Peroxisomes in molluscs, characterization by subcellular fractionation combined with western blotting, immunohistochemistry, and immunocytochemistry

Peroxisomes of the digestive glands of mussels, Mytilus galloprovincialis Lmk, were investigated by immunoblotting and immunohistochemistry using rabbit antibodies against several mammalian hepatic peroxisomal proteins. Western blot analysis of main subcellular fractions revealed immunoreactive polypeptides with molecular weights comparable to those of the corresponding mammalian hepatic proteins. They could be localized to the peroxisomal matrix in the case of catalase, multifunctional enzyme (PH), and palmitoyl-CoA oxidase (AOX), and to the peroxisomal membrane in respect to PMP 70. The purification of peroxisomes by metrizamide density gradient centrifugation revealed the existence of two subpopulations with densities of 1.16 and 1.20 g cm^–3 exhibiting different protein compositions. In paraffin sections, positive immunolabeling for catalase was distributed along the apical cytoplasm of the epithelia of digestive ducts and stomach and throughout the cytoplasm of digestive tubule cells. The peroxisomal β-oxidation enzymes, AOX and PH, also appeared predominantly in the ducts and the stomach epithelia with a weaker immunolabeling in the tubules. At the electron microscopic level a clear labeling with gold particles was observed in the peroxisomal matrix with the anti-guinea pig catalase antibody. In addition to peroxisomes, the anti-PH antibody also labeled the mitochondria. The similarity in the protein composition of molluscan and mammalian peroxisomes as revealed by the present study indicates that those proteins have been well conserved in evolution suggesting that functionally peroxisomes in molluscs could also be involved in the metabolism of lipids and in detoxification of xenobiotics. Thus, the antibodies tested could provide useful tools for detection of peroxisomal induction in molluscan biomonitoring programs for the assessment of aquatic environmental pollution. Accepted: 19 October 1999     

D-aspartate oxidase in rat, bovine and sheep kidney cortex is localized in peroxisomes.

D-Aspartate oxidase (EC 1.4.3.1) was assayed in subcellular fractions and in highly purified peroxisomes from rat, bovine and sheep kidney cortex as well as from rat liver. During all steps of subcellular-fractionation procedures, D-aspartate oxidase co-fractionated with peroxisomal marker enzymes. In highly purified preparations of peroxisomes, the enrichment of D-aspartate oxidase activity over the homogenate is about 32-fold, being comparable with that of the peroxisomal marker enzymes catalase and D-amino acid oxidase. Disruption of the peroxisomes by freezing and thawing released more than 90% of the enzyme activity, which is typical for soluble peroxisomal-matrix proteins. Our findings provide strong evidence that in these tissues D-aspartate oxidase is a peroxisomal-matrix protein and should be added as an additional flavoprotein oxidase to the known set of peroxisomal oxidases.     

Alcohol oxidase and catalase in peroxisomes of methanol-grown Candida boidinii.

Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from methanol-grown cells of Candida boidinii. Spheroplast lysates were separated on non-continuous Ficoll density gradients, resulting in a mitochondrial fraction and a peroxisome fraction. Estimates of purity using the mitochondrial enzyme markers suggested that the contamination of mitochondria in the peroxisome fraction was about 2-3%. As shown by electron microscopy the peroxisomes were 0.4-0.6 mum in diameter and contained crystalloid inclusions. Alcohol oxidase and catalase, which catalyse the oxidation of methanol to formaldehyde in Candida boidinii, could be localized within the peroxisomes. Gel-electrophoretic studies of the peroxisome fraction demonstrated that it contained only two predominant protein bands consistent with alcohol oxidase and catalase. No alcohol oxidase and catalase activity was found in mitochondria.     

Cytochemical and immunocytochemical study on the peroxisomes of rat liver after administration of a hypolipidemic drug, MLM-160

After administration of a hypolipidemic drug, MLM-160, to male rats, liver peroxisomes were studied by biochemical, cytochemical, and immunocytochemical methods. The activities of D-amino acid oxidase, glycolate oxidase, and urate oxidase increased 2 to 3-fold by the treatment. The increase of the oxidases was confirmed by immunoblotting analysis. By light microscopy, immunoreaction for catalase was present in the cytoplasmic granules of hepatocytes. The stained granules formed some clusters and overlapped each other after MLM-160 treatment. However, immunostaining for D-amino acid oxidase and urate oxidase was present in discrete fine granules which did not overlap each other. By electron microscopy, many peroxisomes showed ring-like extensions and cavitation of the matrix, often giving the appearance of a peroxisome-within-a-peroxisome. In many cases, these unusual peroxisomes seemed to be interconnected with each other. Within the peroxisomes, the catalase was localized in the matrix. Urate oxidase was associated with the crystalloid cores. D-amino acid oxidase was localized focally in a small part of the matrix where the catalase was mostly negative. In conclusion, the administration of MLM-160 to male rats induces some peroxisomal oxidases, accompanying the appearance of unusual peroxisomes. The precise localization of peroxisomal enzymes suggested that there are subcompartments within the liver peroxisomes as shown in rat kidney peroxisomes.     

