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PubMed1985

DNA modification by chemical carcinogens.

The chemistry and molecular biology of DNA adducts is only one part of the carcinogenic process. Many other factors will determine whether a particular chemical will exert a carcinogenic effect. For example, the size of particles upon which a carcinogenic may be adsorbed will influence whether or not, and if so where, deposition within the lung will occur. The simultaneous exposure to several different agents may enhance or inhibit the metabolism of a chemical to its ultimate carcinogenic form (Rice et al., 1984; Smolarek and Baird, 1984). The ultimate carcinogenic metabolites may be influenced in their ability to react with DNA by a number of factors such as internal levels of detoxifying enzymes, the presence of other metabolic intermediates such as glutathione with which they could react either enzymatically or non-enzymatically, and the state of DNA which is probably most heavily influenced by whether or not the cell is undergoing replication or particular sequences being expressed. Replicating forks have been shown to be more extensively modified than other areas of DNA. Another critical factor which can influence the final outcome of the DNA damage is whether or not the modifications can be repaired. If this occurs with high fidelity and the cell has not previously undergone replication then the effect of the damage by the carcinogen is likely to be minimal. The major area in which progress is needed is an understanding of what this damage really does to the cell such that after an additional period of time, which may be as long as twenty or more years, these prior events are expressed and cell proliferation occurs. Clearly additional stimulatory factors, for example tumor promoting agents such as the phorbol esters or phenobarbital, are often needed. After such prolonged periods it seems likely that the DNA adducts would no longer be present. However, the way in which their earlier presence is remembered is not clear. Simple mutations do not explain all the characteristics of tumor progression and, when it occurs, regression. Even if a specific site mutation does occur then its expression must be under other types of control. Any explanation of the action of DNA modification at the molecular level also requires that account be taken of the diverse nature of the DNA adducts from simple modifications such as methylation to bulkier adducts such as benzo[a]pyrene, aflatoxin or aromatic amines.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Nitroquinoline-1-oxide↗

PubMed1996

Modification of Lowry's method for collagen concentration measurement.

A procedure for the determination of collagen concentration is described. It is simple and sensitive and is based on a modification of Lowry's method. The modification includes an initial heating of collagen samples in alkaline solution and changes in the concentrations of reagents used. It may be used for different collagen types, i.e. I, II, V and XI, from various sources as well as for gelatine. The influence of different buffers, salts, detergents, etc. on the precision of the method was studied. The precision of the measurements was not influenced by pH, the turbidity of the sample and the presence of certain salts, osmolytes and preservatives, routinely used in biochemical, microbiological and immunological experimental protocols. The modification is suitable for routine determinations of collagen concentration in a wide variety of samples.

Animals↗

PubMed1996

Nonspecific modification of cellular immunity by Yersinia enterocolitica.

An isogenic pair of virulent and avirulent Yersinia enterocolitica O9 strains was used to examine the influence of the virulence plasmid on the non-specific modification of the cellular immunity in BALB/c mice after experimental infection with yersiniae. The modification of contact hypersensitivity response to dinitrofluorobenzene, resistance to the syngeneic lymphoma LSTRA, and resistance to Listeria monocytogenes was heavily influenced by the presence of the virulence plasmid. As a general rule for the modification of cellular immunity by yersiniae, the plasmid-bearing strain induced a short-term suppression followed by a potentiation, whereas the isogenic plasmid-less derivative induced only a short-term potentiation. The Yersinia-mediated enhancement of cellular immunity resulted in protection against infection with Listeria and partial protection against LSTRA transplantation. Results of Concanavalin A-induced proliferation of splenocytes from Yersinia-infected mice suggested a role for cytokines as gamma-interferon in the Yersinia-mediated immunopotentiation.

Animals↗

PubMed1983

A simple modification of the Salmonella liquid-incubation assay. Increased sensitivity for detecting mutagens in human urine.

A simple modification of the Salmonella/microsome liquid-incubation procedure improves the sensitivity of the assay for detecting mutagens in human urine. Extracts from cigarette smokers' urine were used as a model complex mutagenic mixture for validation of the assay. The modification consists of adding increased numbers of bacterial cells (approximately 10(9] in a concentrated suspension to liver homogenate mix and urine extract, all in 0.2-ml volume. After 90 min incubation at 37 degrees C, the mixture is processed according to the standard Ames test protocol. This procedure is 20 times more sensitive than the standard plate-incorporation test and 13 times more sensitive than a previously reported liquid-incubation protocol. The number of spontaneous revertants did not increase under these conditions and, compared to the plate-incorporation test, 10-fold less liver homogenate and 5-fold less enzymatic cofactors were needed per plate. The procedure was approximately 14 times more sensitive in detecting the mutagenic activity of benzo[ a ]pyrene. We also used the modification to determine mutagenic activity in urine from a group of nonsmokers. The method may be generally useful for investigations of mutagenic activity in human urine samples.

