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D E Stevenson

Publications and source records attributed to D E Stevenson.

At least 19 recordsLinked to original sources
PubMed1992

Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase.

Rhodococcus ATCC 39484 produced a nitrilase when induced with isovaleronitrile. The enzyme was obtainable pure in milligram amounts, had a subunit Mr of 40 kDa, and demonstrated a substrate-induced activation related to aggregation of subunits to form a 560-kDa complex. The enzyme had a broad substrate specificity, had a pH optimum of 7.5, was stable up to 40 degrees C, and had one disulfide bridge and two free cysteine residues, one of which appeared to be catalytically essential. The N-terminal sequence was determined and found to have 78.3% homology, in a 23-residue overlap, with Klebsiella ozaenae nitrilase. The enzyme was inhibited competitively by benzylamine and benzaldehyde and irreversibly by benzyl bromide. However, benzyl bromide was shown to be nonspecific, causing multiple alkylation. Acid quenching of enzyme-substrate mixtures allowed for the detection of covalent enzyme-substrate complexes using mass spectrometry. The covalent intermediate is suggested to be either a thioimidate or an acylenzyme and a reaction mechanism consistent with this observation and also the inhibitor results is proposed. The rate of breakdown of the covalent intermediates was found to be rate limiting even for substrates with undetectable rates of hydrolysis or those with very slow rates of intermediate formation. For phenylacetonitrile, a poor substrate, in addition to acid, approximately 2% of the product was the corresponding amide. This result suggests that a tetrahedral intermediate is formed which, for selected substrates, can break down anomalously to produce amide in place of the normal acid product. Under the conditions used in this study all other substrates tested were converted to acid.

Amino Acid Sequence↗
PubMed1992

Evaluation of sustained hyperplasia and other short-term tests as predictors of tumorigenic potential in oil products.

Some oil products are known to cause skin tumors following long-term application while others do not. The ability to predict which ones might cause tumors is important. Development of reliable short term tests which can accurately predict tumorigenic potential of oil products is needed to avoid the high cost and long time required for traditional animal bioassays. Several short term tests were evaluated for their ability to predict tumorigenic potential of 10 coded oil samples and results were compared to results of mouse bioassays. Analytical determinations of PAC content (DMSO extraction) and 3-6 ring PAC content were also made for each of the 10 samples for further comparison. Tests which showed good correlation with bioassay results and thus were considered good predictors of tumorigenic potential were: Sustained Epidermal Hyperplasia as measured by epidermal thickness, Nuclear Area of epidermal basal cells, Modified Ames Test and DMSO extraction for PAC content. Tests which did not show good correlation with bioassay results and which were not considered good predictors of tumorigenic potential were: Polymorphonuclear leukocyte (PMN) infiltration into the dermis, Unscheduled DNA Synthesis in epidermal cells and Changes in Nuclear DNA Content of CHO cells.

Animals↗
PubMed1990

Detection of covalent enzyme-substrate complexes of nitrilase by ion-spray mass spectroscopy.

Nitrilase from Rhodococcus ATCC 39484 was found to consist of two species of Mr 40,258 +/- 2 and 40,388 +/- 2 Da. When the enzyme was incubated with nitrile substrates and the reaction quenched with acid, higher Mr species were observed. The mass differences were consistent with addition of a substrate molecule to each species. These results represent the first reported demonstration that this, or any other nitrilase forms a covalent intermediate with its substrates. The observation that the intermediate, suggested to be either a thioimidate or an acylenzyme, can be trapped by acidification indicates that the rate of breakdown of the intermediate is rate-limiting.

Aminohydrolases↗
PubMed1990

L-methionine decarboxylase from Dryopteris filix-mas: purification, characterization, substrate specificity, abortive transamination of the coenzyme, and stereochemical courses of substrate decarboxylation and coenzyme transamination.

