Distinct long chain and very long chain fatty acyl CoA synthetases in rat liver peroxisomes and microsomes

Mitochondria, peroxisomes, and microsomes were isolated from rat liver homogenates, and stearic acid and lignoceric acid beta-oxidation, as well as stearoyl CoA synthetase and lignoceroyl CoA synthetase activities in the three organelles, were compared. Stearic acid beta-oxidation in peroxisomes was sixfold greater compared to the oxidation in mitochondria. Lignoceric acid beta-oxidation, observed only in peroxisomes, was fivefold lower compared to stearic acid beta-oxidation. Stearoyl CoA synthetase was present whereas lignoceroyl CoA synthetase was absent in mitochondria. Stearoyl CoA synthetase and lignoceroyl CoA synthetase activities were present in microsomes and peroxisomes, but the activity of stearoyl CoA synthetase was several-fold greater compared to lignoceroyl CoA synthetase in both organelles. The differing responses to detergents and phospholipids of stearoyl CoA and lignoceroyl CoA synthetase activities in microsomes as well as peroxisomes indicated that each activity was catalyzed by a separate enzyme. Differences in detergent and phospholipid response were also noted when either stearoyl CoA or lignoceroyl CoA synthetase activity in one organelle was compared with the corresponding activity in the other organelle, suggesting that the same activity in different organelles may be catalyzed by separate enzyme proteins.     

Acyl-CoA synthetase and the peroxisomal enzymes of beta-oxidation in human liver. Quantitative analysis of their subcellular localization.

The presence of acyl-CoA synthetase (EC 6.2.1.3) in peroxisomes and the subcellular distribution of beta-oxidation enzymes in human liver were investigated by using a single-step fractionation method of whole liver homogenates in metrizamide continuous density gradients and a novel procedure of computer analysis of results. Peroxisomes were found to contain 16% of the liver palmitoyl-CoA synthetase activity, and 21% and 60% of the enzyme activity was localized in mitochondria and microsomal fractions respectively. Fatty acyl-CoA oxidase was localized exclusively in peroxisomes, confirming previous results. Human liver peroxisomes were found to contribute 13%, 17% and 11% of the liver activities of crotonase, beta-hydroxyacyl-CoA dehydrogenase and thiolase respectively. The absolute activities found in peroxisomes for the enzymes investigated suggest that in human liver fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal beta-oxidation pathway, when palmitic acid is the substrate.     

Characteristics and subcellular localization of pristanoyl-CoA synthetase in rat liver.

We have investigated the activation of pristanic acid to its CoA-ester in rat liver. The results show that peroxisomes, mitochondria as well as microsomes contain pristanoyl-CoA synthetase activity. On the basis of competition experiments and immunoprecipitation studies using antibodies raised against rat liver microsomal long-chain fatty acyl-CoA synthetase (EC 6.2.1.3) we conclude that pristanic acid is activated by the same enzyme which activates long-chain fatty acids, i.e., long-chain fatty acyl-CoA synthetase.     

Identity of purified monoacylglycerol lipase, palmitoyl-CoA hydrolase and aspirin-metabolizing carboxylesterase from rat liver microsomal fractions. A comparative study with enzymes purified in different laboratories.

Two purified carboxylesterases that were isolated from a rat liver microsomal fraction in a Norwegian and a German laboratory were compared. The Norwegian enzyme preparation was classified as palmitoyl-CoA hydrolase (EC 3.1.2.2) in many earlier papers, whereas the German preparation was termed monoacylglycerol lipase (EC 3.1.1.23) or esterase pI 6.2/6.4 (non-specific carboxylesterase, EC 3.1.1.1). Antisera against the two purified enzyme preparations were cross-reactive. The two proteins co-migrate in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Both enzymes exhibit identical inhibition characteristics with Mg2+, Ca2+ and bis-(4-nitrophenyl) phosphate if assayed with the two substrates palmitoyl-CoA and phenyl butyrate. It is concluded that the two esterase preparations are identical. However, immunoprecipitation and inhibition experiments confirm that this microsomal lipase differs from the palmitoyl-CoA hydrolases of rat liver cytosol and mitochondria.     

