either lost during the crosslinking reaction or sterically hindered from binding to the column matrix. The amino acid composition of the ascorbic acid-modified proteins was identical to controls except for a 15% decrease in lysine. Amino acid analysis after borohydride reduction, however, showed a 25% decrease in lysine, a 7% decrease in arginine and an additional peak which eluted between phenylalanine and histidine. Extensive browning occurred during the ascorbic acid-modification reaction. This resulted in protein-bound chromophores with a broad absorption spectrum from 300 to 400 nm, and protein-bound fluorophores with excitation/emission maxima of 350/450 nm. A 4 week incubation of dialyzed crude lens extract with [1-14C]ascorbic acid showed increased incorporation for 2 weeks, followed by a decrease over the next 2 weeks as crosslinking was initiated. The addition of cyanoborohydride to the reaction mixture completely inhibited crosslinking and increased [1-14C]ascorbic acid incorporation to a plateau value of 180 nmol per mg protein. Amino acid analysis showed a 50% loss of lysine, and 8% decrease in arginine and the presence of a new peak which eluted slightly earlier than methionine. These data are consistent with the non-enzymatic glycation of lens proteins by either ascorbic acid or an oxidation product of ascorbic acid via a Maillard-type reaction.
Amino Acids↗
Effects of ascorbate on insoluble elastin accumulation and cross-link formation in rabbit pulmonary artery smooth muscle cultures.
Cultured pulmonary artery smooth muscle cells derived from the medial vessel layer of weanling rabbits were grown in the presence or absence of sodium ascorbate. The connective tissue elements insoluble elastin and collagen were identified and quantified. Formation and accumulation of alpha-aminoadipic acid gamma-semialdehyde (allysine) and the intermolecular cross-links desmosine (Des), isodesmosine (Ides), and aldol condensation product (Aldol) were evaluated from [14C]lysine pulse-chase experiments. [14C]Des, [14C]Ides, peptide-bound [14C]lysine, [14C]allysine, and [14C]Aldol were determined from amino acid analysis. The latter two components were determined after reduction with NaBH4. [14C]Proline conversion to hydroxy[14C]proline and collagenase susceptibility were used to identify and quantify collagen synthesis. Ascorbate dramatically affects insoluble elastin synthesis, accumulation, and cross-link formation. Cells grown in the presence of ascorbate synthesize and accumulate significantly less insoluble elastin than non-ascorbate cultures. Those elastin molecules which do become incorporated into the extracellular matrix in the presence of ascorbate contain a slightly elevated content of hydroxyproline and lysine and, most importantly, are turned over more rapidly.
Animals↗
Lipid peroxidation of the microsomal fraction and extracted microsomal lipids from DAB-induced hepatomas.
NADPH- and ascorbic acid-induced microsomal lipid peroxidation was almost absent in subcutaneously implanted DAB-induced hepatomas D23, D30 and D192A, and present at greatly reduced levels in DAB-induced primary hepatomas when compared with normal liver controls. Fatty acid analysis of the microsomal lipid from passaged tumours demonstrated adequate levels of substrate in the phospholipid fractions to support lipid peroxidation. Lipid extracted from hepatoma microsomal fractions was shown to undergo ascorbic acid-induced lipid peroxidation, but to a lesser extent that the corresponding liver extract. This may be partially explained by a decrease in the phospholipid content of hepatoma microsomal membranes. However, phospholipid extracted from microsomal fractions of hepatoma and liver supported lipid peroxidation to a similar extent. The possible role of the non-lipid component of the membrane in the process of lipid peroxidation is discussed.
Animals↗
Site-specific oxidation of angiotensin I by copper(II) and L-ascorbate: conversion of histidine residues to 2-imidazolones.
