Differential apoprotein loss of rat liver cytochromes P450 after their inactivation by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine: a case for distinct proteolytic mechanisms?

Abstract

Suicide inactivation of hepatic cytochrome P450 (P450) enzymes 2C11, 2C6, and 3A1/A2 by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) in intact rats results in prosthetic heme destruction, albeit by apparently distinct mechanisms. Such heme destruction is now shown to be associated with the loss of immunochemically detectable apoprotein of P450s 2C11 and 3A but with little of that of P450 2C6, in spite of their comparable DDEP-mediated functional inactivation. The loss of a approximately 50-kDa hepatic microsomal protein band along with the immunoreactive P450 3A loss strengthens the concept that such an in vivo loss indeed reflects proteolysis of the DDEP-inactivated P450. Furthermore, this propensity of DDEP-inactivated P450s 3A for proteolysis appears to correlate with the relative degree of prosthetic heme alkylation of their apoprotein rather than their functional inactivation per se. Thus, rapid degradation of apoP450s 3A was seen after DDEP treatment, which promoted extensive irreversible heme binding to apoP450s 3A, but not after exposure to allylisopropylacetamide (AIA), which inactivates these isozymes comparably, but induced minimal apoP450 3A heme alkylation. In addition, differences were observed in the relative sensitivities of proteolysis of DDEP-inactivated P450s 2C11 and 3A to hemin, which largely prevented the DDEP-induced proteolytic loss of P450 2C11 but apparently failed to prevent the loss of DDEP-inactivated P450s 3A, when coadministered with DDEP. This differential hemin sensitivity of the proteolysis of DDEP-inactivated P450 2C11, coupled with the observation that immunochemically detectable P450 2C11 loss occurs after its inactivation by both AIA and DDEP, provides compelling support for the existence of distinct proteolytic pathways for individual suicidally inactivated P450s.