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.     

Apparent Catalase Synthesis in Sunflower Cotyledons during the Change in Microbody Function: A Mathematical Approach for the Quantitative Evaluation of Density-labeling Data

Density-labeling with 10 mm K¹⁵NO₃/70% ²H₂O has been used to investigate catalase synthesis in different developmental stages of sunflower (Helianthus annuus L.) cotyledons. A mathematical approach is introduced for the quantitative evaluation of the density-labeling data. The method allows, in the presence of preexisting enzyme activity, calculation of this synthesized activity (apparent enzyme synthesis) which results from the balance between actual enzyme synthesis and the degradation of newly synthesized enzyme at a given time. During greening of the cotyledons, when the catalase activity declines and the population of leaf peroxisomes is formed, the apparent catalase synthesis is lower than, or at best equal to, that occurring during a developmental stage when the leaf peroxisome population is established and catalase synthesis and degradation of total catalase are in equilibrium. This result suggests a formation, in fatty cotyledons, of the leaf peroxisomes by transformation of the glyoxysomes rather than by de novo synthesis.     

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.     

Catalase protects HepG2 cells from apoptosis induced by DNA-damaging agents by accelerating the degradation of p53

Oxidants such as H(2)O(2) play a role in the toxicity of certain DNA-damaging agents, a process that often involves the tumor suppressor p53. H(2)O(2) is rapidly degraded by catalase, which protects cells against oxidant injury. To study the effect of catalase on apoptosis induced by DNA-damaging agents, HepG2 cells were infected with adenovirus containing the cDNA of catalase (Ad-Cat). Forty-eight hours after infection, catalase protein and activity was increased 7-10-fold compared with control cells infected with Ad-LacZ. After treatment with Vp16 or mitomycin C, control cells underwent apoptosis in a p53-dependent manner; however, overexpression of catalase inhibited this apoptosis. Basal levels as well as Vp16- or mitomycin C-stimulated levels of p53 and p21 protein were decreased in the catalase-overexpressing cells as compared with control cells; however, p53 mRNA levels were not decreased by catalase. There was no difference in p53 protein synthesis between catalase-overexpressing cells and control cells. However, pulse-chase experiments indicated that p53 protein degradation was enhanced in the catalase-overexpressing cells. Proteasome inhibitors but not calpeptin prevented the catalase-mediated decrease of p53 content. Whereas Vp16 increased, catalase overexpression decreased the phosphorylation of p53. The protein phosphatase inhibitor okadaic acid did not prevent the catalase-mediated down-regulation of p53 or phosphorylated p53. These results demonstrate that catalase protects HepG2 cells from apoptosis induced by DNA-damaging agents in association with decreasing p53 phosphorylation; the latter may lead to an acceleration in the degradation of p53 protein by the proteasome complex. This suggests that the level of catalase may play a critical role in cell-induced resistance to the effects of anti-cancer drugs which up-regulate p53.     

Effect of cobalt on the synthesis and degradation of hepatic catalse in vivo

1. The administration of CoCl(2) to rats caused a decrease in hepatic catalase activity as well as a decrease in the amount of catalase protein as measured by immunological assay. The mitochondrial enzyme decreased progressively over 2 days, whereas the cytosol enzyme decreased over 12h and then remained essentially unchanged for 2 days after a single injection of cobalt. 2. Incorporation of [(14)C]glycine into catalase haem was dramatically decreased by a single injection of cobalt, but that into catalase protein remained essentially unaltered. 3. Incorporation of [(3)H]leucine into liver protein increased in rats in a steady state receiving a daily injection of cobalt, which was in contrast with a marked inhibition observed in 5-amino[(3)H]laevulinate incorporation. 4. The initial rate of [(3)H]leucine incorporation into mitochondrial and cytosol catalase did not alter or was slightly depressed in the cobalt-treated animals, whereas the incorporation of 5-amino[(3)H]laevulinate into mitochondrial and cytosol catalase was conspicuously decreased, indicating that haem synthesis was limiting catalase formation. 5. The degradation rate of catalase protein, as measured by a double-labelling method, was not changed by the cobalt treatment.     

