Clues on the role of Beauveria bassiana catalases in alkane degradation events

Entomopathogenic fungi adapt to growth in a culture medium containing an insect-like hydrocarbon as the sole carbon source inducing the beta-oxidation pathway during the alkane degradation. The effect of two carbon sources on the catalase activity was studied in the entomopathogenic fungus Beauveria bassiana. Catalase activity was detected both in the peroxisomal and cytosolic fraction. A significant increment in the specific activity of the peroxisomal fraction (12.6-fold) was observed when glucose was replaced by an insect-like hydrocarbon, whereas the specific activity in the cytosol diminished more than 1.2-fold in the same culture condition. After purification to homogeneity by gel filtration and strong anion exchange chromatography, an apparent molecular mass of 54.7 and 84.0 kDa per subunit were determined respectively for the peroxisomal and cytosolic catalase. The enzymes showed different biochemical and kinetic characteristics, but both were inhibited by 3-amino-1,2,4 triazole. Peroxisomal catalase was sensitive to pH, heat and high concentration of the hydrogen peroxide substrate. Inversely the cytosolic isoform exhibited a broad range of optimal pH (6.0-10.0), high thermostability (<55 C) and remained fully active at least up to 70 mM hydrogen peroxide. Measurement of catalase activity is a new approach for evaluating fungal ability to degrade hydrocarbons.     

Is the cytosolic catalase induced by peroxisome proliferators in mouse liver on its way to the peroxisomes?

Dietary treatment of male C57B1/6 mice with clofibrate, nafenopin or WY-14.643 resulted in a modest (at most 2-fold) increase in the total catalase activity in the whole homogenate and mitochondrial fraction prepared from the livers of these animals. On the other hand, the catalase activity recovered in the cytosolic fraction was increased 12- to 18-fold, i.e. 30-35% of the total catalase activity in the hepatic homogenate was present in the high-speed supernatant fraction after treatment with these peroxisome proliferators. A study of the time course of the changes in peroxisomal and cytosolic catalase activities demonstrated that the peroxisomal activity both increased upon initiation of exposure and decreased after termination of treatment several days after the increase and decrease, respectively, in the corresponding cytosolic activity. This finding suggests that the cytosolic catalase may be on its way to incorporation into peroxisomes.     

Proteolytic modification of mouse liver catalase

When catalase was immunoprecipitated from different subfractions of mouse liver homogenates, the enzyme which was obtained from extracts of the large granular fraction exhibited a lower molecular weight than that from either the cytosol or purified peroxisomal fractions, as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis. This modification of the enzyme could be prevented by the addition of proteolytic inhibitors to extraction buffers; and consequently, unmodified catalase was able to be purified in the presence of 5 mM iodoacetamide. Electrophoretic comparison of the catalases against standards of known molecular sizes indicated that the unmodified enzyme had a subunit mass approximately 2,000 daltons larger than the modified enzyme. The significance of these proteolytic modifications has been discussed in relation to the involvements of catalase and peroxisome turnover.     

Immunological detection of alkaline-diaminobenzidine-negativeperoxisomes of the nematode Caenorhabditis elegans purification and unique pH optima of peroxisomal catalase.

We purified catalase-2 of the nematode Caenorhabditis elegans and identified peroxisomes in this organism. The peroxisomes of C. elegans were not detectable by cytochemical staining using 3, 3'-diaminobenzidine, a commonly used method depending on the peroxidase activity of peroxisomal catalase at pH 9 in which genuine peroxidases are inactive. The cDNA sequences of C. elegans predict two catalases very similar to each other throughout the molecule, except for the short C-terminal sequence; catalase-2 (500 residues long) carries a peroxisomal targeting signal 1-like sequence (Ser-His-Ile), whereas catalase-1 does not. The catalase purified to near homogeneity from the homogenate of C. elegans cells consisted of a subunit of 57 kDa and was specifically recognized by anti-(catalase-2) serum but not by anti-(catalase-1) serum. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected catalase-2 inside vesicles judged to be peroxisomes using morphological criteria. The purified enzyme (220 kDa) was tetrameric, similar to many catalases from various sources, but exhibited unique pH optima for catalase (pH 6) and peroxidase (pH 4) activities; the latter value is unusually low and explains why the peroxidase activity was undetectable using the standard alkaline diaminobenzidine-staining method. These results indicate that catalase-2 is peroxisomal and verify that it can be used as a marker enzyme for C. elegans peroxisomes.     

