Immunoelectron microscopic localization of the isozymes of L-alpha-hydroxyacid oxidase in renal peroxisomes of beef and sheep: evidence of distinct intraorganellar subcompartmentation

L-alpha-hydroxyacid oxidase (HAOX), a peroxisomal marker enzyme in mammals, exists in two isozymic forms, HAOX A (EC 1.1.3.1) and HAOX B (EC 1.3.4.2), which differ in their substrate specificity. In rat tissues HAOX A is found exclusively in hepatocyte peroxisomes and HAOX B in renal peroxisomes. Recently we found enzymatic evidence that highly purified peroxisome preparations from beef and sheep kidney cortex contain both isozymes. In situ, the peroxisomes in the proximal tubule cells of both species exhibit peculiar angular outlines apparently due to the presence of multiple marginal plates. Marginal plates are plate-like crystalline matrix inclusions which are apposed to the inner aspect of the peroxisomal membrane. In this study monospecific antibodies against HAOX A and B proteins purified from rat liver and kidney, respectively, were raised in rabbits and used to study the intraorganellar localization of each isozyme in beef and sheep kidney cortex peroxisomes. Incubation of ultra-thin sections of LR White-embedded tissue with anti-HAOX A or B followed by protein A-gold revealed that in both species HAOX A is present diffusely in the peroxisomal matrix, whereas HAOX B is localized almost exclusively in the membrane associated marginal plates. This is the first report on the in situ immunocytochemical characterization of marginal plates, which are the most common inclusions in the matrix of renal peroxisomes.     

Immunocytochemical localization of L-α-hydroxyacid oxidase in dense bar of dumb-bell-shaped peroxisomes of monkey kidney

Localization of the B of L-hydroxyacid oxidase (HOX-B) in monkey kidney peroxisomes was investigated by immunoelectron microscopic techniques. Kidneys of Japanese monkeys,Macaca fuscata, were fixed with 4% paraformaldehyde+0.25% glutaraldehyde and embedded in LR White resin. Thin sections were stained for HOX-B and catalase by the immunogold technique. HOX-B was localized in the marginal plates of normal peroxisomes and the dense bar of dumb-bellshaped peroxisomes. Catalase was detected in the matrix of normal peroxisomes and in the terminal dilatations of dumb-bell-shaped peroxisomes. There were no gold particles indicating presence of catalase associated with the marginal plates or with the dense bars. Immunoblot analysis of monkey kidney homogenate showed that HOX-B has a molecular mass of 42 kDa that was slightly larger than that of rat kidney HOX-B (39 kDa). The results show that the dense bar of dumb-bell-shaped peroxisomes in monkey kidney is composed of at least HOX-B and is a variation of the marginal plates.     

A freeze-etch study of angular marginal-plate-containing peroxisomes in the proximal tubules of bovine kidney

Summary The ultrastructure of peroxisomes in the proximal nephron tubules of bovine kidney cortex was studied using ultrathin-sectioning, diaminobenzidine cytochemistry for the visualization of catalase, and by freeze-fracture. Peroxisomes in this nephron segment are up to 1.5 m in diameter and exhibit a peculiar angular shape, which is probably related to the occurrence of multiple straight plate-like inclusions (marginal plates) in the matrix of peroxisomes; they lie directly underneath the peroxisomal membranes. The peroxisomal membrane in such regions follows the outline of the marginal plate. The peculiar shape of peroxisomes allows their unequivocal identification in freeze-fracture preparations. Peroxisomal membranes are recognized by their flat, often rectangular appearance. Intramembrane particles are much more numerous on P-fracture faces than on E-fracture faces. A crystalline lattice-structure with a periodicity of approximately 10 nm can be observed on the flat rectangular areas of E-fracture faces. This lattice structure is intensified after prolonged freeze-etching. Intramembranous particles seem to be superimposed over this pattern. The crystalline pattern on the E-fracture faces of peroxisomal membranes is probably not a membrane structure but it reveals the structure of the membrane-associated marginal plates. A cast of the marginal-plate surface may be generated by a collapse of the peroxisomal membrane half onto the immediately underlying matrix inclusion.     

