Cytochemical detection of catalase with 3,3′-diaminobenzidine

Summary The influence of various parameters of fixation and incubation upon the oxidation of DAB by catalase have been analyzed. Crystalline beef liver catalase was fixed with different concentrations of glutaraldehyde and peroxidatic activity was determined spectrophotometrically using DAB as hydrogen donor. Although aldehyde fixation appeared to be important in elicitation of the peroxidatic activity of catalase, the final pigment production after 60 min incubation was optimal with the lowest concentration of glutaraldehyde (1%), after the shortest fixation period (30 min), and at the lowest temperature (5° C) tested. Similarly cytochemical studies with rat kidney sections incubated for 10 min confirmed that the staining of peroxisomes in proximal tubules was strongest after the mildest fixation conditions. The pH and the temperature of incubation were closely interrelated, so that at room temperature (25° C) the maximal pigment production was obtained at pH 10.5 but incubation at 45° C gave the strongest staining at pH 8.5. The production of pigment increased with higher DAB concentrations which required larger amounts of H₂O₂ in the incubation medium. Cytochemical studies on renal peroxisomes were in agreement with these biochemical findings. The observations indicate that there are several options for the localization of catalase depending on the fixation and incubation conditions. Hence, these conditions should be selected according to the tissue and the purpose of the study. Examples for such selective applications are presented.     

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

Summary In 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 Karnovsky's 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.     

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

In 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 specificity 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 Karnovsky's 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 cacodylte 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) buffer 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.     

Intracellular distinction between peroxidase and catalase in exocrine cells of rat lacrimal gland: a biochemical and cytochemical study.

The lacrimal gland (Glandula orbitalis externa) of rat contains both peroxidase and catalase and was used as a model for biochemical and cytochemical distinction between peroxidase and catalase. Both enzymes were isolated by ammonium sulfate precipitation from tissue homogenates, and the effects of fixation with glutaraldehyde and various conditions of incubation were investigated colorimetrically using DAB as hydrogen donor. The lacrimal gland peroxidase is strongly inhibited by glutaraldehyde treatment. In contrast, for catalase the fixation with glutaraldehyde is the prerequistie for demonstration of its peroxidatic activity. The maximal peroxidatic activity was obtained after treatment of catalase with 3% glutaraldehyde, higher concentrations being inhibitory. For lacrimal gland peroxidase, the maximal rate of oxidation of DAB is at pH 6.5, whereas for catalase it is at pH 10.5. The optimal concentration of H2O2 for lacrimal gland peroxidase is at 10(-3)M and for peroxidatic activity of catalase at 10(-1)M. These optimal conditions obtained biochemically were applied to tissue sections of rat lacrimal gland. After the fixation of tissue with a low concentration of glutaraldehyde and incubation in the DAB medium at neutral pH containing 10(-3)M H2O2 (Peroxidase medium), the reaction product was localized in the cisternae of the rough endoplasmic reticulum, in elements of the Golgi apparatus, and in secretory granules. After the fixation of tissue with 3% glutaraldehyde and incubation in the DAB-medium containing 10(-1)M H2O2 and at pH 10.5 (catalase medium), the staining in the endoplasmic reticulum, the Golgi-apparatus and in secretory granules was completely inhibited and reaction product was localized exclusively in small (0.2-0.5 mu) particles similar to small peroxisomes described in various other cell-types.     

Cytochemistry of human catalase. The demonstration of hepatic and renal peroxisomes by a high temperature procedure.

The cytochemical demonstration of marker enzymes for subcellular organelles permits light microscopic analysis of their structure and function in normal and diseased tissues. Currently available staining procedures for the peroxidatic activity of catalase in peroxisomes of plant and animal cells yield weak and inconsistent light microscopic staining when applied to human tissues. We have developed a simple and sensitive high temperature procedure that clearly and reproducibly stains these abundant, but poorly understood, organelles in biopsy specimens of human liver and kidney. This method utilizes formaldehyde fixation, a modified diaminobenzidine (DAB) medium, incubation at 45 degrees C and postosmication for both light and electron microscopy.     

