Activity and hematin content of catalase from greening sunflower cotyledons

The specific activity of catalase purified from the peroxisomes of sunflower cotyledons declines in parallel with the total cotyledonary catalase activity during the transition of peroxisomes from glyoxysomal to leaf peroxisomal function. The hematin content of the purified catalase however, remains constant at 4 hematin groups per catalase molecule. The absorbance coefficients of catalase at 404 and 280 nm were determined to be 372 and 540/mM/cm, respectively.     

Immunocytochemical Analysis Shows that Glyoxysomes Are Directly Transformed to Leaf Peroxisomes during Greening of Pumpkin Cotyledons

The functional transition of glyoxysomes to leaf peroxisomes occurs during greening of germinating pumpkin cotyledons (Cucurbita sp. Amakuri Nankin). The immunocytochemical protein A-gold method was employed in the analysis of the transition using glyoxysomal specific citrate synthase immunoglobulin G and leaf peroxisomal specific glycolate oxidase immunoglobulin G. The labeling density of citrate synthase was decreased in the microbodies during the greening, whereas that of glycolate oxidase was dramatically increased. Double labeling experiments using different sizes of protein A-gold particles show that both the glyoxysomal and the leaf peroxisomal enzymes coexist in the microbody of the transitional stage indicating that glyoxysomes are directly transformed to leaf peroxisomes during greening.     

cDNA cloning and differential gene expression of three catalases in pumpkin

Three cDNA clones (cat1, cat2, cat3) for catalase (EC 1.11.1.6) were isolated from a cDNA library of pumpkin (Cucurbita sp.) cotyledons. In northern blotting using the cDNA-specific probe, the cat1 mRNA levels were high in seeds and early seedlings of pumpkin. The expression pattern of cat1 was similar to that of malate synthase, a characteristic enzyme of glyoxysomes. These data suggest that cat1 might encode a catalase associated with glyoxysomal functions. Furthermore, immunocytochemical analysis using cat1-specific anti-peptide antibody directly showed that cat1 encoding catalase is located in glyoxysomes. The cat2 mRNA was present at high levels in green cotyledons, mature leaf, stem and green hypocotyl of light-grown pumpkin plant, and correlated with chlorophyll content in the tissues. The tissue-specific expression of cat2 had a strong resemblance to that of glycolate oxidase, a characteristic enzyme of leaf peroxisomes. During germination of pumpkin seeds, cat2 mRNA levels increased in response to light, although the increase in cat2 mRNA by light was less than that of glycolate oxidase. cat3 mRNA was abundant in green cotyledons, etiolated cotyledons, green hypocotyl and root, but not in young leaf. cat3 mRNA expression was not dependent on light, but was constitutive in mature tissues. Interestingly, cat1 mRNA levels increased during senescence of pumpkin cotyledons, whereas cat2 and cat3 mRNAs disappeared during senescence, suggesting that cat1 encoding catalase may be involved in the senescence process. Thus, in pumpkin, three catalase genes are differentially regulated and may exhibit different functions.     

Intraorganellar distribution of superoxide dismutase in plant peroxisomes (glyoxysomes and leaf peroxisomes)

