Citrate synthases from germinating castor bean seeds – II. Time course of synthesis and immunochemical comparison

The time course of total citrate synthase activity in castor bean ( Ricinus communis L., type Sanzibariensis) endosperm showed a 7-fold increase during the initial 5 days of germination and a decrease thereafter. All citrate synthase activity in the ungerminated seeds was due to the mitochondrial isoenzyme. After two days of germination the glyoxysomal isoenzyme began to appear. After 5 days the glyoxysomal citrate synthase represented 50 to 55% of the total activity and the mitochondrial enzyme the remainder. This was estimated from (a) inactivation of the glyoxysomal citrate synthase by 5,5'-dithiobis(2-nitrobenzoic acid); (b) solid phase adsorption of the glyoxysomal synthase by a specific antiserum; (c) separation of isoenzymes by (NH₄)₂SO₄ gradient solubilization.
The increase of both citrate synthases during the initial 4 days of germination could be prevented by 10 μg cycloheximide ml⁻¹, but not by 40 or 400 μg chloramphenicol ml⁻¹, indicating a synthesis on 80 S ribosomes. Actinomycin D completely inhibited the appearance of the glyoxysomal enzyme while the mitochondrial enzyme was not affected. Antisera against the two isoenzymes revealed major structural differences between two citrate synthases, however, also some common determinants. No cross-reaction was observed with the citrate synthase from pig heart or E. coli.     

Citrate synthases from germinating castor bean seeds – I. Purification and properties

The mitochondrial and glyoxysomal citrate synthase (EC 4.1.3.7) from the endosperm of germinating castor beans ( Ricinus communis L., type Sanzibaricnsis) were purified to a final specific activity of 76 and 78 U (mg protein)⁻¹, respectively. Both citrate synthases could be bound to ATP-Sepharose. However, only the mitochondrial enzyme could be eluted by either 100 μ M oxaloacetate or 100 μ M coenzyme A (indicative of affinity chromatography), while the glyoxysomal enzyme was only eluted by 0.5 M KCI (indicative of ion-exchange chromatography). Many properties of the two isoenzymes were similar including the pH dependence and temperature dependence of activity, the pH stability, and the inactivation of the enzyme at elevated temperatures. The most pronounced differences between the two citrate synthases were the isolelectric points of pH 5.9 for the mitochondrial and of pH 9.1 for the glyoxysomal enzyme. Both citrate synthases are dimers in the native form with a molecular weight of 95000 each, as determined by gel filtration on Sepharose CL-6B and by polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. However, the glyoxysomal citrate synthase existed also as a tetramer with a molecular weight of 200000 in the presence of 10 m M MgCl₂.     

Evidence that glyoxysomal malate synthase is segregated by the endoplasmic reticulum

At the onset of castor bean (Ricinus communis) germination, 76% of the cellular malate synthase activity of the endosperm tissue was located in the microsomal fraction, with the remainder in the glyoxysomal fraction. During later developmental stages, when rapid malate synthase synthesis was occurring, an increasing proportion of the enzyme was recovered in glyoxysomes. The kinetics of [³⁵S]methionine incorporation into microsomal and glyoxysomal malate synthase in 2-day-old endosperm tissue was followed by employing antiserum raised against glyoxysomal malate synthase to precipitate specifically the enzyme from KCl extracts of these organelle fractions. This experiment showed that microsomal malate synthase was labeled before the glyoxysomal enzyme. When such kinetic experiments were interrupted by the addition of an excess of unlabeled methionine, ³⁵S-labeled malate synthase was rapidly lost from the microsomal fraction and was quantitatively recovered in the glyoxysomal fraction.

Free cytoplasmic ribosomes were separated from bound ribosomes (rough microsomes) using endosperm tissue labeled with [³⁵S]methionine or ¹⁴C-amino-acids. Nascent polypeptide chains were released from polysome fractions using a puromycin-high salt treatment, and radioactive malate synthase was shown to be exclusively associated with bound polysomes.

Together these data establish that malate synthase is synthesized on bound ribosomes and vectorially discharged into the endoplasmic reticulum cisternae prior to its ultimate sequestration in glyoxysomes.