Evidence that peroxisomal acyl-CoA synthetase is located at the cytoplasmic side of the peroxisomal membrane.

1. Subfractionation by isopycnic density-gradient centrifugation in self-generating Percoll gradients of peroxisome-rich fractions prepared by differential centrifugation confirmed the presence of acyl-CoA synthetase in peroxisomes. Peroxisomes did not contain nicotinamide or adenine nucleotides other than CoA. 2. The gradient fractions most enriched in peroxisomes were pooled and the peroxisomes sedimented by centrifugation, resulting in a 50-fold-purified peroxisomal preparation as revealed by marker enzyme analysis. 3. Palmitate oxidation by intact purified peroxisomes was CoA-dependent, whereas palmitoyl-CoA oxidation was not, demonstrating that the peroxisomal CoA was available for the thiolase reaction, located in the peroxisomal matrix, but not for acyl-CoA synthetase. This suggests that the latter enzyme is located at the cytoplasmic side of the peroxisomal membrane. 4. Additional evidence for this location of peroxisomal acyl-CoA synthetase was as follows. Mechanical disruption of purified peroxisomes resulted in the release of catalase from the broken organelles, but not of acyl-CoA synthetase, indicating that the enzyme was membrane-bound. Acyl-CoA synthetase was not latent, despite the fact that at least one of its substrates appears to have a limited membrane permeability, as evidenced by the presence of CoA in purified peroxisomes. Finally, Pronase, a proteinase that does not penetrate the peroxisomal membrane, almost completely inactivated the acyl-CoA synthetase of intact peroxisomes.     

Proteolytic cleavage of plant proteins by peroxisomal endoproteases from senescent pea leaves

The degradation of peroxisomal and nonperoxisomal proteins by endoproteases of purified peroxisomes from senescent pea (Pisum sativum L.) leaves has been investigated. In our experimental conditions, most peroxisomal proteins were endoproteolytically degraded. This cleavage was prevented, to some extent, by incubation with 2 mM phenylmethylsulfonylfluoride, an inhibitor of serine proteinases. The peroxisomal enzymes glycolate oxidase (EC 1.1.3.1), catalase (EC 1.11.1.6) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were susceptible to proteolytic degradation by peroxisomal endoproteases, whereas peroxisomal manganese superoxide dismutase (EC 1.15.1.1) was not. Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from spinach and urease (EC 3.5.1.5) from jack bean were strongly degraded in the presence of peroxisomal matrices. These results indicate that proteases from plant peroxisomes might play an important role in the turnover of peroxisomal proteins during senescence, as well as in the turnover of proteins located in other cell compartments during advanced stages of senescence. On the other hand, our data show that peroxisomal endoproteases could potentially carry out the partial proteolysis which results in the irreversible conversion of xanthine dehydrogenase into the superoxide-generating xanthine oxidase (EC 1.1.3.22). This suggests a possible involvement of the peroxisomal endoproteases in a regulated modification of proteins. Received: 25 January 1999 / Accepted: 3 June 1999     

Behavior of rat liver catalase during electrophoresis in a pH gradient.