Activity of peroxisomal enzymes and intracellular distribution of catalase in Zellweger syndrome

The activity of peroxisomal enzymes was studied in human liver and cultured human skin fibroblasts in relation to the finding (Goldfischer, S. et al. (1973) Science 182, 62-64) that morphologically distinct peroxisomes are not detectable in patients with the cerebro-hepato-renal (Zellweger) syndrome. In homogenates of liver from the patients, dihydroxyacetone phosphate acyltransferase, a membrane-bound peroxisomal enzyme, is deficient (Schutgens, R.B.H., et al. (1984) Biochem. Biophys. Res. Commun. 120, 179-184). In contrast, there is no deficiency of the soluble peroxisomal matrix enzymes catalase, L-alpha-hydroxyacid oxidase and E-aminoacid oxidase. Catalase is also not deficient in homogenates of cultured skin fibroblasts from the patients. The results of digitonin titration experiments showed that in control fibroblasts at least 70% of the catalase activity is present in subcellular particles distinct from mitochondria or lysosomes. In contrast, all of the catalase activity in fibroblasts from Zellweger patients is found in the same compartment as the cytosolic marker enzyme lactate dehydrogenase.     

Tyrosine codon corresponds to topa quinone at the active site of copper amine oxidases.

The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J. P. (1990) Science 248, 981-987). The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P. G., Evers, M., Waterham, H. R., Kuipers, J., Arnberg, A. C., and Geert, A. B. (1989) Biochim. Biophys. Acta 1008, 157-167). In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H. polymorpha. Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with [14C]phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography. Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor. By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein. In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals.     

Light and electron microscopic localization of L-alpha-hydroxyacid oxidase in rat kidney revealed by immunocytochemical techniques

Light and electron microscopic localization of L-alpha-hydroxyacid oxidase (L-HOX) in rat kidney was studied by means of immunocytochemical techniques. Isozymes A and B of L-HOX were purified from rat liver and kidney, respectively. The apparent molecular weights of the subunits of the isozymes A and B were 35,800 and 33,500 daltons, respectively, by a slab gel electrophoresis. Antibodies to the isozymes were raised in rabbits. Anti(isozyme A) is not cross-reactive with the isozyme B and vice versa anti(isozyme B) not with the isozyme A. Using anti-isozyme B, semithin sections of Epon-embedded material and ultrathin sections of Lowicryl K4M-embedded material were stained by immunoenzyme and protein A-gold techniques, respectively. By light microscopy, fine discrete granular staining was noted in proximal tubules, but not in distal tubules including thick and thin limbs of Henle and collecting tubules. By electron microscopy, gold particles representing the antigen sites for L-HOX B were confined exclusively to peroxisomes, in which most of the gold particles were localized in electron dense peripheral matrix, but little in central matrix with low electron density. The results indicate that L-HOX B does not homogeneously distribute in peroxisomes of rat kidney but might be associated with some substructure within peroxisome matrix.     

Presence of three acyl-CoA oxidases in rat liver peroxisomes. An inducible fatty acyl-CoA oxidase, a noninducible fatty acyl-CoA oxidase, and a noninducible trihydroxycoprostanoyl-CoA oxidase

Mammalian liver peroxisomes are capable of beta-oxidizing a variety of substrates including very long chain fatty acids and the side chains of the bile acid intermediates di- and trihydroxycoprostanic acid. The first enzyme of peroxisomal beta-oxidation is acyl-CoA oxidase. It remains unknown whether peroxisomes possess one or several acyl-CoA oxidases. Peroxisomal oxidases from rat liver were partially purified by (NH4)2SO4 precipitation and heat treatment, and the preparation was subjected to chromatofocusing, chromatography on hydroxylapatite and dye affinity matrices, and gel filtration. The column eluates were assayed for palmitoyl-CoA and trihydroxycoprostanoyl-CoA oxidase activities and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results revealed the presence of three acyl-CoA oxidases: 1) a fatty acyl-CoA oxidase with a pI of 8.3 and an apparent molecular mass of 145 kDa. The enzyme consisted mainly of 52- and 22.5-kDa subunits and could be induced by clofibrate treatment; 2) a noninducible fatty acyl-CoA oxidase with a pI of 7.1 and an apparent molecular mass of 427 kDa. It consisted mainly, if not exclusively, of one polypeptide component of 71 kDa; and 3) a noninducile trihydroxycoprostanoyl-CoA oxidase with a pI of 7.1 and an apparent molecular mass of 139 kDa. It consisted mainly, if not exclusively, of one polypeptide component of 69 kDa. Our findings are probably related to the recent discovery of two species of acyl-CoA oxidase mRNA in rat liver (Miyazawa, S., Hayashi, H., Hijikata, M., Ishii, N., Furata, S., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8131-8137) and they probably also explain why in human peroxisomal beta-oxidation defects an accumulation of very long chain fatty acids is not always accompanied by an excretion of bile acid intermediates and vice versa.     