The relationship between δ-aminolaevulinate synthetase induction and the concentrations of cytochrome P-450 and catalase in rat liver

The porphyrinogenic drug 2-allyl-2-isopropylacetamide causes the degradation of microsomal cytochrome P-450 and inhibits the synthesis of catalase in rat liver. The inhibition of catalase synthesis follows the induction of delta-aminolaevulinate synthetase and the consequent overproduction of haem. The allylisopropylacetamide-mediated breakdown of cytochrome P-450 is a rapid event and has a reciprocal relationship to the pattern of delta-aminolaevulinate synthetase induction. Breakdown of cytochrome P-450 appears to be one of the conditions leading to the ;derepression' of delta-aminolaevulinate synthetase.     

On the turnover and proteolysis of catalase in tissues of the guinea pig and acatalasemic mice.

Turnover characteristics (half-lives and rate constants for synthesis and degradation) have been determined for the catalases of guinea pig and three different strains of mice by means of the kinetics of return of enzyme activity after inhibition with 3-amino-1,2,4-triazole. The catalase of hypocatalasemic mice (strain C_sD) did not display an appreciably different half-life to that of the wild-type mice, but catalase in the tissues of acatalasemic mice (strain C_sB) exhibited a half-life which was only half that of the wild type, while the half-life of guinea pig catalase was more than twice that of wild-type mice. Significant differences were also noticed in regard to the in vitro susceptibility of the catalases of these animals to protease inactivation. Large-granule (lysosomal, mitochondrial and peroxisomal) extracts proved far more susceptible to protease inactivation than cytosol extracts, and marked changes in the heteromorph pattern of mouse liver cytosol catalase were observed to accompany limited proteolysis. These results support the conclusion that the in vitro susceptibility of proteases may be an important determining factor in the rate of degradation of an enzyme in vivo.     

Murine catalase phenotypes

An examination of three inbred strains of mice differing with respect to liver and kidney catalase activity reveals two distinct genetic factors controlling the level of liver catalase activity. The first genetic factor controls the catalytic activity of the enzyme. Specific activity of purified enzyme from C57BL/6 and C57BL/Ha strains is 60% of that of the DBA/2 strain. The second factor controls the content of liver catalase. Liver catalase of C57BL/Ha is degraded in vivo at a rate one half that of liver catalase of DBA/2 and C57BL/6, resulting in the accumulation of twice as many catalase molecules in C57BL/Ha. The factor affecting turnover of catalase is apparently specific for catalase of liver since no differences exist in kidney catalase levels between C57BL/Ha and C57BL/6. Furthermore, this factor does not appear to alter the metabolism of total liver protein since no substantial difference in the turnover rate of liver protein is observed among the strains. It is particularly significant that the genetic factor affecting the amount of liver catalase does so by altering the rate of catalase degradation rather than the rate of synthesis, confirming the previously published report of Rechcigl and Heston (1967). Thus, these studies emphasize that the quantity of an enzyme in animal cells is a balance between the rate of synthesis and the rate of degradation of the enzyme.This paper was presented at a symposium entitled Genetic Control of Mammalian Metabolism held at The Jackson Laboratory, Bar Harbor, Maine, June 30–July 2, 1969. The symposium was supported in part by an allocation from NIH General Research Support Grant FR 05545 from the Division of Research Resources to The Jackson Laboratory.This investigation was supported by USPHS Research Grant GM 14931 from the Division of General Medical Sciences, and Grants PF-373 and P-427 from the American Cancer Society.     

On the synthesis and degradation of the multiple forms of catalase in mouse liver

The incorporation of ⁵⁵Fe-labeled ferrous sulfate and ³H-labeled γ-aminolaevulinic acid into the catalase of mouse liver was measured at intervals up to 96 hr after intraperitoneal injection, and the intracellular location of radioactive catalase followed, as well as the distribution of radiolabel between the multiple forms of this enzyme. At 10 min, catalase radioactivity was present in all the cellular fractions studied, but after this time, label began to disappear from the microsomal fraction and from the peroxisomal detergent extract. By comparison, catalase incorporation reached a peak at about 6 hr in the peroxisomal aqueous extract, and rose to a broad peak after about 30 hr in the cytosol fraction. On resolving the multiple forms of catalase in the supernatant fraction by electrophoresis, it was found that label first appeared in the fastest moving heteromorph, and appeared sequentially in the other multiple forms over a period of 96 hr.The sequence of degradation of catalase was also studied by examination of residual catalase activity subsequent to the injection of allyl-isopropyl acetamide, a heme synthesis antagonist which blocks catalase synthesis. Blood catalase levels did not seem to be significantly affected by this treatment, but in the liver, the decay rates of catalase activity were appreciable, and varied significantly between the intracellular pools. The rate of decrease was greatest in the peroxisomal detergent extract, and least in the supernatant fraction.These findings have been discussed in relation to current understanding of the subcellular disposition, multiplicity, and turnover of hepatic catalase.     