Purification and characterization of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylase from female rat liver mitochondria

5 beta-Cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylase (5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, NADPH:oxygen oxidoreductase (26-hydroxylating), EC 1.14.13.15) was purified from female rat liver mitochondria based on its catalytic activity. The final preparation of the enzyme showed a single major band on the sodium dodecyl sulfate-polyacrylamide gel electrophoretogram. The content of purified enzyme was 12 nmol/mg of protein, and the specific activity was 431 nmol/min/mg of protein. The molecular weight of the enzyme was determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 52,500. The absorption spectra of the purified enzyme and that of the dithionite-reduced CO complex showed peaks at 417 and 450 nm, respectively, indicating the enzyme belongs to the cytochrome P-450 family. Upon reconstitution with the electron-transferring system of the adrenal (adrenodoxin and NADPH-adrenodoxin reductase), the enzyme showed high activity hydroxylating 5 beta-cholestane-3 alpha,7 alpha-12-triol at position 27 with a turnover number of 35.5 min-1 and Km of 6.3 microM. The enzyme activity was completely lost when the electron-transferring system was replaced by that of microsomes (NADPH-cytochrome P-450 reductase purified from rat liver microsomes), confirming that the P-450 enzyme was of the mitochondrial type, but not of the microsomal. The omission of cytochrome P-450, adrenodoxin, or NADPH-adrenodoxin reductase resulted in complete loss of enzyme activity. The specific activity toward 5 beta-cholestane-3 alpha, 7 alpha-diol was less than one-half that toward cholestanetriol and that toward cholesterol was about one-fiftieth. The enzyme showed no activity toward xenobiotics such as benzphetamine, 7-ethoxycoumarin, and benzo[a]pyrene. Its activity was not inhibited by metyrapone and slightly inhibited by aminoglutethimide. The enzyme activity was markedly lowered in an atmosphere of CO/O2/N2, 40/20/40.     

Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase.

Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa.     

Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. I. Use of inhibitors

The effects of various inhibitors of carnitine palmitoyltransferase I were examined in mitochondria from rat liver and skeletal muscle. Three types of inhibitors were used: malonyl-CoA (reversible), tetradecylglycidyl-CoA and three of its analogues (irreversible), and 2-bromopalmitoyl-CoA (essentially irreversible when added with carnitine). Competitive binding studies between labeled and unlabeled ligands together with electrophoretic analysis of sodium dodecyl sulfate-solubilized membranes revealed that in mitochondria from both tissues all of the inhibitors interacted with a single protein. While the binding capacity for inhibitors was similar in liver and muscle (6-8 pmol/mg of mitochondrial protein) the proteins involved were of different monomeric size (Mr 94,000 and 86,000, respectively). Treatment of mitochondria with the detergent, octyl glucoside, yielded a soluble form of carnitine palmitoyltransferase and residual membranes that were devoid of enzyme activity. The solubilized enzyme displayed the same activity regardless of whether carnitine palmitoyltransferase I of the original mitochondria had first been exposed to an irreversible inhibitor or destroyed by chymotrypsin. It eluted as a single activity peak through four purification steps. The final product from both liver and muscle migrated as single band on sodium dodecyl sulfate-polyacrylamide electrophoresis with Mr of approximately 80,000. The data are consistent with the following model. The inhibitor binding protein is carnitine palmitoyltransferase I itself (as opposed to a regulatory subunit). The hepatic monomer is larger than the muscle enzyme. Each inhibitor interacts via its thioester group at the palmitoyl-CoA binding site of the enzyme but also at a second locus that is probably different for each agent and dictated by the chemical substituent on carbon 2. Disruption of the mitochondrial inner membrane by octyl glucoside causes inactivation of carnitine palmitoyltransferase I while releasing carnitine palmitoyltransferase II in active form. The latter is readily purified, is a smaller protein than carnitine palmitoyltransferase I, and has the same molecular weight in liver and muscle. It is insensitive to inhibitors where on or off the mitochondrial membrane.     

Radiation inactivation of microsomal glutathione S-transferase

Radiation inactivation analysis was used to determine the target size of rat liver microsomal glutathione S-transferase both in situ and following purification. When Tris-HCl-washed microsomes were irradiated, there was a 1.5-2.0-fold increase in enzymatic activity over the first 3-6 megarads followed by a decrease in enzymatic activity. Above 48 megarads the radiation inactivation curve of the Tris-HCl-washed microsomes was described by a monoexponential function which gave a target size of 48 kDa. The enzymatic activity of the microsomal enzyme was selectively increased by treating the Tris-HCl-washed microsomes either with N-ethylmaleimide or washing the microsomes with small unilamellar vesicles made from phosphatidylcholine. The inactivation curves obtained with both types of treated microsomes were simple monoexponential decays in enzymatic activity with target sizes of 46 kDa (N-ethylmaleimide) and 44 kDa (unilamellar vesicles). The microsomal enzyme was detergent solubilized and purified. The Mr value of the purified protein was 15,500 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). These data suggest that the functional unit of the microsomal form of glutathione S-transferase in situ is a trimer. The target size of the purified enzyme solubilized in Triton X-100 was 85 kDa, and no increase in activity was observed at the lower radiation doses. The increase in the target size of the purified enzyme could not be ascribed solely to the presence of the detergent. This result suggests that the microsomal form of this enzyme can exist as catalytically active oligomers of different sizes depending on its environment.     