Intracellular distinction between peroxidase and catalase in exocrine cells of rat lacrimal gland: A biochemical and cytochemical study

Summary The lacrimal gland (Glandula orbitalis externa) of rat contains both peroxidase and catalase and was used as a model for biochemical and cytochemical distinction between peroxidase and catalase. Both enzymes were isolated by ammonium sulfate precipitation from tissue homogenates, and the effects of fixation with glutaraldehyde and various conditions of incubation were investigated colorimetrically using DAB as hydrogen donor. The lacrimal gland peroxidase is strongly inhibited by glutaraldehyde treatment. In contrast, for catalase the fixation with glutaraldehyde is the prerequisite for demonstration of its peroxidatic activity. The maximal peroxidatic activity was obtained after treatment of catalase with 3% glutaraldehyde, higher concentrations being inhibitory. For lacrimal gland peroxidase, the maximal rate of oxidation of DAB is at pH 6.5, whereas for catalase it is at pH 10.5. The optimal concentration of H₂O₂ for lacrimal gland peroxidase is at 10⁻³ M and for peroxidatic activity of catalase at 10⁻¹ M. These optimal conditions obtained biochemically were applied to tissue sections of rat lacrimal gland. After the fixation of tissue with a low concentration of glutaraldehyde and incubation in the DAB medium at neutral pH containing 10⁻³ M H₂O₂ (Peroxidase medium), the reaction product was localized in the cisternae of the rough endoplasmic reticulum, in elements of the Golgi apparatus, and in secretory granules. After the fixation of tissue with 3% glutaraldehyde and incubation in the DAB-medium containing 10⁻¹ M H₂O₂ and at pH 10.5 (catalase medium), the staining in the endoplasmic reticulum, the Golgi-apparatus and in secretory granules was completely inhibited and reaction product was localized exclusively in small (0.2–0.5 μ) particles similar to small peroxisomes described in various other cell-types. This work was presented in part at the twenty-fifth Annual Meeting of the Histochemical Society, April 5–6, 1974. Atlantic City, N.J., J. Histochem. Cytochem.22, 288 (1974).     

Peroxisomes: 40 years of histochemical staining,personal reminiscences

The historical circumstances that led to the discovery of the 3,3′-diamino-benzidine (DAB) method for staining of peroxisomes 40 years ago are reviewed. In the course of studies on the uptake and absorption of horse radish peroxidase in mammalian liver, in sections incubated for detection of peroxidase activity in DAB, it was noted that peroxisomes also stained positively for peroxidase activity. Subsequently, it was revealed that the peroxidatic activity of catalase, which is abundantly present in peroxisomes, is responsible for that staining. This notion was confirmed in quantitative biochemical studies with crystalline beef liver catalase and in tracer studies using catalase as an ultrastructural tracer. The application of the DAB method led to the discovery of peroxisomes as a ubiquitous eukaryotic cell organelle, attracting great interest in their investigation in biomedical research.     

Cytochemical discrimination between catalases and peroxidases using diaminobenzidine.

The influence on diaminobenzidine staining of four variables: prefixation in aldehyde, temperature and pH of incubation, and H2O2 concentration, was investigated in catalase-, as well as in peroxydase-containing material. Catalase from five different sources and five types of peroxidase were examined. It is concluded: (a) when cells are incubated without prior fixation, in a DAB medium at room temperature and pH 7.3 with 0.003% H2O2, peroxidases produce a visible cytochemical stain, while catalases do not; (b) the cytochemical reaction elicited by catalases is stimulated by prior aldehyde fixation in specified conditions, and incubation at 45 degrees C and pH 9.7 with 0.06% H2O2; (c) under the latter circumstances several peroxidases also stain. Ultrastructural preservation is satisfactory in tissues incubated prior to fixation.     

Schistosoma mansoni and Biomphalaria glabrata: Ultrastructural localization of enzymes with diaminobenzidine in larvae and host digestive glands.

