Localization of the N-terminal methionine of rat liver cytochrome P-450 in the lumen of the endoplasmic reticulum

Recent cumulative evidence suggests that liver microsomal cytochrome P-450 (P-450) is exposed to the cytosol with the exception of the N-terminal peptide (amino acid residues 1 to 21), or two peptides (residues 1 to 60). We tested the localization of the N-terminal methionine residue of P-450IIB1 of rat liver microsomes in the natural membrane with the site-specific reagent fluorescein isothiocyanate. The N-terminus of isolated P-450 was stoichiometrically modified in solution with fluorescein isothiocyanate. In intact microsomes, the N-terminus was not modified but became accessible to the reagent when the membrane was dissolved with Triton X-100. Our results indicate that the N-terminus faces the lumen of the endoplasmic reticulum, and we propose that P-450 spans the membrane only once with amino acid residues 1 to 21.     

Biosynthesis of rat liver cytochrome P-450 in mitochondria-associated rough endoplasmic reticulum and in rough microsomes in vivo

The hypothesis of a preferential biosynthesis of a major phenobarbital inducible form of hepatic cytochrome P-450 (P-450b) in mitochondria-associated rough endoplasmic reticulum (RERmito) was tested by measuring incorporation rates of [35S]methionine and delta-amino[3H]levulinate into the hemoprotein in adult rats. RERmito, rough microsomes (RM representing RER not associated with mitochondria) and smooth microsomes (SM) were quantitatively isolated from the same homogenate by rate zonal centrifugation and their content of P-450b determined by rocket immunoelectrophoresis. P-450b was isolated by immunoprecipitation from detergent-solubilized membrane fractions. The time course and rate of incorporation of [35S] methionine into immunoprecipitable P-450b of RERmito and of RM were similar at all time points studied (2-15 min) both under conditions of maximal induction (4 injections of phenobarbital in 4 days) and after a single injection of phenobarbital. The incorporation of [35S]methionine into P-450b of SM was slower at early time points (2-8 min) but similar to RERmito and RM after 15 min. In contrast, at short labeling periods (less than 8 min) more delta-amino[3H]levulinate was incorporated into P-450b of RERmito than into P-450b of RM and SM. No significant accumulation of free apocytochrome P-450b was found in either membrane fraction. These data indicate a close coordination of the biosynthesis and assembly of apocytochrome P-450b and its prosthetic heme but do not support the hypothesis of a major functional role of MITO X RER complexes in the synthesis of microsomal cytochrome P-450b.     

The role of components of the endoplasmic reticulum in the biosynthesis of cytochrome P-450.

1. Antibodies have been prepared to rat hepatic cytochrome P-450 and their specificity demonstrated. These antibodies have been used to investigate the biosynthesis of cytochrome P-450 in vitro and in situ in various components of the endoplasmic reticulum. 2. A preparation of heavy rough endoplasmic reticulum translocates proteins newly biosynthesized in vitro vectorially into the luminal space and these are released by low concentrations of deoxycholate. A significant proportion of the radioactivity found in this released fraction is incorporated into cytochrome P-450. 3. Following incorporation of [14C]leucine by perfused rat liver, radioactively labelled cytochrome P-450 can be found in the intrascisternal content of heavy rough, light rough and smooth endopalsmic reticulum and also in a solublized Golgi preparation. 4. We suggest that at least part of the newly biosynthesized cytochrome P-450 is translocated into the intracisternal space of the rough endoplasmic and then passes through the other components of the endoplasmic reticulum before insertion at its ultimate membrane locus.     

Phospholipase D activity of cytochrome P450 in human liver endoplasmic reticulum.