Adult↗

PubMed1982

Modification of tyrosine residues in dihydrofolate reductase from Lactobacillus casei.

Dihydrofolate reductase from Lactobacillus casei was inactivated by reaction with tetranitromethane and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Loss of activity occurred with modification of four of the five tyrosine residues present in the enzyme. The presence of either substrate, NADPH or 7,8-dihydrofolate, as well as NADP and folate, provided extensive protection against inactivation, while NADH and tetrahydrofolate exhibited none. This protection from inactivation occurred on protection of two of the four susceptible tyrosines from modification. Nitration of the enzyme adversely affected its ability to bind substrates. Restoration of the pKa of the nitrated tyrosines by reduction of the nitro group to an amino group did not result in a regeneration of enzymatic activity. However, fluorotyrosine-containing enzyme, prepared by growing the bacterium in the presence of fluorotyrosine, exhibited specific activity identical to that of native enzyme over the pH range of 4.5-8. These results suggest that inactivation of dihydrofolate reductase by tyrosine modification occurs primarily due to a steric effect and that the active site tyrosines may participate in substrate binding.

4-Chloro-7-nitrobenzofurazan↗

PubMed1984

Chemical modification of adrenocortical cytochrome P-450scc with tetranitromethane.

Selective chemical modification of adrenocortical cytochrome P-450scc, responsible for key stages of steroid biogenesis, with tetranitromethane has been carried out. Nitration of the cytochrome P-450scc tyrosine residues results in heme protein inactivation with syncatalytic loss of enzyme activity. Analysis of the cytochrome P-450scc inactivation kinetics indicates that there are several pools of tyrosine residues, differing in their accessibility to tetranitromethane. The modification of cytochrome P-450scc results in changes in the hemeprotein spectral properties and its conformation which indicates to the involvement of essential tyrosine residue(s) in the heme-protein interaction. Cholesterol and adrenodoxin (high-spin effectors) prevent the inactivation of cytochrome P-450scc with tetranitromethane, i.e., protect the essential tyrosine residue(s) from modification. Possible functions of the tyrosine residues in the cytochrome P-450scc molecule are discussed.

Adrenal Cortex↗

PubMed1984

Effect on the equilibrium between the Na+-form and the K+-form of the (Na+ + K+)-ATPase of modification of the enzyme with pyridoxal 5-phosphate.

An increase in pH shifts the equilibrium between the K+-form and the Na+-form of the (Na+ + K+)-ATPase towards the Na+-form. pK for the proton effect on the equilibrium is decreased by modification of the enzyme with pyridoxal 5-phosphate. The reactivity of the enzyme towards pyridoxal 5-phosphate is increased by an increase in pH. Modification by pyridoxal 5-phosphate of epsilon-amino groups on lysine, which has a pK of about 8 with the enzyme in the K+-form and of about 7.4 in the Na+-form, shifts the equilibrium between E1Na+ and E2 towards E2, and the equilibrium between E2(K+occ) and E2 towards E2, but has no effect on the overall equilibrium between E1Na+ and E2(K+occ). An additional modification of epsilon-amino groups on lysine, which has a pK of 9.5-10 with the enzyme in the K+-form and of about 7.7 with the enzyme in the Na+-form, shifts the equilibrium between E2(K+occ) and E1Na+ towards E1Na+; this is due to a shift in the equilibrium between E2(K+occ) and E2 towards E2, but with no effect on the equilibrium between E1Na+ and E2. The results show that the transition from the K+-form to the Na+-form decreases the pK of lysine epsilon-amino groups on the enzyme, and that the protonation of these groups influences the equilibrium between the two conformations.

Animals↗

PubMed1987

Purification and active site modification studies on glyoxalase I from monkey intestinal mucosa.