The intraorganellar distribution of superoxide dismutase (SOD) (EC 1.15.1.1) in two types of plant peroxisomes (glyoxysomes and leaf peroxisomes) was studied by determinations of SOD latency in intact organelles and by solubilization assays with 0.2 molar KCl. Glyoxysomes were purified from watermelon (Citrullus vulgaris Schrad.) cotyledons, and their integrity, calculated on the basis of glyoxysomal marker enzymes, was about 60%. Under the same conditions, the latency of SOD activity determined in glyoxysomes was 40%. The difference between glyoxysomal intactness and SOD latency was very close to the percentage of isozyme Mn-SOD previously determined in glyoxysomes (LM Sandalio, LA Del Río 1987 J Plant Physiol 127: 395-409). In matrix and membrane fractions of glyoxysomes, SOD exhibited a solubilization pattern very similar to catalase, a typical soluble enzyme of glyoxysomes. The analysis of the distribution of individual SOD isozymes in glyoxysomal fractions treated with KCl showed that Cu,Zn-SOD II, the major SOD isozyme in glyoxysomes, was present in the soluble fraction of these organelles, whereas Mn-SOD was bound to the glyoxysomal membrane. These data in conjunction with those of latency of SOD activity in intact glyoxysomes suggest that Mn-SOD is bound to the external side of the membrane of glyoxysomes. On the other hand, in intact leaf peroxisomes where only a Mn-containing SOD is present (LM Sandalio, JM Palma, LA Del Río 1987 Plant Sci 51: 1-8), this isozyme was found in the peroxisomal matrix. The physiological meaning of SOD localization in matrix and membrane fractions of glyoxysomes and the possibility of new roles for plant peroxisomes in cellular metabolism related to activated oxygen species is discussed.     

Pumpkin peroxisomal ascorbate peroxidase is localized on peroxisomal membranes and unknown membranous structures

To investigate the roles of peroxisomal membrane proteins in the reversible conversion of glyoxysomes to leaf peroxisomes, we characterized several membrane proteins of glyoxysomes. One of them was identified as an ascorbate peroxidase (pAPX) that is localized on glyoxysomal membranes. Its cDNA was isolated by immunoscreening. The deduced amino acid sequence encoded by the cDNA insert does not have a peroxisomal targeting signal (PTS), suggesting that pAPX is imported by one or more PTS-independent pathways. Subcellular fractionation of 3- and 5-d-old cotyledons of pumpkin revealed that pAPX was localized not only in the glyoxysomal fraction, but also in the ER fraction. A magnesium shift experiment showed that the density of pAPX in the ER fraction did not increase in the presence of Mg(2+), indicating that pAPX is not localized in the rough ER. Immunocytochemical analysis using a transgenic Arabidopsis which expressed pumpkin pAPX showed that pAPX was localized on peroxisomal membranes, and also on a unknown membranous structure in green cotyledons. The overall results suggested that pAPX is transported to glyoxysomal membranes via this unknown membranous structure.     

Developmental analysis of a putative ATP/ADP carrier protein localized on glyoxysomal membranes during the peroxisome transition in pumpkin cotyledons

In order to clarify the peroxisomal membrane proteins (PMPs), we characterized one of the major PMPs, PMP38. The deduced amino acid sequence for its cDNA in Arabidopsis thaliana contained polypeptides with 331 amino acids and had high similarity with those of Homo sapiens PMP34 and Candida boidinii PMP47 known as homologues of mitochondrial ATP/ADP carrier protein. We expected PMP38 to be localized on peroxisomal membranes, because it had the membrane peroxisomal targeting signal. Cell fractionation and immunocytochemical analysis using pumpkin cotyledons revealed that PMP38 is localized on peroxisomal membranes as an integral membrane protein. The amount of PMP38 in pumpkin cotyledons increased and reached the maximum protein level after 6 d in the dark but decreased thereafter. Illumination of the seedlings caused a significant decrease in the amount of the protein. These results clearly showed that the membrane protein PMP38 in glyoxysomes changes dramatically during the transformation of glyoxysomes to leaf peroxisomes, as do the other glyoxysomal enzymes, especially enzymes of the fatty acid beta-oxidation cycle, that are localized in the matrix of glyoxysomes.     

