Terminal olefins frequently are transformed by metabolic processes to chemically-reactive, short-lived metabolites which may cause toxic effects through covalent interactions with cellular macromolecules. Examples of such toxicities may be found in the mechanism-based inactivation of several enzymes involved in the beta-oxidation of fatty acids and in the oxidation of endogenous and exogenous substances by cytochromes P450. In addition, many terminal olefins are genotoxic due to alkylation of DNA by reactive metabolites thereof. To date, most studies on terminal olefins have focused on preformed unsaturated compounds of pharmaceutical or environmental importance. However, there is now a growing body of evidence which indicates that certain olefins can themselves be formed by metabolic desaturation of aliphatic substrates, and that the responsible enzyme system is the cytochrome P450-dependent mixed function oxidase. A case in point is 4-ene-VPA, a potent hepatotoxic metabolite of the antiepileptic drug valproic acid (VPA) which is formed from the parent drug by the action of p450. In light of these recent observations, it becomes important to understand, at the molecular level, which factors control the conversion of compounds such as aliphatic carboxylic acids to their corresponding alkenes, and how the regiochemistry of the desaturation process is determined. (Recent work has shown that the isomeric 3-ene-VPA also is formed from VPA by P450). In particular, there is a need for basic information on the mechanism by which p450 catalyzes these novel desaturation reactions (which accompany the more traditional oxygenation events). The overall goal of the proposed research is to define the chemical and enzymatic mechanisms of P450-dependent desaturation reactions. This will be accomplished using VPA and the endogenous molecules, lauric acid and palmitic acid, as model substrates. A comprehensive series of studies is proposed to identify specific isoforms of animal and human liver P450 responsible for terminal and sub-terminal desaturation of these substrates and to elucidate the underlying reaction mechanisms.
The specific aims of the research are as follows; (1) To identify and purify specific rat liver forms of p450 responsible for 3-ene-VPA formation; (2) To identify the rate-limiting step(s) in the desaturation of VPA to 3- and 4-ene-VPA; (3) To establish that P450 is responsible for the terminal desaturation of long-chain monocarboxylic acids; (4) To correlate relative w:w-2 fatty acid hydroxylase activities with terminal:subterminal olefin formation using cDNA-expressed forms of human P450; and (5) To probe the possible involvement of carbocations as intermediates in P450-mediated fatty acid desaturation. The fundamental knowledge gained from these investigations will be employed to construct a unified model for P450-catalyzed oxidation reactions, and will provide a working framework for the prediction of the oxidized metabolites expected to arise from catalysis by this important enzyme system. GRANTS=R43GM50044 Energy-dispersive x-ray detectors (EDDs) are heavily used in biological research at synchrotron radiation facilities, particularly for absorption spectroscopy in fluorescence mode on dilute samples. Current research relies on semiconductor EDD spectrometers, which typically become pileup limited at fairly low count rates for the biologically important elements P, S and C1, due to the long electronic processing times required to obtain acceptable energy resolution for these soft x-rays. The present research proposes to develop an alternative detector based on electron multiplication technology. Although electron multipliers are very low noise amplifiers and are capable of very high counting rates, presently available scintillator-photomultiplier (PMT) detectors are not competitive with semiconductor EDDs due to their extremely poor energy resolution, which arises from statistical fluctuations in the inefficient conversion between x-rays and electrons for amplification by the PMT. We are therefore proposing to investigate a novel technology for increasing the efficiency of the conversion process. Initial calculations suggest that if this conversion process can be made to work close to its theoretical efficiency, it would be possible to construct detectors having energy resolution superior to semiconductor EDDs for energies below 4 ke V and capable of throughputs in excess of 106 counts/sec. Because no complex electronics would be required to process these detectors' output pulses, they would also be relatively inexpensive to array for even higher countrate capability.
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