The reaction of a histidine-containing peptide (angiotensin I) with copper (II)/ascorbate under physiological conditions has been studied chemically. In the presence of a catalytic amount of copper(II) ion, ascorbate mediated the oxidative damage to the peptide via selective loss of the histidine residue. Furthermore, the reaction of copper(II)/ascorbate with the peptide gave two products (AGT-1 and AGT-2) selectively. From amino acid analysis of the modified peptides, it was found that either of the two histidine residues within the native peptide was modified. Amino-terminal sequence analysis indicated that AGT-1 and AGT-2 were modified at the His9 and the His6, respectively. In addition, the data of FAB-MS and 1H NMR suggested that the unknown residues (modified histidine) within AGT-1 and AGT-2 should have the 2-imidazolone structure. In order to confirm the 2-imidazolone residue in both modified peptides, they were hydrolyzed and analyzed by reverse-phase HPLC. The result demonstrated that the acid hydrolysis of modified peptides gave a product which was identical to authentic 2-imidazolone residue. Consequently, it was confirmed that the reaction of Cu(II)/ascorbate occurs specifically at the C-2 position of the imidazole ring of the histidine residue within a peptide.
Amino Acid Sequence↗
Serum folates in man.
In an aseptic microbiological assay of folate compounds and their breakdown compounds, using Lactobacillus casei, Streptococcus faecalis, and Pediococcus cerevisiae, 4a-hydroxy-5methyl-4,5,6,7-tetrahydrofolate and 5-methyl-5,8-dihydrofolate were inactive under all conditions to all three organisms and 5-methyl-5,6-dihydrofolate was inactive unless ascorbate was present in the incubation medium, and then only to L. casei. 5-Methyltetrahydrofolate was active only for L. casei, and activity in purified samples to S. faecalis was due to trace amounts of folic acid. Analysis of S. faecalis values in the serum in normal subjects and in patients with various disorders showed that levels of 10-formyltetrahydrofolate are raised in coeliac disease, leukaemia, rheumatoid arthritis, and schizophrenia. 5-Methyltetrahydrofolate is readily absorbed by normal human subjects and by patients with pernicious anaemia but poorly absorbed by patients with coeliac disease or leukaemia. 5-Methyl-5,6-dihydrofolate was quickly absorbed by normal human subjects, being reflected by a considerably raised level of 5-methyltetrahydrofolate in serum when sodium bicarbonate was given by mouth before the 5-methyl-5,6-dihydrofolate. These higher levels were comparable to those in patients with pernicious anaemia after oral administration of 5-methyl-5,6-dihydrofolate. Oral 5-methyl-5,8-dihydrofolate and 4a-hydroxy-5-methyl-tetrahydrofolate did not appear as microbiologically active folates in the serum. The findings of this study suggest that the availability for biological utilisation of the major dietary folate compounds will depend on the amount of gastric acidity and of ascorbate in the intestinal chyme. Many may be unavailable for metabolic utilization in the body.
Anemia, Pernicious↗
The glycation and cross-linking of isolated lens crystallins by ascorbic acid.
Individual lens crystallins were isolated from calf lens extracts and incubated in the presence of ascorbic acid for 3 weeks under aerobic conditions. Both alpha-crystallin and beta H-crystallin rapidly cross-linked to form high molecular weight proteins, which did not enter the resolving gel on SDS-PAGE. Beta L-crystallin was somewhat less reactive, but gamma-crystallin showed little or no crosslinking. Gamma-crystallin, however, was almost equivalent to the other crystallins as a substrate for glycation. This was measured by: (a) the binding of protein to a boronate affinity column; (b) the incorporation of 3H from NaB3H4 into protein; (c) amino acid analysis of the modified proteins to estimate the extent of lysine modification; and (d) the incorporation of [1-14C]ASA into individual crystallins. When the separated crystallins were combined with [125I]gamma-crystallin and incubated with ascorbic acid, radioactivity was readily incorporated into the cross-linked products with other crystallins, but again not with gamma-crystallin itself. Gel filtration chromatography of a mixture of [125I]gamma-crystallin and alpha-crystallin showed the formation of a complex between gamma- and alpha-crystallins. These data suggest that all crystallins are glycated, but that cross-linking occurs preferentially between proteins, which are already bound together non-covalently.
Amino Acids↗
Methionine-80-sulfoxide cytochrome c: preparation, purification and electron-transfer capabilities.