Leaf peroxisomes are directly transformed to glyoxysomes during senescence of pumpkin cotyledons

Summary After the functional transition of glyoxysomes to leaf peroxisomes during the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Immunocytochemical labeling with protein A-gold was performed to analyze the reverse microbody transition using antibodies against a leaf-peroxisomal enzyme, glycolate oxidase, and against two glyoxysomal enzymes, namely, malate synthase and isocitrate lyase. The intensity of labeling for glycolate oxidase decreased in the microbodies during senescence whereas in the case of malate synthase and isocitrate lyase intensities increased strikingly. Double labeling experiments with protein A-gold particles of different sizes showed that the leaf-peroxisomal enzymes and the glyoxysomal enzymes coexist in the microbodies of senescing pumpkin cotyledons, indicating that leaf peroxisomes are directly transformed to glyoxysomes during senescence.     

Purification and properties of glyoxysomal lipase from castor bean

The alkaline lipase in the glyoxysomes from the endosperm of young castor bean seedlings, an integral membrane component, was solubilized in deoxycholate:KCl and purified to apparent homogeneity. The molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 62,000 daltons. The enzyme reaction was markedly stimulated by salts and inhibited by detergents. Triricinolein, the endogenous storage lipid, was hydrolyzed by the purified enzyme which is therefore a true lipase. Treatment of intact glyoxysomes with trypsin strongly diminished the lipase activity but did not affect matrix enzymes. An antibody preparation raised in a rabbit against the purified enzyme inhibited the purified enzyme and that in glyoxysomal membranes.     

Novel glyoxysomal protein kinase, GPK1, identified by proteomic analysis of glyoxysomes in etiolated cotyledons of Arabidopsis thaliana

Glyoxysomes are present in etiolated cotyledons and contain enzymes for gluconeogenesis, which constitutes the major function of glyoxysomes. However, 281 genes seemingly related to peroxisomal functions occur in the Arabidopsis genome, implying that many unidentified proteins are present in glyoxysomes. To better understand the functions of glyoxysomes, we performed glyoxysomal proteomic analysis of etiolated Arabidopsis cotyledons. Nineteen proteins were identified as glyoxysomal proteins, including 13 novel proteins, one of which is glyoxysomal protein kinase 1 (GPK1). We cloned GPK1 cDNA by RT-PCR and characterized GPK1. The amino acid sequence deduced from GPK1 cDNA has a hydrophobic region, a putative protein kinase domain, and a possible PTS1 motif. Immunoblot analysis using fractions collected on a Percoll density gradient confirmed that GPK1 is localized in glyoxysomes. Analysis of suborganellar localization and protease sensitivity showed that GPK1 is localized on glyoxysomal membranes as a peripheral membrane protein and that the putative kinase domain is located inside the glyoxysomes. Glyoxysomal proteins are phosphorylated well in the presence of various metal ions and [g-32P]ATP, and one of them is identified as thiolase by immunoprecipitation. Immuno-inhibition of phosphorylation in glyoxysomes suggested that GPK1 phosphorylates a 40-kDa protein. These results show that protein phosphorylation systems are operating in glyoxysomes.     

The C-terminal domain of plant catalases. Implications for a glyoxysomal targeting sequence.

A castor bean (Ricinus communis cv. Hale) cDNA encoding catalase was cloned and sequenced. The cDNA encoding the carboxy-terminal domain of catalase was compared to the corresponding sequences of six other plant catalases. The deduced amino acid sequences were compared according to the chemical attributes of each amino acid within each carboxy-terminal domain. A tripeptide sequence having the chemical attributes of the peroxisomal targeting sequence [Gould, S.J., Keller, G.-A., Hosken, N., Wilkinson, J. & Subramani, S. (1989) J. Cell Biol. 108, 1657-1664] was common to all the glyoxysomal/peroxisomal plant catalases. This sequence motif was located six amino acids from the carboxy terminus of each of the plant catalases. An identical motif was also found within the carboxy-terminal domain of three mammalian catalases previously sequenced. We hypothesize that these motifs are at least part of the targeting mechanism for catalase entry into plant glyoxysomes/peroxisomes.     

Plant microbody proteins, I. Purification and characterization of catalase from leaves of lens culinaris.