Stoichiometric analysis of the specific interaction of the glucocorticoid receptor with DNA

Purified preparations of activated glucocorticoid X receptor complex (GR) contain a Mr 94,000 hormone-binding polypeptide co-purifying together with a Mr 72,000 non-hormone-binding polypeptide (Wrange, O., Okret, S., Radojcic, M., Carlstedt-Duke, J., and Gustafsson, J.-A. (1984) J. Biol. Chem. 259, 4534-4541). GR binds selectively to discrete regions of DNA in mouse mammary tumor virus (Payvar, F., DeFranco, D., Firestone, G.L., Edgar, B., Wrange, O., Okret, S., Gustafsson, J.-A., and Yamamoto, K. R. (1983) Cell 35, 381-392). Such GR-binding DNA fragments were used to measure the stoichiometry of GR to DNA. Quantitative DNaseI protection "footprinting" analysis was used to ensure that saturation conditions for specific DNA-binding were achieved. Glycerol density gradient centrifugation was used to quantitate Mr 94,000 binding to specific and nonspecific DNA sites. One Mr 94,000 entity was bound per specific DNA site. A modified GR purification procedure resulted in increased amounts of Mr 72,000 polypeptide (1.6:1, 94,000:72,000 molar ratio), compared to previous GR preparations. Glycerol gradient centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the specific GR X DNA complex contained similar amounts of Mr 94,000 and Mr 72,000 polypeptide. It is as yet uncertain if the Mr 72,000 polypeptide is a functional subunit of GR or a co-purifying contaminant only.     

Oxidation of oxalate and polyamines by rat peroxisomes

During renal failure, polyamines and oxalate levels are elevated in the serum and the glomerular filtrate and are dumped by the kidney. Both of these compounds can be catabolized by oxidative reactions. We have, therefore, investigated the intracellular distribution of oxalate oxidase and of a polyamine oxidase in normal female rat kidney and liver. Polyamine oxidase was demonstrable, using spermidine as substrate in the cerous peroxyhydrate procedure of Briggs et al., in peroxisomes of kidney tubule cells and of hepatocytes. Oxalate oxidase could not be studied with this technique due to precipitation of cerium oxalate in the incubation medium. To demonstrate oxalate oxidase, and to confirm the polyamine oxidase localization, we incubated aldehyde-fixed tissue in a diaminobenzidine medium at pH 8, following the approach of Veenhuis et al., in which oxidases are demonstrated by virtue of their production of H2O2, which then serves as a substrate for endogenous catalase. Using oxalate or spermidine as substrate with this approach, we found reaction product in typical renal peroxisomes; we also found reaction product, with the polyamine substrate, in hepatocyte peroxisomes. To strengthen the conclusion that the oxidases themselves are present in peroxisomes, we used a light microscopic method, based on the tetrazolium procedures of Allen and Beard to demonstrate polyamine and oxalate oxidase activities in bodies with the distribution of renal peroxisomes.     

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 reviewed. The problem of extraperoxisomal 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 and d-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. Accepted: May 5, 1996     

Molecular cloning and sequence analysis of cDNAs encoding porcine kidney D-amino acid oxidase

