Bioactivation of organohalogen xenobiotics produces reactive intermediates which alkylate macromolecules. The activation of carbon tetrachloride, trichloroethylene, and methylene chloride was studied in isolated rat hepatocytes by examining alkylation of lipid, protein, RNA, and DNA. All organohalogens alkylated lipid and protein. Carbon tetrachloride and trichloroethylene, but not methylene chloride, alkylated RNA and DNA. Methylene chloride was more highly activated in an oxygen containing atmosphere by hepatocytes, consistent with a proposed formation of formyl chloride as its reactive intermediate. Trichloroethylene was also shown to be more highly activated in an oxygen containing atmosphere, consistent with a proposed trichloroethylene epoxide reactive intermediate. Carbon tetrachloride was shown to be more highly activated in an oxygen-free atmosphere, consistent with a proposed trichloromethyl free radical reactive intermediate. Hepatocytes from rats pretreated with phenobarbital to induce cytochrome P-450 mixed function oxidase activated carbon tetrachloride and trichloroethylene to alkylating intermediates greater than did hepatocytes from non-induced rats. The interaction of carbon tetrachloride metabolites with fatty acids was studied in a chemical activation model system. The thermal decomposition of benzoyl peroxide produced free radicals which activated carbon tetrachloride. The resulting trichloromethyl free radicals abstracted a hydrogen from methyl stearate resulting in chloroform and fatty acid free radicals. Using chemical ionization mass spectrometry, it was discovered that the fatty acid free radical abstracted a chlorine from carbon tetrachloride resulting in chlorinated fatty acid esters. When methyl oleate was used as a substrate in the benzoyl peroxide model system, it was discovered that the trichloromethyl free radical binds covalently, resulting in a fatty acid adduct radical. This radical then abstracted a chlorine to produce chloro, trichloromethyl stearic acid methyl ester, identified by chemical ionization mass spectrometry. Carbon tetrachloride radiolabeled with ¹⁴C or ³⁶Cl in dual label binding experiments in the benzoyl peroxide model system confirmed the mass spectral data. Methyl stearate bound ³⁶Cl- and ¹⁴C-carbon tetrachloride in the ratio of approximately 10 to 1, whereas methyl oleate bound in the ratio of approximately 3.5 to 1. The existence of fatty acid radicals due to hydrogen abstraction or covalent binding by trichloromethyl free radicals was demonstrated in microsomal preparations. In the presence of tritiated water and ¹⁴C-carbon tetrachloride, dual-label analysis demonstrated that the tritium incorporation into microsomal lipids approximately equalled the sum of carbon tetrachloride metabolites bound covalently to microsomal lipids and chloroform production.

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Leishmania parasites are reliant on salvage mechanisms to acquire purines from the extracellular environment. GMP reductase (GMPR) catalyzes the conversion of GMP to IMP, an integral reaction for maintaining purine nucleotide balance. Enzymatically active L. major GMPR (LmGMPR) has been cloned, expressed and purified. The LmGMPR gene complements GMPR deficiency in E. coli strains. Quaternary structure analysis indicates that LmGMPR forms tetramers and higher order complexes under reducing conditions. Kinetic assays reveal that the enzyme deviates from hyperbolic behaviour with regard to GMP but conforms to typical Michaelis-Menten kinetics for NADPH. Sequence analysis indicates that LmGMPR contains CBS domains and an MPA binding site. MPA competes for the NADPH binding site with a K i of 20 muM. ATP and GTP regulate enzymatic activity through inhibition and activation, respectively. This data indicates that LmGMPR is a novel enzyme that performs a highly regulated step in Leishmania purine metabolism.