L-Methionine decarboxylase from the male fern Dryopteris filix-mas has been purified 256-fold from acetone powder extracts to very near homogeneity. The enzyme is membrane-associated and requires detergent for solubilization during the initial extraction. The enzyme is a homodimer of subunit Mr 57,000 and shows a pH optimum at approximately 5.0 with 20 mM (2S)-methionine as substrate. The specific activity, kcat, for methionine is approximately 50 mol s(-1) (mol of active site)(-1) at pH 4.5 and below. A wide range of straight- and branched-chain (2S)-alkylamino acids are substrates for the enzyme. The values for the rate of decarboxylation, Vmax, and for the apparent Michaelis constant, Km, however, vary with structure and with the chirality at C-3. The pH dependence of V and V/K has been examined for three substrates: (2S)-methionine, valine, and leucine. Pyridoxal 5'-phosphate (PLP) is required for activity, and in the absence of excess PLP, the activity of the enzyme in incubations reduced with respect to time. The addition of PLP fully restores the activity, indicating that an abortive decarboxylation-transamination accompanies the normal decarboxylation reaction. The occurrence of the abortive reaction was confirmed by showing that [35S]methionine is converted to labeled 3-(methylthio)propionaldehyde while [4'-3H]PLP is converted to labeled pyridoxamine 5'-phosphate (PMP). The decarboxylation of (2S)-methionine gave 3-(methylthio)-1-aminopropane. Preparation of the N-camphanamide derivative of the amine allowed the C-1 methylene protons to be distinguished by 1H NMR spectroscopy. Synthetic samples of the camphanamide were prepared in which each of the C-1 methylene protons was replaced by deuterium. When (2S)-methionine and the C-2 deuteriated isotopomer were incubated with the enzyme in deuterium oxide and protium oxide, respectively, and the products were converted to their camphanamide derivatives and analyzed by 1H NMR spectroscopy, it was evident that decarboxylation occurred with retention of configuration at C-2. When the decarboxylation of six other substrates was studied, examination of the N-camphanamide derivatives of the amines indicated that decarboxylation occurred stereospecifically and, by analogy, with retention of configuration at C-2. When tritiated pyridoxal phosphate was incubated with the enzyme, tritiated pyridoxamine phosphate was formed. Analysis of the chirality of the methylene group at C-4' indicated that, during abortive transamination, protonation occurred from the 4'-si face of the coenzyme, the same stereochemical result as that obtained for several bona fide transaminase enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Carboxy-Lyases↗
PubMed1990

Fern L-methionine decarboxylase: kinetics and mechanism of decarboxylation and abortive transamination.

L-Methionine decarboxylase from Dryopteris filix-mas catalyzes the decarboxylation of L-methionine and a range of straight- and branched-chain L-amino acids to give the corresponding amine products. The deuterium solvent isotope effects for the decarboxylation of (2S)-methionine are D(V/K) = 6.5 and DV = 2.3, for (2S)-valine are D(V/K) = 1.9 and DV = 2.6, and for (2S)-leucine are D(V/K) = 2.5 and DV = 1.0 at pL 5.5. At pL 6.0 and above, where the value of kcat for all of the substrates is low, the solvent isotope effects on Vmax for methionine are 1.1-1.2 whereas the effects on V/K remain unchanged, indicating that the solvent-sensitive transition state occurs before the first irreversible step, carbon dioxide desorption. The enzyme also catalyzes an abortive decarboxylation-transamination reaction in which the coenzyme is converted to pyridoxamine phosphate [Stevenson, D. E., Akhtar, M., & Gani, D. (1990a) Biochemistry (first paper of three in this issue)]. At very high concentration, the product amine can promote transamination of the coenzyme. However, the reaction occurs infrequently and does not influence the partitioning between decarboxylation and substrate-mediated abortive transamination under steady-state turnover conditions. The partition ratio, normal catalytic versus abortive events, can be determined from the amount of substrate consumed by a known amount of enzyme at infinite time, and the rate of inactivation can be determined by measuring the decrease in enzyme activity with respect to time. For methionine, the values of Km as determined from double-reciprocal plots of concentration versus inactivation rate are the same as those calculated from initial catalytic (decarboxylation) rate data, indicating that a single common intermediate partitions between product formation and slow transamination. The partition ratio is sensitive to changes in pH and is also dependent upon the structure of the substrate; methionine causes less frequent inactivation than either valine or leucine. The pH dependence of the partition ratio with methionine as substrate is very similar to that for V/K. Both curves show a sharp increase at approximately pH 6.25, indicating that a catalytic group on the enzyme simultaneously suppresses the abortive reaction and enhances physiological reaction in its unprotonated state. Experiments conducted in deuterium oxide allowed the solvent isotope effects for the partition ratio and the abortive reaction to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)

Amines↗
PubMed1990

Streptomyces L-methionine decarboxylase: purification and properties of the enzyme and stereochemical course of substrate decarboxylation.