Subcellular distribution and properties of acyl/alkyl dihydroxyacetone phosphate reductase in rodent livers

On subcellular fractionation, the enzyme acyl/alkyl dihydroxyacetone phosphate (DHAP) reductase (EC 1.1.1.101) in guinea pig and rat liver was found to be present in both the light mitochondrial (L) and microsomal fractions. By using metrizamide density gradient centrifugation, it was shown that the alkyl DHAP reductase activity in the "L" fraction is localized mainly in peroxisomes. From the distribution of the marker enzymes it was calculated that about two-thirds of the liver reductase activity is in the peroxisomes and the rest in the microsomes. The properties of this enzyme in peroxisomes and microsomes are similar with respect to heat inactivation, pH optima, sensitivity to trypsin, and inhibition by NADP+ and acyl CoA. The enzyme activity in the peroxisomes and microsomes from mouse liver is increased to the same extent by chronically feeding the animals clofibrate, a hypolipidemic drug. The kinetic properties of this enzyme in these two different organelles are also similar. From these results it is concluded that the same enzyme is present in two different subcellular compartments of liver.     

The phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system is a 36.5-kilodalton polypeptide

The phosphohydrolase component of the microsomal glucose-6-phosphatase system has been identified as a 36.5-kDa polypeptide by 32P-labeling of the phosphoryl-enzyme intermediate formed during steady-state hydrolysis. A 36.5-kDa polypeptide was labeled when disrupted rat hepatic microsomes were incubated with three different 32P-labeled substrates for the enzyme (glucose-6-P, mannose-6-P, and PPi) and the reaction terminated with trichloroacetic acid. Labeling of the phosphoryl-enzyme intermediate with [32P]glucose-6-P was blocked by several well-characterized competitive inhibitors of glucose-6-phosphatase activity (e.g. Al(F)-4 and Pi) and by thermal inactivation, and labeling was not seen following incubations with 32Pi and [U-14C]glucose-6-P. In agreement with steady-state dictates, the amount of [32P]phosphoryl intermediate was directly and quantitatively proportional to the steady-state glucose-6-phosphatase activity measured under a variety of conditions in both intact and disrupted hepatic microsomes. The labeled 36.5-kDa polypeptide was specifically immunostained by antiserum raised in sheep against the partially purified rat hepatic enzyme, and the antiserum quantitatively immunoprecipitated glucose-6-phosphatase activity from cholate-solubilized rat hepatic microsomes. [32P]Glucose-6-P also labeled a similar-sized polypeptide in hepatic microsomes from sheep, rabbit, guinea pig, and mouse and rat renal microsomes. The glucose-6-phosphatase enzyme appears to be a minor protein of the hepatic endoplasmic reticulum, comprising about 0.1% of the total microsomal membrane proteins. The centrifugation of sodium dodecyl sulfate-solubilized membrane proteins was found to be a crucial step in the resolution of radiolabeled microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Ubiquinone biosynthesis in rat liver peroxisomes

The possibility that ubiquinone biosynthesis is present in rat liver peroxisomes was investigated. The specific activity of trans-prenyltransferase was 30% that of microsomes, with a pH optimum of around 8. trans-Geranyl pyrophosphate was required as a substrate and maximum activity was achieved with Mn(2+). Several detergents specifically inactivated the peroxisomal enzyme. The peroxisomal transferase is present in the luminal soluble contents, in contrast to the microsomal enzyme which is a membrane component. The treatment of rats with a number of drugs has demonstrated that the activities in the two organelles are subjected to separate regulation. Nonaprenyl-4-hydroxybenzoate transferase has about the same specific activity in peroxisomes as in microsomes and like the transferase activity, its regulation differs from the microsomal enzyme. The results demonstrate that peroxisomes are involved in ubiquinone biosynthesis, and at least two enzymes of the biosynthetic sequence are present in this organelle.     