In order to explore the electron-transferring properties of methionine-80-sulfoxide cytochrome c, the pure, chromatographically homogeneous methionine-80-sulfoxide cytochrome c was previously published procedure (Ivanetich, K.M., Bradshaw, J.J. and Kaminsky, L.S. (1976) Biochemistry 15, 1144-1153) was found to produce a mixture of products. In the pure derivative, visible spectroscopy indicates that the 695 nm band indicative of the Met-80-Fe coordination is missing, amino acid analysis indicates that only one methionine is modified to the sulfoxide, and the E0' is found to be 240 mV vs. N.H.E. For succinate cytochrome c reductase activity, the Km for modified cytochrome was about one-ninth that of the native protein, while the maximum turnover number of the reductase with the modified protein was only about 54% of that with native protein. In contrast, the activity with cytochrome oxidase measured polarographically using ascorbate and TMPD under two different buffer/pH conditions, gave Km values that were very similar for both the native and modified cytochromes c, but the maximum turnover numbers of the oxidase with the modified protein were less than 40% of native in either buffer. It is concluded that the Met-80-sulfoxide cytochrome c in the reduced form is able to maintain substantially its heme crevice structure and thus maintain Km values similar to those of native protein. However, the low maximum turnover numbers for oxidase activity with the modified protein in the reduced state indicate that electron transfer itself has been significantly decreased, probably because the parity of acid/base and electrostatic interactions of Met-80 sulfur with the Fe in the two redox states has been disrupted.
Animals↗
The effect of zinc on iron-induced lipid peroxidation in different lipid systems including liposomes and micelles.
The effect of zinc on FeSO4/ascorbic acid-induced lipid peroxidation was measured by the thiobarbituric acid assay in various lipid systems including small unilamellar liposomes prepared from egg phosphatidylcholine (EPC), ionic micelles prepared from arachidonic acid (C20:4), non-ionic monocomponent micelles prepared from EPC-derived, methylated fatty acids, and an eicosatetrene emulsion. With the exception of C20:4 micelles, zinc inhibited lipid peroxidation in each of the above systems in a similar dose-related fashion, with 0.5 mM zinc having maximal effect. Gas-chromatographic fatty acid analysis too indicated a protective effect of zinc against FeCl3-induced lipid peroxidation in soybean PC vesicles, which do not contain C20:4 moieties. These findings, in particular the inhibition of lipid peroxidation in eicosatetrene emulsion, suggest that the presence of uncharged polar head groups, or packing of lipid molecules into ordered self-assemblages (membranes and micelles) have no critical influence on the antioxidant effect of zinc. The results with Fe2+ are compatible with the concept that zinc interferes with the formation of Fe2+-oxygen-enoic complexes. This mechanism, however, cannot account for the inhibition by zinc of the Fe#+-induced lipid peroxidation, suggesting the involvement of other types of zinc effects in these systems.
Ascorbic Acid↗
Glycation of MP26 and MP22 in bovine lens membranes.
Alkali treated membranes were isolated from mature bovine lenses and incubated with different sugars for 3 weeks to study the effect of glycation on the lens intrinsic membrane proteins, MP26 and MP22. The obtained results show that a) [1-14C] ascorbic acid (ASA) was able to glycate the intrinsic membrane proteins as rapidly as soluble lens proteins; b) on 15% acrylamide gels in SDS, glucose, fructose, galactose and ribose exhibited low activity for crosslinking membrane proteins; whereas ASA, dehydroascorbate (DHA), diketogulonate (DKG), xylosone and threose, all showed not only the formation of protein multimers, but also highly crosslinked products, which did not enter the spacer gel; c) except glycated MP22, all of the crosslinks of MP26 or MP22, and also the glycated MP26, showed cross reactivity with polyclonal MP26 antibody; d) the extent of crosslinking correlated with an equal loss of lysine and arginine contents by amino acid analysis.
Animals↗
Formation and identification of glutathione conjugates from 2-nitrosofluorene and N-hydroxy-2-aminofluorene.
