Two-dimensional electrophoresis of membrane proteins factors affecting resolution of rat-liver microsomal proteins

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI > 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112–120) gives good resolution of proetins of pI 5–8, including cytochromes P-450, but fails to resolve a major acidic protein of pI < 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R·N⁺(CH₃)₂·CH₂CH₂CH₂SO₃^? are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C₁₄ or C₁₆) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.     

Interaction of epoxide hydrolase with itself and other microsomal proteins

The interactions of rat liver epoxide hydrolase (EC 3.3.2.3) with itself and with cytochromes P-450 and NADPH-cytochrome P-450 reductase were investigated in microsomal preparations and in reconstituted systems in which all of the enzymes are functionally active. Hydrodynamic measurements indicated that purified epoxide hydrolase behaves as a single aggregate of approximately 16 monomeric units and that further aggregation of the protein only occurs in the presence of high concentrations of phospholipid. Neither guanidine-HCl nor the nonionic detergent Lubrol PX was able to completely dissociate the aggregate into monomers. The interactions of epoxide hydrolase with NADPH-cytochrome P-450 reductase and the major forms of cytochrome P-450 isolated from phenobarbital- and 5,6-benzoflavone-treated rats were studied by Soret difference spectroscopy, by perturbation of the fluorescence of NADPH-cytochrome P-450 reductase and fluorescein-labeled epoxide hydrolase, and by CD spectroscopy. The spectra provided evidence that binding of the proteins to each other occurs and some of the results suggest that affinity constants are on the order of 10⁷, m^?1. The spectral perturbations were not observed with other intrinsic membrane proteins. When microsomes were treated with the crosslinking reagent dimethylsuberimidate and solubilized with detergents, epoxide hydrolase could be precipitated with antibodies raised to cytochromes P-450 or NADPH-cytochrome P-450 reductase. Transient times were determined for the conversion of 1-octene to octene-1,2-dihydrodiol in a reconstituted enzyme system and for the conversion of naphthalene to naphthalene-1,2-dihydrodiol in rat liver microscomes and compared to the transient times predicted from the enzymatic rates of hydrolysis of the intermediate epoxides. In all cases the observed transient times were shorter than expected, in support of the view that coupling of epoxide hydrolase with cytochromes P-450 occurs. These results support the view that epoxide hydrolase couples with cytochrome P-450-containing mixed-function oxidase systems and may have relevance to the metabolism of potentially harmful xenobiotics by these enzymes.     

Detergents for the stabilization and crystallization of membrane proteins

The use of detergents for the structural study of membrane proteins is discussed with an emphasis on practical issues relating to membrane solubilization, protein aggregation, detergent purity and detergent quantitation. Detergents are useful reagents as mimics of lipid bilayers because of their self-assembling properties, but as a result, they have complex properties in solution. It can be difficult to maintain a solubilized membrane protein in a native conformational state, and the non-specific aggregation of detergent-solubilized proteins is a common problem. Empirical "stability screens" can be helpful in choosing which detergents, and which detergent concentrations, may be optimal for a given system.     

Solubilization of bacterial membrane proteins using alkyl glucosides and dioctanoyl phosphatidylcholine

The non-ionic detergent octyl glucoside solubilizes a substantial amount of Streptococcus faecalis membrane protein without loss of the monitored enzyme activities. A secondary detergent, dioctanoyl phosphatidylcholine, appears to increase the yield of solubilized material. In addition, the effect of ionic strength indicates that it may be possible to selectively extract groups of membrane proteins by their characteristic solubility at different ionic strengths.The solubilized membrane-associated enzymes, ATPase and NADH dehydrogenase enter polyacrylamide gels as distinct species. Electrophoretic studies suggest that there are two membrane-associated ATPases in the Streptococcus faecalis, one which dissociates from the membrane in the absence of Mg²⁺ ions and the other which remains particulate until solubilized by detergents.Octyl glucoside can be easily removed from a solution containing solubilized proteins and lipid by dialysis.     

Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination

Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent-solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein-detergent complexes were characterized with small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross-linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent-solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein-detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein-detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein-detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins.     