All mitochondria contained reaction product when daughter sporocysts of Schistosoma mansoni and digestive glands of the snail host, Biomphalaria glabrata, were cytochemically incubated for 45 or 60 min with alkaline 3, 3′-diaminobenzidine (DAB) at pH 7.4 and 9.0. The pigment marked the presence of cytochrome c-cytochrome oxidase activity, and was not found in parasite or gland tissues incubated with DAB and KCN at pH 7.4, 9.0, and 9.8.After incubation for 45 min in the pH 7.4 DAB medium, tegumental mitochondria in young intrasporocyst cercariae showed DAB reaction product, but little or none of the pigment was found in tegumental mitochondria of older, glycocalyx-covered cercariae. In contrast, mitochondria of subtegumental cells were strongly DAB positive at all stages of intrasporocyst cercarial development. No differences in DAB reactivity were detected in mitochondria of sporocysts, or of infected and uninfected host gland cells.Reaction product was found in certain vacuoles of digestive cells incubated in the pH 9.8 DAB medium with KCN, but not in the pH 9.8 DAB medium with amino triazole, or in the pH 7.4 DAB medium. No peroxisomes or microperoxisomes were found in the tissues studied.     

The reaction of mitochondria in the coleoptiles of rice (Oryza sativa L.) with diaminobenzidine.

Diaminobenzidine, DAB, was applied to segments of aerobically and anaerobically grown coleoptiles of rice, Oryza sativa L., with the object of studying the location of cytochrome oxidase at the electron-microscope level. A specific staining of mitochondrial cristae and inner membrane was obtained, with no reaction in other organelles; with extended periods of incubation, the reaction product filled the mitochondria completely. In anaerobically grown coleoptiles, the reaction was much slower and the difference was particularly marked in vascular bundle companion cells and parenchyma, which gave the strongest reaction in aerobic tissue, but in the anaerobic stained even less than the cortical parenchyma. The reaction was inhibited by boiling and slowed very much by lowering of the incubation temperature from 27 to 4 degrees C. This indicated the involvement of an enzymic reaction and cyanide inhibition indicated that a haem enzyme was involved. The catalase inhibitor aminotriazole did not inhibit DAB oxidation. Nevertheless the specificity of the reaction for cytochrome oxidase must be questioned, because preheating of the tissue to 60 degrees C before incubation, which would be expected to destroy cytochrome oxidase activity, failed to decrease the oxidation, at least in aerobically grown coleoptiles. It is concluded that DAB is oxidized in the rice coleoptile tissue by a cytochrome system, and the development of this system is inhibited by anaerobiosis, but the oxidation cannot be claimed to represent cytochrome oxidase activity exclusively. Perhaps other autoxidizable, more heat-stable cytochromes participate in the reaction.     

Ultrastructural cytochemistry of the diaminobenzidine reaction in the aquatic fungus Entophlyctis variabilis

Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.Abbreviations DAB 3,3-diaminobenzidine-HCl - ER endoplasmic reticulum     

Ultrastructural cytochemical localization of uricase in peroxisomes of rat liver

Ultrastructural localization of uricase (urate: oxygen oxidoreductase, E.C.1.7.3.3.) in rat liver parenchymal cells has been studied with the cerium technique. The cerous ions react with H2O2 generated by the activity of the enzyme in the presence of urate, forming the electron-dense reaction product of cerous perhydroxide. Tissue fixation is carried out by perfusion for 5 min with a low concentration (0.25%) of glutaraldehyde. Since in a biochemical assay it was found that the activity of uricase determined in Trismaleate buffer is substantially weaker than in the Pipes buffer, the classical medium of Briggs et al. (6) was modified, and the latter buffer was substituted for the Trismaleate. Vibratome sectons are incubated at 37 degrees C for 60 min in 0.1 M Pipes buffer, pH 7.8, containing 3 mM cerium chloride and 0.1 mM sodium urate. Under these conditions, the reaction product is localized in the crystalline cores of hepatic peroxisomes. The intensity of the staining is dependent on the concentration of the substrate and the incubation time. In control preparations incubated without urate or with 2,6,8-trichloropurine, a specific inhibitor of uricase, staining is almost completely abolished. In sections incubated with 5 mM cerium and 0.1 mM sodium urate, fine granules with a distribution corresponding to peroxisomes are also visible at the light microscopic level. This latter observation is invaluable for correlative light and electron microscopic studies.     