     

Purification and comparative properties of microsomal and glyoxysomal malate synthase from castor bean endosperm

Sucrose density gradient centrifugation was employed to separate microsomes, mitochondria, and glyoxysomes from homogenates prepared from castor bean (Ricinus communis) endosperm. In the case of tissue removed from young seedlings, a significant proportion of the characteristic glyoxysomal enzyme malate synthase was recovered in the microsomal fraction. Malate synthase was purified from both isolated microsomes and glyoxysomes by a procedure involving osmotic shock, KCI solubilization, and sucrose density gradient centrifugation. All physical and catalytic properties examined were identical for the enzyme isolated from both organelle fractions. These properties include a molecular weight of 575,000, with a single subunit type of molecular weight 64,000, a pH optimum of 8, apparent K_m for acetyl-CoA of 10 μm and glyoxylate of 2 mm. Microsomal and glyoxysomal malate synthases showed identical responses to various inhibitors. Adenine nucleotides were competitive inhibitors with respect to acetyl-CoA, and oxalate (K_i 110 μm) and glycolate (K_i 150 μm) were competitive inhibitors with respect to glyoxylate. Antiserum raised in rabbits against purified glyoxysomal malate synthase was used to confirm serological identity between the microsomal and glyoxysomal enzymes, and was capable of specifically precipitating ³⁵S-labeled malate synthase from KCI extracts of both microsomes and glyoxysomes isolated from [³⁵S]methionine-labeled endosperm tissue.     

Microbody Malate Dehydrogenase Isozyme in Cotyledons of Cucumis sativus L. during Development

The properties of the microbody malate dehydrogenase (EC 1.1.1.37) (MDH) isozyme from cotyledons of Cucumus sativus L. were compared during development. It is concluded that the isozyme remains unaltered, despite the transition from glyoxysomal to peroxisomal function that occurs during greening of the cotyledons. This conclusion is based on electrophoretic behavior, chromatographic elution from DEAE-cellulose, molecular weight, kinetic behavior, and immunological identity. In most cases, the distinct properties of the other MDH isozymes in the tissue during development provide additional support for an unchanging microbody isozyme. A method for assaying specifically the microbody isozyme was developed; a diluted preparation was assayed spectrophotometrically before and after complete immunological precipitation. The turnover of the microbody MDH isozyme was investigated by a radioactive labeling study. There is incorporation into both glyoxysomal and peroxisomal MDH. Degradation rates do not correspond with either decline of glyoxysomal activity or the continuation of peroxisomal activity. Apparently, the microbody MDH isozyme is continually turned over throughout cotyledon development.     

Citrate synthetase in mitochondria and glyoxysomes of maize scutellum

Mitochondria and glyoxysomes were isolated from scutella of maize (Zea mays L.) by density gradient centrifugation. Citrate synthetase was partly solubilized from the organelles by sonication. The sonicated organelle suspensions were centrifuged at high speed, and the supernatants were used as enzyme preparations without further purification. The enzymes of the two organelles differ in all properties examined (pH activity curve, Km for substrates, elution volume on Sephadex G-100, mobility on starch gel at pH 7). Both enzymes are inhibited by ATP, but the inhibition is stronger for the mitochondrial enzyme. The inhibition is competitive for the mitochondrial enzyme and noncompetitive for the glyoxysomal enzyme. The glyoxysomal, but not the mitochondrial enzyme, is inhibited 40% with 1 mm ADP and cytidine triphosphate.     