Investigations were conducted on the distribution of rat liver catalase subsequent to electrofocusing in a pH gradient. Differences were observed depending on the enzyme being extracted from the total mitochondrial fraction, from the supernatant of the homogenate or from purified peroxisomes. Catalase solubilized from the total mitochondrial fraction exhibits an apparent isoelectric point lower than that of catalase derived from the supernatant. Catalase released from purified peroxisomes shows a behavior similar to that of the supernatant catalase. It has been concluded that, in a total mitochondrial fraction, a factor is present that alters the electric charge of the catalase molecule during or after the extraction of the enzyme. This factor is probably associated with lysosomes existing together with peroxisomes and mitochondria in a total mitochondrial fraction. As a matter of fact, the addition of an extract of purified lysosomes to purified peroxisomes or to supernatant will cause a shift towards a more acid pH of catalase distribution subsequent to electrofocalization.     

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)     

Changes to the integral membrane protein composition of mouse liver peroxisomes in response to the peroxisome proliferators clofibrate,Wy-14,643 and Di(2-ethyl-hexyl)phthalate

Peroxisomes were purified from livers of control mice and from mice treated with three agents which induce proliferation of hepatic peroxisomes — namely two structurally unrelated hypolipidemic drugs, clofibrate (ethyl--p-chlorophenoxyisobutyrate) and Wy-14,643 (4-chloro-6[2,3-xylidino)-2-pyrimidinylthio] acetic acid), and a plasticizer, DEHP (di-(2-ethylhexyl)phthalate).Membranes were isolated from these purified peroxisomes and analysed by SDS-polyacrylamide gel electrophoresis. All membranes which were tested, displayed two predominant integral membrane proteins of apparent molecular weights of 68 kDa and 70 kDa respectively, as well as a number of minor components. Treatment of animals with clofibrate, Wy-14,643 and DEHP was observed to result in each case in an increased proportion of the 70 kDa protein in the peroxisomal membranes. These treatments also resulted in increased peroxisomal fatty acid oxidation in livers and an increase in the proportion of catalase activity in the cytosolic fraction of liver cells.These results have been discussed in relation to alterations in the molecular composition of the membranes, the mechanisms of peroxisome proliferation and the inducibility of peroxisomal membrane proteins.     

On the synthesis and degradation of the multiple forms of catalase in mouse liver

The incorporation of ⁵⁵Fe-labeled ferrous sulfate and ³H-labeled γ-aminolaevulinic acid into the catalase of mouse liver was measured at intervals up to 96 hr after intraperitoneal injection, and the intracellular location of radioactive catalase followed, as well as the distribution of radiolabel between the multiple forms of this enzyme. At 10 min, catalase radioactivity was present in all the cellular fractions studied, but after this time, label began to disappear from the microsomal fraction and from the peroxisomal detergent extract. By comparison, catalase incorporation reached a peak at about 6 hr in the peroxisomal aqueous extract, and rose to a broad peak after about 30 hr in the cytosol fraction. On resolving the multiple forms of catalase in the supernatant fraction by electrophoresis, it was found that label first appeared in the fastest moving heteromorph, and appeared sequentially in the other multiple forms over a period of 96 hr.The sequence of degradation of catalase was also studied by examination of residual catalase activity subsequent to the injection of allyl-isopropyl acetamide, a heme synthesis antagonist which blocks catalase synthesis. Blood catalase levels did not seem to be significantly affected by this treatment, but in the liver, the decay rates of catalase activity were appreciable, and varied significantly between the intracellular pools. The rate of decrease was greatest in the peroxisomal detergent extract, and least in the supernatant fraction.These findings have been discussed in relation to current understanding of the subcellular disposition, multiplicity, and turnover of hepatic catalase.     

Catalase gene of the yeast Candida tropicalis. Sequence analysis and comparison with peroxisomal and cytosolic catalases from other sources

A clone harbouring the genomic DNA sequence for the peroxisomal catalase of an n-alkane-utilizable yeast, Candida tropicalis, has been isolated by the hybrid-selection method and confirmed with a probe of catalase partial cDNA. Nucleotide sequence analysis of the cloned DNA disclosed that the gene fragment coding for catalase had a length of 1455 base pairs (corresponding to 485 amino acids; m = 54937 Da), and that the size of this enzyme was the smallest among all catalases reported hitherto. No intervening sequence was found in this coding region and some portions coincided with the amino acid sequences obtained from the analysis of the purified catalase. The comparison with three peroxisomal catalases from rat liver, bovine liver and human kidney, and one cytosolic catalase from Saccharomyces cerevisiae has revealed that catalase from C. tropicalis was more homologous to the peroxisomal enzymes than to the cytosolic one. C. tropicalis used the codons of the high-expression type. Amino acid residues were all conserved at the active and heme-binding sites. In the N and C-terminal regions there was no characteristic signal sequence or consensus sequence. However, a noticeable region, which can be discriminated between peroxisomal and cytosolic catalases, was proposed.     

