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According to Poole et al. (1970, J. Cell Biol. 45:408-415), newly synthesized peroxisomal proteins are incorporated uniformly into peroxisomes (PO) of different size classes, suggesting that rat hepatic PO form a homogeneous population. There is however increasing cytochemical and biochemical evidence that PO in rat liver are heterogenous, undergoing significant modulations in shape and size in process of PO morphogenesis (Yamamoto and Fahimi, 1987. J. Cell Biol. 105:713-722). In the present study, the kinetics of incorporation of newly synthesized proteins into distinct PO-subpopulations have been studied using short-term in vivo labeling (5-90 min). Two distinct "heavy" and "light" crude PO fractions were prepared by differential pelleting from normal and regenerating liver, and highly purified PO were subsequently isolated by density-dependent metrizamide gradient centrifugation according to Völkl and Fahimi (1985. Eur. J. Biochem. 149:257-265). The peroxisomal fractions banded at 1.20 and 1.24 g x cm-3. They differed in their mean diameters and form-factors and particularly in respect to the activity of beta-oxidation enzymes which was higher in the "light" PO. Whereas the "light" PO exhibited a single immunoreactive band with the antibody to the 70-kD peroxisomal membrane protein the "heavy" PO contained an additional (68 kD) band. In pulse-labeling experiments "light" PO showed clearly a higher initial rate of incorporation than the "heavy" PO. The relative specific activity in the "heavy" PO fraction, however increased progressively reaching that of "light" PO by 90 min. These observations provide evidence for the existence of different PO populations in rat liver which differ in their morphological and biochemical properties as well as in their rates of incorporation of new proteins.

The matrix of mammalian peroxisomes frequently contains crystalline inclusions. The most common inclusions are membrane associated plate-like "marginal plates" of hitherto unknown nature in renal peroxisomes and central polytubular "cores" composed of urate oxidase in hepatic peroxisomes. In bovine kidney, peroxisomes of proximal tubules exhibit peculiar angular shapes that are caused by multiple marginal plates (Zaar, K., and H.D. Fahimi. 1990. Cell Tissue Res. 260:409-414). Enriched or highly purified peroxisome preparations from this source were used to purify and characterize marginal plates. By SDS-PAGE, one major polypeptide of Mr 33,500 was observed that corresponded to the marginal plate protein. This polypeptide was identified by its enzymatic activity as well as by immunoblotting and preembedding immunocytochemistry as the isozyme B of L-alpha-hydroxyacid oxidase (EC 1.4.3.2). Morphologically, marginal plates were revealed to consist of rectangular straight-edged sheets, exhibiting a defined crystalline lattice structure. The sheets apparently are composed of a single layer of protomers which associate laterally to form a plate-like structure. As deduced from the negative staining results and the additional information of the thickness of marginal plates, each protomer seems to consist of eight subunits forming a cube-like array. The tendency of L-alpha-hydroxyacid oxidase B to self-associate in vitro (Philips, D.R., J.A. Duley, D.J. Fennell, and R.S. Holmes. 1976. Biochim. Biophys. Acta. 427:679-687) corresponds to the mode of association of cubical protomers to form the so-called marginal plates in renal peroxisomes.

Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554-564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment.

The three-dimensional (3-D) form and the interrelationship of peroxisomes (Po) in the model of regenerating rat liver after partial hepatectomy were studied by computer-assisted 3-D reconstruction of serial ultrathin sections. Po were labeled cytochemically for either catalase, which stains them all uniformly, or for D-amino acid oxidase (DAA-OX), which gives a heterogeneous reaction with lightly and darkly stained PO. In regenerating rat liver, Po exhibit marked pleomorphism with some budding forms and dumbbell-shaped ones. The 3-D reconstruction revealed many single spherical Po measuring 0.15-0.8 micron in diameter. In addition, two to five Po were found interconnected with each other via narrow 30-50-nm hourglass-shaped bridges forming a reticulum. Such aggregates of Po measured 1.5-2.5 microns across. Whereas all segments of this reticulum stained homogeneously for catalase, they exhibited a marked difference in the intensity of the DAA-OX reaction. These observations are consistent with the view of peroxisomal proliferation by budding or fragmentation from preexisting ones. Under such conditions of rapid growth as in regenerating liver, Po may be interconnected forming a reticulum. The interconnections between Po with differing DAA-OX activities suggest that they originate from the same parent organelle.