2-Nitrosofluorene (NOF) and N-hydroxy-2-aminofluorene (N-HO-AF) are potent direct-acting mutagens, derived from metabolic activation of the carcinogen, N-acetyl-2-aminofluorene (AAF). To assess the ability of cellular glutathione (GSH) to detoxify these electrophilic derivatives, we examined the reaction of NOF and N-HO-AF with GSH in vitro. Two reaction products were isolated and identified as glutathionyl derivatives of 2-aminofluorene (AF) containing an N-S linkage. Amino acid analysis, infrared and NMR (500 MHz) spectroscopy, fast atom bombardment mass spectrometry and analysis of reaction characteristics and hydrolysis products established their structures as N-(glutathion-S-yl)-2-aminofluorene S-oxide (GS-AFI) and N-(glutathion-S-yl)-2-aminofluorene (GS-AFII). Ascorbic acid, which reduces NOF to N-HO-AF, was used to modify reaction yields. These results indicated that GS-AFI was derived from reaction with NOF and that GS-AFII could be formed from both NOF and N-HO-AF. A reaction scheme is proposed in which NOF reacts with GSH to form an intermediate addition product that can rearrange either to GS-AFI or be reduced to GS-AFII. The latter could also be formed by direct reaction with N-HO-AF.
Amino Acids↗
The extent of N epsilon-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid.
The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.
2,3-Diketogulonic Acid↗
Site-specific glycation of lens crystallins by ascorbic acid.
The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.
Amino Acid Sequence↗
A novel non-heme iron-containing dioxygenase. Chloridazon-catechol dioxygenase from Phenylobacterium immobilis DSM 1986.
Previously we purified an enzyme from Phenylobacterium immobilis DSM 1986, which cleaves the catechol derivative of the herbicide Chloridazon [5-amino-4-chloro-2-phenyl-3 (2H)-pyridazinone] in the meta position. The enzyme, which could be crystallized, proved in Ouchterlony double-diffusion tests to consist of a single protein species. No cross-reaction was observed with other meta-cleaving enzymes. Its light absorption spectrum showed a maximum at 279 nm (epsilon = 310 mM -1 cm -1), shoulders at 289 nm and 275 nm and a very weak band at around 430 nm (epsilon = 1.14 mM -1 cm -1). The amino acid analysis showed a slight excess of acidic amino acids, in agreement with the pl of 4.5. Surprisingly the enzyme per se is completely inactive, although it contains one non-dialysable iron atom per submit. It has to be activated by preincubation with ferrous ions or ascorbate. The enzyme activated this way is autoxidizable and returns to its non-activated state in the presence of oxygen. During the reaction with the substrate, this inactivation seems to be enhanced about 100 times. Since this kind of activation and inactivation is not observed in other meta-cleaving enzymes, this enzyme seems to represent a new type of a non-heme iron dioxygenase. We tentatively propose the name Chloridazon-catechol dioxygenase for this enzyme.
Amino Acids↗
Polymorphonuclear leukocyte and monocyte chemoattractants produced by human fibroblasts.
The elaboration of leukocyte chemotactic factors by human fibroblasts was studied. 12 lines of normal fibroblasts obtained by skin biopsy and then cultured in vitro produced chemoattractants (assessed by modified Boyden-chamber techniques) for both peripheral blood polymorphonuclear leukocytes and monocytes (obtained by Hypaque-Ficoll and dextran sedimentation). Chemotactic activity was not present performed in fibroblasts, and cycloheximide blocked its elaboration. The chemotactic activity of crude-culture supernate was heat stable (56 degrees C for 30 min), trypsin- and pronase-sensitive, and neuraminidase resistant. Characterization of the chemotactic activity by gel filtration (Sephadex G-75) showed two active fractions, one with mol wt greater than 100,000 and the other less than 10,000. In studies designed to relate these chemotactic factors to collagen, we have confirmed that type I collagen and alpha 1-chain; are chemotactically active for monocytes but not polymorphonuclear leukocytes. However, the chemotactic activity in fibroblast-culture media was media was distinct from collagen in that it attracted neutrophils, it was not precipitated by 25% ammonium sulfate, and it was resistant to collagenase treatment; ascorbic acid, in concentrations known to stimulate fibroblast collagen synthesis, had no effect on the elaboration of the chemotactic factors. Furthermore, amino acid analysis of Sephadex G-75 fractions with chemotactic activity failed to reveal amino acids such as hydroxyproline characteristic of collagen. In addition to the chemotactic factors secreted by fibroblasts, a heat-resistant factor (30 min at 56 degrees C) which generated the chemotactically active fragment of C5 (C5a) from human serum was also secreted. The elaboration of mediators of the inflammatory and immune responses by fibroblasts may initiate and(or) modulate local skin inflammatory reactions and play a protective role in vivo.
Cell Line↗