The liver cytochromes P-450 catalyze the biotransformation of a large number of lipophilic compounds. The metabolic response of a person to a xenobiotic challenge depends on the type and amount of the cytochromes P-450 present which are under genetic control and may be either induced or repressed by environmental factors. The goal of the project is to characterize human livers for their cytochromes P-450 contents. We will purify and characterize human liver cytochromes P-450 homologous to the rat cytochromes P-450a, P-450b, P-450c, P-450-d, P-450g, and p-450p. In addition, the two isozymes that metabolize theophylline will be purified. The purified isozymes will be characterized with respect to molecular weight, spectral properties, NH2-terminal amino acid sequence and metabolic activities. Isozyme specific polyclonal and monoclonal antibodies will then be prepared and immunoinhibition studies will be carried out to determine the relative contribution of that each cytochromes makes to the overall metabolism of a compound in human liver. In addition, Western immunoblot analyses will be used to quantitate the levels of these proteins in human liver samples. Finally, the antibodies will be used to obtain cDNA probes to the mRNA of the cytochromes P-450 by screening a human liver cDNA library cloned in the expression vector Lambdagt11. The nucleotide sequence of the probes will then be determined and the amino acid sequence from them will be deduced. The cDNA probes will also be used in Northern blot analyses to determine the size and quantity of the mRNA for the isolated cytochromes P-450. Finally, the cDNA probe will be used in Southern blot analyses to determine the amount of genomic DNA related to the cDNA. It is expected that the results of these experiments will provide for the first time a comprehensive profile of the metabolic activities, cytochrome P-450 protein concentration and mRNA levels in human liver. Because this information will be correlated with the medical history and environmental exposures of the patients, important new information on the regulation and function of the human liver cytochromes P-450 will be obtained.