Effect of Triton WR-1339 on the rates of synthesis and degradation of hepatic catalase of rat.

The effect of Triton WR-1339 on the rates of synthesis and degradation of hepatic catalase was examined. Triton WR-1339 was injected intraperitoneally into rats at a dose of 200 mg per 100 g body weight. Catalase activity decreased to about 35% of that of the control at 42-48 h after the injection and recovered to the normal level at 96 h. Other peroxisomal enzymes, D-amino acid oxidase and urate oxidase, showed similar patterns of the activities to those of catalase. During the first 48 h after the injection of Triton WR-1339, the rate of catalase synthesis (ks) fell to below a detectable value, while that of the degradation (kd) did not show any significant change. On the other hand, during the period 48-96 h after the injection, the rate of the synthesis (ks) returned to the normal level though that of the degradation (kd) decreased to about 50% of the control.     

Peroxisomes of alkane-utilizing yeasts metabolic functions and practical aspects

One of the most striking features of alkane-grown yeast cells is conspicuous appearance of peroxisomes in harmony with a high level of catalase. This unique phenomenon was first demonstrated in the authors′ laboratory, and the metabolic functions of peroxisomes in yeasts utilizing alkanes has been estabilished with intact peroxisomes isolated by density gradient centrifugation. The organelles participate in the degradation of fatty acids derived from alkanes to C²-units and the synthesis of gluconeogenic intermediates from C²-units. The abundant appearance of peroxisomes in alkane-utilizing cells has allowed successful production of several useful enzymes including catalase, D-amino acid oxidase, uricase, acyl-CoA oxidase etc. Yeast cells will be an excellent system for investigation the functions and development of peroxisomes because biogenesis of the organelles is induced only by transferring the cells into alkane medium from glucose or ethanol medium.     

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.     

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.     

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).     

Preferential photoinactivation of catalase and photoinhibition of photosystem II are common early symptoms under various osmotic and chemical stress conditions

Activity of catalase (EC 1.11.1.6) and variable fluorescence (F) were measured in sections of rye leaves (Secale cereale L. cv. Halo) that were exposed for 24 h to moderately high irradiance under osmotic or chemical stress conditions (paraquat, DCMU, mannitol, NaCl, CdCl₂, CuSO₄, Pb(NO₃)₂, KNO₂, or K₂SO₃). Changes of the chlorophyll content and of enzyme activities related to peroxide metabolism, such as glycolate oxidase, glutathione reductase, and peroxidase, were assayed for comparison. In the presence of the herbicides paraquat and low DCMU concentrations that exert only partial inhibition of photosynthesis, as well as after most treatments with osmotic or chemical stress factors, catalase markedly declined due to a preferential photoinactivation. At higher DCMU levels catalase did not decline. At low KNO₂ concentrations catalase activity was preferentially increased. In general, photoinactivation of catalase was accompanied by a decline of the F/F_m ratio, indicating photoinhibition of photosystem II, while other parameters were much more stable. Inasmuch as both catalase and the D1 reaction center protein of photosystem II have a rapid turnover in light, their steady state levels appear to decline whenever stress effects either excessively enhance deleterious oxidative conditions and degradation (e. g. Paraquat, low DCMU), or inhibit repair synthesis. Photoinactivation of catalase and of photosystem II represent specific and widely occurring early symptoms of incipient photodamage indicating stress conditions where the repair capacity is not sufficient. During prolonged exposures, e. g. to NaCl and CuSO₄, chlorophyll was bleached in light and the rate of its photodegradation increased in proportion as the catalase level had declined. The results suggest that the enhanced susceptibility of leaf tissues to photooxidative damage which is widely observed in stressed plants is related to the early loss of catalase.     