Complementary DNAs encoding D-amino acid oxidase (EC 1.4.3.3, DAO), one of the principal and characteristic enzymes of the peroxisomes of porcine kidney, have been isolated from the porcine kidney cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of two clones revealed a complete 3211-nucleotide sequence with a 5'-terminal untranslated region of 198 nucleotides, 1041 nucleotides of an open reading frame that encoded 347 amino acids, and a 3'-terminal untranslated region of 1972 nucleotides. The deduced amino acid sequence was completely identical with the reported sequence of the mature enzyme [Ronchi, S., Minchiotti, L., Galliano, M., Curti, B., Swenson, R. P., Williams, C. H. J., & Massey, V. (1982) J. Biol. Chem. 257, 8824-8834]. These results indicate that the primary translation product does not contain a signal peptide at its amino-terminal region for its translocation into peroxisomes. RNA blot hybridization analysis suggests that porcine kidney D-amino acid oxidase is encoded by three mRNAs that differ in size: 3.3, 2.7, and 1.5 kilobases. Comparison of the sequences of the two cDNA clones revealed that multiple polyadenylation signal sequences (ATTAAA and AACAAA) are present in the 3'-untranslated region, making the different mRNA species. The efficiency of 3' processing of the RNA was quite different between the two signal sequences ATTAAA and AACAAA. Southern blot analysis showed the presence of a unique gene for D-amino acid oxidase in the porcine genome.     

Identification of S-hydroxylysyl-methionine as the covalent cross-link of the noncollagenous (NC1) hexamer of the alpha1alpha1alpha2 collagen IV network: a role for the post-translational modification of lysine 211 to hydroxylysine 211 in hexamer assembly

Collagen IV networks are present in all metazoans as components of basement membranes that underlie epithelia. They are assembled by the oligomerization of triple-helical protomers, composed of three alpha-chains. The trimeric noncollagenous domains (NC1) of each protomer interact forming a hexamer structure. Upon exposure to acidic pH or denaturants, the hexamer dissociates into monomer and dimer subunits, the latter reflect distinct interactions that reinforce/cross-link the quaternary structure of hexamer. Recently, the cross-link site of the alpha1alpha1alpha2 network was identified, on the basis of x-ray crystal structures at 1.9-A resolution, in which the side chains of Met93 and Lys211 were proposed to be connected by a novel thioether bond (Than, M. E., Henrich, S., Huber, R., Ries, A., Mann, K., Kuhn, K., Timpl, R., Bourenkov, G. P., Bartunik, H. D., and Bode, W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6607-6612); however, at the higher resolution of 1.5 A, we found no evidence for this cross-link (Vanacore, R. M., Shanmugasundararaj, S., Friedman, D. B., Bondar, O., Hudson, B. G., and Sundaramoorthy, M. (2004) J. Biol. Chem. 279, 44723-44730). Given this discrepancy in crystallographic findings, we sought chemical evidence for the location and nature of the reinforcement/cross-link site. Trypsin digestion of monomer and dimer subunits excised a approximately 5,000-Da complex that distinguished dimers from monomers; the complex was characterized by mass spectrometry, Edman degradation, and amino acid composition analyses. The tryptic complex, composed of two peptides of 44 residues derived from two alpha1 NC1 monomers, contained Met93 and Lys211 post-translationally modified to hydroxylysine (Hyl211). Truncation of the tryptic complex with post-proline endopeptidase reduced its size to 14 residues to facilitate characterization by tandem mass spectrometry, which revealed a covalent linkage between Met93 and Hyl211. The novel cross-link, termed S-hydroxylysyl-methionine, reflects at least two post-translational events in its formation: the hydroxylation of Lys211 to Hyl211 within the NC1 domain during the biosynthesis of alpha-chains and the connection of Hyl211 to Met93 between the trimeric NC1 domains of two adjoining triple-helical protomers, reinforcing the stability of collagen IV networks.     

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.     

A terminal energy acceptor of the phycobilisome: the 75,000-dalton polypeptide of Synechococcus 6301 phycobilisomes--a new biliprotein

A rapid procedure is described for the isolation of "linker" polypeptides (Lundell, D. J., R. C. Williams, and A. N. Glazer. 1981. J. Biol. Chem. 256:3580-3592) of cyanobacterial phycobilisomes. The 75,000-dalton component of the core of Synechococcus 6301 phycobilisomes isolated by this procedure has been shown to carry a bilin similar in spectroscopic properties to phycocyanobilin. "Renatured" 75,000-dalton polypeptide has absorption maxima at 610 and 665 nm and a fluorescence emission maximum at 676 nm, similar to that of intact phycobilisomes. A complex of allophycocyanin and a 40,000- dalton bilin-carrying fragment of the 75,000-dalton polypeptide, obtained by limited tryptic digestion, is described. This complex, which lacks allophycocyanin B, shows a fluorescence emission maximum at 676 nm. The above data indicate that the 75,000-dalton polypeptide functions as a terminal energy acceptor in the phycobilisome.     

