The overall objectives of this research are to understand the structural basis for the cellular localization and enzymatic activity of microsomal cytochromes P450. Cytochrome P450 is the terminal oxidase in the liver microsomal drug metabolizing system and is responsible for the oxidation of hundreds of exogenous and endogenous compounds. Rabbit cytochrome P450IIC subfamily cDNAs will be introduced into cultured mammalian cells with vaccinia virus expression vectors. Variants of cytochrome P450IIC2 with N- terminal regions modified such that the expressed protein is either not inserted into or is translocated tb the luminal side of the endoplasmic reticulum membrane will be compared with native cytochrome P450, which is anchored to the membrane on the cytoplas- mic side, to determine the influence of membrane orientation on localization and function of the enzyme. The effects of deletions and site specific mutations on the subcellular localization of cytochrome P450IIC2 will be used to define the signal for retention of the enzyme in the endoplasmic reticulum. Localization of the proteins will be determined by subcellular fractionation followed by gel electrophoresis and autoradiography or western blotting. Cross-linking techniques will be used to attempt to detect a endo- plasmic reticulum """"""""receptor"""""""" for cytochrome P450. To determine the regions important for the substrate specificity of cytochrome P450, substrate profiles will be determined for the expressed cytochromes P450IIC1 and P450IIC2. Regions of cytochrome P450IIC1 will be substituted for corresponding regions of cytochrome P450IIC2 and substrate profiles of the hybrid proteins compared to the parental profiles. Selected amino acids within the regions determined to be important for substrate specificity will be altered by site specific mutagenesis to define individual amino acids involved in substrate recognition. Cytochromes P450 play an important role in a variety of clinical problems. Species differences in metabolic pathways of drugs may be a serious problem in the preclinical testing of therapeutic agents and differences among humans may greatly affect the clinical response to a drug. The induction of cytochromes P450 with broad substrate specificity underlies clinically important drug interactions. Cytochrome P450s may also activate rather than inactivate compounds, carcinogens being an outstanding example. Knowledge of the factors involved in generating a functional cytochrome P450, such as those that determine its cellular location and substrate specificity, should increase our understanding of these important clinical problems.
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