H. D. Fahimi

Selective cytochemical localization of peroxidase, cytochrome oxidase and catalase in rat liver with 3,3'-diaminobenzidine.

SummaryIn rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specifity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovskys glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15–30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylate buffer pH 6.5 and 0.02% H2O2. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) butffer at pH 10.5 and 0.15% H2O2. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.

L-Lactate Dehydrogenase A- and AB Isoforms Are Bona Fide Peroxisomal Enzymes in Rat Liver EVIDENCE FOR INVOLVEMENT IN INTRAPEROXISOMAL NADH REOXIDATION

The subcellular localization of L-lactate dehydrogenase (LDH) in rat hepatocytes has been studied by analytical subcellular fractionation combined with the immunodetection of LDH in isolated subcellular fractions and liver sections by immunoblotting and immunoelectron microscopy. The results clearly demonstrate the presence of LDH in the matrix of peroxisomes in addition to the cytosol. Both cytosolic and peroxisomal LDH subunits have the same molecular mass (35.0 kDa) and show comparable cross-reactivity with an anti-cytosolic LDH antibody. As revealed by activity staining or immunoblotting after isoelectric focussing, both intracellular compartments contain the same liver-specific LDH-isoforms (LDH-A > LDH-AB) with the peroxisomes comprising relatively more LDH-AB than the cytosol. Selective KCl extraction as well as resistance to proteinase K and immunoelectron microscopy revealed that at least 80% of the LDH activity measured in highly purified peroxisomal fractions is due to LDH as a bona fide peroxisomal matrix enzyme. In combination with the data of cell fractionation, this implies that at least 0.5% of the total LDH activity in hepatocytes is present in peroxisomes. Since no other enzymes of the glycolytic pathway (such as phosphoglucomutase, phosphoglucoisomerase, and glyceraldehyde-3-phosphate dehydrogenase) were found in highly purified peroxisomal fractions, it does not seem that LDH in peroxisomes participates in glycolysis. Instead, the marked elevation of LDH in peroxisomes of rats treated with the hypolipidemic drug bezafibrate, concomitantly to the induction of the peroxisomal β-oxidation enzymes, strongly suggests that intraperoxisomal LDH may be involved in the reoxidation of NADH generated by the β-oxidation pathway. The interaction of LDH and the peroxisomal palmitoyl-CoA β-oxidation system could be verified in a modified β-oxidation assay by adding increasing amounts of pyruvate to the standard assay mixture and recording the change of NADH production rates. A dose-dependent decrease of NADH produced was simulated with the lowest NADH value found at maximal LDH activity. The addition of oxamic acid, a specific inhibitor of LDH, to the system or inhibition of LDH by high pyruvate levels (up to 20 mM) restored the NADH values to control levels. A direct effect of pyruvate on palmitoyl-CoA oxidase and enoyl-CoA hydratase was excluded by measuring those enzymes individually in separate assays. An LDH-based shuttle across the peroxisomal membrane should provide an efficient system to regulate intraperoxisomal NAD/NADH levels and maintain the flux of fatty acids through the peroxisomal β-oxidation spiral.

Light microscopic immunocytochemical demonstration of peroxisomal enzymes in epon sections

SummaryA procedure is described for light microscopic immunocytochemical localization of catalase and three enzymes of peroxisomal lipid β-oxidation: acyl-CoA oxidase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase in semithin sections of rat liver processed for routine electron microscopy. Satisfactory immunostaining required the removal of the epoxy resin with sodium ethoxide, controlled digestion of deplasticized sections with proteases and, in case of osmiumfixed tissue, bleaching with oxidants. Resin removal was essential for successful immunostaining, and protease treatment enhanced markedly the intensity of the reaction. This study shows that tissues processed for conventional ultrastructural studies can be used for postembedding immunocytochemical demonstration of various peroxisomal enzymes.

Immunoelectron Microscopy of Peroxisomal Enzymes; Their Substructural Association and Compartmentalization in Rat Kidney Peroxisomes

The rat renal peroxisomes exhibit by electron microscopy three substructural components 1) the limiting membrane, 2) the peripheral dense matrix containing circular and tubular profiles with vague filamentous structures, and 3) a central clear matrix. In this study, we have investigated the association of peroxisomal enzymes with these substructures by quantitative immunoelectron microscopy. Vibratome sections (lOOpim thick) of the kidney were embedded in Lowicryl KUM at -20°C. Ultrathin sections were labeled by the protein A-gold technique for following enzymes: catal- ase (CAT), acyl-CoA oxidase (AOX), bifunctional enzyme (PH), thiolase (PT), D-ami- no acid oxidase (DAO), L- ℒ-hydroxy acid oxidase (HOX), acyl-CoA synthetase (PCS), and serine:pyruvate aminotransferase (SPT). Labeling density in the substructures was quantitatively analyzed by a semicomputing system. All of the enzymes examined were visualized on the Lowicryl K4M sections as gold particles. The β oxidation enzymes, CAT, HOX, and SPT were localized mainly in the peripheral dense matrix. A few gold particles present in the central clear matrix were frequently associated with filamentous structures extending into this region. DAO was detected exclusively on the central clear matrix. PCS was associated with the inner side of the peroxisome membrane. Quantitative analysis also confirmed these data. The results indicate that the various enzymes in peroxisomes are localized in specific domains, thus suggesting a subcompartmentalization in this organelle.