1) Catalase from green leaves of Lens culinaris (lentils) was investigated with respect to isoenzyme patterns. In contrast to other plants, which have been reported to contain multiple forms of catalase, only one form of this enzyme was revealed when crude extracts were subjected to starch gel electrophoresis or to polyacrylamide disc-gel electrophoresis. Furthermore, catalases from leaves, stems and cotyledons were electrophoretically identical. 2) The leaf enzyme has been purified by conventional methods to apparent homogeneity. It has a molecular weight of 225 000 (ultracentrifuge) and is composed of four identical subunits of molecular weight 54 000 (sodium dodecylsulphate gel electrophoresis). The ratio A280/A405 of the pure enzyme was found to be 1.5. The isoelectric point is at pH 5.5. The enzyme, very labile at pH-values below 7.0, is stable in Tris chloride and potassium phosphate buffers between pH 7.5 and 9.5. It is slowly inactivated by 1mM dithiothreitol and is rapidly inactivated by 1mM mercaptoethanol. 3) The catalase was shown to be the major protein component of the peroxisomal matrix. It could not be detected at the membranes of the leaf peroxisomes.     

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.     

Development of Microbody Membrane Proteins during the Transformation of Glyoxysomes to Leaf Peroxisomes in Pumpkin Cotyledons

The microbody transition observed in the cotyledons of somefatty seedlings involves the conversion of glyoxysomes to leafperoxisomes. To clarify the molecular mechanisms underlyingthe microbody transition, we established a method for the preparationof highly purified microbodies. SDS-PAGE and immunoblot analysisof isolated microbodies from pumpkin cotyledons at various stagesshowed that glyoxysomal enzymes are replaced by leaf-peroxisomalenzymes during the microbody transition. Two proteins in glyoxysomalmembranes, with molecular masses of 31 kDa and 28 kDa, werenot solubilized from the membranes with 0.2 M KCl, an indicationthat these proteins are bound tightly with glyoxysomal membranes.Their polyclonal antibodies were raised against the respectivepurified protein. Immunoblot analysis of subcellular fractionsand immunogold analysis confirmed that these proteins were specificallylocalized on glyoxysomal membranes. Analysis of these membraneproteins during development revealed that the amounts of thesemembrane proteins decreased during the microbody transitionand that the large one was retained in leaf peroxisomes, whereasthe small one could not be found in leaf peroxisomes after completionof the microbody transition. The results clearly showed thatmembrane proteins in glyoxysomes change dramatically duringthe microbody transition, as do the enzymes in the matrix. ¹Present address: School of Agriculture, Nagoya University Chikusa,Nagoya, 464-01 Japan.     

Thiolase mRNA translated in vitro yields a peptide with a putative N-terminal presequence

Thiolase is part of the fatty acid oxidation machinery which in plants is located within glyoxysomes or peroxisomes. In cucumber cotyledons, proteolytic modification of thiolase takes place during the transfer of the cytosolic precursor into glyoxysomes prior to the intraorganellar assembly of the mature enzyme. This was shown by size comparison of the in vitro synthesized precursor and the 45 kDa subunit of the homodimeric glyoxysomal form. We isolated a full-length cDNA clone encoding the 48 539 Da precursor of thiolase. This plant protein displayed 40% and 47% identity with the precursor of fungal peroxisomal thiolase and human peroxisomal thiolase, respectively. Compared to bacterial thiolases, the precursor of the plant enzyme was distinguished by an N-terminal extension of 34 amino acid residues. This putative targeting sequence of cucumber thiolase shows similarities with the cleavable presequences of rat peroxisomal thiolase and plant peroxisomal malate dehydrogenase.     