Induction of a novel long-chain acyl-CoA hydrolase in rat liver by administration of peroxisome proliferators

The activity of long-chain acyl-CoA hydrolase in rat liver was increased by the administration of peroxisome proliferators, such as ethyl p-chlorophenoxyisobutyrate, di(2-ethylhexyl)phthalate or acetylsalicylic acid. The induced activity was mainly confined in the soluble fluid after the subcellular fractionation. The enzyme was purified nearly to homogeneity from livers of rats treated with di(2-ethylhexyl)phthalate. The specific activity of the final preparation was 247 mumol palmitoyl-CoA hydrolyzed min-1 mg protein-1. The molecular weight of the native enzyme was estimated to be 150 000 by gel filtration and that of the subunits was 41 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The activity of the enzyme was not increased but inhibited by bovine serum albumin or Triton X-100. The molecular and catalytic properties of the enzyme suggest that the induced enzyme was different from mitochondrial and microsomal long-chain acyl-CoA hydrolyses in liver.     

Bile acid: CoASH ligases from guinea pig and porcine liver microsomes. Purification and characterization

A procedure for the purification of the enzyme bile acid:CoA ligase from guinea pig liver microsomes was developed. Activity toward chenodeoxycholate, cholate, deoxycholate, and lithocholate co-purified suggesting that a single enzyme form catalyzes the activation of all four bile acids. Activity toward lithocholate could not be accurately assayed during the earlier stages of purification due to a protein which interfered with the assay. The purified ligase had a specific activity that was 333-fold enriched relative to the microsomal cell fraction. The purification procedure successfully removed several enzymes that could potentially interfere with assay procedures for ligase activity, i.e. ATPase, AMPase, inorganic pyrophosphatase, and bile acid-CoA thiolase. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified ligase gave a single band of approximately 63,000 Mr. A molecular size of 116,000 +/- 4,000 daltons was obtained by radiation inactivation analysis of the ligase in its native microsomal environment, suggesting that the functional unit of the ligase is a dimer. The purified enzyme was extensively delipidated by adsorption to alumina. The delipidated enzyme was extremely unstable but could be partially stabilized by the addition of phospholipid vesicles or detergent. However, such additions did not enhance enzymatic activity. Kinetic analysis revealed that chenodeoxycholate, cholate, deoxycholate, and lithocholate were all relatively good substrates for the purified enzyme. The trihydroxy bile acid cholate was the least efficient substrate due to its relatively low affinity for the enzyme. Bile acid:CoA ligase could also be solubilized from porcine liver microsomes and purified 180-fold by a modification of the above procedure. The final preparation contains three polypeptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three peptides range in size from 50,000 to 59,000, somewhat smaller than the guinea pig enzyme. The functional size of the porcine enzyme in its native microsomal environment was determined by the technique of radiation inactivation analysis to be 108,000 +/- 5,000 daltons. Thus, the functional form of the porcine enzyme also appears to be a dimer.     

CDP-diglyceride:inositol transferase from rat liver. Purification and properties.

CDP-diglyceride:inositol transferase, which catalyzes the final step of the de novo synthesis of phosphatidylinositol, was solubilized by sodium cholate from microsomes prepared from rat liver and purified by ammonium sulfate fractionation, sucrose density gradient centrifugation, and DEAE-cellulose column chromatography. Addition of phospholipid during the purification and the assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The recovery of the purified enzyme from the microsomal fraction was 3 to 3.3% with respect to activity and 0.12% with respect to amount of protein. The molecular weight of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified enzyme required exogenous phospholipds for its activity. Various phospholipid classes activated the enzyme rather nonspecifically. The Km for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X 10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for activity. The optimal concentration of Mn2+ for activation was 0.5 mM, while the activity in the presence of Mg2+ increased up to 20 mM. The enzyme was inhibited by thiol-reactive reagents. There was a competition for inositol by inosose-2 but not by scyllitol.     

Acyl-Coenzyme A synthetase and fatty acid oxidation in rat liver peroxisomes.

Rat liver peroxisomes oxidized palmitate in the presence of ATP, CoA and NAD+, and the rate of palmitate oxidation exceeded that of palmitoyl-CoA oxidation. Acyl-CoA synthetase [acid: CoA ligase (AMP-forming); EC 6.2.1.3] was found in peroxisomes. The substrate specificity of the peroxisomal synthetase towards fatty acids with various carbon chain lengths was similar to that of the microsomal enzyme. The peroxisomal synthetase activity toward palmitate (40--100 nmol/min per mg protein) was higher than the rate of palmitate oxidation by the peroxisomal system (0.7--1.7 nmol/min per mg protein). The data show that peroxisomes activate long chain fatty acids and oxidize their acyl-CoA derivatives.     