Peroxisomes of the nematode Caenorhabditis elegans: distribution and morphological characteristics

We analyzed the distribution and morphological characteristics of peroxisomes in the nematode Caenorhabditis elegans by routine electron microscopy, immunoelectron microscopy, and morphometry. Peroxisomes were mainly contained in the epithelial cells of the digestive tract and pharyngeal gland, but some were observed in other cells. Their shape varied from round to twisted. The matrix of most peroxisomes was coarse and uneven, and contained electron-dense nucleoids and frequently tubular substructures. The diameter of peroxisomes in the gut (0.185 micro m) was smaller than that in pharyngeal gland (0.262 micro m). The volume density of peroxisomes per 100 micro m(2) of cytoplasm was 1.86 in the gut and 1.75 in the pharyngeal gland. After treatment with clofibrate, the diameter of peroxisomes increased approximately 1.11-fold in the gut and 1.2-fold in the pharyngeal gland. The volume density of peroxisomes also increased by 2.2-fold in the gut and 2.6-fold in the pharyngeal gland. The labeling density for catalase-2 was almost identical between gut and pharyngeal gland peroxisomes. The results show that in C. elegans peroxisomes mainly distribute in the epithelial cells of the gut and pharyngeal gland. Peroxisomes of the pharyngeal gland are larger than those of the gut, but peroxisomes of both tissues contain catalase-2 at similar concentrations.     

Ultrastructural, immunocytochemical and morphometric characterization of liver peroxisomes in gray mullet, Mugil cephalus

Peroxisomes of the hepatocytes of gray mullets, Mugil cephalus, were characterized cytochemically and immunocytochemically using antibodies against the peroxisomal proteins catalase and palmitoyl-coenzyme A (CoA) oxidase. In addition, morphometric parameters of peroxisomes were investigated depending on the hepatic zonation, the age of the animals and the sampling season. Mullet liver peroxisomes were reactive for diaminobenzidine, but presented a marked heterogeneity in staining intensity. Most of the peroxisomes were spherical or oval in shape, although irregular forms were also observed. Their size was heterogeneous, with profile diameters ranging from 0.2 to 3 microm. Peroxisomes tended to occur in clusters, usually near the mitochondria and lipid droplets. They also showed a very close topographical relationship to the smooth endoplasmic reticulum. Mullet liver peroxisomes did not contain cores or nucleoids as rodent liver peroxisomes, but internal substructures were observed in the matrix, consisting of small tubules about 60 nm in diameter and larger semicircles 120 nm in diameter. The volume density of peroxisomes was higher in periportal hepatocytes of mullets sampled in summer than in pericentral hepatocytes, indicating that mullet peroxisomes vary depending on physiological and environmental conditions. By immunoblotting, the mammalian antibodies cross-react with the corresponding proteins in whole homogenates of mullet liver. Paraffin sections immunostained with the antibodies against catalase and palmitoyl-CoA oxidase showed a positive reaction corresponding to peroxisomes localized in the hepatocyte cytoplasm. In agreement, the ultrastructural study revealed that catalase and palmitoyl-CoA oxidase are exclusively localized in the peroxisomal matrix in fish hepatocytes, showing a dense gold labeling. The presence of the peroxisomal beta-oxidation enzyme palmitoyl-CoA oxidase in peroxisomes indicated that these organelles play a key role in the lipid metabolism of fish liver.     

Proliferation of myocardial peroxisomes in experimental rat diabetes: a biochemical and immunocytochemical study.

Myocardial peroxisomes were investigated in normal and diabetic rats. Catalase and acyl-CoA oxidase activities were increased in the diabetic rat heart and immunoblot analysis showed that both enzyme proteins were markedly enhanced in diabetic heart homogenates. After immunoenzyme staining, catalase and acyl-CoA oxidase were localized in fine granules in the myocardium, which were increased in number in diabetic rats. The numerical density of the granules stained for catalase was increased 1.7 times and that for acyl-CoA oxidase 1.8 times, compared with controls. Protein A-gold labeling for catalase and acyl-CoA oxidase was present in myocardial peroxisomes. The labeling density for both enzymes was increased in diabetic rats by 1.6 times for catalase and 1.5 times for acyl-CoA oxidase, compared with controls. The results indicate that myocardial peroxisomes are increased in the diabetic rat and that this proliferation is accompanied by an increase in catalase and acyl-CoA oxidase activities.     