Glyoxalase I ((R)-S-lactoylglutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) from monkey intestinal mucosa was purified to homogeneity. The purified enzyme had a molecular weight of 48,000, composed of two apparently identical subunits. Active-site modification was carried out on the purified enzyme in presence and absence of S-hexylglutathione, a reversible competitive inhibitor of glyoxalase I. Modification by tetranitromethane and N-acetylimidazole caused inactivation of the enzyme. Inactivation by N-acetylimidazole was reversible with hydroxylamine treatment, suggesting the importance of tyrosine residues for the activity of the enzyme. The enzyme was inactivated by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, 2,4,6-trinitrobenzenesulphonic acid, pyridoxal phosphate and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, indicating the importance of tryptophan, lysine and glutamic acid/aspartic acid residues for the activity of the enzyme. The enzyme was inactivated by diethyl pyrocarbonate and the activity was not restored by hydroxylamine treatment, suggesting that histidine residues may not be important for activity. Modification by N-ethylmaleimide and p-hydroxymercuribenzoate did not affect its activity, indicating that sulphydryl groups may not be important for activity. These studies indicated that the amino acids present in the active site of glyoxalase I from intestinal mucosa which may be important for activity are tyrosine, tryptophan, lysine and glutamic acid/aspartic acid residues.

Amino Acids, Dicarboxylic↗

PubMed1988

Age-related modifications in rat cardiac phosphoglycerate kinase. Rejuvenation of the old enzyme by unfolding-refolding.

The occurrence of age-related modifications in functional and structural properties of several enzymes has been documented; however, the molecular basis of this phenomenon is still mostly unexplained. In the present work a comparative study of phosphoglycerate kinase preparations isolated from hearts of young and old rats was undertaken. Marked age-related effects were revealed in the heat-inactivation kinetics of the enzyme, similar to the ones previously found in purified muscle phosphoglycerate kinase. In view of the previously reported failure of immunotitration to distinguish between phosphoglycerate kinase forms in crude heart extracts from young and old rats, it appears likely that the modifications in old rat heart phosphoglycerate kinase are in a domain which is not involved in antibody binding, and may be localized in the interior of the enzyme. These age-related modifications were completely relieved by extensive unfolding of the enzyme in 2 M guanidine hydrochloride, followed by enzyme reactivation upon dilution of the denaturant. The refolding products of young and old enzymes displayed identical heat-inactivation kinetics as native young phosphoglycerate kinase. It is concluded that the age-related alterations in rat cardiac phosphoglycerate kinase, like those found in the muscle enzyme, are purely conformational and hence develop postsynthetically.

Aging↗

PubMed1990

Irreversible thermoinactivation of glucoamylase from Aspergillus niger and thermostabilization by chemical modification of carboxyl groups.

The incubation of glucoamylase from Aspergillus niger at 70 degrees C induced its rapid and irreversible inactivation. The covalent modifications of the protein structure involved in the thermoinactivation depended on the pH of the medium. We observed the formation of a low amount of disulfide-linked oligomers showing that disulfide exchange takes place at pH 5.5. Hydrolysis of peptide bonds at pH 3.5 and 4.5 was also detected. The chemical modification of carboxyl groups with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) decreased the rate of appearance of low-molecular-weight peptides at pH 3.5 and 4.5 upon heating at 70 degrees C. However, the rate of inactivation at such pH values was not modified. Modification of carboxyl groups with EDC in the presence of ethylenediamine leading to the transformation of three carboxyl groups to amino groups increased the thermostability of the enzyme for temperatures above the temperature of compensation, Tc, which is 60 degrees C.

Aspergillus niger↗

PubMed1991

Specific modification of Escherichia coli RNA polymerase with monomercury derivative of fluorescein acetate.

The method of specific modification of RNA polymerase with a monomercuric fluorescein derivative, fluorescein-monomercuriacetate (FMMA), is proposed. Under an appropriate condition of modification, FMMA is capable of mercaptid bonding with one of the alpha-subunits. It is shown that covalent modification with FMMA does not affect the kinetic parameters (KB and k2) of RNA synthesis nor does it lead to the inhibition of the overall RNA synthesis. The spectral characteristics of FMMA covalently bound to RNA polymerase were found to be sensitive to some temperature-induced conformational alterations of RNA polymerase, indicating that the labeled enzyme allows study of conformational behaviour of RNA polymerase during its functioning.

Binding Sites↗

PubMed1992

Chemical modification of buried lysine residues of bovine serum albumin and its influence on protein conformation and bilirubin binding.

