1971 — 1982 |
Coon, Minor |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Detoxication of Hydrocarbons, Drugs, and Related Compounds @ University of Michigan Ann Arbor |
0.915 |
1981 — 1984 |
Coon, Minor |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mechanism of Oxygen Activation @ University of Michigan Ann Arbor |
0.915 |
1985 — 1996 |
Coon, Minor J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Alcohol Metabolism--Role of P-450 Oxygenases @ University of Michigan At Ann Arbor
The overall goal of the reseach project is to determine the role of P-450 oxygenases in alcohol metabolism and toxicity. Our laboratory has recently isolated a new form of P-450 (isozyme 3a) from liver microsomes of rabbits chronically treated with ethanol and shown that this cytochrome has relatively high activity in ethanol oxidation in the reconstituted enzyme system. The specific aims are: (a) to characterize ethanol-inducible P-450 from various sources and determine its quantitative role in ethanol oxidation; (b) to determine whether alcohol-inducible P-450 catalyzes the oxidation of other hydroxy compounds of biological or toxicological importance; (c) to examine other aspects of the P-450 oxygenase system such as alterations in biosynthesis or interaction of the components as an explanation of the metabolic effects of ethanol; and (d) to elucidate the mechanism of ethanol oxidation by P-450 isozyme 3a in comparison to that of the other less active isozymes and of the reductase alone. The extent to which ethanol-inducible P-450 participates in ethanol oxidation will be determined by the inhibitory effect of isozyme 3a antibodies added to microsomal suspensions. An attempt will be made to find a specific inhibitor of ethanol oxidation by P-450-LM3a for studies in tissue slices and intact animals, and blood ethanol clearance in vivo in normal and alcohol-induced rabbits will be compared with the in vitro data on P-450-mediated alcohol oxidation. Although catalysis by P-450 is probably not a major pathway, this cytochrome may play a significant role in ethanol oxidation in individual organelles, tissues, or species, or in animals or man chronically exposed to ethanol. Studies to determine the mechanism of electron transfer and oxygen activation by the various P-450 isozymes will include attempts to account for the O2 and NADPH consumed in excess of known products formed, stopped flow spectral identification of active oxygen species derived from the O2-ferrous complex, examination of the role of peroxy compounds substituted for NADPH and O2, determination of the rate-limiting step(s), and site-directed mutagenesis to determine the function of specific amino acid residues in the enzyme.
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0.994 |
1985 — 2002 |
Coon, Minor J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Function of Hydroxylases and Other Oxidative Enzymes @ University of Michigan At Ann Arbor
Of the known biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. Rapid progress in recent years as revealed a P450 gene superfamily with numerous members in bacteria, fungi, plants, invertebrates, and vertebrates, including the human. These enzymes are of great interest from a fundamental point of view because of their remarkable versatility and reaction mechanisms involving the generation of a powerful oxidant from molecular oxygen, and also from the viewpoint of their biomedical relevance. Because the substrates include physiologically important compounds such as steroids, bile acids, fatty acids, prostaglandins, retinoids, biogenic amines, and lipid hydroperoxides, as well as a host of "environmental chemicals," it is no exaggeration to say that improved knowledge of P450 function will contribute to progress in drug metabolism and design, as well as to better insights into chemical carcinogenesis, alcoholism, endocrine disorders, and oxidative stress. Our main objectives are: 1. To obtain detailed evidence on the important question of whether multiple oxygenating species contribute to substrate oxidation by cytochrome P450. Mutant P450s blocked in proton delivery to the active site will be examined for their rates of oxidation of various substrates as an indication of the role of peroxo-hydroperoxo- and oxenoid-iron as discrete oxidants. Attempts will be made to characterize these labile species by chemical and physical methods in order to correlate the apparent steady state level of a particular oxidant with the catalytic rates. In addition, the possibility will be examined that the levels of the oxidants are subject to regulation by effectors, including cytochrome b5 and flavonoids. 2. To continue our attempts to obtain one or more mammalian P450s in a crystalline form suitable for structure determination by x-ray diffraction. Various full-length and truncated P450s will be studied for this purpose. 3. To determine the role of phospholipids and other membrane components in influencing the formation of binary and ternary complexes of P450, NADPH-P450 reductase, and cytochrome b5, and in altering the rates and specificities of substrate oxidation in lipid bilayers. The effect of lipid composition in membrane bilayers. The effect of lipid composition in membrane bilayers will be examined with respect to single-phase fluid membranes with co-existing solid-phase obstacles to protein diffusion.
