Ghazi A. Dannan

Purification and characterization of microsomal cytochrome P-450s

1. Eight different forms of cytochrome P-450 have been purified to electrophoretic homogeneity. Electrophoretic, spectral and catalytic properties of these cytochrome P-450s are presented and comparison is made with preparations presented elsewhere in the literature. 2. The levels of these forms of cytochrome P-450 present in liver microsomes of rats treated with various compounds have now been quantified. Several forms of cytochrome P-450 are induced, in a more or less coordinate manner, while levels of other cytochrome P-450s are lowered, during administration of commonly used inducing agents. 3. The role of cytochrome P-450 purification and characterization studies in the understanding of the total field is discussed, along with directions in which future research is needed.

Comparisons of warfarin metabolism by liver microsomes of rats treated with a series of polybrominated biphenyl congeners and by the component-purified cytochrome P-450 isozymes☆

R- and S-warfarin metabolite profiles (regio- and stereoselectivity) has been determined with hepatic microsomes from untreated rats and rats treated with nine individual polybrominated biphenyl (PBB) congeners, a commercial mixture of PBBs, and, for comparison with phenobarbital and 3-methylcholanthrene. The metabolic rates have been correlated with cytochrome P-450 (P-450) isozyme concentrations in the microsomes determined by immunochemical quantitation techniques (G.A. Dannan, F.P. Guengerich, L.S. Kaminsky, and S.D. Aust, (1983) J. Biol. Chem. 258, 1282-1288). The warfarin hydroxylase activities of the P-450 isozyme components of the various microsomal preparations (F.P. Guengerich, G.A. Dannan, S.T. Wright, M.V. Martin, and L.S. Kaminsky (1982) Biochemistry 21, 6019-6030) were multiplied by the corresponding isozyme concentrations to obtain an assessment of the potential warfarin hydroxylase capacity of the microsomes, and the results were compared with actual activities. The results of these studies and comparisons indicate that substrate regio- and stereoselectivities of microsomal-bound P-450s are essentially retained on purification of the isozymes to homogeneity and reconstitution, that warfarin metabolism by microsomal preparations can be used to predict microsomal P-450 isozyme compositions, and that microsomal warfarin hydroxylase activity is greater than would be predicted based on the approx 20:1 ratio of P-450 to NADPH-P-450 reductase in the microsomes and on the known activities of constituent isozymes. Two P-450 isozymes which are induced by treatment of rats with phenobarbital appear to be more tightly linked to NADPH-P-450 reductase than does an isozyme induced by beta-naphthoflavone.

Intestinal Maturation: Calcium Transport by Basolateral Membranes

ABSTRACT: The overall characteristics of calcium transport across the intestine have been defined using in vivo perfusion techniques and in vitro everted gut sacs. However, calcium transport represents three separate processes: entry at the brush border membranes, movement across the cytoplasm, and exit at the basolateral membranes (BLM). Studies in adult animals indicated that the active step in calcium transport is located at the BLM. The present studies describe for the first time the maturational aspects of calcium transport across the BLM of the enterocyte. We utilized a percoll density gradient to prepare enriched BLMs from suckling and adolescent rats. Calcium uptake into BLMs represented mainly transport into the intravesicular space. Calcium transport in both age groups was driven by ATP; the calcium transport in the presence and absence of ATP was significantly greater in suckling rats compared to adolescent rats. Kinetics of calcium uptake calculated from uptake values in the presence of ATP minus no ATP conditions showed a Km of 0.06 ± 0.01 and 0.03 ± 0.01 μM for adolescent and suckling rats respectively (p < 0.05). Vmax was 1.5 ± 0.1 and 0.8 ± 0.08 nmol/mg protein/min for adolescent and suckling rats respectively (p < 0.01). Calcium/sodium exchange mechanisms was also present in both age groups. However, the magnitude of sodium-dependent calcium exchange was smaller in suckling rats compared to adolescent rats. These data suggest that calcium exit at the BLMs of enterocytes of suckling and adolescent rats occurs by an ATP-dependent and a calcium/sodium exchange mechanism. Both mechanisms exhibit maturational changes.