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.     

Participation of peroxisomes in lipid biosynthesis in the harderian gland of guinea pig.

Peroxisomal enzyme activities in the guinea-pig harderian gland, which has a unique lipid composition, were studied. Activities of catalase, acyl-CoA oxidase and the cyanide-insensitive acyl-CoA beta-oxidation system in this tissue were comparable with those in rat liver. The activities of dihydroxyacetone phosphate acyltransferase (DHAPAT, EC 2.3.1.42) and alkyl-DHAP synthase (EC 2.5.1.26) were appreciable, and the distributions of both activities were consistent with that of sedimentable catalase activity. Glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15), which is localized in both microsomes (microsomal fractions) and mitochondria in the rat liver, was a peroxisomal enzyme in the harderian gland, though the activity was only about one-tenth of the DHAPAT activity. These enzymes had different pH profiles and substrate specificity. The existence of high activities of enzymes of the acyl-DHAP pathway in peroxisomes suggests the physiological significance of peroxisomes in the biosynthesis of glycerol ether phospholipid and 1-alkyl-2,3-diacylglycerol in the guinea-pig harderian gland.     

Immunocytochemical localization of urate oxidase, fatty acyl-CoA oxidase, and catalase in bovine kidney peroxisomes

We investigated the localization of urate oxidase, peroxisomal fatty acyl-CoA oxidase, and catalase in bovine kidney by immunoblot analysis and protein A-gold immunocytochemistry, using the respective polyclonal monospecific antibodies raised against the enzymes purified from rat liver. By immunoblot analysis, these three proteins were detected in bovine kidney and bovine liver homogenates. Subcellular localization of these three enzymes in kidney was ascertained by protein A-gold immunocytochemical staining of Lowicryl K4M-embedded tissue. Peroxisomes in bovine kidney cortical epithelium possessed crystalloid cores or nucleoids, which were found to be the exclusive sites of urate oxidase localization. The limiting membrane, the marginal plate, and the matrix of renal peroxisomes were negative for urate oxidase staining. In contrast, catalase and fatty acyl-CoA oxidase were found in the peroxisome matrix. These results demonstrate that, unlike rat kidney peroxisomes which lack urate oxidase, peroxisomes of bovine kidney contain this enzyme as well as peroxisomal fatty acyl-CoA oxidase.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

Substructure of crystalline peroxisomes in methanol-grown Hansenula polymorpha: evidence for an in vivo crystal of alcohol oxidase.

The substructural organization of completely crystalline peroxisomes present in Hansenula polymorpha cells grown under methanol limitation in a chemostat was investigated by different cytochemical and ultrastructural techniques. Time-dependent cytochemical staining experiments indicated that activities of the two main constituents of these organelles, namely, alcohol oxidase and catalase, were present throughout the crystalline matrix. Catalase was completely removed from isolated peroxisomes by osmotic shock treatment. After such treatment, the ultrastructure of the crystalline matrix of the organelles remained virtually intact. Because alcohol oxidase activity was still present in this matrix, it was concluded that alcohol oxidase protein is the only structural element of the peroxisomal crystalloids. The molecular architecture of the crystalloids was investigated in ultrathin cryosections which permitted recognition of individual molecules in the crystalline matrix. Depending on the plane of sectioning, different crystalline patterns were observed. Tilting experiments indicated that these images were caused by superposition of octameric alcohol oxidase molecules arranged in a tetragonal lattice. A three-dimensional model of the crystalloid is presented. The repeating unit of this structure is composed of four alcohol oxidase molecules. The crystalloid represents an open structure, which may explain the observed free mobility of catalase molecules.     