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0.994 |
1987 — 1991 |
Coon, Minor J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Alcohol Metabolism: Role of P-450 Oxygenases @ University of Michigan At Ann Arbor
The chronic consumption of alcohol is known to be associated with the toxicity of numerous foreign compounds, including drugs, that undergo metabolic activation by cytochrome P-450. This laboratory purified and characterized the ethanol-inducible form of the cytochrome (termed P-450 isozyme 3a on the basis of eleccrophoretic behavior or P-450ALC to indicate high activity in alcohol oxygenation) and has since shown the central role of this catalyst in alcohol-related toxicities. The specific aims of this proposal are: (a) to complete the characterization of various forms of P-450's involved in alcohol oxidation, including hepatic isozyme 3a, a non-inducible but immunochemically similar cytochrome in nasal membranes, and a variant nasal cytochrome having different electrophoretic behavior and much higher catalytic activities; (b) to determine the mechanism(s) by which diverse agents and treatments cause induction of P-450ALC; and (c) to determine whether the variable responses of different animals to the inducing effects of ethanol can be predicted. The purified enzymes will be sequenced and examined for post-translational modifications, and an attempt will be made to correlate substrate specificity with any structural differences found. Other organelles such as mitochondria and nuclei, and other tissues such as esophagus, stomach, aorta, endocrine tissues, and brain will be examined for interesting new forms of P-450ALC. Variants will also be identified by screening of an appropriate cDNA library. Determination of the level(s) of which P-450ALC biosynthesis is regulated will involve measurement of rates of transcription in isolated nuclei and quantitation of intranuclear precursors and cytoplasmic mRNA transcripts using cloned cDNA as a hybridization probe, as well as quantitation of cellular enzyme levels by radioimmunoassay. The P-450ALC genes will be identified and characterized by restriction mapping, Southern blotting, and sequence determination, and regulatory elements will be identified in the f'-flanking region or elsewhere. With an improved understanding of P-450ALC variants and of the control of P-450ALC gene expression in various tissues, attempts will be made to show by the techniques of molecular genetics whether an animal has been chronically exposed to ethanol and to predict whether a particular animal will be highly induced by such exposure.
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0.994 |
1993 |
Coon, Minor J |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Function of Hydroxylases and Other Oxidative @ University of Michigan At Ann Arbor
The overall goal of the research project is an improved understanding of the structure-function relationships and control mechanisms for mammalian cytochrome P-450. Such information is essential for an adequate evaluation of the role of this large and versatile family of cytochromes in the metabolism of steroids and prostaglandins and in the conversion of environmental chemicals either to more toxic substances, including carcinogens, or to detoxified products. The specific aims are as follows: (a) to isolate and characterize the remaining unidentified or inadequately identified P-450 isozymes of rabbit liver and determine their physical and chemical properties, including amino acid sequences. The cytochromes to be purified include one for which this laboratory has already obtained the predicted sequence from an isolated gene (P-450z) and other P-450's that are induced by clofibrate, flavonoids, cholestyramine, debrisoquine, or starvation, are sex-specific, or are responsible for the oxygenation of steroids and prostaglandins in various positions. Attempts to crystallize these membrane-bound enzymes for the purpose of X-ray diffraction will be continued, and additional genes, particularly of the "phenobarbital family," will be isolated and characterized. A related goal is to carry out site-specific mutagenesis of selected P-450 amino acid residues that are likely to play a role in P-450 catalysis or the binding of substrates and electron donors; (b) to determine the substrate specificity of liver microsomal P-450's under conditions more typical of the in vivo situation, with partially limiting substrate and electron carrier concentrations. The rates of competing reactions in which 0-2 is converted to H-20-2 or undergoes 4-electron reduction with the presumed formation of water will also be determined; and (c) to study factors that control the activity of the P-450 system, such as induction and repression, possible allosteric effectors, and cytochrome b-5. The effects of b-5 are striking in that it stimulates some reactions and inhibits others. The mechanism, which apparently involves electron transfer to or from b-5 by the Fe-II-0-2 complexes of the P-450 isozymes, will be studied by stopped flow spectrophotometry. Preliminary results indicate that b-5 reacts differently with different P-450's and controls the rate of formation of several other oxygen-containing species from the ferrous dioxygen complex.