On the topology of the catalase biosynthesis and -degradation in the guinea pig liver. A cytochemical study

The biosynthesis, transport and degradation of catalase have been studied in the guinea pig liver parenchymal cell using 2-allyl-2-isopropylacetamide (AIA) as an inhibitor of de novo formation of catalase. Total catalase activity was assayed biochemically; cytoplasmic catalase was measured microspectrophotometrically after quantitative diaminobenzidine staining of the liver. By morphometry, number and size of peroxisomes in catalase stained sections were determined. From our data we conclude that (1) the final step in the catalase formation takes place inside peroxisomes, (2) catalase is transported from the peroxisomes into the cytoplasm, (3) in the cytoplasm catalase is degraded. These conclusions in part confirm the topological model on the intracellular catalase biosynthesis pathway of Lazarow and de Duve (1973) except for the presence of cytoplasmic catalase which is released from the peroxisomes as proposed earlier by Jones and Masters (1975).     

Enhanced potential for oxidative stress in hyperinsulinemic rats: imbalance between hepatic peroxisomal hydrogen peroxide production and decomposition due to hyperinsulinemia.

Oxidative stress is involved in aging and age-related diseases. Several metabolic alterations similar to those encountered with aging and age-related diseases have been observed in response to hyperinsulinemia. Surprisingly, this metabolic derangement diminished hepatic peroxisomal beta-oxidation which is a major source of H2O2 production in the liver, suggesting a protective effect against oxidative stress. However, the impact of hyperinsulinemia on the balance between H2O2 production and elimination in the liver is not known. Consequently, this study was undertaken to evaluate the effect of sustained high serum insulin levels on the activity of hepatic catalase, a peroxisomal antioxidant enzyme involved in the decomposition of H2O2. Male Sprague-Dawley rats received intravenous infusion of either 30% glucose, 30% galactose or normal saline for seven days. Activity of hepatic peroxisomal beta-oxidation and catalase decreased 58% and 74%, respectively, in glucose-infused rats compared with galactose- or saline-infused animals. When infused simultaneously with glucose, diazoxide blocked glucose-enhanced insulin secretion and prevented the decrease in peroxisomal enzyme activities, without altering blood glucose concentration. Neither diazoxide alone nor galactose, which did not alter serum insulin levels, had any effect on enzyme activities. These results suggest that hyperinsulinemia is responsible for the decreased enzyme activities observed in glucose-infused rats. Indeed, a strong negative correlation between serum insulin levels and hepatic peroxisomal enzyme activities was found. To investigate the mechanism by which insulin modulates catalase activity, we studied rates of synthesis and degradation of catalase in saline- and glucose-infused rats. Data show that insulin diminishes rates of catalase synthesis, while exhibiting no effect on its degradation. Upsetting the balance between the cellular capacity to produce and eliminate H2O2 may be a contributing factor to the known deleterious effects of hyperinsulinemia.     

On the topology of the catalase biosynthesis and-degradation in the guinea pig liver

Summary The biosynthesis, transport and degradation of catalase have been studied in the guinea pig liver parenchymal cell using 2-allyl-2-isopropylacetamide (AIA) as an inhibitor of de novo formation of catalase. Total catalase activity was assayed biochemically; cytoplasmic catalase was measured microspectrophotometrically after quantitative diaminobenzidine staining of the liver. By morphometry, number and size of peroxisomes in catalase stained sections were determined. From our data we conclude that (1) the final step in the catalase formation takes place inside peroxisomes, (2) catalase is transported from the peroxisomes into the cytoplasm, (3) in the cytoplasm catalase is degraded. These conclusions in part confirm the topological model on the intracellular catalase biosynthesis pathway of Lazarow and de Duve (1973) except for the presence of cytoplasmic catalase which is released from the peroxisomes as proposed earlier by Jones and Masters (1975).     

Inactivation and degradation of rat liver catalase by mitochondrial and lysosomal proteinases

The initial phases of catalase degradation in rat hepatocytes were studied. Preparations of highly purified fractions of lysosomes and mitochondria from rat liver were obtained. The proteinase activity was measured by the radio-isotope method by the increase of the free amino groups or by the decrease of the catalase activity, using labelled catalase as a substrate. It was found that the initial step of catalase degradation occurs in the enzyme localized in the inner membrane as well as in the mitochondrial matrix and that the total degradation of catalase is completed in the lysosomal fraction of rat liver.