Phospholipase D (PLD) activity in mammalian liver endoplasmic reticulum (ER) has not been characterized. Purified human liver microsomal cytochromes P450 (P450)-P450 1A2 and P450 2E1-were shown to have appreciable PLD activity, hydrolyzing phosphatidylcholine but not other phospholipids, generating PA and choline. The activity was confirmed using recombinant and mutated human P450s expressed in bacteria. In human liver microsomes, immunoinhibition of PLD activity was observed with anti-P450 1A2 > anti-P450 2C > anti-P450 2E1. Thus, P450 may act as a significant PLD in human liver ER and exert its biological effects by several mechanisms, including signaling functions and change of membrane properties.     

Testosterone metabolism by microsomal cytochrome P-450 in liver of rats treated with some inducers

1. The stereoselective hydroxylation of testosterone by microsomal cytochrome P-450 and the changes in level of components participated in the microsomal electron transport system were observed in the microsomes induced unique P-450 isozymes. 2. Flavone- and hesperetin-inducible P-450 catalyzed the hydroxylation of testosterone more effectively than other chemicals-inducible ones. 3. The P-450 in all the microsomal preparations tested most rapidly oxidized testosterone to 6 beta-monohydroxy form. 4. Particularly, MC- and BNF-inducible P-450 showed high stereoselectivity on C6-position of testosterone, and PB-, flavone- and hesperetin-inducible one showed that on C2-position of this compound, respectively. 5. This specificity of two flavonoid-inducible P-450 for the formation of 2 alpha- and 2 beta-epimer of monohydroxytestosterone was opposite to each other. 6. The content of P-450 and the activity of NADPH-cytochrome P-450 reductase were high in PB-, MC- and BNF-microsomes, whereas NADH-cytochrome b5 reductase activity was high in two flavonoid-microsomes and the content of cytochrome b5 was not changed except the PB-treated rats. 7. It is suggested that the increasing activities of testosterone hydroxylases in flavonoid-microsomes seems to be closely related to NADH-cytochrome b5 reductase.     

Aryl hydrocarbons as inducers of cytochrome P-450 in Acinetobacter calcoaceticus

Summary The inducibility of cytochrome P-450 in Acinetobacter calcoaceticus by some compounds known as typical inducers of hepatic cytochromes P-450 was investigated. Besides biphenyl also indene and phenanthrene are inducers, whereas compounds of the so-called phenobarbital type are not. Biphenyl appears to be the most effective inducer with regard to the yield of cytochrome P-450/mg of cell protein. By addition of the compounds in the vapour phase an induction of the protein by naphthalene could be demonstrated. The results are indicative of the existence of bacterial cytochromes P-450 that resemble hepatic cytochromes.     

Differential oxidase activity of hepatic and pulmonary microsomal cytochrome P-450 isozymes after treatment with cytochrome P-450 inducers.

Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used as inducers of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxide-generating activity. The contribution of cytochromes CYP 1A, CYP 2B and CYP 2E1 to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2E1 inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies 1-7-1 and 2-66-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome P450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP 1A1, induced by 3-MC, demonstrates an unusually low oxidase activity.     

Antioxidants as cytochrome stabilizers in endoplasmic reticulum membranes of rat liver in vivo]

It has been shown that synthetic antioxidant 4-methyl-2, 6-ditrebuthylphenole (ionole) inhibits free radical oxidation of phospholipids in the membranes of endoplasmic reticulum, increases the life time of cytochrome P-448 after its induction with 20 methylcholantrene.     

The differentiation of rat liver endoplasmic reticulum membranes: Apo–cytochrome P450 as a membrane protein

The proteins of washed microsomal membranes from adult rat liver were solubilized by 2% SDS and electrophoresed on polyacrylamide gels. Confirming earlier reports, a large Coomassie-Blue staining band in the ～50,000 MW region was identified as cytochrome P₄₅₀ by four criteria: similar electrophoretic mobility to a purified cytochrome P₄₅₀ preparation, an increase in this band after in vivo phenobarbital administration, a decrease in this band after in vivo allylisopropylacetamide administration, and direct specific binding of added purified heme to this region of a washed, unfixed gel. Although cyt P₄₅₀ is not spectrally evident until just at the time of birth of the rats, a large band in this region was detectable in gels of microsomal membrane protein at all times, from three days before birth onward; this band also bound added heme after membrane proteins from fetal rat liver microsomes were electrophoresed on the gels. The conclusion was that apo-cyt P₄₅₀ is present in microsomal membranes at these times during differentiation, and that, regarding this protein, during differentiation heme is bound to the apo-protein already there, concomitant with a synthesis of more cyt P₄₅₀ molecules. The process of differentiation of this membrane type is also discussed.     

