MICROBODIES IN EXPERIMENTALLY ALTERED CELLS : II. The Relationship of Microbody Proliferation to Endocrine Glands

The liver cells of intact male rats given ethyl-α-p-chlorophenoxyisobutyrate (CPIB) characteristically show a marked increase in microbodies and in catalase activity, while those of intact female rats do not. In castrated males given estradiol benzoate and CPIB the increase in catalase activity and microbody proliferation is abolished, while in castrated females given testosterone propionate and CPIB the livers show a marked increase in microbodies and in catalase activity. No sex difference in microbody and catalase response is apparent in fetal and neonatal rats. Both sexes show a sharp rise in catalase activity on the day of birth, with a rapid decline at 5 days after birth. Thyroidectomy abolishes the hypolipidemic effect of CPIB in rats, but microbody proliferation and increase in catalase activity persists in thyroidectomized male rats, indicating that microbody proliferation can be independent of hypolipidemia. Adrenalectomy does not alter appreciably the microbody-catalase response to CPIB. These experiments demonstrate that (1) in adult rats, hepatic microbody proliferation is dependent to a significant degree upon male sex hormone but is largely independent of thyroid or adrenal gland hormones; (2) hepatic microbody proliferation is independent of the hypolipidemic effect of CPIB; (3) displacement of thyroxine from serum protein may not be sufficient cause for stimulation of microbody formation.     

NAFENOPIN-INDUCED HEPATIC MICROBODY (PEROXISOME) PROLIFERATION AND CATALASE SYNTHESIS IN RATS AND MICE : Absence of Sex Difference in Response

Nafenopin (2-methyl-2[p-(1,2,3,4-tetrahydro-1-naphthyl)phenoxy]-propionic acid; Su-13437), a potent hypolipidemic compound, was administered in varying concentrations in ground Purina Chow to male and female rats, wild type (Cs^a strain) mice and acatalasemic (Cs^b strain) mice to determine the hepatic microbody proliferative and catalase-inducing effects. In all groups of animals, administration of nafenopin at dietary levels of 0.125% and 0.25% produced a significant and sustained increase in the number of peroxisomes. The hepatic microbody proliferation in both male and female rats and wild type Cs^a strain mice treated with nafenopin was of the same magnitude and was associated with a two-fold increase in catalase activity and in the concentration of catalase protein. The increase in microbody population in acatalasemic mice, although not accompanied by increase in catalase activity, was associated with a twofold increase in the amount of catalase protein. The absence of sex difference in microbody proliferative response in nafenopin-treated rats and wild type mice is of particular significance, since ethyl-α-p-chlorophenoxyisobutyrate (CPIB)-induced microbody proliferation and increase in catalase activity occurred only in males. Nafenopin can, therefore, be used as an inducer of microbody proliferation and of catalase synthesis in both sexes of rats and mice. The serum glycerol-glycerides were markedly lowered in all the animals given nafenopin, which paralleled the increase in liver catalase. All the above effects of nafenopin were fully reversed when the drug was withdrawn from the diet of male rats. During reversal, several microbody nucleoids were seen free in the hyaloplasm or in the dilated endoplasmic reticulum channels resulting from a rapid reduction in microbody matrix proteins after the withdrawal of nafenopin from the diet. Because of microbody proliferation and catalase induction with increasing number of hypolipidemic compounds, additional studies are necessary to determine the interrelationships of microbody proliferation, catalase induction, and hypolipidemia.     

How proteins get into microbodies (peroxisomes, glyoxysomes, glycosomes)