Using a double modification technique about 20% of the lysine residues of bovine serum albumin (BSA) which are not easily accessible in the native protein have been modified. The technique involved approximately 80% modification of lysine residues of BSA with citraconic anhydride followed by chemical modification of the remaining lysine residues with acetic anhydride, succinic anhydride, potassium cyanate, or O-methylisourea. Finally, these preparations were decitraconylated under mild acidic conditions to yield acetylated, succinylated, carbomylated or guanidinated BSA. All of these preparations were found to be homogeneous with respect to charge and size. The spectral, hydrodynamic and bilirubin binding properties of these preparations are described. In contrast to most of the highly modified proteins these preparations with the exception of succinylated BSA are very similar to native BSA in their spectral and hydrodynamic properties. However, the equilibrium association constant (Ka) with bilirubin measured by fluorescence quenching was decreased by about 100-fold in acetylated, carbamylated and succinylated BSA, but only 3-fold in guanidinated BSA. Since conformationally acetylated and carbamylated BSAs are identical to guanidinated BSA we conclude that the decrease in Ka in these preparations is solely due to loss of positive charge on 'critical' lysine residues. The results support a binding model for BSA in which bilirubin binding site is buried and the protein undergoes a series of relaxational changes in conformation upon interaction with bilirubin.

Animals↗

PubMed1993

Chemical modification of bile acid: CoA ligase.

The effect of chemical modification of bile acid:CoA ligase on its enzymatic activity was examined. Reagents which modify tyrosine and carboxyl groups did not affect the activity of either the purified enzyme or the enzyme in its native microsomal environment. The modification of arginine residues with either diacetyl or phenylglyoxal resulted in a loss of activity for both the purified and microsomal forms of the enzyme. ATP was able to protect the enzyme from inactivation. Neither cholate nor CoA were able to alter the time-course of inactivation. The sulfhydryl reagent N-ethylmaleimide (MalNEt) produced a biphasic effect on both the purified and microsomal forms of the enzyme. At short reaction times ligase activity increased, but further reaction lead to nearly complete inactivation. With the purified enzyme, ATP increased the extent of activation by MalNEt and decreased the rate of inactivation. With microsomes, ATP did not affect the extent of activation by MalNEt, but did slow the rate of inactivation. For both the purified and microsomal forms cholate provided no protection. Treatment of both forms of the enzyme with the sulfhydryl reagent iodoacetic acid produced a similar biphasic activation/inactivation of the ligase. It was hypothesized that modification of a fast-reacting cysteine leads to activation while a slower-reacting cysteine leads to inactivation. This latter cysteine appeared to be in the ATP-binding site on the enzyme.

Adenosine Triphosphate↗

PubMed1993

Chemical modification of tissue inhibitor of metalloproteinases-1 and its inactivation by diethyl pyrocarbonate.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) was treated with a range of chemical modification reagents in order to identify amino acid residues essential for inhibitory activity. Diethyl pyrocarbonate (DEPC) was found to be a potent inactivator at low reagent/TIMP molar concentrations. The extent of modification at 50% inactivation was determined as 1.5 sites/molecule. The DEPC-modified inhibitor did not form stable complexes with stromelysin, but was shown to retain native structure as judged by conformational stability to denaturation by guanidine hydrochloride. Peptide mapping experiments were used to find the sites of DEPC incorporation within the primary structure of TIMP and three residues were identified (His-95, His-144 and His-164). Mutant TIMPs in which histidine residues have been substituted or deleted retain inhibitory activity and were found to be equally as sensitive to DEPC inactivation as the wild-type. No new sites of DEPC modification in the mutant proteins were detected. The possible contribution made by His residues 95, 144 and 164 to the inhibitory activity of TIMP is discussed.

Amino Acid Sequence↗

PubMed1994

Active-site residues of procarboxypeptidase Y are accessible to chemical modification.

The accessibility of the active-site cleft of procarboxypeptidase Y from Saccharomyces cerevisiae has been studied by chemical modifications of two specific amino-acid residues. Previous studies have shown that these residues, Cys-341 and Met-398 in the mature enzyme, are located in the S1 and S'1 substrate binding sites, respectively, of carboxypeptidase Y. We have found that these residues also in proCPY are accessible to modification with fairly bulky reagents and in the case of Met-398 the rate of modification is even faster than in carboxypeptidase Y. While the catalytic serine in the mature enzyme reacts with diisopropylfluorophosphate, this is not the case for procarboxypeptidase Y.