Selective induction of peroxisomal enzymes by the hypolipidemic drug bezafibrate. Detection of modulations by automatic image analysis in conjunction with immunoelectron microscopy and immunoblotting

Quantitative immunoelectron microscopy in conjunction with quantitative analysis of immunoblots have been used to study the effects of bezafibrate (BF), a peroxisome-proliferating hypolipidemic drug, upon six different enzyme proteins in rat liver peroxisomes (Po). Antibodies against following peroxisomal enzymes: catalase, urate oxidase, alpha-hydroxy acid oxidase, acyl-CoA oxidase, bifunctional enzyme (hydratase-dehydrogenase) and thiolase, were raised in rabbits, and their monospecificities were confirmed by immunoblotting. Female Sprague-Dawley rats were treated for 7 days with 250 mg/kg/day bezafibrate and liver sections were incubated with the appropriate antibodies followed by the protein A-gold complex. The labeling density for each enzyme was estimated by automatic image analysis. In parallel experiments immunoblots prepared from highly purified peroxisome fractions of normal and BF-treated rats were incubated with the same antibodies. The antigens were visualized by an improved protein A-gold method including an anti-protein A step and silver amplification. The immunoblots were also quantitated by an image analyzer. The results revealed a selective induction of beta-oxidation enzymes by bezafibrate with thiolase showing the most increase followed by bifunctional protein and acyl-CoA oxidase. The labeling density for catalase and alpha-hydroxy acid oxidase was reduced, confirming fully the quantitative analysis of immunoblots which in addition revealed reduction of uricase. These observations demonstrate that hypolipidemic drugs induce selectively the beta-oxidation enzymes while other peroxisomal enzymes are reduced. The quantitative immunoelectron microscopy with automatic image analysis provides a versatile, highly sensitive and efficient method for rapid detection of modulations of individual proteins in peroxisomes.     

Microsomal and cytosolic epoxide hydrolases, the peroxisomal fatty acid beta-oxidation system and catalase. Activities, distribution and induction in rat liver parenchymal and non-parenchymal cells

A number of structurally unrelated hypolipidaemic agents and certain phthalate-ester plasticizers induce hepatomegaly and proliferation of peroxisomes in rodent liver, but there is relatively limited data regarding the specific effects of these drugs on liver non-parenchymal cells. In the present study, liver parenchymal, Kupffer and endothelial cells from untreated and fenofibrate-fed rats were isolated and the activities of two enzymes associated with peroxisomes (catalase and the peroxisomal fatty acid beta-oxidation system) as well as cytosolic and microsomal epoxide hydrolase were measured. Microsomal epoxide hydrolase, cytosolic epoxide hydrolase and catalase activities were 7-12-fold higher in parenchymal cells than in Kupffer or endothelial cells from untreated rats; the peroxisomal fatty acid beta-oxidation activity was only detected in parenchymal cells. Fenofibrate increased catalase, cytosolic epoxide hydrolase and peroxisomal fatty acid beta-oxidation activities in parenchymal cells by about 1.5-, 3.5- and 20-fold, respectively. The induction of catalase (2-3-fold) and cytosolic epoxide hydrolase (3-5-fold) was also observed in Kupffer and endothelial cells; furthermore, a low peroxisomal fatty acid beta-oxidation activity was detected in endothelial cells. Morphological examination by electron microscopy showed that peroxisomes were confined to liver parenchymal cells in untreated animals, but could also be observed in endothelial cells after administration of fenofibrate.     

Biochemical, electrophoretic and immunological characterization of peroxisomal enzymes in three soybean organs

Biochemical, electrophoretic and immunological studies were made among peroxisomal enzymes in three organs of soybean [Glycine max (L.) Merr. cv. Centennial] to compare the enzyme distribution and characteristics of specialized peroxisomes in one species. Leaves, nodules and etiolated cotyledons were compared with regard to several enzymes localized solely in their peroxisomes: catalase (EC 1.11.1.6), malate synthase (EC 4.1.3.2), glycolate oxidase (EC 1.1.3.1), and urate oxidase (EC 1.7.3.3). Catalase activity was found in all tissue extracts. Electrophoresis on native polyacrylamide gels indicated that leaf catalase migrated more anodally than nodule or cotyledon catalase as shown by both activity staining and Western blotting. Malate synthase activity and immunologically detectable protein were present only in the cotyledon extracts. Western blots of denaturing (lithium dodecyl sulfate) gels probed with anti-cotton malate synthase antiserum, reveal a single subunit of 63 kDa in both cotton and soybean cotyledons. Glycolic acid oxidase activity was present in all three organs, but ca 20-fold lower (per mg protein) in both nodule and cotyledon extracts compared to leaf extracts. Electrophoresis followed by activity staining on native gels indicated one enzyme form with the same mobility in nodule, cotyledon and leaf preparations. Urate oxidase activity was found in nodule extracts only. Native gel electrophoresis showed a single band of activity. Novel electrophoretic systems had to be developed to resolve the urate oxidase and glycolate oxidase activities; both of these enzymes moved cathodally in the gel system employed while most other proteins moved anodally. This multifaceted study of enzymes located within three specialized types of peroxisomes in a single species has not been undertaken previously, and the results indicate that previous comparisons between the enzyme content of specialized peroxisomes from different organisms are mostly consistent with that for a single species, soybean.     