All microbody proteins studies, including one microbody membrane protein, are made on free polysomes and imported post-translationally. This holds for animal tissues, plants, and fungi. The majority of microbody protein sub-units are synthesized in a form not detectably different from mature sub-units. In five cases a larger precursor protein has been found. The position of the extra piece in this precursor is not known. In two of the five cases, processing of the precursor is not coupled to import; in the other three this remains to be determined. It is not even known whether information in the prepiece contributes to topogenesis, or serves other purposes. Microbody preparations from Neurospora, plant tissue and rat liver can take up some newly synthesized microbody proteins in vitro. In most cases uptake is inefficient. No special requirements for uptake have been established and whether a receptor is involved is not yet known. Several examples have been reported of peroxisomal enzymes with a counterpart in another cell compartment. With the exception of catalase, no direct evidence is available in any of these cases for two isoenzymes specified by the same gene. In the Zellweger syndrome, a lethal hereditary disease of man, characterized by a lack of peroxisomes, the levels of several enzymes of lipid metabolism are strongly decreased. In contrast, D-amino-acid oxidase, L-alpha-hydroxyacid oxidase and catalase levels are normal. The catalase resides in the cytosol. Since there is no separate gene for cytosolic catalase, the normal catalase levels in Zellweger cells show that some peroxisomal enzymes can mature and survive stably in the cytosol. It is possible that maturation of the peroxisomal enzyme in the cytoplasm can account for the finding of cytosolic catalase in some normal mammalian cells. The glycosomes of trypanosomes are microbodies that contain a glycolytic system. Comparison of the glycosomal phosphoglycerate kinase with its cytosolic counterpart has shown that these isoenzymes are 93% homologous in amino-acid sequence, but less than 50% homologous to the corresponding enzymes of yeast and mammals. This implies that few alterations are required to direct a protein into microbodies. This interpretation is supported by the evidence for homology between some microbody and mitochondrial isoenzymes in other organisms mentioned under point 4. The major changes of the glycosomal phosphoglycerate kinase relative to the cytosolic enzyme are a large increase in positive charge and a C-terminal extension of 20 amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Microbody proliferation and segregation cycle in the single-microbody alga Cyanidioschyzon merolae

The proliferation cycle of the microbody was studied in the primitive red alga Cyanidioschyzon merolae, which contains one microbody per cell. Cells were synchronized with a dark/light cycle, and the morphology of the microbody and its interaction with other organelles were observed three-dimensionally by fluorescence microscopy, transmission electron microscopy, and computer-assisted three-dimensional reconstruction of serial thin sections. The microbody in interphase cells is a sphere of 0.3 μm in diameter without a core. In M-phase, the microbody passes through a series of irregular shapes, in the order rod, worm, branched, H-shaped and dumbbell, and symmetric fission occurs just before cytokinesis. The microbody duplicates its volume in M-phase and three-dimensional quantitative analysis revealed that its surface area increases before its volume does. The microbody touches the mitochondrion and the chloroplast throughout its proliferation cycle, except briefly in interphase cells, winding around the divisional plane of the mitochondrion at one phase. Immunocytochemical labeling of catalase as a marker of matrix proteins of the microbody revealed that the duplication of catalase occurs in tandem with the volume increase. While no specific apparatus was identified in the microbody divisional areas, we identified an electron-dense apparatus about 30–50 nm in diameter between the microbody and the mitochondrion that may play a role in segregating the daughter microbodies. These results are the first characterization to show the morphological changes of one microbody in a one-microbody alga without proliferation-inducing substrates, which have been used in many studies, and clearly show that two daughter microbodies arise by binary fission of the pre-existing microbody. Received: 11 November 1998 / Accepted: 22 December 1998     

Microbody proliferation in liver induced by nafenopin, a new hypolipidemic drug: comparison with CPIB

Nafenopin (2-methyl-2[P-(1,2,3,4-tetrahydro-1 naphthyl) phenoxy]- propionic acid, a phenolic ether with hypolipidemic properties, when administered by gavage at 100 mg/kg b wt daily for 1 to 2 weeks, caused a significant increase in the number of microbody profiles and simultaneous increase in catalase activity in livers of male rats. The concentration of catalase protein and the rate of incorporation of H³-δ-aminolevulinic acid into catalase fraction, as determined by immunochemical methods were approximately twice that of controls. The microbody proliferation resulting from nafenopin treatment was comparable to that induced by CPIB.     

Developmental Studies on Microbodies in Wheat Leaves : II. Ontogeny of Particulate Enzyme Associations

Crude particulate fractions from wheat leaves (Triticum vulgare L.) were separated on continuous sucrose density gradients, resulting in: broken chloroplasts, a mitochondrial fraction (indicated by cytochrome c oxidase), and microbodies. The visible band of the microbody fraction from adult leaves appears at a buoyant density of 1.25 grams per cm³ and contains most of the activities of catalase, glycolate oxidase, and hydroxypyruvate reductase on the gradient. In the shoots of freshly soaked seeds, catalase is already highly particulate. During further development in light or in darkness, 40 to 60% of the total activities of catalase and glycolate oxidase and 25 to 40% of the total activity of hydroxypyruvate reductase are always found in the particulate fractions of the leaves. In young developmental stages, the peaks of the activity profiles of the microbody enzymes appear on sucrose gradients at relatively low densities, first between 1.17 to 1.20 grams per cm³. During development in light, the buoyant density of the microbody fraction shifts to the final value of 1.25 grams per cm³. However, even after 1 week of growth in the dark, the microbody fraction from etiolated leaves was observed at buoyant densitites 1.17 to 1.24 grams per cm³ and did not appear as a defined visible band. A characteristic visible microbody band at a buoyant density 1.24 grams per cm³ was found when the dark-grown seedlings received only three separate 5-minute exposures to white light. A similar peak was also obtained from light-grown leaves in which chloroplast development had been blocked by 3-amino-1,2,4-triazole.     

The peroxisome (microbody) membrane: effects of detergents and lipid solvents on its ultrastructure and permeability to catalase

Synopsis The effects of detergents, organic lipid solvents, and several adjuvants used in cell fractionation on the ultrastructure of the peroxisomal (microbody) membrane and its permeability to catalase have been investigated. Chopper sections of glutaraldehyde-fixed liver were incubated in the presence of various agents, followed by cytochemical staining for catalase and processed for electron microscopy. Catalase activity was also determined biochemically in the incubation medium. Marked catalase diffusion was found after treatment with 1% or 0.5% Triton X-100 or deoxycholate, as well as with 50% ethanol or acetone or 20% propanol ort-butanol. In contrast, 1% digitonin and lower concentrations of the above agents, as well as sucrose or glycerine caused selective diffusion of catalase from a limited population of peroxisomes. Tieatment with 10% polyvinylpyrrolidone (PVP), which has been used as a protective agent in the isolation of microbodies, did not produce any alteration in the fine structure and cytochemical appearance of peroxisomes. These findings concur with earlier biochemical studies on freshly isolated peroxisomes and demonstrate the susceptibility of microbodies, even in glutaraldehyde-fixed rat liver to the effects of various agents which affect the microbody membrane. A close correlation between the ultrastructural integrity of the microbody membrane and its permeability to catalase has been found. The significance of these observations for the assessment of the permeability characteristics of the microbody membrane is discussed.     

Effects of catalase inhibitors on the ultrastructure and peroxidase activity of proliferating microbodies

Summary The relationship between the formation of microbodies and catalase synthesis in the hepatic cells of male rats was examined with conventional electron microscopy and with the peroxidase staining technic for demonstrating catalase. Daily intraperitoneal injections of ethyl--p-chlorophenoxyisobutyrate (CPIB) for 5 days caused a profound increase in microbody numbers without markedly affecting the appearance of the matrix material and all microbodies retained peroxidase activity. A single injection 5 days before sacrifice of 3-amino-1,2,4-triazole (AT), an inhibitor of catalase activity but not catalase synthesis, did not affect their numbers, appearance of matrix material or peroxidase staining. Twice daily injection for 5 days of allylisopropylacetamide (AIA), an inhibitor of catalase synthesis, also did not affect microbody numbers but lowered the electron-density of the microbody matrix and abolished peroxidase staining. After combined administration of these drugs, the number of hepatic microbodies increased but they did not contain peroxidase activity. The results suggest strongly that microbody proliferation is dependent not on catalase synthesis but on synthesis of non-enzymatic protein.This study was supported by research grant HD-01337 from the Institute of Child Health and Human Development, United States Public Health Service. The authors thank Mrs. Judith Henrickson, and Mr. Gerald Haiden for technical assistance. Dr. Legg is at present on leave from the Department of Anatomy, Monash University, Melbourne, Australia.     

PEROXIDASE ACTIVITY IN RAT LIVER MICROBODIES AFTER AMINO-TRIAZOLE INHIBITION

The in vivo effects of 3-amino-1,2,4-triazole (AT) on the fine structure of microbodies in hepatic cells of male rats has been studied by the peroxidase-staining technique. Within 1 hr of intraperitoneal injection AT abolishes microbody peroxidase-staining, and the return of staining coincides temporally with the known pattern of return of catalase activity following AT inhibition; this is further evidence that the peroxidase staining of microbodies is due to catalase activity. Peroxidase staining reappears in the microbody matrix without evidence of either massive degradation or rapid proliferation of the organelles. Furthermore, during the period of return of activity, ribosomal staining occurs adjacent to microbodies whose matrix shows little or no peroxidase staining. These observations are interpreted as evidence that (a) catalase is capable of entering preexisting microbodies without traversing the cisternae of the rough endoplasmic reticulum or the Golgi apparatus, and that (b) the ribosomal staining is probably not cytochemical diffusion artifact and may represent a localized site of synthesis or activation of catalase.     

NEW OBSERVATIONS ON MICROBODIES : A Cytochemical Study on CPIB-Treated Rat Liver

The liver of male rats has been studied after CPIB stimulation by using the peroxidase reaction for localizing catalase in hepatic cells. CPIB administration leads to an increase in the number of microbodies, and it is suggested that one mechanism by which microbody proliferation occurs is a process of fragmentation or budding from preexisting microbodies. Reaction product was observed not only within the microbody matrix, but outside the limiting membrane of the microbody and in association with ribosomes of adjacent rough endoplasmic reticulum. This localization of reaction product is interpreted as evidence that catalase after synthesis on rough endoplasmic reticulum may accumulate near microbodies and may be transferred directly into these organelles without traversing the cisternae of the endoplasmic reticulum or Golgi apparatus.     

Developmental studies on microbodies in wheat leaves : I. Conditions influencing enzyme development

Catalase, glycolate oxidase, and hydroxypyruvate reductase, enzymes which are located in the microbodies of leaves, show different developmental patterns in the shoots of wheat seedlings. Catalase and hydroxypyruvate reductase are already present in the shoots of ungerminated seeds. Glycolate oxidase appears later. All three enzymes develop in the dark, but glycolate oxidase and hydroxypyruvate reductase have only low activities. On exposure of the seedlings to continuous white light (14.8 × 10³ ergs cm⁻² sec⁻¹), the activity of catalase is doubled, and glycolate oxidase and hydroxypyruvate reductase activities increase by 4- to 7-fold. Under a higher light intensity, the activities of all three enzymes are considerably further increased. The activities of other enzymes (cytochrome oxidase, fumarase, glucose-6-phosphate dehydrogenase) are unchanged or only slightly influenced by light. After transfer of etiolated seedlings to white light, the induced increase of total catalase activity shows a much longer lag-phase than that of glycolate oxidase and hydroxypyruvate reductase. It is concluded that the light-induced increases of the microbody enzymes are due to enzyme synthesis. The light effect on the microbody enzymes is independent of chlorophyll formation or the concomitant development of functional chloroplasts. Short repeated light exposures which do not lead to greening are very effective. High activities of glycolate oxidase and hydroxypyruvate reductase develop in the presence of 3-amino-1,2,4-triazole which blocks chloroplast development. The effect of light is not exerted through induced glycolate formation and appears instead to be photomorphogenetic in character.     

Identification of glycosomes and metabolic end products in pathogenic and nonpathogenic strains of Cryptobia salmositica (Kinetoplastida: Bodonidae)

Whole cell lysates of pathogenic and nonpathogenic strains of Cryptobia salmositica were subjected to subcellular fractionation using differential and isopycnic centrifugation in sucrose. The glycolytic enzymes hexokinase, fructose-1,6-biphosphate aldolase, triosephosphate isomerase, glucosephosphate isomerase and glyceraldehyde-3-phosphate-dehydrogenase and the peroxisomal enzyme catalase were associated with a microbody that had a buoyant density in sucrose of 1.21 g cm-3. Lactate dehydrogenase was detected in whole cell lysates, but not in purified organelles. A microbody with a positive reaction for catalase was detected in electron microscope sections of the pathogenic and nonpathogenic strains. These catalase-containing microbodies fused with lipid bodies and vacuoles, arose by division from pre-existing microbodies and expelled their contents into the cytoplasm of the cell. Both strains also modified the catalase content in their microbodies. Under aerobic conditions, they metabolized glucose to pyruvate and lactate. We conclude that part of the glycolytic pathway in C. salmositica is compartmentalized in a microbody called the glycosome.     

Untersuchungen zur Funktionsänderung der Microbodies in den Keimblättern von Helianthus annuus L.

Summary The enzyme patterns in sunflower cotyledons indicate that the glyoxysomal function of microbodies is replaced by the peroxisomal function of these organelles during the transition from fat degradation to photosynthesis. The separation of the microbody population into glyoxysomes and peroxisomes during this transition period is reported. The mean difference in density between the activity peaks of glyoxysomal and peroxisomal marker enzymes on a sucrose gradient was calculated to be 0.007±0.004 g/cm³ and turned out to be significant (t=7.8>4.04=t _5;0.01). The activity peak of catalase coincides with that of isocitrate lyase in early stages of development, but shifts to the activity peak of peroxisomal marker enzymes during the transition period. No isozymes of the catalase could be detected by gel electrophoresis in the microbodies with the two different functions.During the rise of the peroxisomal marker enzymes no synthesis of the common microbody marker, catalase, could be demonstrated using the inhibitor allylisopropylacetamide. Using D₂) for density labeling of newly-formed catalase, no difference is observed between the density of catalase from cotyledons grown on 99.8% D₂O during the transition period and the density of enzyme from cotyledons grown on H₂O. The activity of particulate glycolate oxidase is reduced 30–50% by allylisopropylacetamide, but is not affected by D₂O. The chlorophyll formation in the cotyledons is strongly inhibited by both substances.     

Microbody of methanol-grown yeasts. Localization of catalase and flavin-dependent alcohol oxidase in the isolated microbody.

Profuse appearance of microbodies was observed in the cells of methanol-utilizing yeasts in connection with the enhanced catalase activity. These microbodies were isolated successfully by means of sucrose gradient centrifugation from the methanol-grown cells of Kloeckera sp. no. 2201. Localization of a flavin-dependent alcohol oxidase as well as characteristic microbody enzymes (catalase and D-amino acid oxidase) were ascertained in the isolated microbodies, whereas formaldehyde and formate dehydrogenases were detected in the cytoplasmic region. Localization of catalase in the isolated microbody was also demonstrated by the cytochemical technique with 3,3'-diaminobenzidine.     

Close association of centrosomes to the distal ends of the microbody during its growth, division and partitioning in the green alga Klebsormidium flaccidum

Summary. Division and partitioning of microbodies (peroxisomes) of the green alga Klebsormidium flaccidum, whose cells contain a single microbody, were investigated by electron microscopy. In interphase, the rod-shaped microbody is present between the nucleus and the single chloroplast, oriented perpendicular to the pole-to-pole direction of the future spindle. A centriole pair associates with one distal end of the microbody. In prophase, the microbody changes not only in shape, from a rodlike to a branched form, but also in orientation, from perpendicular to parallel to the future pole-to-pole direction. Duplicated centriole pairs are localized in close proximity to both distal ends of the microbody. In metaphase, the elongated microbody flanks the open spindle, with both distal ends close to the centriole pair at either spindle pole. The microbody further elongates in telophase and divides after septum formation (cytokinesis) has started. The association between the centrioles and both distal ends of the microbody is maintained throughout mitosis, resulting in the distal ends of the elongated microbody being fixed at the cellular poles. This configuration of the microbody may be favorable for faithful transmission of the organelle during cell division. After cytokinesis is completed, the microbody reverts to the perpendicular orientation by changing its shape. Microtubules radiating from the centrosomes flank the side of the microbody throughout mitosis. The close association of centrosomes and microtubules with the microbody is discussed in respect to the partitioning of the microbody in this alga. Correspondence: H. Hashimoto, Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan. Present address: M. Honda, Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan.     

CYTOCHEMICAL LOCALIZATION OF PEROXIDATIC ACTIVITY OF CATALASE IN RAT HEPATIC MICROBODIES (PEROXISOMES)

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 H₂O₂, 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 H₂O₂ appreciable staining of microbodies was noted only after prolonged incubation. Addition of sodium pyruvate, which inhibits endogenous generation of H₂O₂ by tissue oxidases, or of crystalline catalase, which decomposes such tissue-generated H₂O₂, completely abolished microbody staining in the absence of H₂O₂. 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.     

Ordered Regions of Channel Nucleoporins Nup62, Nup54, and Nup58 Form Dynamic Complexes in Solution