In situ measurements of the pH of mammalian peroxisomes using the fluorescent protein pHluorin

Peroxisomes are metabolically active organelles that participate in the oxidation of long-chain fatty acids and in the biosynthesis of bile acids, cholesterol, and ether phospholipids. Even though maintenance of a stable acid-base milieu is essential for proper peroxisomal function, the determination of the peroxisomal pH (pH(p)) remains inconclusive, and little is known about its regulation. To measure the pH of intact peroxisomes in situ, we used the peroxisome-specific carboxyl-terminal targeting sequence, SKL, to deliver a pH-sensitive mutant of the green fluorescent protein (pHluorin-SKL) selectively into peroxisomes. Proper targeting was verified by colocalization with the peroxisomal marker catalase. Peroxisomes were visualized by imaging fluorescence microscopy, and ratiometric measurements were combined with calibration using ionophores or a null-point method to estimate pH(p). The pH(p) was between 6.9 and 7.1, resembling the cytosolic pH. Manipulation of the cytosolic pH in intact cells or after permeabilization of the plasmalemma with streptolysin O revealed that pH(p) changed in parallel, suggesting that the peroxisomal membrane is highly permeable to H(+) (equivalents). We conclude that peroxisomes do not regulate their pH independently, but instead their large H(+) permeability effectively connects them with the buffer reservoir of the cytoplasm and with the homeostatic mechanisms that control cytosolic pH.     

Degradation of peroxisomal catalase and urate oxidase of rat liver.

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5--9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation. After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37 degrees C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process. From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.     

Purification of glyoxysomal catalase and immunochemical comparison of glyoxysomal and leaf peroxisomal catalase in germinating pumpkin cotyledons

As a step to study the mechanism of the microbody transition (glyoxysomes to leaf peroxisomes) in pumpkin (Cucurbita sp. Amakuri Nankin) cotyledons, catalase was purified from glyoxysomes. The molecular weight of the purified catalase was determined to be 230,000 to 250,000 daltons. The enzyme was judged to consist of four identical pieces of the monomeric subunit with molecular weight of 55,000 daltons. Absorption spectrum of the catalase molecule gave two major peaks at 280 and 405 nanometers, showing that the pumpkin enzyme contains heme. The ratio of absorption at 405 and 280 nanometers was 1.0, the value being lower than that obtained for catalase from other plant sources. These results indicate that the pumpkin glyoxysomal catalase contains the higher content of heme in comparison with other plant catalase.

The immunochemical resemblance between glyoxysomal and leaf peroxisomal catalase was examined by using the antiserum specific against the purified enzyme preparation from pumpkin glyoxysomes. Ouchterlony double diffusion and immunoelectrophoretic analysis demonstrated that catalase from both types of microbodies cross-reacted completely whereas the immunotitration analysis showed that the specific activity of the glyoxysomal catalase was 2.5-fold higher than that of leaf peroxisomal catalase. Single radial immunodiffusion analysis showed that the specific activity of catalase decreased during the greening of pumpkin cotyledons.