The limitations of the conventional histochemical methods for localization of lactic dehydrogenase (LDH) in white skeletal muscle have been analyzed quantitatively. It is demonstrated that more than 80 per cent of LDH diffuses into the incubation medium within the first 10 minutes of incubation. Furthermore, it is confirmed that the addition of phenazine methosulfate (PMS) to the ingredients of the histochemical reaction for LDH increases substantially the capacity of the white muscle extract to reduce Nitro-BT. Based on these observations, a modified method of cytochemical localization of LDH has been developed. This method prevents the leakage of LDH from tissue sections by the application of all the ingredients of the histochemical reaction to tissue sections in a thin gelatin film. The incubation mixture contains PMS so that the staining system is independent of tissue diaphorase. The application of this method to the adductor magnus muscle of the rabbit revealed a fine reticulum in the sarcoplasm of all muscle fibers, in addition to the staining of mitochondria. The distribution of the staining suggests that LDH is localized in the sarcoplasmic reticulum.

The cytochemical localization, by conventional methods, of lactate and glyceraldehyde-3-phosphate dehydrogenases is limited, firstly, by the solubility of these enzymes in aqueous media and, secondly, by the dependence of the final electron flow from reduced nicotinamide-adenine dinucleotide (NADH) to the tetrazolium on tissue diaphorase activity: localization is therefore that of the diaphorase, which in rabbit adductor magnus is mitochondrial. NADH has been found to have great affinity to bind in the sarcoplasmic reticulum, and, therefore, if it is generated freely in the incubation media containing 2,2',5,5'-tetra-p-nitrophenyl-3,3'-(3,3'-dimethoxy-4,4'-phenylene)-ditetrazolium chloride (TNBT) and N-methyl phenazonium methyl sulfate (PMS), it can bind there and cause a false staining. Since such a production of NADH can readily occur in the incubation media for glycolytic dehydrogenases due to diffusion of these soluble enzymes from tissue sections, the prevention of enzyme solubilization is extremely important. Fixation in formaldehyde prevented such enzyme diffusion, while at the same time sufficient activity persisted to allow for adequate staining. The incubation media contained PMS, so that the staining system was largely independent of tissue diaphorase activity. Application of these methods to adductor magnus of rabbit revealed by light microscopy, for both enzymes, a fine network which was shown by electron microscopy to represent staining of the sarcoplasmic reticulum. Mitochondria also reacted. These findings add further support for the notion that the sarcoplasmic reticulum is probably involved in glycolytic activity.

Prominent staining of rat hepatic microbodies was obtained by incubating sections of aldehyde-fixed rat liver in a modified Graham and Karnovsky's medium for ultrastructural demonstration of peroxidase activity. The electron-opaque reaction product was deposited uniformly over the matrix of the microbodies. The microbodies were identified by their size, shape, presence of tubular nucleoids, and other morphologic characteristics, and by their relative numerical counts. The staining reaction was inhibited by the catalase inhibitor, aminotriazole, and by KCN, azide, high concentrations of H(2)O(2), and by boiling of sections. These inhibition studies suggest that the peroxidatic activity of microbody catalase is responsible for the staining reaction. In the absence of exogenous H(2)O(2) appreciable staining of microbodies was noted only after prolonged incubation. Addition of sodium pyruvate, which inhibits endogenous generation of H(2)O(2) by tissue oxidases, or of crystalline catalase, which decomposes such tissue-generated H(2)O(2), completely abolished microbody staining in the absence of H(2)O(2). Neither diaminobenzidine nor the product of its oxidation had any affinity to bind nonenzymatically to microbody catalase and thus stain these organelles. The staining of microbodies was optimal at alkaline pH of 8.5. The biological significance of this alkaline pH in relation to the similar pH optima of several microbody oxidases is discussed. In addition to staining of microbodies, a heat-resistant peroxidase activity is seen in some of the peribiliary dense bodies. The relation of this reaction to the peroxidase activity of lipofuscin pigment granules is discussed.

