Degradation of cytochrome P450 isozymes after their suicidal inactivation: a role for cytosolic proteases in the rat liver?

1375 mitochondrial membrane, as well as MAO from Triton X-100 pretreated prevarations. This might be acceptable for empirical, comparative evaluation, but should certainly be considered in any absolute evaluation of MAO activity. Differences between MAO-A and lV~.O-B are yet to be investigated. Table 1 Extent of inhibition of MAO activity by (NH4)2SO4 and NaCI

Mitochondrial MAO Triton X-IO0treated MAO

Concentration of (NH4)2SO4 1~ 10~ 25~ 55~$ 18~$

60~ 80~

43~$

Concentration of NaCi 1~ 3~ 70~ 905 60~ 70~

I P.~'e.242 [

Degradation of cytochrome P450 |sozymes after ¢he|r suicidal inactivation: a role [or cytosol|c proteases in the rat l|ver? Berry, L.A. and Correia, M.A. Department of Pharmacology and the Liver Center, University of California, San Francisco, San Francisco, CA 94143, U.S.A.

~ o u s "in vitro" studies have shown that aerobic incubation of NADPH-supplemented rat fiver microsomes ~ t h the suicide substrate 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-l,4-dihydropyridine (DDEP) results in inactivation of cytochrome P450 (P450) p, h and k isozymes and that P450-dependent metabolism of DDEP is necessary for this event. Inactivation also occurs "in vivo" in DDEP-treated rats, and is accompanied by a marked decrease in immunochemically detectable levels of hepatic microsomal apocytochrome P450 p and h. In vitro as well as in vivo, P450p destruction is also accompanied by prosthetic heine alkylation of the apoprotein. In contrast, cytochrome P450k which is inactivated largely through DDEP-mediated prosthetic heine N-ethylation, appears to be relatively resistant to such DDEP-mediated proteolytic loss. Thus, it appears that DDEP-mediated structural damage of the susceptible isozymes results in accelerated proteolysis of their apoproteins. No appreciable contribution of the autophagic/ lysosomal pathway to such degradation is detected when the process is probed with lysosomotropic inhibitors (propylamine), lysosomal protease inhibitors (leupeptin) or autophagic suppressors (3-methyladenine). Many cellular proteins of short and medium half-fife are degraded by a host of cytosolic proteolytic systems, several of which are ATP-dependent. In many, but not all of these systems, proteins are targetted for proteolysis by conjugation with the peptide ubiquitin in a process which is also ATP.dependent. To investigate whether such cytosolic systems are responsible for the proteolytic loss of DDEP-susceptible isozymes, rats pretreated with the P450p inducer, dexamethasone, were administered [14C]NaHCO3 to label intracelluiar proteins. Liver microsomes were incubated (37°C, 30 rain) with DDEP :t: NADPH to inactivate microsomal P450 isozymes. These incubations also included bovine serum albumin as an external trap for DDEP.derived ethyl radicals to circumvent structural damage to the microsomal membrane through protein alkylation. The excess DDEP was "washed" off, and the DDEP-incubated microsomes further incubated at 370 for varying lengths of time in a reconstituted proteolytic system [consisting of ~iver cytosol (passed through Sephadex G-50, to remove endogenous ATP and ubiquitin), Mg 2+, ATP-regenerating system, ± ATP, 4-ubiquitin]. Our findings indicate that DDEP-inactivated cytochrome P450 isozymes are degraded by cytosolic proteases by at least two separate mechanisms. One mechanism is dependent on ubiquitin and may involve its binding to the damaged apoprotein thereby targetting it for proteolysis. The second mechanism occurs independently of ubiquitin, but is dependent on ATP, and may involve direct degradation of the apoprotein by ATP-dependent proteases. Immunochemical quantitation of individual apocytochrome P450s h, k and p, indicates that after their DDEP-mediated inactivation, all of these isozymes are degraded in part by the ubiquitin-independent mechanism. In addition, P450h and k but not p are also degraded by a process that is ubiquitin-dependent. These findings indicate that proteolysis of

1376 die dm-aaged ~pocytochrome P450 by cytosolic proteases may be one mechanism by which DDEP-mediated loss of susceptible P450 isozymes occurs "in vivo". Supported by NIH grants DX 26506 and GM 44037. I P.we.243 1

Rat liver cytochrome P-450 and ageing Horbach, G.J.M.J., Van Asten, J.G., Rietjens *, I.C.M. and Van Bezooijen, C.F.A. TNO Institute for Experimental Gerontology, P.O. Box 5815, 2280 HV Rijswijk and * Agricultural University Wageningen, The Netherlands

Age-related changes in the cytochrome P-450 syst~,n in the ra~ are often described and generally a decrease in both content and activity are reported, although increases have been described as well. The age-re~ated changes are more pronounced in male rats than in female ones, dl:,, to changes in gender-specific, constitutive forms of cvtochrome P-450, which in male rats are dependent on testosterone levels. As far as the inducible types of cytochrome P-45~ ~ e concerned, little 6ata are available on the influence of age. In this study, data will be presented on the levels of r;:;~ssenger RNA and enzymes for the cytochromes P-450 IA1, IA2, IIBl. and lIB2 in male rats either untreated or maximally induc~:~ ~,ith phenobarbital, 3-methylcholanthrene or isosafrole. Also the specific activities of P-450 enzymes were m~asurea using the highly specific substrates, pentoxyresorufin and ethoxyresorufin. The results of this study show that after maximal ~,~duction there are considerable changes with age in the levels of mRNAs for F-4501A1, IA2, liB1 and liB2. The inducibility of the mRNAs for P-45011B1 and lIB2 by both phenobarbital and isosafrole shows a marked decrease between 12 and 36 months of age. A decrease in the inducibility of the mRNAs for P-4501A1 and IA2 is only observed when using isosafrole as the inducer and not when using 3-methylcholanthrene. From these observations, it can be concluded that changes with age in the mRNA levels are different for the different P-450 enzymes and are dependent on the inducer used. However, after maximal induction no age-related changes were observed in the amount and activity of the cytochrome P-4501A1, IA2, IIB1 and lIB2. Therefore, the lower mRNA levels with age are not reflected in lower enzyme levels, but rather result in a decreased synthesis rate of these enzymes. I P.we.244 ]

Different roles of protein kinase C isozymes in differentiation of PCI2h cells Kunugi, Y.-U., Tamura, H., Shimohama *, S., Saitoh * *, T. Taniguchi, T. and K i m u r a *, J. Department of Neurobiology, Kyoto Pharmaceutical University, Kyoto, 607, • Department of Neurology, Kyoto University School of Medicine, Kyoto, 606, Japan and * * Department of Neurosciences, School of Medicine, University o.f California, San Diego, U.S.A.

Protein kinase C has been implicated in the regulation of various cellular processes including growth, differentiation, hormone and neurotransmitter release, gene expression and cellular n'Letabolism. However, the role of each protein kinase C isozyme (a, ill, flII or v type) in the regulation of different cellular functions remains unknown. Rat pheochromocytoma PC12 cells can be induced to differentiate into cells with typical morphology by a number of substrates including nerve growth factor (NGF), cAMP derivatives and TPA, an activator of protein kinase C. Although this differentiation process is well known to be accompanied by various physiological responses, the molecular mechanisms of their signal transductions are not clear. In order to clarify the contribution of protein kinase C-related signal transduction pathway, and moreover, to corelate the presence of a protein kinase C isozyme with