The HLB dependency for detergent solubilization of hormonally sensitive adenylate cyclase

The HLB dependency for the solubilization of membrane proteins and adenylate cyclase activity from a plasma membrane-enriched fraction from rat liver has been determined. The HLB (hydrophilic/lipophilic/balance) number of a detergent is an empirical measure of its relative hydrophobicity. Detergent HLB numbers vary systematically with the length of the ethylene oxide chain for a homologous series of detergents such as the Triton X series. These detergents have a constant hydrophobic moiety, octylphenyl, and a variable polar portion, polyethoxyethanol. Basal-NaF-epine-phrine-, and glucagon-stimulated adenylate cyclase activities were solubilized in the HLB range of 16.8–17.4. Solubilization was most effective in 0.01 M Tris buffers at pH 7.5 containing 1–5 mM mercaptoethanol, 1 mM MgCl₂, and 0.1% Triton X-305. The detergent to membrane protein ratio used in these studies was 3:1. Criteria for solubilization included lack of sedimentation at 100,000 × g, the absence of particulate material in the supernatant when examined by electron microscopy, and inclusion of hormonally sensitive adenylate cylcase activity in Sephadex G-200 gels. The apparent molecular weight of the solubilized enzyme was approximately 200,000 in the presence of Triton X-305. The solubilized enzyme was stimulated 5-fold by NaF, 7-fold by glucagon, and 20-fold by epinephrine compared to the particulate enzyme used in this study which was stimulated 10-fold, 3,4-fold, and 4-fold by NaF, epinephrine, and glucagon, respectively. The solubilized enzyme is stable for several weeks when stored at ?60° C.     

Comparative study of two highly purified forms of liver microsomal cytochrome P-450: circular dichroism and other properties.

The two major forms of rabbit liver microsomal cytochrome P-450, P-450_LM2 and P-450_LM4, which were previously shown to differ in their absorption spectra, electrophoretic and immunochemical properties, and substrate specificities, have been further characterized by several methods, (a) The two cytochromes have different CD spectra in the ferric state but similar spectra when reduced. Upon conversion of P-450_LM2 to P-420 by treatment with sodium dodecyl sulfate, the CD spectrum is greatly diminished except in the far ultraviolet region, whereas the conversion of P-450_LM4 toP-420 with this detergent results in a spectrum with a new positive band in the visible region, (b) Although P-450_LM4 has a much higher tryptophan content than P-450_LM2, the fluorescence spectra of these proteins are similar in magnitude. Upon denaturation, the fluorescence of P-450_LM4 increases, thereby indicating a large quenching effect in the native protein, (c) Studies on the interaction of dilauroylglyceryl-3-phosphorylcholine with the cytochromes showed that P-450_LM2 gives a much stronger Type I difference spectrum than does P-450_LM4. This phospholipid has no significant effect on the state of aggregation of these cytochromes as judged by calibrated gel filtration. The CD spectra of P-450_LM2 and P-450_LM4 are unchanged in the visible region but are enhanced in the far ultraviolet region upon the addition of phosphatidylcholine. The results appear to indicate an increase in α-helical content, particularly with P-450_LM4, in the presence of the phospholipid.     

Reconstitution of the reduced nicotinamide adenine dinucleotide phosphate- and reduced nicotinamide adenine dinucleotide-peroxidase activities from solubilized components of rat liver microsomes.

The liver microsomal enzyme system that catalyzes the oxidation of NADPH by organic hydroperoxides has been solubilized and resolved by the use of detergents into fractions containing NADPH-cytochrome c reductase, cytochrome P-450 (or P-448), and microsomal lipid. Partially purified cytochromes P-450 and P-448, free of the reductase and of cytochrome b₅, were prepared from liver microsomes of rats pretreated with phenobarbital (PB) and 3-methylcholanthrene (3-MC), respectively, and reconstituted separately with the reductase and lipid fractions prepared from PB-treated animals to yield enzymically active preparations functional in cumene hydroperoxide-dependent NADPH oxidation. The reductase, cytochrome P-450 (or P-448), and lipid fractions were all required for maximal catalytic activity. Detergent-purified cytochrome b₅ when added to the complete system did not enhance the reaction rate. However, the partially purified cytochrome P-450 (or P-448) preparation was by itself capable of supporting the NADPH-peroxidase reaction but at a lower rate (25% of the maximal velocity) than the complete system. Other heme compounds such as hematin, methemoglobin, metmyoglobin, and ferricytochrome c could also act as comparable catalysts for the peroxidation of NADPH by cumene hydroperoxide and in these reactions, NADH was able to substitute for NADPH. The microsomal NADH-dependent peroxidase activity was also reconstituted from solubilized components of liver microsomes and was found to require NADH-cytochrome b₅ reductase, cytochrome P-450 (or P-448), lipid, and cytochrome b₅ for maximal catalytic activity. These results lend support to our earlier hypothesis that two distinct electron transport pathways operate in NADPH- and NADH-dependent hydroperoxide decomposition in liver microsomes.     