Alterations in the integrity of peroxisomal membranes in livers of mice treated with peroxisome proliferators

Catalase leakage from its particulate compartment within the light mitochondrial fraction of liver was used as an index of the integrity of peroxisomes in untreated mice and in mice treated with the peroxisome proliferators clofibrate(ethyl-p-chlorophenoxyisobutyrate), Wy-14,643(4-chloro-6[2,3-xylidino)-2-pyrimidinylthio]acetic acid) and DEHP(di-(2-ethylhexyl)phthalate).Catalase leakage represented about 2% of the total catalase activity when fractions from untreated mice were incubated at 4°C, increasing to about 5% during 60 min incubation at 37°C. In fractions from livers of mice treated with peroxisome proliferators, catalase leakage was significantly higher, being 7–11% at 4°C and increasing to approximately 20% after 60 min incubation at 37°C. The pattern of release was similar for all proliferators. Parallel data were obtained for catalase latency in these fractions, i.e. following 60 min incubation at 37°C, free (non-latent) catalase activity was 18% in control mice and 65, 67, and 83% in fractions from clofibrate-, Wy-14,643- and DEHP-treated mice, respectively. Differences in catalase leakage from peroxisomes in fractions from untreated mice and clofibrate-treated mice were also apparent following treatments designed to effect membrane permeabilization, as in freeze-thawing, osmotic rupture, and extraction with Triton X-100 and lysophosphatidylcholine.These data are consistent with a significant alteration in the integrity of the membranes of peroxisomes in livers of mice which have been treated with peroxisome proliferators, and furthermore indicate a commonality of effect of these agents.     

Production of laccase by a newly isolated strain of Trametes modesta

The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by Trametes modesta were evaluated using the one-factor-at-a-time method. The final optimisation was done using a central composite design resulting in a four-fold increase of the laccase activity to 178 nkat ml(-1). Response-surface analysis showed that 7.34 g l(-1) wheat bran, 0.87 g l(-1) glucose, 2.9 g l(-1) yeast extract, 0.25 g l(-1) ammonium chloride, an initial pH of 6.95 and an incubation temperature of 30.26 degrees C were the optimal conditions for laccase production. Laccase produced by T. modesta was fully active at pH 4 and at 50 degrees C. The laccase was very stable at pH 4.5 and at 40 degrees C but half-lives decreased to 120 and 125 min at higher temperature (60 degrees C) and lower pH (pH 3).     

Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes.

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.     

Zonal heterogeneity of peroxisome proliferation and morphology in rat liver after gemfibrozil treatment

The effect of gemfibrozil on the fine structure of peroxisomes across the rat liver lobule was investigated by light and electron microscopy using the alkaline diaminobenzidine (DAB) medium for the visualization of catalase peroxidatic activity. The oral administration of gemfibrozil for 2 weeks induces a striking heterogeneity in the lobular distribution of peroxisomes. The size and shape of peroxisomes, variety of matrix modifications, catalase content, and position within the cell, are functions of the zonal localization of the hepatocytes. The largest and most numerous peroxisomes were found in the centrilobular region indicating that these cells are most sensitive to peroxisome proliferation. On the other hand, the greatest variety of peroxisome shapes and matrix alterations (tubules and plates) was seen more peripherally in the mid-zonal and periportal regions. The larger, round centrilobular peroxisomes stained less intensely than the elongated peroxisomes found more peripherally, indicating a discrepancy between peroxisome size and catalase content. A distinct population of small irregularly shaped peroxisomes, lacking matrix specializations and containing variable catalase content, was found in the mid-zonal region. Peroxisomes in the centrilobular region were located within areas of the cell containing SER and glycogen while those in the more peripheral region were relegated to areas of the cytoplasm separate from RER and SER. In addition to modifications of peroxisomes, gemfibrozil treatment resulted in a proliferation and formation of whorled configurations of SER. This was particularly evident in the mid-zonal region, where single peroxisomal profiles could be seen surrounded by whorls of SER membranes. The results suggest that rat liver hepatocytes of the centrilobular region are the most sensitive to peroxisome proliferation and those of the periportal area are most susceptible to peroxisome matrix alterations after gemfibrozil treatment.     