Peroxisomes of soybean (Glycine max) root nodule vascular parenchyma cells contain a "nodule—specific" urate oxidase

The cortex of soybean ( Glycine max L. cv. Centennial) nodules contain an organellerich layer of vascular parenchyma tissue, which encircles the elaborate vascular tissue of the nodule. Peroxisomes with small, electron-opaque nucleoids are found in the vascular parenchyma cells. Positive cytochemical staining for catalase (EC 1.11.1.6) confirms their morphological identification as peroxisomes. Activities of both glycolate oxidase (EC 1.1.3.1) and urate oxidase (EC 1.7.3.3) were detected cytochemically in these peroxisomes. Nodule-specific urate oxidase was localized principally in the nucleoid region of these vascular parenchyma peroxisomes, as indicated by immunogold labelling using antibodies against nodulin-35, the nodule-specific urate oxidase. The density of urate oxidase immunogold labelling in the vascular parenchyma peroxisome nucleoid is similar to that of the more well-characterized interstitial cell peroxisomes of the infected zone. These results show that the induction of nodule-specific urate oxidase may be induced in tissue outside of the infected zone.     

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.     

Peroxisomes of alkane-utilizing yeasts metabolic functions and practical aspects

One of the most striking features of alkane-grown yeast cells is conspicuous appearance of peroxisomes in harmony with a high level of catalase. This unique phenomenon was first demonstrated in the authors′ laboratory, and the metabolic functions of peroxisomes in yeasts utilizing alkanes has been estabilished with intact peroxisomes isolated by density gradient centrifugation. The organelles participate in the degradation of fatty acids derived from alkanes to C²-units and the synthesis of gluconeogenic intermediates from C²-units. The abundant appearance of peroxisomes in alkane-utilizing cells has allowed successful production of several useful enzymes including catalase, D-amino acid oxidase, uricase, acyl-CoA oxidase etc. Yeast cells will be an excellent system for investigation the functions and development of peroxisomes because biogenesis of the organelles is induced only by transferring the cells into alkane medium from glucose or ethanol medium.     

Cytochemical localization of catalase and several hydrogen peroxide-producing oxidases in the nucleoids and matrix of rat liver peroxisomes

Synopsis The distribution of catalase, amino acid oxidase, -hydroxy acid oxidase, urate oxidase and alcohol oxidase was studied cytochemically in rat hepatocytes. The presence of catalase was demonstrated with the conventional diaminobenzidine technique. Oxidase activities were visualized with methods based on the enzymatic or chemical trapping of the hydrogen peroxide produced by these enzymes during aerobic incubations.All enzymes investigated were found to be present in peroxisomes. Catalase activity was found in the peroxisomal matrix, but also associated with the nucleoid. After staining for oxidase activities the stain deposits occurred invariably in the peroxisomal matrix as well as in the nucleoids. In all experiments the activity of both catalase and the oxidases was confined to the peroxisomes. The presence of a hydrogen peroxide-producing alcohol oxidase was demonstrated for the first time in peroxisomes in liver cells.The results imply that the enzyme activity of the nucleoids of rat liver peroxisomes is not exclusively due to urate oxidase. The nucleoids obviously contain a variety of other enzymes that may be more or less loosely associated with the insoluble components of these structures.     

Peroxisome development in the metanephric kidney of mouse.

The relationship of enzymatic activity to organelle development and organelle number during differentiation of the metanephric kidney in the mouse was approached from several experimental directions. Biochemical analyses of marker enzymes for peroxisomes (catalase and D-amino acid oxidase), mitochondria (cytochrome oxidase) and lysosomes (acid phosphatase) were performed on kidneys at ages from 17 days prenatal to adult. These data were correlated with a morphometric analysis of populations of peroxisomes and mitochondria in differentiating cells of the proximal tubule. Postnatal development of the metanephric kidney was found to be accompanied by a rapid increase in both the specific activity of catalase and the number of peroxisomes per 100 mu2 in the proximal tubule during the first 4 weeks of postnatal growth. Elaboration of the endoplasmic reticulum (ER) was seen to parallel the increase in number of peroxisomes to which segments of ER were often in close apposition. Extensive interactions between segments of ER and peroxisomes were readily visible in 0.5-mu sections viewed in the high voltage electron microscope. In contrast to peroxisomes, neither mitochondria nor lysosomes followed a similar pattern of net organelle increase, suggesting that a defined population density of mitochondria and lysosomes may exist in the proximal tubule at birth, prior to complete development of the kidney.