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.     

Immobilized cytochrome P-450LM2: Dissociation and reassociation of oligomers

Subunit interactions in the purified hexameric cytochrome P-450_LM2 have been studied using covalent binding of one of the 6 protomers to an insoluble matrix. High ionic strength, large-scale pH changes, guanidine chloride and sodium cholate taken at membrane-solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6-fold decrease in the amount of the bound cytochrome. Non-ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (> 0.2%), monomers and immobilized dimers were obtained as cytochrome P-450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P-450_LM2 demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P-450 when it is bound to the native membranes.     

Purification and Properties of the Plasma Membrane H-Translocating Adenosine Triphosphatase of Phaseolus mungo L. Roots

The plasma membrane ATPase of mung bean (Phaseolus mungo L.) roots has been solubilized with a two-step procedure using the anionic detergent, deoxycholate (DOC) and the zwitterionic detergent, zwittergent 3-14 as follows: (a) loosely bound membrane proteins are removed by treatment with 0.1% DOC; (b) The ATPase is solubilized with 0.1% zwittergent in the presence of 1% DOC; (c) the solubilized material is further purified by centrifugation through a glycerol gradient (45-70%). Typically, about 10% of the ATPase activity is recovered, and the specific activity increases about 11-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the peak fraction from the glycerol gradient contains three major polypeptides of M_r = 105,000, 67,000, and 57,000 daltons. The properties of the purified ATPase are essentially the same as those of membrane-bound ATPase, with respect to pH optimum, substrate specificity, inhibitor sensitivity, and ion stimulation.     

Blue native electrophoresis as a tool for detection of PIP-containing protein complexes in the plasma membranes

Blue native electrophoresis (BN-PAGE) is presently considered as one of effective methods for the identification of membrane protein complexes. The choice of a nonionic detergent and the detergent to protein ratio are critically important. Our experiments with plasma membranes of etiolated pea (Pisum sativum L.) seedlings showed that various nonionic detergents—digitonin, dodecyl maltoside, and Triton X-100—solubilized similar assortments of protein complexes. Irrespective of the detergent type, PIP aquaporins were always observed in the 440-kD protein complex. Only in the case of dodecyl maltoside, the PIP aquaporins were also revealed in the complexes with the lower and higher molecular weights when the detergent/protein ratio increased.     

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.     

Photosynthate partitioning in split-root citrus seedlings with mycorrhizal and nonmycorrhizal root systems

The plasma membrane ATP-phosphohydrolase (ATPase) from red beet (Beta vulgaris L.) storage tissue was solubilized with the zwitterionic detergent Zwittergent 3-14 from a plasma membrane-enriched fraction which was extracted with the anionic detergent, sodium deoxycholate. For both the extraction of extraneous proteins by deoxycholate and the solubilization of active plasma membrane ATPase by Zwittergent 3-14, the optimal concentration of detergent was 0.1% (weight per volume) with a detergent to protein ratio of 1.0 (milligram per milligram). The properties of the solubilized ATPase were found to be similar to the membrane-bound enzyme with respect to pH optimum, substrate specificity, inhibitor sensitivity, and kinetics of K⁺ stimulation. The solubilized ATPase preparation formed a rapidly turning over phosphoenzyme, the breakdown velocity of which was increased in the presence of 50 millimolar KCl. Solubilization with 0.1% Zwittergent 3-14 following extraction with 0.1% deoxycholate resulted in an increase in both ATPase activity and steady state phosphoenzyme level; however, a direct correspondence between the increase in ATPase activity and phosphorylation level did not exist. It is proposed that this discrepancy may be the result of a detergent-mediated modification of kinetic rate constants in the mechanism of the enzyme.     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

The state of association of band 3 protein of the human erythrocyte membrane in solutions of nonionic detergents

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was solubilized and purified in aqueous solutions of two nonionic detergents: Ammonyx-LO (dimethyl laurylamine oxide) and C12E9 (nonaethylene glycol lauryl ether). The state of association of the purified protein was studied by analytical ultracentrifugation. Band 3 protein solubilized and studied in solutions of Ammonyx-LO was found to be in a monomer/dimer/tetramer association equilibrium. Band 3 protein freshly prepared in C12 E9 showed the same behaviour; however, during aging the protein was converted into stable noncovalent dimers. The conversion was retarded by the presence of beta-mercaptoethanol or by treatment of the samples with iodoacetamide; it seems to be due to oxidation of the protein by degradation products of the detergent. It is concluded that a monomer/dimer/tetramer association equilibrium is the native state of association of band 3 protein solubilized by nonionic detergents. Since nonionic detergents are assumed not to interfere with protein-protein interactions among membrane proteins, the results strongly support the claim that, in the erythrocyte membrane, band 3 is in a monomer/dimer/tetramer association equilibrium (Dorst, H.-J. and Schubert, D. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1605-1618).     