L-Methionine decarboxylase from Streptomyces species ATCC 21020 has been purified to near homogeneity, characterized, and compared to the enzyme from the fern Dryopteris filix-mas [Stevenson, D.E., Akhtar, M., & Gani, D. (1990) Biochemistry (first paper of three in this issue)]. The enzyme catalyzes the decarboxylation of a range of alkylamino acid substrates, but the substrate specificity is different from that for the fern enzyme. In accord with the properties of the fern enzyme, the Streptomyces enzyme is also a homodimer of Mr 100,000 +/- 5000 and requires PLP for activity. At low pH where the value of Vmax for both enzymes is maximal and essentially pH independent, kcat for the Streptomyces enzyme with (2S)-methionine as substrate is slightly higher (60 s-1) than the value for the eukaryotic protein (50 s-1). The pH optimum for V/K is much higher than that for the fern enzyme although many features of the pH dependence are similar, including the shape of the curve for the pH dependence of Km. When the decarboxylations of (2S-methionine, (2S)-norleucine, and (2R)-S-ethyl-L-cysteine were conducted on a preparative scale in protium and deuterium oxide, unlabeled and deuteriated amines were formed. 1H NMR spectroscopic analysis of the stereochemistry at C-1 of the camphanamide derivatives of the products [Stevenson, D. E., Akhtar, M., & Gani, D. (1990) Biochemistry (first paper of three in this issue)] indicated that each conversion was stereospecific and occurred with retention of configuration at C-2 of the substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Carboxy-Lyases↗
PubMed1989

IARC and HHS lists of carcinogens: regulatory use based on misunderstanding of the scope and purpose of the lists.

In the last two decades there has been a tremendous increase in data on carcinogenic activity in experimental animals. While there have been few additions to the list of human carcinogens based on human data, the number of carcinogens based on animal data continues to increase unabated. The International Agency for Research on Cancer (IARC) list of carcinogens grew out of the IARC Monograph Series. The evidence classification system used to prepare the IARC lists in 1980, 1982, and 1988 is based on the sufficiency, i.e., strength, of the evidence of carcinogenic activity in one or more studies, not a full weight-of-the-evidence evaluation of all relevant data. Titles of categories of animal evidence referring to human risk potential were based on a presumption: "for practical purposes . . . as if." No evaluation was made of the predictive relevance of animal data to human risk. The IARC listing did not involve evaluation of potency or mechanism and was intended as a useful input but not as a basis for regulatory or legislative decisions. The Department of Health and Human Services (HHS) lists in the Annual Reports on Carcinogens are selected from the IARC lists and from reports of positive bioassay experiments conducted by the National Toxicology Program (NTP). The reports on the NTP bioassays relate to the strength of the evidence in each experiment and recognize that a "wider analysis" is necessary for determination of human risk. Because of a misunderstanding of the limited scope of the analysis involved, the IARC and HHS lists have recently been used as a basis for legislative and regulatory decisions. Examples of unanticipated use of the lists as triggers for regulatory and legislative decisions will be discussed. Some recommendations to mitigate the consequences of past unanticipated use of the lists and to prevent further misuse are discussed.

Animals↗
PubMed1988

Policy principles for utilizing science in decision-making on chronic health issues.

Scientific advances will continue to contribute to our understanding of latent chronic diseases related to chemical exposure. Regulatory agencies must deal with a complex matrix of emerging scientific information, a diversity of potential risk situations, and a variety of statutory prescriptions for protecting public health. Seven policy principles are proposed for facilitating integration of the latest scientific thought into the administrative decision-making process. The principles relate to distinguishing between risk assessment and risk management, analysis of all relevant information in developing a risk assessment, consideration of weight-of-the-evidence and more probable than not criterion on key assumptions, scientific peer review of assessments, scoping scientific input appropriately with the nature of a specific regulatory activity, emphasizing research which enhances the basis of risk assessment, and education and communication on risk matters. The policy principles are interdependent; collectively they need endorsement and promotion by the scientific and regulatory communities and by policy leaders in federal and state governments in the interest of establishing a framework for further improving the basis of critical decisions for protecting public health.