A new cerium-based method for cytochemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes

SummaryThe use of cerium chloride for the localization of thiamine-pyrophosphatase (TPPase) in rat liver parenchymal cells has been investigated and the results are compared with the classical lead capture method. A medium containing 3 mM cerium chloride gave the most uniform and consistent results with a homogenous electron dense reaction product in the first trans lamella of the Golgi complex and a weak staining of endoplasmic reticulum. The fine deposits of cerium phosphate filled completely the first trans Golgi cisterna. In contrast the reaction product of the lead-based method appeared clumpy and aggregated with an irregular distribution over both Golgi complex and endoplasmic reticulum. Higher and lower concentrations of cerium chloride than 3 mM gave inconsistent results. The present study demonstrates that the cerium-based method is superior to the classical lead-technique for the localization of TPPase.

Detection of peroxisomes in human liver and kidney fixed with formalin and embedded in paraffin: The use of catalase and lipid β-oxidation enzymes as immunocytochemical markers

SummaryWe describe the immunocytochemical localization of four peroxisomal enzymes by light microscopy in human liver and kidney processed routinely by formalin fixation and paraffin embedding. Monospecific antisera against catalase and three enzymes of peroxisomal lipid β-oxidation (acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase) were used in conjunction with either the indirect immunoperoxidase method or the protein A—gold technique followed by silver intensification. The sections of formalin-fixed paraffin-embedded tissue had to be deparaffinized and subjected to controlled proteolysis in order to obtain satisfactory immunostaining. Under the conditions employed, peroxisomes were distinctly visualized in liver parenchymal cells with no reaction in bile duct epithelial or sinusoidal lining cells. In the kidney, peroxisomes were confined to the proximal tubular epithelial cells with negative staining of glomeruli, distal tubules and collecting ducts. A positive immunocytochemical reaction was obtained even in paraffin blocks stored for several years. The method offers a simple approach for detection of peroxisomes and evaluation of their various enzyme proteins in material processed routinely in histopathology laboratories and should prove useful in the investigation of the role of peroxisomes in human pathology for both prospective and retrospective studies.

Electron microscopic cytochemical localization ofα-hydroxyacid oxidase in rat liver

The substrate specificity and the intraperoxisomal localization of alpha-hydroxyacid oxidase in rat liver has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay. Rat liver is fixed by perfusion with a low concentration (0.25%) of glutaraldehyde and vibratome sections are incubated for 60 min at 37 degrees C in a medium containing 3 mM CeCl3, 100 mM NaN3 and 5 mM of an alpha-hydroxyacid in 0.1 M of one of the following buffers: Pipes, Mops, Na-cacodylate, Tris-maleate, all adjusted to pH 7.8. Ten different alpha-hydroxyacids with a chain length between 2 and 8 carbon atoms were tested. The best results were obtained with glycolic, argininic and L-alpha-isocaproic acids. These cytochemical findings were confirmed also biochemically using purified peroxisomal fractions isolated by gradient centrifugation in metrizamide. The pattern of the intraperoxisomal localization of the enzyme was influenced markedly by the type of buffer used for the cytochemical incubation. Whereas in the Tris-maleate medium both the cores and the matrix stained with the same intensity, with all other buffers the reaction in cores was more prominent. The staining of cores was abolished by pretreating sections in Tris-maleate (pH 7.8) or alkaline pyrophosphate buffers. These observations establish the substrate specificity of alpha-hydroxyacid oxidase in rat liver and demonstrate the delicate association of this enzyme with the crystalline cores and the matrix of peroxisomes in rat liver.SummaryThe substrate specificity and the intraperoxisomal localization of α-hydroxyacid oxidase in rat liver has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay. Rat liver is fixed by perfusion with a low concentration (0.25%) of glutaraldehyde and vibratome sections are incubated for 60 min at 37°C in a medium containing 3 mM CeCl3, 100 mM NaN3 and 5 mM of an α-hydroxyacid in 0.1M of one of the following buffers: Pipes, Mops, Na-cacodylate,Tris-maleate, all adjusted to pH 7.8. Ten different α-hydroxyacids with a chain length between 2 and 8 carbon atoms were tested. The best results were obtained with glycolic, argininic andl-α-isocaproic acids. These cytochemical findings were confirmed also biochemically using purified peroxisomal fractions isolated by gradient centrifugation in metrizamide. The pattern of the intraperoxisomal localization of the enzyme was influenced markedly by the type of buffer used for the cytochemical incubation. Whereas in theTris-maleate medium both the cores and the matrix stained with the same intensity, with all other buffers the reaction in cores was more prominent. The staining of cores was abolished by pretreating sections inTris-maleate (pH 7.8) or alkaline pyrophosphate buffers. These observations establish the substrate specificity of α-hydroxyacid oxidase in rat liver and demonstrate the delicate association of this enzyme with the crystalline cores and the matrix of peroxisomes in rat liver.