Glycoproteins in the matrix of glyoxysomes in endosperm of castor bean seedlings

The matrix of glyoxysomes from endosperm of castor bean (Ricinus communis cv Hale) seedlings has been analyzed for the presence of glycosylated proteins. Glyoxysome preparations were monitored for organelle homogeneity by electron microscopy and enzyme marker activities. Glyoxysomes were essentially free of endoplasmic reticulum, mitochondria, and protein bodies. At least eight glyoxysomal matrix glycopeptides ranging in size from 39 to 160 kilodaltons were identified by their affinity for concanavalin A. The glyoxysomal glycoproteins were shown to be radioactively labeled when endosperm was allowed to incorporate glucosamine. Incorporation of glucosamine was inhibited by tunicamycin under conditions which did not inhibit protein synthesis. Hydrolysis of glyoxysomal extracts and subsequent analysis by paper chromatography showed that the labeled precursor was incorporated into the glycoprotein without prior dispersion of the label into amino acids. The present data demonstrate the occurrence of N-linked, high mannose oligosaccharides on polypeptides of the glyoxysomal matrix. This finding is discussed in relation to pathways of protein maturation and transport during glyoxysomal biogenesis.     

Evidence for the Presence of a Porin in the Membrane of Glyoxysomes of Castor Bean

Glyoxysomes of endosperm tissue of castor bean (Ricinus communis L.) seedlings were solubilized in a detergent and added to a lipid bilayer. Conductivity measurements revealed that the glyoxysomal preparation contained a porin-like channel. Using an electrophysiological method, which we established for semiquantitative determination of porin activity, we were able to demonstrate that glyoxysomal membranes purified by sucrose density gradient centrifugation contain an integral membrane protein with porin activity. The porin of glyoxysomes was shown to have a relatively small single-channel conductance of about 330 picosiemens in 1 M KCl and to be strongly anion selective. Thus, the glyoxysomal porin differs from the other previously characterized porins in the outer membrane of mitochondria or plastids, but is similar to the porin of spinach (Spinacia oleracea L.) leaf peroxisomes. Our results suggest that, in analogy to the porin of leaf peroxisomes, the glyoxysomal porin facilitates the passage of small metabolites, such as succinate, citrate, malate, and aspartate, through the membrane.     

Development of Microbodies in Sunflower Cotyledons and Castor Bean Endosperm during Germination

In cotyledons of sunflower seedlings glyoxysomal and peroxisomal enzymes exhibit different rates of development during germination. The total activity of isocitrate lyase, a glyoxysomal marker enzyme, rapidly increased during the first 3 days, and then decreased 89% by day 9. Exposure to light accelerated this decrease only slightly. The specific activity of glyoxysomal enzymes (malate synthetase, isocitrate lyase, citrate synthetase, and aconitase) in the microbody fraction from sucrose density gradients increased between days 2 and 4 about 2- to 3-fold, and thereafter it remained about constant in light or darkness.     

Inactivation of the glyoxysomal citrate synthase from castor bean endosperm by 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)

Summary Two isoenzymes of citrate synthase were found in the endosperm of germinating castor bean seeds. One isoenzyme is restricted to mitochondria and the other to glyoxysomes. The two citrate synthases can be separated by (NH₄)₂SO₄ gradient solubilization, eluting at 58 and 43% (NH₄)₂SO₄, respectively. They are easily distinguished by the sensitivity to 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of oxalacetate: the glyoxysomal enzyme is completely inactivated within 15 seconds, while the mitochondrial enzyme remains unaffected. The time course of inactivation is a first order reaction. Oxalacetate prevents inactivation in high concentrations. The differences in DTNB sensitivity of the two citrate synthases can, in turn, easily be used to distinguish between the two isoenzymes. Since DTNB is a chromogenic compound in the assay for citrate synthase, it interfers with the assay at low concentrations of oxalacetate during K_m determinations. This can be avoided by other assays which do not include DTNB. The inactivation of the glyoxysomal citrate synthase of castor bean endosperm is similar to the known inactivation of prokaryotic citrate synthases.Abbreviation DTNB 5,5-dithiobis(2-nitrobenzoic acid)     

Disruption of the peroxisomal citrate synthase CshA affects cell growth and multicellular development in Dictyostelium discoideum