Chronic Disease↗
PubMed1979

Current problems in the choice of animals for toxicity testing.

Animal models for the study of toxicity are chosen more for pragmatic reasons such as life-span, ease of handling, and economics than because of a comprehensive process of validation. There is also a tendency to seek the answers to nonspecific questions and then to use those answers in situations where they cannot lead to proper judgments. In some cases, such as in carcinogenicity testing, an uneasy compromise exists between the sensitivity of the model and the background noise. There is scope, by increased attention to both the control of environmental factors and the genetics of the animal models, to increase the sensitivity and validity of the systems that are used in toxicology.

Animals↗
PubMed1978

Intestinal disaccharidase activities in relation to age, race, and mucosal damage.

Studies were undertaken to determine the relationship of intestinal disaccharidase activity to age and race, and the relationship of mucosal damage to a primary low lactase activity. The first study consisted of data on 399 persons (339 whites, 53 blacks, and 7 American Indians) ages 1 month to 93 years, with normal intestinal histology. Among whites, all 117 children 5 years old or under had high lactase levels, whereas low levels were found only in subjects over 5 years of age. No low lactase levels were identified among the 11 black children 3 years old or under, but in comparison to coetaneous white children, their mean lactase activity was signficantly less. The majority of older blacks had low lactases. In whites and blacks alpha-disaccharidases did not participate in the age-related changes demonstrated with lactase. Of the 7 American Indians, none under 26 months old had low lactase levels, whereas the 4 over 10 years old had low activities. Heterozygotes for sucrase-isomaltase deficiency were identified only among whites. Low lactase levels developed during childhood in all races studied, however, many for unknown reasons maintained their lactose tolerance until adulthood. In the second study of 13 additional children with secondary disaccharidase deficiencies, emergence of a primary low lactase was related to age and race, rather than to mucosal damage. It appears that primary low intestinal lactase levels are absent or rare in whites under 5 and blacks under 3 years of age, and the deficiency is not related to mucosal damage.

Adolescent↗
PubMed1976

Dichlorvos -- a 2-year inhalation carcinogenesis study in rats.

To determine the effects of dichlorvos vapour on the tumour incidence in rats, 5 week old Carworth Farm E strain rats weighing between 94 and 150 g were exposed to 0, 0.05, 0.5 and 5.0 mg/m3 in a 2-year inhalation study. The growth rate of all treated rats was depressed, particularly in the males. There was increased survival of the rats exposed to 5 mg/m3. There were no consistent differences in food intakes, organ weights, haematological or blood chemistry estimations, except in cholinesterase activites, amongst the various groups of rats. No compound-related differences were seen in acetylcholine and choline estimations carried out on a small number of female rats' brain tissues after two years' exposure. There were no gross or microscopical compound-related changes in the rats' tissues. Ultrastructural examination of the respiratory tissues of the rats from the control and 5 mg/m3 group showed no changes attributable to dichlorvos. The results of a relative risk analysis of the tumour data showed that no dose-related increase in tumour risk was established for rats of either sex. These data confirm the results of earlier st.udies supporting the safety of insecticidal uses of dichlorvos.

Animals↗
PubMed1975

Pesticides and domestic animals.

Domestic animals are quite often poisoned by pesticides despite the considerable effort made through legislation and Ministry recommendations to provide adequate safeguards for the use of agricultural chemicals. All pesticides have to be registered and recommendations and restrictions for use are printed on the labels. But a number of cases involving the misuse of certain poisons as bait for vermin has caused concern recently to both Government and industry. Special problems face the veterinary surgeon who has to treat a pet animal with suspected esticide poisoning. This paper provides possible sources of information from which the veterinary surgeon may obtain help with diagnosis, advice on treatment and laboratory aid.

Animals↗