The juxtamembrane sequence of cytochrome P-450 2C1 contains an endoplasmic reticulum retention signal

The N-terminal signal anchor of cytochrome P-450 2C1 mediates retention in the endoplasmic reticulum (ER) membrane of several reporter proteins. The same sequence fused to the C terminus of the extracellular domain of the epidermal growth factor receptor permits transport of the chimeric protein to the plasma membrane. In the N-terminal position, the ER retention function of this signal depends on the polarity of the hydrophobic domain and the sequence KQS in the short hydrophilic linker immediately following the transmembrane domain. To determine what properties are required for the ER retention function of the signal anchor in a position other than the N terminus, the effect of mutations in the linker and hydrophobic domains on subcellular localization in COS1 cells of chimeric proteins with the P-450 signal anchor in an internal or C-terminal position was analyzed. For the C-terminal position, the signal anchor was fused to the end of the luminal domain of epidermal growth factor receptor, and green fluorescent protein was additionally fused at the C terminus of the signal anchor for the internal position. In these chimeras, the ER retention function of the signal anchor was rescued by deletion of three leucines at the C-terminal side of its hydrophobic domain; however, deletion of three valines from the N-terminal side did not affect transport to the cell surface. ER retention of the C-terminal deletion mutants was eliminated by substitution of alanines for glutamine and serine in the linker sequence. These data are consistent with a model in which the position of the linker sequence at the membrane surface, which is critical for ER retention, is dependent on the transmembrane domain.     

Immobilization of liver microsomal cytochrome P-450

Rabbit liver microsomal cytochrome P-450 was immobilized by entrapment in calcium alginate gel. Aminopyrine demethylation experiments showed that the immobilized enzyme system is highly active and exhibits an unimpaired functional stability as compared with crude microsomes. The alginate entrapped microsomes were employed in a fixed bed recirculation reactor, where aminopyrine was continuously demethylated. Such model enzyme reactor can be a useful tool for studying extracorporeal drug detoxification or preparative substrate conversion with microsomal enzyme systems.     

Induction of hepatic cytochrome P-450 mediated alkoxyresorufin O-dealkylase activities in different species by prototype P-450 inducers

The induction of cytochrome P-450-mediated alkoxyresorufin O-dealkylase activities by various xenobiotics was examined in liver from a variety of animal species in order to gain insights into the substrate specificities of the induced P-450s. We found that forms of cytochrome P-450 capable of mediating the O-dealkylation of the short-chain phenoxazone ethers methoxy-, ethoxy- and propoxyresorufin were highly induced by 3-methylcholanthrene-type inducers and by Aroclor-1254 in all species tested, although there were species differences in the relative turnover rates for the various substrates. For example, in hamster liver the turnover rates for the short-chain resorufin ethers decreased in the following order: methoxy greater than ethoxy much greater than propoxy, while in the rat liver almost the exact opposite order was observed: ethoxy = propoxy much greater than methoxy. In contrast, the degree of induction by phenobarbital-type inducers of isozymes catalyzing the O-dealkylation of pentoxy- or benzyloxyresorufin was highly species-dependent. Thus, F344/NCr rats, B6C3F1 mice and NZB rabbits showed the greatest (greater than 20-fold) induction of these activities, either by phenobarbital or Aroclor-1254, while Mongolian gerbils showed intermediate levels of induction and Syrian golden hamsters exhibited very low induction. In the Japanese quail, phenobarbital- or DDT-treatment resulted in minimal induction of pentoxy- or benzyloxyresorufin O-dealkylase activity, although significant induction of the latter activity occurred following treatment with 5,6-benzoflavone or with Aroclor-1254. Since substrate specificities of most enzymes can be rationalized based upon differences in the steric requirements at the enzyme active site, we employed molecular modeling techniques to calculate the molecular dimensions of the alkoxyresorufins. Surprisingly, the minimal energy conformations in vacuo of each of the resorufin ethers examined are essentially planar. However, alternative configurations, especially for the pentoxy- and benxyloxy-ethers, having greater three-dimensional bulk are also energetically possible.     