Peroxisomes in human colon carcinomas. A cytochemical and biochemical study.

The presence of peroxisomes and their enzymic content were investigated and compared in healthy and neoplastic human colon epithelial cells using cytochemical studies at the ultrastructural level as well as biochemical analyses. Catalase-positive organelles were found to be more numerous in normal than in colonic neoplastic cells. Biochemical assays revealed that no D-aminoacid oxidase or L-alpha-hydroxyacid oxidase activity was detected in normal or tumor tissues. The specific activities of catalase, fatty-acyl CoA oxidase and enoyl-CoA hydratase/3 hydroxyacyl-CoA dehydrogenase (the so-called peroxisomal bifunctional enzyme of the beta-oxidation system) were found to be diminished in carcinoma cells compared with the control tissue. The fall in catalase activity correlated well with tumor stage according to Dukes, suggesting that this peroxisomal enzyme could be used as a potential prognostic marker.     

Stable complexes of axoplasmic vesicles and microtubules: protein composition and ATPase activity

Fast transport of axonal vesicles and organelles is a microtubule-associated movement (Griffin, J. W., K. E. Fahnestock, L. Price, and P. N. Hoffman, 1983, J. Neuroscience, 3:557-566; Schnapp, B. J., R. D. Vale, M. P. Sheetz, and T. S. Reese, 1984, Cell, 40:455-462; Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson, 1985, J. Cell Biol., 100:1736-1752). Proteins that mediate the interactions of axoplasmic vesicles and microtubules were studied using stable complexes of microtubules and vesicles (MtVC). These complexes formed spontaneously in vitro when taxol-stabilized microtubules were mixed with sonically disrupted axoplasm from the giant axon of the squid Loligo pealei. The isolated MtVCs contain a distinct subset of axoplasmic proteins, and are composed primarily of microtubules and attached membranous vesicles. The MtVC also contains nonmitochondrial ATPase activity. The binding of one high molecular mass polypeptide to the complex is significantly enhanced by ATP or adenyl imidodiphosphate. All of the axoplasmic proteins and ATPase activity that bind to microtubules are found in macromolecular complexes and appear to be vesicle-associated. These data allow the identification of several vesicle-associated proteins of the squid giant axon and suggest that one or more of these polypeptides mediates vesicle binding to microtubules.     

Development of crystalline peroxisomes in methanol-grown cells of the yeast Hansenula polymorpha and its relation to environmental conditions

The development of peroxisomes has been studied in cells of the yeast Hansenula polymorpha during growth on methanol in batch and chemostat cultures. During bud formation, new peroxisomes were generated by the separation of small peroxisomes from mature organelles in the mother cells. The number of peroxisomes migrating to the buds was dependent upon environmental conditions. Aging of cells was accompanied by an increase in size of the peroxisomes and a subsequent increase in their numbers per cell. Their ultimate shape and substructure as well as their number per cell was dependent upon the physiological state of the culture. The change in number and volume density of peroxisomes was related to the level of alcohol oxidase in the cells. Development of peroxisomes in cells of batch cultures was accompanied by an increase in size of the crystalline inclusions in the organelles; they had become completely crystalline when the cells were in the stationary phase. Peroxisomes in cells from methanol-limited chemostat cultures were completely crystalline, irrespective of growth rate. Results of biochemical and cytochemical experiments suggested that alcohol oxidase is a major component of the crystalline inclusions in the peroxisomes of methanol-grown Hansenula polymorpha. Possible mechanisms involved in the ultrastructural changes in peroxisomes during their development have been discussed.Abbreviations DAB 3,3-diaminobenzidine - OD optical density (663 nm)     

COX8, the structural gene for yeast cytochrome c oxidase subunit VIII. DNA sequence and gene disruption indicate that subunit VIII is required for maximal levels of cellular respiration and is derived from a precursor which is extended at both its NH2 and COOH termini