Detection of proliferating cell nuclear antigen in diagnostic histopathology.

We describe the effects of tissue preservation, fixation time, and hydrolytic treatment on the detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining with three commercial anti-PCNA antibodies (19A2, 19F4, PC10). Our goal was to provide guidelines for PCNA immunohistochemistry in formalin-fixed, paraffin-embedded specimens. In proliferative cell compartments, nuclear staining was achieved with all three antibodies. In some cases PCNA was also expressed in non-proliferative, histologically normal tissues associated with tumors or other lesions elsewhere. In most autopsy specimens PNCA immunoreactivity was markedly diminished as compared with similar surgical specimens. Incubation overnight with primary antibody at 4 degrees C enhanced PCNA immunoreactivity over incubation at 42 degrees C for 45 min. Pre-treatment with 2 N HCl did not increase staining. Staining with the PC10 antibody was much better preserved than staining with the antibodies 19A2 and 19F4 after prolonged formalin fixation of surgical specimens and in tissues obtained at autopsy. With all three antibodies, however, PCNA immunoreactivity was well preserved during formalin fixation for 8-24 hr and during fixation delays for 8 hr at room temperature. This indicates that PCNA is stable under conditions routinely encountered in diagnostic surgical pathology and facilitates its potential use as a diagnostic proliferation marker.     

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.     

Peroxisomes of the rat cardiac and soleus muscles increase after starvation. A biochemical and immunocytochemical study

We have investigated the change of catalase activity in the homogenates of rat cardiac and skeletal muscles. After 7 days' starvation, the catalase activity of heart increased about 3-fold and that of soleus muscle enhanced 2-fold higher than that of control rats. Immunoblot analysis of catalase showed a single band in the homogenates of cardiac and soleus muscles and increase of catalase antigen after starvation. Light microscopic immunoenzyme staining showed that after starvation catalase positive granules markedly increased in both the cardiac and soleus muscle. Quantitative analysis of the staining showed that number of the granules per 100 microns 2 of tissue section was about 1.4-fold in the soleus muscle and 1.7-fold in the cardiac muscle after starvation. By electron microscopy of alkaline DAB staining, we confirmed that the granules were peroxisomes, which increased in both number and size. Furthermore, we stained the peroxisomes for catalase by a protein A-gold technique. Labeling density (gold particles/micron 2) of the cardiac and soleus muscles from the starved rat increased approximately 1.4 times as much as that of normal animal. When the numerical density is multiplied by the labeling density, the values are largely consistent with the enhancement of catalase activity. These results show that increase in the catalase activity of the muscle tissue after starvation is caused by increase in number and size of peroxisomes.     

Actin localization and function in higher plants

Summary Two different cytochemical methods were used to study the localization of uricase (EC 1.7.3.3) and catalase (EC 1.11.1.6) in developing root nodules of soybean (Glycine max) inoculated as seeds withBradyrhizobium japonicum. One of the methods employs DAB (3,3-diaminobenzidine) and detects uricase activity indirectly by coupling it to endogenous catalase activity. The other method utilizes cerium chloride to detect uricase activity directly. These methods were modified to obtain not only a strong staining reaction but also improved ultrastructural preservation. With the indirect DAB method, intense staining indicative of both uricase and catalase activity was obtained in the enlarged peroxisomes of older uninfected cells. Similar staining was observed in enlarging peroxisomes of younger uninfected cells, and in the material of associated sacs whose bounding membranes appear to arise as distensions of the ER. The observations are discussed in relation to the controversial role of the ER in peroxisome biogenesis. Although the small peroxisome-like organelles of infected cells did not give a clearly positive reaction in the indirect DAB method, they reacted positively in the cerium chloride method, and are considered to be peroxisomes.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum