Catalase Synthesis and Turnover during Peroxisome Transition in the Cotyledons of Helianthus annuus L

Based on measurements of total catalase hematin and the degradation constants of catalase hematin, zero order rate constants for the synthesis of catalase were determined during the development of sunflower cotyledons (Helianthus annuus L.). Catalase synthesis reached a sharp maximum of about 400 picomoles hematin per day per cotyledon at day 1.5 during the elaboration of glyoxysomes in the dark. During the transition of glyoxysomes to leaf peroxisomes (greening cotyledons, day 2.5 to 5) catalase synthesis was constant at a level of about 30 to 40 picomoles hematin per day per cotyledon. In the cotyledons of seedlings kept in the dark (day 2.5 to 5) catalase synthesis did not exceed 10 picomoles hematin per day per cotyledon. During the peroxisome transition in the light, total catalase hematin was maintained at a high level, whereas total catalase activity rapidly decreased. In continuous darkness, total catalase hematin decreased considerably from a peak at day 2. The results show that both catalase synthesis and catalase degradation are regulated by light. The turnover characteristics of catalase are in accordance with the concept that glyoxysomes are transformed to leaf peroxisomes as described by the one population model and contradict the two population model and the enzyme synthesis changeover model which both postulate de novo formation of the leaf peroxisome population and degradation of the glyoxysome population.     

Catalase Degradation in Sunflower Cotyledons during Peroxisome Transition from Glyoxysomal to Leaf Peroxisomal Function

First order rate constants for the degradation (degradation constants) of catalase in the cotyledons of sunflower (Helianthus annuus L.) were determined by measuring the loss of catalase containing ¹⁴C-labeled heme. During greening of the cotyledons, a period when peroxisomes change from glyoxysomal to leaf peroxisomal function, the degradation of glyoxysomal catalase is significantly (P = 0.05) slower than during all other stages of cotyledon development in light or darkness. The degradation constant during the transition stage of peroxisome function amounts to 0.205 day⁻¹ in contrast to the constants ranging from 0.304 day⁻¹ to 0.515 day⁻¹ during the other developmental stages. Density labeling experiments comprising labeling of catalase with ²H₂O and its isopycnic centrifugation on CsCl gradients demonstrated that the determinations of the degradation constants were not substantially affected by reutilization of ¹⁴C-labeled compounds for catalase synthesis. The degradation constants for both glyoxysomal catalase and catalase synthesized during the transition of peroxisome function do not differ. This was shown by labeling the catalases with different isotopes and measuring the isotope ratio during the development of the cotyledons. The results are inconsistent with the concept that an accelerated and selective degradation of glyoxysomes underlies the change in peroxisome function. The data suggest that catalase degradation is at least partially due to an individual turnover of catalase and does not only result from a turnover of the whole peroxisomes.     

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.     

Glycerol phosphate dehydrogenase in mammalian peroxisomes

Peroxisomes isolated on sucrose density gradients from homogenates of rat, chicken, or dog livers and rat kidney contained NAD⁺:α-glycerol phosphate dehydrogenase. Since the amount of sucrose in the peroxisomal fraction inhibited the enzyme activity about 70%, it was necessary to remove the sucrose by dialysis. About 8.4% of the total dehydrogenase of rat livers was in the surviving intact peroxisomes after homogenation. If corrected for particle breakage, this represented approximately 21% of the total activity. About 9.5% of the total enzyme was isolated in rat kidney peroxisomes, and because of severe particle rupture may represent over half of the total activity. No glycerol phosphate dehydrogenase was found in spinach leaf peroxisomes. A specific activity of 326 nmoles min^?1 mg^?1 protein in the rat liver peroxisomal fraction was at least twice that in the cytoplasm. NAD⁺:α-glycerol phosphate dehydrogenase was also present in a membrane fraction which was not identified, but none was in the mitochondria. The liver peroxisomal and cytoplasmic NAD⁺:α-glycerol phosphate dehydrogenase moved similarly on polyacrylamide gels and each resolved into two adjacent bands.Malate dehydrogenase was not found in peroxisomes from liver and kidney of rats and pigs, but 1–2% of the total particulate malate dehydrogenase was present in the peroxisomal area of the gradient from dog livers. However, this malate dehydrogenase in dog peroxisomal fractions did not exactly coincide with the peroxisomal marker, catalase. Malate dehydrogenase in dog liver mitochondria and in the peroxisomal fraction had similar pH optima and K_m values and migrated similarly to the anode at pH 6.5 on starch gels as a major and a minor band. The cytoplasmic malate dehydrogenase had a different pH optimum and K_m value and resolved into five different isoenzymes by electrophoresis. It is concluded that NAD⁺:α-glycerol phosphate dehydrogenase is in peroxisomes of liver and kidney, whereas malate dehydrogenase, present in peroxisomes of plants, is apparently absent in animal peroxisomes.     

Purification of glyoxysomal catalase and immunochemical comparison of glyoxysomal and leaf peroxisomal catalase in germinating pumpkin cotyledons

As a step to study the mechanism of the microbody transition (glyoxysomes to leaf peroxisomes) in pumpkin (Cucurbita sp. Amakuri Nankin) cotyledons, catalase was purified from glyoxysomes. The molecular weight of the purified catalase was determined to be 230,000 to 250,000 daltons. The enzyme was judged to consist of four identical pieces of the monomeric subunit with molecular weight of 55,000 daltons. Absorption spectrum of the catalase molecule gave two major peaks at 280 and 405 nanometers, showing that the pumpkin enzyme contains heme. The ratio of absorption at 405 and 280 nanometers was 1.0, the value being lower than that obtained for catalase from other plant sources. These results indicate that the pumpkin glyoxysomal catalase contains the higher content of heme in comparison with other plant catalase.

The immunochemical resemblance between glyoxysomal and leaf peroxisomal catalase was examined by using the antiserum specific against the purified enzyme preparation from pumpkin glyoxysomes. Ouchterlony double diffusion and immunoelectrophoretic analysis demonstrated that catalase from both types of microbodies cross-reacted completely whereas the immunotitration analysis showed that the specific activity of the glyoxysomal catalase was 2.5-fold higher than that of leaf peroxisomal catalase. Single radial immunodiffusion analysis showed that the specific activity of catalase decreased during the greening of pumpkin cotyledons.

     

Characterization of biotin and 3-methylcrotonyl-coenzyme a carboxylase in higher plant mitochondria

Mitochondria from green pea (Pisum sativum) leaves were purified free of peroxisomes and chlorophyll contamination and examined for their biotin content. The bulk of the bound biotin detected in plant mitochondria was shown to be associated with the matrix space to a concentration of about 13 micromolar, and no free biotin was detected. Western blot analysis of mitochondrial polypeptides using horseradish peroxidase-labeled streptavidin revealed a unique biotin-containing polypeptide with a molecular weight of 76,000. This polypeptide was implicated as being the biotinylated subunit of 3-methylcrotonyl-coenzyme A (CoA) carboxylase. Fractionation of pea leaf protoplasts demonstrated that this enzyme activity was located largely in mitochondria. The 3-methylcrotonyl-CoA carboxylase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of mitochondria. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8.3 in the presence of Mg²⁺. Kinetic constants (apparent K_m values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 0.05 millimolar; ATP, 0.16 millimolar; HCO₃⁻, 2.2 millimolar. The involvement of 3-methylcrotonyl-CoA carboxylase in the leucine degradation pathway in plant mitochondria is proposed.     

Synthesis of Isocitrate Lyase in Sunflower Cotyledons during the Transition in Cotyledonary Microbody Function

Density-labeling with 10 millimolar K¹⁵NO₃/70% ²H₂O has been used to investigate isocitrate lyase synthesis during greening of sunflower (Helianthus annuus L.) cotyledons when the glyoxysomal enzyme activities sharply decline and the transition in cotyledonary microbody function occurs. A density shift of 0.0054 (kilograms per liter) was obtained for the profile of isocitrate lyase activity in the CsCl gradient with respect to the ¹H₂O control. Quantitative evaluation of the density-labeling data indicates that about 50% of the isocitrate lyase activity present towards the end of the transition stage in microbody function is due to enzyme molecules newly synthesized during this stage.     

Lecithin synthesis during microbody biogenesis in watermelon cotyledons

During the normal development of watermelon seedlings, leaf peroxisomes succeed glyoxysomes as the major microbody component in the cotyledons. The possibility has thus been raised that the two organelles are ontogenetically related; that leaf peroxisomes are derived from glyoxysomes. The behavior of lecithin, an important constituent of the membranes of both kinds of organelle was examined in this study. Using labeled choline as a precursor of lecithin, its incorporation into various membrane fractions was followed during the period when glyoxysomal activity was declining and that of leaf peroxisomes increasing after exposure to light. The results showed that glyoxysomal membrane was selectively destroyed during this period. Furthermore, from double-labeling experiments using [¹⁴C]- and [³H]choline it was shown that newly synthesized lecithin was incorporated into the membranes of the developing leaf peroxisomes. These results support the thesis that leaf peroxisomes are not derived from glyoxysomes and instead represent two distinct microbody populations.     

In Vivo Synthesis and Turnover of alpha-Amylase in Attached and Detached Cotyledons of Vigna mungo Seeds

α-Amylase activity increased in attached cotyledons of germinated Vigna mungo seeds until the 5th day after imbibition and decreased thereafter, whereas in detached and incubated cotyledons the activity continuously increased and, at the 6th day, reached the value more than three times that of the maximum activity of attached cotyledons. Zymograms of the activities and Ouchterlony double immunodiffusion test on the activities of attached and detached cotyledons showed that the increase of activity in detached cotyledons was due to the identical enzyme as in attached tissues. α-Amylase contents, determined by single radial immunodiffusion method, changed in parallel with enzyme activity in both attached and detached cotyledons, which also suggested the de novo synthesis of α-amylase in V. mungo cotyledons.

The rate of incorporation of the label from [³H]leucine into α-amylase and the ratios of dpm in α-amylase/dpm in trichloroacetic acid-insoluble fraction did not show significant difference between attached and detached cotyledons. The results indicated that in attached cotyledons fluctuation of α-amylase activity was regulated by both synthesis and degradation of the enzyme, whereas in detached cotyledons α-amylase was synthesized and accumulated, because of low degrading activity during incubation.

     

Biochemical, electrophoretic and immunological characterization of peroxisomal enzymes in three soybean organs

Biochemical, electrophoretic and immunological studies were made among peroxisomal enzymes in three organs of soybean [Glycine max (L.) Merr. cv. Centennial] to compare the enzyme distribution and characteristics of specialized peroxisomes in one species. Leaves, nodules and etiolated cotyledons were compared with regard to several enzymes localized solely in their peroxisomes: catalase (EC 1.11.1.6), malate synthase (EC 4.1.3.2), glycolate oxidase (EC 1.1.3.1), and urate oxidase (EC 1.7.3.3). Catalase activity was found in all tissue extracts. Electrophoresis on native polyacrylamide gels indicated that leaf catalase migrated more anodally than nodule or cotyledon catalase as shown by both activity staining and Western blotting. Malate synthase activity and immunologically detectable protein were present only in the cotyledon extracts. Western blots of denaturing (lithium dodecyl sulfate) gels probed with anti-cotton malate synthase antiserum, reveal a single subunit of 63 kDa in both cotton and soybean cotyledons. Glycolic acid oxidase activity was present in all three organs, but ca 20-fold lower (per mg protein) in both nodule and cotyledon extracts compared to leaf extracts. Electrophoresis followed by activity staining on native gels indicated one enzyme form with the same mobility in nodule, cotyledon and leaf preparations. Urate oxidase activity was found in nodule extracts only. Native gel electrophoresis showed a single band of activity. Novel electrophoretic systems had to be developed to resolve the urate oxidase and glycolate oxidase activities; both of these enzymes moved cathodally in the gel system employed while most other proteins moved anodally. This multifaceted study of enzymes located within three specialized types of peroxisomes in a single species has not been undertaken previously, and the results indicate that previous comparisons between the enzyme content of specialized peroxisomes from different organisms are mostly consistent with that for a single species, soybean.     

The biosynthesis of ribonuclease and its accumulation in protein bodies in the cotyledons of mung bean seedlings

Germination and seedling growth of mung bean are accompanied by a 7- to 10-fold increase in the ribonuclease content of the cotyledons. The increase occurs during the first 4 days of seedling growth and precedes the senescence of the cotyledons. Separation of the RNases in the cotyledons by polyacrylamide gel electrophoresis indicates the presence of several minor bands in seeds imbibed for 24 hr. On the second day of seedling growth a new major band with an R_f of 0.76 is present. In 4- to 5-day old seedlings this major band accounts for nearly all the RNase activity in the tissue. The characteristics of this RNase show that it is a plant ribonuclease I (pH optimum of 5.0; MW 16,000; activity preferentially inhibited by purine nucleotides; no activity toward DNA; no phosphodiesterase activity). When the seedlings are grown in 66% D₂O the RNase activity undergoes a density shift of 0.61% indicating that the increase in enzyme activity is due to the de novo synthesis of the enzyme molecules. A method is described for the isolation of protein bodies from protoplasts of storage parenchyma cells. Fractionation of protoplast lysates on Ficoll gradients results in the recovery of a high proportion (75%) of intact protein bodies. On these gradients RNase activity comigrates with α-mannosidase, a protein body marker enzyme indicating that the newly synthesized RNase accumulates in the protein bodies. We suggest that the synthesis of RNase in the cotyledons and its accumulation in the protein bodies indicates that protein bodies may function in the degradation of cellular macromolecules other than the reserves stored within them.     

Turnover of catalase heme and apoprotein moieties in cotyledons of sunflower seedlings

The turnover of catalase apoprotein and catalase heme was studied in cotyledons of sunflower (Helianthus annuus L.) seedlings by density labeling of apoprotein and radioactive labeling of heme moieties. The heavy isotope (50% ²H₂O) and the radioactive isotope ([¹⁴C]5-aminolevulinic acid) were applied either during growth in the dark (day 0-2.5) or in the light (day 2.5 and 5). Following isopycnic centrifugation of catalase purified from cotyledons of 5-day-old seedlings, superimposition curve fitting was used to determine the amounts of radioactive heme moieties in native and density-labeled catalase. Data from these determinations indicated that turnover of catalase heme and apoprotein essentially was coordinate. Only small amounts of heme groups were recycled into newly synthesized apoprotein during growth in the light, and no evidence was found for an exchange of heme groups in apoprotein moieties. It followed from these observations that degradation of catalase apoprotein was slightly faster than that of catalase heme. A degradation constant for catalase apoprotein of 0.263 per day was determined from the data on heme recycling and the degradation constant of catalase heme determined previously to be 0.205 per day (R Eising, B Gerhardt [1987] Plant Physiol 84: 225-232).     

Development of enzymes in the cotyledons of watermelon seedlings

Changes in hypocotyl length, cotyledon weight, lipid content, chlorophyll content, and capacity for photosynthesis have been described in seedlings of Citrullus vulgaris, Schrad. (watermelon) growing at 30 C under various light treatments. Corresponding changes in the levels of 19 enzymes in the cotyledons are described, with particular emphasis on enzymes of microbodies, since during normal greening, enzymes of the glyoxysomes are lost and those of leaf peroxisomes appear. In complete darkness enzymes of the glyoxysomes reach a peak at 4 days and decline as the fat is depleted. Enzymes of mitochondria and of glycolytic pathways also peak at 4 to 5 days and either remain unchanged or decline to a lesser extent. Exposure to light at 4 days, when the cotyledons emerge, results in a selectively greater destruction of enzymes of the glyoxylate cycle; chlorophyll synthesis and capacity for photosynthesis increase in parallel, and there is a striking increase in the activities of chloroplast enzymes and in those of the leaf peroxisomes, hydroxypyruvate reductase and glycolate oxidase. The reciprocal changes in enzymes of the glyoxysomes and of leaf peroxisomes can be temporally dissociated, since even after 10 days in darkness, when malate synthetase and isocitrate lyase have reached very low levels, hydroxypyruvate reductase and glycolate oxidase increase strikingly on exposure to light and the cotyledons become photosynthetic. Furthermore, the parallel development of enzymes of leaf peroxisomes and functional chloroplasts is not immutable, since hydroxypyruvate reductase and glycolate oxidase activity can be elicited in darkness following a 5-minute exposure to light at day 4 while chlorophyll does not develop under these conditions.     

Enzymes of beta-Oxidation in Different Types of Algal Microbodies

The algae Mougeotia and Eremosphaera were used for isolation of microbodies with the characteristics of leaf peroxisomes and unspecialized peroxisomes, respectively. In both types of organelles, the following enzymes of the β-oxidation pathway were determined: acyl-CoA oxido-reductase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase. There are indications that the peroxisomal oxidoreductase of both algae is a H₂O₂-forming oxidase rather than a dehydrogenase.

The enzymes enoyl-CoA hydratase and acyl-CoA oxidoreductase are located also in the mitochondria from Eremosphaera but not from Mougeotia. The mitochondrial acyl-CoA oxidizing enzyme was found to be a dehydrogenase. The specific activities of acyl-CoA oxidase and enoyl-CoA hydratase are lower than in spinach leaf peroxisomes. However, the activity of 3-hydroxyacyl-CoA dehydrogenase in the peroxisomes of both algae is almost 2-fold higher. The capability for degradation of fatty acids is a common feature of all different types of peroxisomes from algae.

     

Light dependence of catalase synthesis and degradation in leaves and the influence of interfering stress conditions

The enzyme catalase (EC 1.11.1.6) is light sensitive and subject to a rapid turnover in light, similar to the D1 reaction center protein of photosystem II. After 3 h of preadaptation to darkness or to different light intensities (90 and 520 μmol m⁻² s⁻¹ photosynthetic photon flux density), sections of rye leaves (Secale cereale L.) were labeled for 4 h with l-[³⁵S]methionine. From leaf extracts, catalase was immunoprecipitated with an antiserum prepared against the purified enzyme from rye leaves. Both incorporation into catalase and degradation of the enzyme polypeptide during a subsequent 16-h chase period increased with light intensity. At a photon flux density of 520 μmol m⁻² s⁻¹, the apparent half-time of catalase in rye leaves was 3 to 4 h, whereas that of the D1 protein was much shorter, about 1.5 h. Exposure to stress conditions, such as 0.6 m NaCl or a heat-shock temperature of 40°C, greatly suppressed both total protein synthesis and incorporation of the label into catalase and into the D1 protein. Immunoblotting assays indicated that in light, but not in darkness, steady-state levels of catalase and of the D1 protein strongly declined during treatments with salt, heat shock, or translation inhibitors that block repair synthesis. Because of the common property of rapid photodegradation and the resulting dependence on continuous repair, declines in catalase as well as of the D1 protein represent specific and sensitive indicators for stress conditions that suppress the translational activities of leaves.     

Isolation of microbodies from plant tissues

Specialized microbodies have previously been isolated and characterized from fatty seedling tissues (glyoxysomes) and leaves (leaf peroxisomes). We have now examined 11 other plant tissues, including tubers, fruits, roots, shoots, and petals, and find that all contain particulate catalase, a distinctive common enzyme component of microbodies. On linear sucrose gradients the catalase activity peaks sharply at a higher equilibrium density (1.20 to 1.25 gram per cm³ in the various tissues) than the mitochondria (1.17 to 1.20). Only small amounts of protein are recovered in the fractions containing catalase, although a definite band is visible in preparations from some tissues, e.g., potato. As in the preparations from castor bean endosperm and spinach leaves for which comparable data are provided, the distribution of glycolate oxidase and uricase follows closely that of catalase on the gradients. The preparations from potato lack glyoxylate reductase and the transaminases, typical enzymes of leaf peroxisomes, and the distinctive enzymes of glyoxysomes are missing. Nonspecialized microbodies with limited enzyme composition can thus be isolated from a variety of plant tissues.     

β-galactosidase activity in the germinating seeds of Vigna sinensis

The β-galactosidase activity in cotyledons of Vigna sinensis increases during seed germination and is inhibited by cycloheximide. The increasing activity may be due to the de novo synthesis of enzyme protein. The enzyme has been partially purified by gel filtration and characterized with respect to some biochemical parameters. The optimum pH and optimum temperature are 4.5 and 55°, respectively and the enzymes follows typical Michaelis kinetics with K_m and V_max of 4.5 x 10^?4 M and 2.0 x 10^?5 mol/hr respectively. K_i for galactose and lactose are 4.5 and 220 mM, respectively. The energy of activation of the enzyme for p-nitrophenyl β-D-galactoside is 9.83 kcal/mol. The apparent relative MW of the enzyme as determined by gel filtration was 56000.     

Biogenesis and turnover of peroxisomes involved in the concurrent oxidation of methanol and methylamine in Hansenula polymorpha

Growth of Hansenula polymorpha in shake flasks and chemostat cultures in the presence of methanol as the sole source of carbon and methylamine as the sole source of nitrogen was associated with the development of peroxisomes in the cells. The organelles were involved in the concurrent oxidation of these two compounds, since they contained both alcohol oxidase and amine oxidase, which are key enzymes in methanol and methylamine metabolism, respectively. In addition catalase was present. Peroxisomes with a completely crystalline substructure were observed in methanol-limited chemostat-grown cells. Amine oxidase probably formed an integral part of these crystalloids, whereas catalase was present in a freely diffusable form. Transfer of cells, grown in a methanol-limited chemostat in the presence of methylamine into glucose/ammonium sulphate media resulted in the loss of both alcohol oxidase and amine oxidase activity from the cells. This process was associated with degradation of the crystalline peroxisomes. However, when cells were transferred into glucose/methylamine media, amine oxidase activity only declined during 2 h after the transfer and thereafter increased again. This subsequent rise in amine oxidase activity was associated with the development of new peroxisomes in the cells in which degradation of the crystalline peroxisomes, originally present, continued. These newly formed organelles probably originated from peroxisomes which had not been affected by degradation. When in the methanollimited chemostat methylamine was replaced by ammonium sulphate, repression of the synthesis of amine oxidase was observed. However, inactivation of this enzyme or degradation of peroxisomes was not detected. The decrease of amine oxidase activity in the culture was accounted for by dilution of enzyme as a result of growth and washout.     

Genetics of Catalase in DROSOPHILA MELANOGASTER: Rates of Synthesis and Degradation of the Enzyme in Flies Aneuploid and Euploid for the Structural Gene

A screen for allelic variants of the enzyme catalase indicated that the Cat⁺ locus is essentially monomorphic in D. melanogaster. Segmental aneuploidy was used to screen the genome for a dosage-sensitive region for catalase activity. One region, 75D–78A on the polytene chromosome map of 3L, exhibited a hyperploid/euploid ratio of enzyme activity of 1.5. Further dissection localized the region to 75D–76A. We suggest that this region contains the structural locus for catalase in D. melanogaster.

Simple methods have been developed using the specific inhibitor, 3-amino-1,2,4-triazole, for the direct analysis of rates of synthesis and degradation of the Cat⁺ gene product. Based on kinetic studies of catalase synthesis in flies aneuploid and euploid for region 75D–76B, we suggest that these techniques can be readily applied to an examination of mutants that control the expression of the structural gene for catalase in Drosophila.