Effect of detergents on the enzyme activities of the rat liver plasma membrane

The anionic detergents sodium dodecyl sulfate (SDS) and Alipal CO-433 and the non-ionic detergent Trition X-100 at concentrations of 0.02–0.10% cause a more rapid solubilization of phospholipid than proteins in isolated rat liver plasma membranes. All three detergents cause an increase in membrane turbidity at low detergent concentration (0.01–0.04%) but then decrease the turbidity at higher detergent concentration (0.04–0.10%). Each detergent gives a characteristic turbidity-detergent concentration profile which is pH dependent.The activities of the membrane-bound enzymes Mg²⁺ ATPase, 5′-nucleotidase and acid and aklaline phosphatase were influenced by each detergent to a different extent. Each enzyme gave a characteristic activity-detergent concentration profile. Mg²⁺ ATPase was inhibited by all detergents. 5′-Nucleotidase was stimulated by Triton and Alipal but inhibited by SDS. Alkaline phosphatase was stimulated by Alipal and SDS and not influenced by Triton. Acid phosphatase was stimulated by Triton and inhibited by Alipal and SDS. 56% of the total membrane-bound alkaline phosphatase and 23% of the total membrane-bound 5′-nucleotidase was solubilized in an active form by 0.06% and 0.05% SDS respectively.     

Visualization of detergent solubilization of membranes: implications for the isolation of rafts

Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (l_d) and raft liquid ordered (l_o) lipid phases by selectively solubilizing the l_d phase. A higher concentration of Lubrol was required, and not all the l_d phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some l_d phase and then progressed to the solubilization of both l_d and l_o phases simultaneously. Octyl glucoside simultaneously solubilized both l_o and l_d phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems.     

Detergents as intrinsic P-glycoprotein substrates and inhibitors

We assessed the interaction of three electrically neutral detergents (Triton X-100, C₁₂EO₈, and Tween 80) with P-glycoprotein (ABCB1, MDR1) and identified the molecular elements responsible for this interaction. To this purpose we titrated P-glycoprotein in inside-out plasma membrane vesicles of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) with the detergents below their critical micelle concentration, CMC. The P-glycoprotein ATPase measured as a function of the detergent concentration yielded bell-shaped activity curves which were evaluated with a two-site binding model. The lipid-water partition coefficient and the transporter-water binding constant of the detergents were measured independently. Knowledge of these two parameters allowed assessment of the free energy of detergent binding to P-glycoprotein in the lipid membrane, ΔG_tl⁰, that reflects the direct detergent-transporter affinity. It increased as the number of ethoxyl groups increased, suggesting that these hydrogen bond acceptor groups are the key elements for the detergent-transporter interaction in the lipid membrane. The free energy of binding to P-glycoprotein per ethoxyl group (EO) was determined as approximately ΔG_EO⁰ = − 1.6 kJ/mol. The present findings moreover document that, depending on the concentration applied, detergents are intrinsic substrates for, or inhibitors of P-glycoprotein.     

In vitro biosynthesis of liver cytochrome P450 mature peptide sub-unit by translation of isolated poly(A)+ mRNA from normal and phenobarbital induced rats

The biosynthesis of a cytochrome P₄₅₀ peptide sub-unit by the in vitro translation of total hepatic poly (A)⁺ mRNA in an heterologous cell-free-system is described. The ability of the liver poly (A)⁺ RNA preparations from normal and phenobarbital induced rats to promote protein synthesis and the identification of in vitro synthesized proteins revealed the presence of a cytochrome P₄₅₀ peptide sub-unit presenting the same apparent molecular weight of the native peptide. This fact demonstrates that rat liver poly (A)⁺ mRNA fraction contains an important amount of cytochrome P₄₅₀ peptide messages. Total poly (A)⁺ RNA from rats in an early phenobarbital induction stage exhibits a higher cytochrome P₄₅₀ template activity in good agreement with the enhancement of this hemeprotein concomitantly observed in vivo, in the liver microsomes, it is also concluded that cytochrome P₄₅₀, peptide sub-unit, induced in rat liver by phenobarbital, is translated in its mature form.