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0.994 |
1996 — 1997 |
Coon, Minor |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Symposium On Biological Oxidations At the University of Michigan to Be Held June 28-29,1996 @ University of Michigan Ann Arbor
9514258 Coon The area of biological oxidations, which is of major importance to modern biochemistry, molecular biology, and biophysics, continues to be one of vigorous investigation. This special symposium is being held in honor of Dr.Vincent Massey, a leader in the field of biological oxidations, on his 70th birthday. The symposium will focus on enzyme- catalyzed oxidations and flavins and flavoproteins. The speakers will include 10 from the United States and 6 from foreign countries. All have made significant research contributions in the field of biological oxidation's and many are Dr. Maser's former students and collaborators. Topics that will be discussed include: oxygenases, hydroxylases, oxidases, and dehydrogenases in addition to broader topics such as protein flexibility and biological development of the oxidative proteins. %%% Biological oxidation includes virtually all of the biochemical processes by which energy is derived from foods. A large range of enzymes accomplish the various reactions that are involved. This meeting will bring together investigators who are working on the broad range of enzymes and related topics that are linked by a common theme, i.e., biological oxidation. It is expected that information exchange among these investigators will lead to new insight into this field. ***
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0.915 |
1997 — 2000 |
Coon, Minor J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Alcohol Metabolism--Role of P450 Oxygenases @ University of Michigan At Ann Arbor
APPLICANT'S ABSTRACT: The alcohol-inducible forms of cytochrome P450 contribute to alcohol oxidation and are of biomedical importance because they are involved in the chemical toxicities, mutagenesis, and carcinogenesis associated with alcohol abuse. In addition, they catalyze the reductive cleavage of lipid hydroperoxides and may thereby cause a loss in the integrity of biological membranes. Our goals are as follows: 1. To determine the role of alcohol-inducible P450 oxygenases (rabbit liver microsomal isozymes 2E1 and 2E2) in the oxidative cleavage of esters and amides, with the eventual goal of finding physiologically important substrates for the reaction. Compounds of metabolic importance to be considered include retinyl esters, thiol esters, phosphate esters of carbohydrates, triglycerides, phospholipids, nucleotides, and various peptides. Xenobiotic esters and amides that form aldehydes or other potentially toxic products will also be examined as possible substrates. 2. To compare the activities of P450 2E1 and 2E2 in the reductive beta-scission of various lipid hydroperoxides and determine the metabolic fate and possible function of the products. The long-range aim of the work is to determine whether chronic exposure of animals to ethanol, by causing induction of P450s, leads to enhanced degradation of membrane lipids and eventual loss of membrane integrity. In addition to determining the metabolic fate of the products of reductive cleavage, we will look for the presence of hydroxylated fatty acids, which are alternative products of hydroperoxide reduction. 3. To study the inactivation of ethanol-inducible P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal (HNE) and related products of membrane lipid peroxidation. We will also study the biosynthesis of HNE in mammalian tissues and examine the possible inhibitory effects on P450s 2E1 and 2E2 of a series of other alpha, beta-unsaturated carbonyl compounds produced by ionizing radiation and drug metabolism as well as by lipid peroxidation. 4. To determine the mechanism of induction of P450 2E1 in diabetes, which may involve release of down-regulation of gene expression by insulin. We will first establish a cell line with stably expressed P450 2E1 under the control of its own promoter, in Hep G2, which does not express detectable P450 2E1 endogeneously, and then investigate the effect of insulin in the transfected Hep G2 cells. The mechanism of regulation by insulin will also be examined by nuclear run-on assays and mRNA half-life determination. In addition, we will determine whether induction in animals by the diabetic state and by alcohol administration give additive effects on the P450 2E level in liver microsomes. If so, the oxidative damage and chemical pathology (including nitrosamine activation and acetaminophen toxicity) associated with these cytochromes may be comparably elevated.
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0.994 |