Endogenous peroxidase activity has been demonstrated in sections of rat liver fixed briefly by glutaraldehyde perfusion and incubated in Graham and Karnovsky's medium for cytochemical demonstration of peroxidase activity (29). In 25-40% of sinusoidal cells, an electron-opaque reaction product is localized in segments of the endoplasmic reticulum, including the perinuclear cisternae, a few Golgi vesicles and saccules and in some large membrane-bounded granules. This staining is abolished after prolonged fixation or boiling of tissue sections in glutaraldehyde, and in the absence of H(2)O(2) or DAB from the incubation medium. Furthermore, the reaction is inhibited completely by sodium azide and high concentrations of H(2)O(2), and partially by KCN and aminotriazole. Among the different cells in hepatic sinusoids, the nonphagocytic "fat-storing" cells (39) are always peroxidase negative, whereas the lining cells in process of erythrophagocytosis are consistently peroxidase positive. The possible biological significance of endogenous peroxidase in Kupffer cells is discussed. In addition, the uptake of exogenous horseradish peroxidase by Kupffer cells has been investigated. The exogenous tracer protein, which in contrast to endogenous peroxidase of Kupffer cells is not inhibited by prolonged aldehyde fixation, is taken up by micropinocytosis and remains confined to the lysosomal system of Kupffer cells. The significance of these observations in respect to some recent studies suggesting localization of exogenous peroxidases in the endoplasmic reticulum of Kupffer cells and peritoneal macrophages (22, 23) is briefly discussed.

Beef liver catalase was injected intravenously into mice, and its distribution in the kidney, myocardium, and liver was studied with the electron microscope. A specific and relatively sensitive method was developed for its ultrastructural localization, based on the peroxidatic activity of catalase and employing a modified Graham and Karnovsky incubation medium. The main features of the medium were a higher concentration of diaminobenzidine, barium peroxide as the source of peroxide, and pH of 8.5. Ultrastructurally, the enzyme was seen to permeate the endothelial fenestrae and basement membranes of tubular and glomerular capillaries of the kidney. The urinary space and tubular lumina contained no reaction product. In the myocardial capillaries, the tracer filled the pinocytotic vesicles but did not diffuse across the intercellular clefts of the endothelium. In liver, uptake of catalase was seen both in hepatocytes and in Kupffer cells.

The fine structural characteristics and phagocytic properties of peroxidase-positive and peroxidase-negative cells in rat hepatic sinusoids were investigated. Cells with a positive peroxidase reaction in the endoplasmic reticulum and the nuclear envelope make up approximately 40% of cells in rat hepatic sinusoids and have abundant cytoplasm containing numerous granules and vacuoles, and occasional tubular, vermiform invaginations. After intravenous injection of colloidal carbon, the luminal plasma membrane of these cells shows continuous sticking of carbon, and there is evidence of avid phagocytosis of colloidal carbon particles. Peroxidase-positive cells are the only cells in hepatic sinusoids which phagocytize large (0.8 micro in diameter) latex particles. In contrast, the peroxidase-negative endothelial cells, which make up 48% of cells, have scanty perinuclear cytoplasm and organelles, and their long cytoplasmic extensions that form the lining of the hepatic sinusoids have fenestrations; these cells ingest small amounts of colloidal carbon, principally by micropinocytosis, exhibit no sticking of carbon particles to their plasma membranes, and do not ingest the larger (latex) particles. The so-called fat-storing cells are peroxidase negative and totally nonphagocytic. The peroxidase reaction thus distinguishes the typical mononuclear phagocytes or Kupffer cells of rat liver from the endothelial-lining cells.