Non-mitochondrial citrate synthase catalyses citrate synthesis in the glyoxylate cycle in gluconeogenesis. Screening Dictyostelium discoideum mutants generated by insertional mutagenesis isolated a poor-growing mutant that displayed aberrant developmental morphology on bacterial lawns. Axenically grown mutants developed normally and formed mature fruiting bodies on buffered agar. The affected locus encoded a novel protein (CshA) that was homologous to glyoxysomal citrate synthase. cshA was expressed maximally during vegetative growth and gradually decreased through subsequent developmental stages. An in vitro citrate synthase assay revealed that cshA disruption resulted in a 50% reduction in enzyme activity, implicating CshA as an active citrate synthase. The amino-terminus of CshA was found to have an atypical mitochondrial targeting signal, instead containing a unique nonapeptide sequence (RINILANHL) that was homologous to the conserved peroxisomal targeting signal 2 (PTS2). CshA protein was shown to be localized in the peroxisomes, and the RINILANHL sequence only efficiently targeted the peroxisomal green fluorescent protein. The growth defect of cshA(-) cells was associated with the impairment of phagocytosis and fluid-phase endocytosis, independent from cytokinesis. Disrupted multicellular development on bacterial lawns resulted from the abnormal susceptibility to the environmental conditions, perhaps because of citrate insufficiency. Taken together, these results provide new insights into the function of peroxisomal citrate synthase in cell growth and multicellular development.     

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.     

Structure and Expression of Mitochondrial Citrate Synthases from Higher Plants

Mitochondrial citrate synthase (EC 4.1.3.7 [EC] ) represents the firstenzyme of the tricarboxylic acid cycle, catalyzing the condensationof acetyl-CoA and oxaloacetate, finally yielding citrate andCoA. We report here the isolation of cDNA clones encoding citratesynthase from Nicotiana tabacum, Beta vulgaris and Populus.Nucleotide and deduced amino acid sequences were compared withpreviously published sequences of mitochondrial citrate synthasesfrom Arabidopsis thaliana and potato, as well as with the sequenceof glyoxysomal citrate synthase from pumpkin. Homologies betweenthe various plant mitochondrial enzymes were in the range from77.2% (potato vs. Arabidopsis) to 94.2% (potato vs. tobacco)on the nucleotide level (coding regions only), and in the rangefrom 70.1% to 90.4% (potato vs. Arabidopsis, and potato vs.tobacco, respectively) on the amino acid level. Identities ofthe mitochondrial isozymes to the pumpkin glyoxysomal enzymewere below 30% on the nucleotide and amino acid level. In Northernblot experiments citrate synthase mRNA was detected in all tissuesanalyzed. However, levels of expression showed tissue dependencydespite the fact that citrate synthase is usually considereda house-keeping enzyme. Whether these different levels of expressionreflect tissuespecifc variations with respect to basic metabolismawaits further analysis. (Received May 20, 1996; Accepted August 20, 1996)     

Presence of a Bacterial-Like Citrate Synthase Gene in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>: Recent Lateral Gene Transfers (LGT) or Multiple Gene Losses Subsequent to a Single Ancient LGT?

Citrate synthase is the initial enzyme in the tricarboxylic acid cycle of mitochondria. In plants and fungi, it is the second isozyme in the glyoxylate cycle of peroxisomes (or glyoxysomes), and it is also present in bacteria. Some of the biochemical reactions in the glyoxylate cycle of the ciliated protozoan Tetrahymena pyriformis depend upon mitochondrial enzymes, as T. pyriformis lacks some glyoxysome-specific enzymes. Here we demonstrate a new citrate synthase gene from Tetrahymena thermophila that is different from the mitochondrial counterpart. A potential peroxysome-targeted signal was detected in the N-terminus, suggesting the localization of the enzyme in peroxysomes. Phylogenetic analysis placed the Tetrahymena sequence in a clade consisting of a few sequences from eukaryotes such as cellular slime molds and two land plants, near a green sulfur bacterium and many proteobacteria as a sister group but not in a mitochondrial clade. Southern blot analysis revealed that this type of gene was absent from distantly related ciliates and other species of Tetrahymena except for the closest species, T. mallaccensis. The scattered presence of the bacterial-like genes among distantly related eukaryotes suggests three alternative interpretations of acquisition of the novel glyoxysomal citrate synthase gene via lateral gene transfer (LGT). (1) Some eukaryotes independently acquired the gene from a common bacterium or closely related bacteria via LGT. (2) A hypothetical eukaryote once acquired the gene, which was thereafter independently transferred from the eukaryote to other eukaryotes. (3) A single event of LGT (or duplication) occurred in a certain common ancestor of eukaryotes, followed by multiple losses in many eukaryotic lineages during the subsequent evolution. Considering the monophyly of the bacterial-like eukaryotic citrate synthase genes, the first model is somewhat unlikely, even though it is not impossible. The second and third models can rationally explain the present observation, so these models are discused in some detail.     