Three out of ∼30 nucleoporins, Nup62, Nup54, and Nup58, line the nuclear pore channel. These “channel” nucleoporins each contain an ordered region of ∼150–200 residues, which is predicted to be segmented into 3–4 α-helical regions of ∼40–80 residues. Notably, these segmentations are evolutionarily conserved between uni- and multicellular eukaryotes. Strikingly, the boundaries of these segments match our previously reported mapping and crystal data, which collectively identified two “cognate” segments of Nup54, each interacting with cognate segments, one in Nup58 and the other one in Nup62. Because Nup54 and Nup58 cognate segments form crystallographic hetero- or homo-oligomers, we proposed that these oligomers associate into inter-convertible “mid-plane” rings: a single large ring (40–50 nm diameter, consisting of eight hetero-dodecamers) or three small rings (10–20 nm diameter, each comprising eight homo-tetramers). Each “ring cycle” would recapitulate “dilation” and “constriction” of the nuclear pore complex''s central transport channel. As for the Nup54·Nup62 interactome, it forms a 1:2 triple helix (“finger”), multiples of which project alternately up and down from mid-plane ring(s). Collectively, our previous crystal data suggested a copy number of 128, 64, and 32 for Nup62, Nup54, and Nup58, respectively, that is, a 4:2:1 stoichiometry. Here, we carried out solution analysis utilizing the entire ordered regions of Nup62, Nup54, and Nup58, and demonstrate that they form a dynamic “triple complex” that is heterogeneously formed from our previously characterized Nup54·Nup58 and Nup54·Nup62 interactomes. These data are consistent both with our crystal structure-deduced copy numbers and stoichiometries and also with our ring cycle model for structure and dynamics of the nuclear pore channel.     

Uncovering a Microbial Enigma: Isolation and Characterization of the Streamer-Generating,Iron-Oxidizing,Acidophilic Bacterium “Ferrovum myxofaciens”

A betaproteobacterium, shown by molecular techniques to have widespread global distribution in extremely acidic (pH 2 to 4) ferruginous mine waters and also to be a major component of “acid streamer” growths in mine-impacted water bodies, has proven to be recalcitrant to enrichment and isolation. A modified “overlay” solid medium was devised and used to isolate this bacterium from a number of mine water samples. The physiological and phylogenetic characteristics of a pure culture of an isolate from an abandoned copper mine (“Ferrovum myxofaciens” strain P3G) have been elucidated. “F. myxofaciens” is an extremely acidophilic, psychrotolerant obligate autotroph that appears to use only ferrous iron as an electron donor and oxygen as an electron acceptor. It appears to use the Calvin-Benson-Bassham pathway to fix CO₂ and is diazotrophic. It also produces copious amounts of extracellular polymeric materials that cause cells to attach to each other (and to form small streamer-like growth in vitro) and to different solid surfaces. “F. myxofaciens” can catalyze the oxidative dissolution of pyrite and, like many other acidophiles, is tolerant of many (cationic) transition metals. “F. myxofaciens” and related clone sequences form a monophyletic group within the Betaproteobacteria distantly related to classified orders, with genera of the family Nitrosomonadaceae (lithoautotrophic, ammonium-oxidizing neutrophiles) as the closest relatives. On the basis of the phylogenetic and phenotypic differences of “F. myxofaciens” and other Betaproteobacteria, a new family, “Ferrovaceae,” and order, “Ferrovales,” within the class Betaproteobacteria are proposed. “F. myxofaciens” is the first extreme acidophile to be described in the class Betaproteobacteria.     

Distribution of 59- and 55-kDa Catalase in Dark- and Light-grown Pumpkin and Various Other Plant Tissues

The catalase molecule in germinating pumpkin cotyledons is synthesizedas a precursor (59-kDa) form, whose relative molecular massis larger than the mature enzyme (55-kDa). Although both typesof molecules are localized in the microbodies, the 59-kDa specieshas been shown to be present predominantly in the leaf peroxisomesisolated from green cotyledons, while the 55-kDa species ispredominantly in the glyoxysomes from etiolated cotyledons [Yamaguchiet al. (1984) Proc. Natl. Acad. Sci. USA, 81: 4809]. We examinedthe distribution of the 59- and 55-kDa catalase molecules indark- and light-grown tissues of pumpkin seedlings as well asin other plant species, using the immunoblotting technique.The ratios of the 59- and 55-kDa catalase species differed inthe pumpkin tissues examined. Light interferes with the conversionof the 59-kDa precursor to the 55-kDa form, especially in thecotyledons. The effect of light was less pronounced in the rootsand hypocotyls, indicating that the light regulation of theconversion is tissue-specific. Dark- and light-grown cotyledonsfrom cucumber and watermelon seedlings showed a similar lightregulation, suggesting that cucurbitaceous plants possess similarlight-regulatory mechanism. From the analysis of catalase proteinfrom various plant tissues, a limited correlation between molecularforms of catalase and different microbody populations was observed. (Received September 6, 1986; Accepted December 4, 1986)