From the amino acid sequence of yeast cytochrome c oxidase subunit VIII published previously (Power, S. D., Lochrie, M.A., Patterson, T.E., and Poyton, R.C. (1984) J. Biol. Chem. 259, 6571-6574), we have synthesized a pair of oligonucleotide probes and used them to identify COX8, its structural gene. By genomic Southern blot analysis and disruption of the COX8 chromosomal locus, we have shown that this gene is present in one copy per haploid genome and that its product, subunit VIII, is essential for maximal levels of cellular respiration and cytochrome c oxidase activity. Alignment of the amino acid sequence predicted from the DNA sequence of COX8 with the determined amino acid sequence of subunit VIII indicates that mature subunit VIII is derived from a larger precursor that extends from both the NH2 and COOH termini of the mature polypeptide. Thus, like many other nuclear coded mitochondrial proteins, subunit VIII is derived from a precursor which carries a leader peptide. In addition, this precursor, like that for yeast cytochrome c oxidase subunit VIIa, appears to carry a four-amino acid "trailer peptide" at its COOH terminus.     

Isolation and characterization of peroxisomes from the renal cortex of beef, sheep, and cat

The isolation and characterization of highly purified and structurally well-preserved peroxisomes from the renal cortex of different mammalian species (beef, sheep, and cat) is reported. Renal cortex tissue was homogenized and a peroxisome-enriched light mitochondrial fraction was prepared by differential centrifugation. This was subfractionated by density-dependent banding on a linear gradient of metrizamide (1.12-1.26 g/cm3) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at a mean density of 1.225 cm3. Ultrastructural morphometric examination revealed that peroxisomes made up 97 to 98% of the isolated fractions. By biochemical analysis the contamination with marker enzymes of mitochondria and lysosomes was extremely low. The specific activity of catalase was enriched, depending on the species, between 28- and 38-fold over the homogenate. Peroxisome preparations from all three species exhibited a high but varying level of activity for cyanide-insensitive lipid beta-oxidation. In beef and sheep preparations a small amount of esterase activity cosediments with peroxisomes. These peroxisomes show distinct structural membrane associations with smooth elements of ER. Urate oxidase, a marker enzyme for rat liver peroxisomes, is found only in peroxisomes prepared from beef kidney cortex, with sheep and cat preparations being negative. This correlated with the occurrence of polytubular inclusions in the beef kidney peroxisomes. The large size and the angular shape of isolated peroxisomes as well as the presence of paracrystalline matrical inclusions imply that the majority of peroxisomes are derived from the epithelial cells of the proximal tubule of the kidney cortex. The significant differences found in the characteristics of the renal peroxisomes in three different species investigated, demonstrate the remarkable adaptability and plasticity of this organelle.     

An electron microscopical study of the development of peroxisomes during formation and germination of ascospores in the methylotrophic yeast Hansenula polymorpha

Ascospore formation was studied in liquid cultures of the yeast Hansenula polymorpha, previously grown under conditions in which the synthesis of alcohol oxidase was repressed (glucose as growth substrate) or derepressed (methanol, glycerol and dihydroxyacetone as growth substrates and after growth on malt agar plates). In ascospores obtained from repressed cells, generally one small peroxisome was present. The organelle probably originated from the small peroxisome, originally present in the vegetative cells. They had no crystalline inclusions and cytochemical experiments indicated the presence of catalase, urate oxidase and amino acid oxidase activities in these organelles. In ascospores obtained from derepressed cells, generally 1–3 crystalline peroxisomes were observed. These organelles also originated from the peroxisomes originally present in the vegetative cells by means of fragmentation or division. They contained, in addition to the enzymes characteristic for peroxisomes in spores from repressed cells, also alcohol oxidase. The latter enzyme is probably responsible for the crystalline substructure of these peroxisomes.Peroxisomes had no apparent physiological function in the process of ascosporogenesis. A glyoxysomal function of the organelles during germination of the ascospores was also not observed. Germination of mature ascospores in media containing different sources of carbon and nitrogen showed that the function of the peroxisomes present in ascospores of Hansenula polymorpha is probably identical to that in vegetative haploid cells. They are involved in the oxidative metabolism of different carbon and nitrogen sources. Their enzyme profile is a reflection of that of peroxisomes of vegetative cells and their presence may enable the formation of cells which are optimally adapted to environmental conditions extant during spore germination.