Cloning, expression, and purification of glyoxysomal 3-oxoacyl-CoA thiolase from sunflower cotyledons

The glyoxysomal beta-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the coexistence of two different thiolase isoforms, thiolase I and II. So far, this phenomenon has only been described for glyoxysomes from sunflower cotyledons. Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9) recognizes acetoacetyl-CoA only, while thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16) exhibits a more broad substrate specificity towards 3-oxoacyl-CoA esters of different chain length. Here, we report on the cloning of thiolase II from sunflower cotyledons. The known DNA sequence of Cucumis sativus 3-oxoacyl-CoA thiolase was used to generate primers for cloning the corresponding thiolase from sunflower cotyledons. RT-PCR was then used to generate an internal fragment of the sunflower thiolase gene and the termini were isolated using 5'- and 3'-RACE. Full-length cDNA was generated using RT-PCR with sunflower thiolase-specific primers flanking the coding region. The resultant gene encodes a thiolase sharing at least 80% identity with other plant thiolases at the amino acid level. The recombinant sunflower thiolase II was expressed in a bacterial system in an active form and purified to apparent homogeneity in a single step using Ni-NTA agarose chromatography. The enzyme was purified 53.4-fold and had a specific activity of 235 nkat/mg protein. Pooled fractions from the Ni-NTA column resulted in an 83% yield of active enzyme to be used for further characterization.     

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.     

Transport of chimeric proteins that contain a carboxy-terminal targeting signal into plant microbodies

Malate synthase is a glyoxysome-specific enzyme. The carboxy-terminal tripeptide of the enzyme is Ser—Arg—Leu (SRL), which is known to function as a peroxisomal targeting signal in mammalian cells. To analyze the function of the carboxy-terminal amino acids of pumpkin malate synthase in plant cells, a chimeric gene was constructed that encoded a fusion protein which consisted of β-glucuronidase and the carboxyl terminus of the enzyme. The fusion protein was expressed and accumulated in transgenic Arabidopsis that had been transformed with the chimeric gene. Immunocytochemical analysis of the transgenic plants revealed that the carboxy-terminal five amino acids of pumpkin malate synthase were sufficient for transport of the fusion protein into glyoxysomes in etiolated cotyledons, into leaf peroxisomes in green cotyledons and in mature leaves, and into unspecialized microbodies in roots, although the fusion protein was no longer transported into microbodies when SRL at the carboxyl terminus was deleted. Transport of proteins into glyoxysomes and leaf peroxisomes was also observed when the carboxy-terminal amino acids of the fusion protein were changed from SRL to SKL, SRM, ARL or PRL. The results suggest that tripeprides with S, A or P at the −3 position, K or R at the −2 position, and L or M at the carboxyl terminal position can function as a targeting signal for three kinds of plant microbody.     

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)     

Plant microbody proteins. Purification and glycoprotein nature of glyoxysomal isocitrate lyase from cucumber cotyledons.

1. Isocitrate lyase from cotyledons of cucumber seedlings (Cucumis sativus) has been purified 100-fold. Two methods of preparing the soluble glyoxylate cycle enzyme are described: an elaborated method which used crude extracts of cucumber cotyledons, and another procedure which started with purified glyoxysomes from 4-day-old cotyledons and included a separation of glyoxysomal matrix enzymes by zonal centrifugation. The product behaved as a single species when tested by (a) polyacrylamide gel electrophoresis in the presence of dodecyl sulfate, (b) zonal centrifugation, and (c) double immunodiffusion against rabbit antibody to isocitrate lyase. 2. Isocitrate lyase of cucumber glyoxysomes exhibited a molecular weight of 255,000 and was composed of four apparently identical subunits of Mr 64,000. An isoelectric point of 5.9 was determined. 3. It was shown that isocitrate lyase is a glycoprotein, (a) by Schiff stain on polyacrylamide gels, (b) by periodate oxidation of the enzyme, subsequent reduction with NaB[3H]4 and electrophoretic analysis of the labelled glycoprotein, and (c) by incorporation of [3H]glucosamine in vivo into a protein which could be precipitated with antibodies to isocitrate lyase and revealed a 64,000-Mr band upon electrophoresis.