Induction of cytochrome P-450 in rat liver by the antibiotic troleandomycin: partial purificaton and properties of cytochrome P-450-troleandomycin metabolite complexes

Liver cytochrome P-450 from rats treated intraperitoneally with troleandomycin (TAO) were solubilized and partially purified using DE 52 anion exchange chromatography. The major TAO-induced cytochrome P-450 form appears in fraction A which is not bound on the DE 52 column. It is different from the major form induced in rats by phenobarbital or 3-methylcholanthrene in terms of absolute visible spectroscopy, gel electrophoresis (M 45000) and reactions with antibodies. This TAO-induced form mainly exists $\text{in vivo}$ as an iron-TAO metabolite complex and exhibits a characteristic Soret peak at 456 nm. Reconstitution experiments using this partially purified form, after dissociation of its iron-metabolite bond by ferricyanide treatment, underline its particular ability to demethylate TAO itself. TAO also leads to an important induction of other cytochromes P-450 that are present in fraction B (retained on DE 52 column) like the major phenobarbital-induced form, but are immunologically distinct from it.     

The use of proteinases to determine the topological location of cytochrome P-450 in vesicles derived from smooth endoplasmic reticulum of rat liver.

1. The phospholipid bilayer of intact vesicles from smooth endoplasmic reticulum is impermeable to macromolecules. Specific and non-specific proteinases were used to investigate the site of membrane proteins in the transverse plane of the bilayer. 2. When two proteinases were used in conjunction, denaturing effects additional to proteolysis were observed on cytochrome P-450 content and glucose 6-phosphatase activity which did not depend on the integrity of the phospholipid bilayer. 3. When lipid peroxidation was inhibited, these effects were not observed.     

Isolation and properties of cytochrome P-450 from rabbit liver microsomes]

A purified low-spin form of cytochrome P-450 was isolated from phenobarbital-induced rabbit liver microsomes. The preparation was functionally active and free from cytochromes b5 and P-420 and phospholipids. The specific content of the cytochrome was 18 nmoles per mg of protein. At the molecular weight of the hemoprotein of 50,000, it corresponds to 90% of purification. The purified hemoprotein binds substrates of type II and some substrates of type I. The complexes formed reveal spectral properties, similar to those for the complexes of these substrates with the microsomal form of cytochrome P-450.     

Purification and properties of cytochrome P-450 from untreated monkey liver microsomes

Untreated monkey liver cytochrome P-450 (monkey P-450) has been purified to a specific content of 14.9 n mole/mg protein. The purified preparation was apparently homogeneous and the minimum molecular weight was estimated to be 50,000 by SDS-PAGE. Absolute spectrum of the oxidized form showed peaks at 565, 535 and 417 nm. The monkey P-450 was active in the mixed function oxidation of benzphetamine, aminopyrine, ethylmorphine, aniline and 7-ethoxycoumarin in the presence of rat liver NADPH-cytochrome P-450 reductase and DLPC. Anti monkey P-450 IgG could not inhibit rat P-450s (PB P-450, MC P-448(1) and MC P-448(2] catalyzed 7-ethoxycoumarin O-deethylation activities.