Acetophenones↗

PubMed1995

Modification with tetranitromethane of an essential tyrosine residue in uridine phosphorylase from Escherichia coli.

Treatment with tetranitromethane (TNM) rapidly and irreversibly inactivates uridine phosphorylase (UPase) from E. coli under mildly alkaline conditions. Modification of one of the four tyrosine residues decreases enzyme activity to 10%, while modification of all tyrosines decreases it to 8%. The second-order rate constant for the inactivation is 1250 +/- 50 M-1 min-1 at pH 8.0. Phosphate (0.1 M) does not affect the inactivation rate, while 5 mM uridine, or uridine plus phosphate nearly completely protect the enzyme against inactivation. Free sulfhydryl groups of UPase are not oxidized by TNM. A single modified peptide was isolated from tryptic digest by reverse-phase HPLC. The mass to charge ratio and the sequence determined are consisted with modification of Tyr-169, which corresponds to tryptic peptide 169Tyr-Asp-Thr-Tyr-Ser-Gly-Arg175. Tyrosine nitration leads to a significant decrease in the pKa of the phenolic hydroxy group without significantly affecting enzyme structure. Comparison of the pH dependence of activity and inactivation by diethylpyrocarbonate for the native and modified UPase reveals interaction between the modified tyrosine residue and an essential histidine residue (Drabikowska, A.K. and Wozniak, G (1990) Biochem. J. 270, 319-323). It is suggested that Tyr-169 takes part in the stabilization of the imidazole ring of the essential histidine in UPase.

Amino Acid Sequence↗

PubMed1996

Engineering the C-terminus of firefly luciferase as an indicator of covalent modification of proteins.

Protein kinase recognition sequences and proteinase sites were engineered into the cDNA encoding firefly luciferase from Photinus pyralis in order to establish whether these modified proteins could be developed as bioluminescent indicators of covalent modification of proteins. Two key domains of the luciferase were modified in order to identify regions of the protein in which peptide sequences may be engineered whilst retaining bioluminescent activity; one between amino acids 209 and 227 and the other at the C-terminus, between amino acids 537 and 550. Mutation of amino acids between residues 209 and 227 reduced bioluminescent activity to less than 1% of wild-type recombinant. In contrast engineering peptide sequences at the C-terminus resulted in specific activities ranging from 0.06-120% of the wild-type recombinant. Addition of cyclic AMP dependent protein kinase catalytic subunit, to a variant luciferase incorporating the kinase recognition sequence, LRRASLG, with a serine at amino-acid position 543 resulted in a 30% reduction in activity. Alkaline phosphatase treatment restored activity. The bioluminescent activity of a variant luciferase containing a thrombin recognition sequence, LVPRES, with the cleavage site positioned between amino acid 542 and 543, decreased by 50% when incubated in the presence of thrombin. The results indicate regions within luciferase where peptide sequences may be engineered while retaining bioluminescent activity and have shown changes in bioluminescent activity when these sites are subjected to covalent modification. Changes in secondary structure, charge and length at the C-terminus of luciferase disrupt the microenvironment of the active site, leading to alterations in light emission. This has important implications both in understanding the evolution of beetle bioluminescence and also in development of bioluminescent indicators of the covalent modification of proteins.

Amino Acid Sequence↗

PubMed1985

The effect of chemical modification of Saccharomyces cerevisiae on electrophoretic mobility, cell-wall structure and amphotericin B uptake.

Saccharomyces cerevisiae NCYC 239 suspended in solutions of NaCl showed two distinct plateaus in plots of electrophoretic mobility vs. pH, corresponding to pKa values of approx. 2 and 5. This is in contrast to cells suspended in buffer where only a single pKa (4) can be determined. Modification of cells with KI/I2 or nitrous acid led to altered electrophoretic mobility, indicating the presence of sulphydryl and amino groups, respectively, in the yeast cell surface, whereas uranyl nitrate modification had little effect, suggesting phosphate groups to be absent. Electron micrographs showed visible effects of KI/I2 and nitrous acid modification on cell membrane structure, and in these modified cells amphotericin B uptake was rapid. It is suggested that diffusion through the cell wall is the rate-limiting step for amphotericin B uptake. An activation energy of 20 kJ X mol-1 was determined for uptake of amphotericin B by unmodified cells.

Amphotericin B↗