Purification and characterization of human placental aromatase cytochrome P-450

Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots.     

NADPH-cytochrome c (P-450) reductase has the activity of NADPH-linked aquacobalamin reductase in rat liver microsomes.

To elucidate the mammalian system for synthesis of cobalamin coenzymes, microsomal NADPH-linked aquacobalamin reductase was purified and characterized. The enzyme was purified about 534-fold over rat liver microsomal fraction in a yield of about 32%. The purified enzyme was homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had a monomeric molecular weight of 79,000. The purified aquacobalamin reductase showed a high specific activity (about 55 mumol/min per mg protein) of NADPH-cytochrome c (P-450) reductase. About 33% of the NADPH-cytochrome c reductase activity found in the microsomal fraction was recovered in the final purified preparation. The activity ratio of NADPH-cytochrome c reductase/NADPH-linked aquacobalamin reductase was about 5.0 through the purification steps, indicating that the rat liver microsomal NADPH-linked aquacobalamin reductase is the NADPH-cytochrome c reductase.     

Activation of rat liver microsomal glutathione S-transferase by hydrogen peroxide: role for protein-dimer formation.

The mechanism of oxygen radical-dependent activation of hepatic microsomal glutathione S-transferase by hydrogen peroxide was studied. Glutathione S-transferase activity in liver microsomes was increased 1.5-fold by incubation with 0.75 mM hydrogen peroxide at 37 degrees C for 10 min, and the increase in activity was reversed by incubation with dithiothreitol. Purified glutathione S-transferase was also activated by hydrogen peroxide after incubation at room temperature, and the increase in the activity was also reversed by dithiothreitol. Immunoblotting with anti-microsomal glutathione S-transferase antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hydrogen peroxide-treated microsomes or purified glutathione S-transferase revealed the presence of a glutathione S-transferase dimer. These results indicate that the hydrogen peroxide-dependent activation of the microsomal glutathione S-transferase is associated with the formation of a protein dimer.     

Selenium-containing proteins of rat kidney and liver microsomes

Selenium (Se)-containing proteins in microsomal fractions of rat kidney and liver were investigated after isotopic labeling of rats with [75Se]selenite. More than 85% of the 75Se in the solubilized microsomal extracts precipitated with protein after trichloroacetic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), used to separate the labeled protein subunits in the solubilized microsomal extracts, revealed several 75Se-containing proteins in addition to glutathione peroxidase. 75Se-labeled subunits with molecular weights of 55, 30, 26, 22, 19, and 17 kDa were present in microsomal fractions of kidney and liver. The 75Se-labeled tryptic peptide of the 55 kDa subunit had the same Rf value on a 17% SDS-PAGE gel as the peptide from plasma selenoprotein P. A time-course study of the labeling of individual protein subunits in kidney and liver microsomes from Se-supplemented and Se-deficient rats showed that most of the 75Se was associated with the 55 kDa subunit 3 hr after injection. The amount of 75Se associated with this protein subunit decreased by 12 hr, with a concurrent increase in the labeling of lower molecular-weight subunits. The results support the hypothesis that there is a mechanism for transfer of Se from the 55 kDa subunit to other Se-containing proteins.     

Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro

Total polysomal RNA of rat liver was translated in vitro in a rabbit reticulocyte lysate system. The translation products were mixed with a postnuclear supernatant fraction of rat liver and incubated post-translationally at 26 degrees C for 15-60 min. The import assay mixture was separated into a particulate fraction and supernatant by centrifugation, both of which were analyzed by immunoprecipitation with a goat antibody against rat liver peroxisomal proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. One peroxisomal translation product (Mr 72,000) appeared in the particulate fraction, was partly proteinase K-resistant, and addition of detergents prior to proteolysis abolished this resistance. In isopycnic centrifugation of the uptake assay mixture, the protease-resistant 35S-polypeptide of Mr 72,000 cosedimented with the peroxisomes. This translation product was identified immunochemically as fatty acyl-CoA oxidase; both before and after import it was indistinguishable in size from subunit A of the purified enzyme by prolonged sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the cell-free translation products were incubated with highly purified peroxisomes, 35S-catalase entered peroxisomes (by the criterion of protease resistance), and its entry was stimulated by the addition of a high speed supernatant (cytosolic) fraction of rat liver. These results demonstrate the post-translational import into peroxisomes in vitro of at least two cell-free translation products.