Electron microscopic cytochemical localization ofα-hydroxyacid oxidase in rat kidney cortex

SummaryThe substrate specificity ofα-hydroxyacid oxidase in the rat kidney has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay applied to isolated renal peroxisomes. Rat kidneys were fixed by perfusion via the abdominal aorta with a low concentration (0.25%) of glutaraldehyde. Vibratome sections were incubated for 60 min at 37°C in a medium containing 3 mM CeCl3, 100 mM NaN3 and 5 mM of anα-hydroxyacid in 0.1M Pipes or 0.1M Tris-maleate buffer both adjusted to pH 7.8. Ten aliphatic α-hydroxyacids with chain lengths between 2 and 8 carbon atoms and two aromatic substrates were tested. The α-hydroxyacid oxidase in the kidney exhibited a markedly different substrate specificity than the corresponding enzyme in the liver. Thus glycolate gave a negative reaction while two aromatic substrates, mandelic acid and phenyllactic acid, stained prommently. With aliphatic substrates a stronger reaction was obtained in Pipes than in theTris-maleate buffered incubation media. The best reaction in the kidney was obtained with hydroxybutyric acid. These cytochemical findings were confirmed by the luminometric determination of the oxidase activity in isolated purified peroxisome fractions. By electron microscopy the electron dense reaction product of cerium perhydroxide was found in the matrix of peroxisomes in the proximal tubules. The intensity of reaction varied markedly in neighbouring epithelial cells but also in different peroxisomes within the same cell. Thus heavily stained particles were seen next to lightly reacted ones. These observations establish the substrate specificity of α-hydroxyacid oxidase in the rat kidney and demonstrate the marked heterogeneity in the staining of renal peroxisomes for this enzyme.

Contributions of the immunogold technique to investigation of the biology of peroxisomes.

The immunogold labeling technique has been extremely useful in investigation of the structure and function of peroxisomes. In this report a few examples of the application of this technique with significant implications in the field are briefly reviewd. The problem of extra-peroxisomal catalase, the subject of long controversy between the biochemists and cytochemists, was settled with the immunogold technique, which unequivocally revealed the presence of that enzyme not only in the cytoplasm, but also in the euchromatin region of nucleus, in addition to peroxisomes. On the other hand, lactate dehydrogenase, a typical cytoplasmic protein, has also been shown recently to be present in peroxisomes and to be involved in the reoxidation of NADH produced by the peroxisomal β-oxidation system. The immunogold technique has revealed several distinct compartments in the matrix of mammalian peroxisomes: urate oxidase in the crystalline cores, α-hydroxy acid oxidase B in the marginal plates andd-amino acid oxidase in a non-crystaline condensed region of matrix. The specific alterations of peroxisomal proteins are reflected in their immunolabeling density with gold particles. Quantitation of gold-label by automatic image analysis has revealed that the induction of lipid β-oxidation enzyme proteins by diverse hypolipidemic drugs is initiated and more pronounced in the pericentral region of the liver lobule. Finally, immunogold labeling with an antibody to 70 kDa peroxisomal membrane protein has identified a novel class of small peroxisomes that initially incorporate radioactive amino acids more efficiently than regular peroxisomes and thus may represent early stages in the biogenesis of peroxisomes.

Immunocytochemical localization of two peroxisomal enzymes of lipid β-oxidation in specific granules of rat eosinophils

SummaryPeroxisomes contain a system for β-oxidation of fatty acids which differs from the mitochondrial system and is associated with hydrogen peroxide formation. We show that two enzymes: enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase of the peroxisomal system are present in specific granules of rat eosinophils. Both enzyme proteins were pufiried from rat liver and monospecific antibodies were raised in rabbits. Eosinophils from peripheral blood and tissue eosinophils from the wall of intenstine, fixed by glutaraldehyde and embedded in Epon were investigated. The postembedding immunocytochemical procedure with protein A-gold technique was used. The gold particles representing the antigenic sites for both enzymes were present only in specific granules of eosinophils with no immune deposits in mitochondria, nucleus and the cytoplasm. Although gold particles were found over the entire domain of the granule, the electron dense paracrystalline inclusions contained more gold than the granule matrix. Control preparations incubated with nonspecific IgG and protein A-gold complex alone were negative. These findings indicate that in specific granules of eosinophils both peroxisomal and lysosomal enzymes share the same intracellular compartment. The peroxisomal lipid β-oxidation in eosinophils may be involved in generation of hydrogen peroxide, which has a crucial role in killing of metazoon parasites.