     

Monoclonal antibodies against the membrane-bound, flavin-linked D-lactate dehydrogenase of Escherichia coli: preparation, characterization, and use in immunoaffinity chromatography

Three mouse hybridoma cell lines are described that produce monoclonal antibodies directed against the membrane-bound, flavin adenine dinucleotide linked D-lactate dehydrogenase of Escherichia coli. In contrast to polyclonal antibodies produced in rabbits, none of the monoclonal antibodies inhibits enzyme activity. Immunoblots of D-lactate dehydrogenase proteolytic fragments indicate that each antibody is directed against a different region of the molecule. One monoclonal antibody, 1B2a, reacts with native, undigested D-lactate dehydrogenase only and is used to purify the enzyme in a single step. The protocol involves chromatography of a Triton X-100 extract of membrane vesicles containing D-lactate dehydrogenase on a column made with the monoclonal antibody coupled to a solid support. After the column is washed free of unadsorbed protein, elution at high pH in the presence of guanidine hydrochloride yields a fraction containing highly purified, catalytically active D-lactate dehydrogenase.     

Role of Pex19p in the targeting of PMP70 to peroxisome

Pex19p is a protein required for the peroxisomal membrane synthesis. The 70-kDa peroxisomal membrane protein (PMP70) is synthesized on free cytosolic ribosomes and then inserted posttranslationally into peroxisomal membranes. Pex19p has been shown to play an important role in this process. Using an in vitro translation system, we investigated the role of Pex19p as a chaperone and identified the regions of PMP70 required for the interaction with Pex19p. When PMP70 was translated in the presence of purified Pex19p, a large part of PMP70 existed as soluble form and was co-immunoprecipitated with Pex19p. However, in the absence of Pex19p, PMP70 formed aggregates during translation. To identify the regions that interact with Pex19p, various truncated PMP70 were translated in the presence of Pex19p and subjected to co-immunoprecipitation. The interaction was markedly reduced by the deletion of the NH(2)-terminal 61 amino acids or the region around TMD6. Further, we expressed these deletion constructs of PMP70 in fusion with the green fluorescent protein in CHO cells. Fusion proteins lacking these Pex19p binding sites did not display any peroxisomal localization. These results suggest that Pex19p binds to PMP70 co-translationally and keeps PMP70 as a proper conformation for the localization to peroxisome.     

Degradation of peroxisomal catalase and urate oxidase of rat liver

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5–9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation.After [³H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37°C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process.From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.     

Determination of the cross-points of rat liver peroxisomes,peroxisomal core and the core components by cross-partition

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same crosspoint, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, d-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2:1. The theoretical value of cross-point of the core calculated from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

Determination of the cross-points of rat liver peroxisomes, peroxisomal core and the core components by cross-partition.

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same cross-point, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, D-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2 : 1. The theoretical value of cross-point of the core calculated from from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

Biosynthesis of membrane polypeptides of rat liver peroxisomes

The biosynthesis of three major peroxisomal membrane polypeptides of rat liver was investigated. Total hepatic RNA extracted by the guanidinium/CsCl method from three control and three di(2-ethylhexyl)phthalate (a peroxisomal proliferator)-fed rats was translated in vitro in a rabbit reticulocyte lysate protein-synthesizing system. Translation products were immunoprecipitated by the antibodies against peroxisomal membrane polypeptides, subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and analyzed by fluorography. The in vitro translation products of 70, 26, and 22 kDa peroxisomal membrane polypeptides were apparently of the same size as the respective mature polypeptides. The ratio of translatable mRNA levels for the 70, 26, and 22 kDa polypeptides in di(2-ethylhexyl)phthalate-fed rats to those in control rats were 5.4, 11.4, and 2.7, respectively. The synthesis of these three polypeptides with the free polysome fraction from di(2-ethylhexyl)phthalate-fed rats was more active than that with the membrane-bound polysome fraction, whereas the synthesis of albumin with the free polysome fraction was 27% of that with the membrane-bound polysome fraction. These results indicate that the peroxisomal major membrane polypeptides are synthesized on free polysomes and transported to peroxisomal membrane without any apparent proteolytic processing, and that the induction of these polypeptides by administration of a peroxisomal proliferator corresponds well to the induction of the peroxisomal beta-oxidation enzymes. The data also support the idea that peroxisomes are organized from pre-existing peroxisomes.