Subcellular distribution, multiple forms and development of glutamate-pyruvate (glyoxylate) aminotransferase in plant tissues

According to a sucrose density gradient analysis of cell organelles from homogenates of green leaves of rye, wheat and pea seedlings glutamate-pyruvate aminotransferase was predominantly localized in the leaf microbodies (peroxisomes; 90%) and to a minor extent in the mitochondria (10%) but completely absent from chloroplasts. In etiolated rye leaves the distribution of the enzyme was similar. In other non-green tissues glutamate-pyruvate aminotransferase was predominantly associated with the mitochondria but also present in the microbodies of dark-grown pea roots and in the glyoxysomes of Ricinus endosperm. In the microbodies isolated from potato tubers the enzyme was not detectable. Glutamate-pyruvate aminotransferase activity was not associated with the proplastid fractions of the non-green tissues. The distribution of glutamate-oxaloacetate aminotransferase was different from that of glutamate-pyruvate aminotransferase. Glutamate-oxaloacetate aminotransferase was found in chloroplasts, proplastids, mitochondria, microbodies and in the supernatant. Evidence is presented that glutamate-pyruvate and glutamate-glyoxylate aminotransferase activities were catalyzed by the same enzyme. Both activities showed the same organelle distribution on sucrose gradients and both were eluted at the same salt concentration from DEAE-cellulose. By chromatography of preparations from rye leaf extracts on DEAE-cellulose two forms of glutamate-pyruvate (glyoxylate) aminotransferase were separated. The major fraction eluting at a low salt concentration was identified as peroxisomal form and the minor fraction eluting at a higher salt concentration was identified as a mitochondrial form. Both the glutamate-glyoxylate and the glutamate-pyruvate aminotransferase activities of the peroxisomal as well as of the mitochondrial forms of the enzyme were strongly (about 80%) inhibited by the presence of 10 mM glycidate, previously described as an inhibitor of glutamate-glyoxylate aminotransferase in tobacco tissue. Pig heart glutamate-pyruvate aminotransferase exhibited no glutamate-glyoxylate aminotransferase activity and was only slightly inhibited by glycidate. The development of glutamate-pyruvate aminotransferase activity in the leaves of rye seedlings was strongly increased in the light, relative to dark-grown seedlings, and very similar to that of catalase activity while the development of glutamate-oxaloacetate aminotransferase was, in close coincidence with the behavior of leaf growth, only slightly enhanced by light. It is discussed that in green leaves an extrachloroplastic synthesis of alanine is of considerable advantage for the metabolic flow during photosynthesis.     

Characterization of the activity of fatty-acid epoxide hydrolase in seeds of castor bean (Ricinus communis L.)

Epoxide hydrolase (EC 3.3.2.3) activity was measured with [1-¹⁴C]cis-9,10-epoxystearic acid as the substrate. Homogenates were prepared from the endosperm tissue of germinating seeds of castor bean (Ricinus communis L. zanzibariensis). The activity of fatty-acid epoxide hydrolase was characterized with respect to dependence on time, amount of protein, pH and temperature. Analyses of enzyme distribution in endosperm, cotyledons, root and hypocotyl showed the highest total activity in the endosperm, less in the cotyledons and low activity in the root and hypocotyl. The specific activity was similar for cotyledons and endosperm. Analysis of the temporal expression of the enzyme in the endosperm during germination revealed high activity already in the imbibed seed. Activity was maximal between days four to six and then decreased at the end of one week. Subcellular fractionation of endosperm revealed a dual distribution of activity between the glyoxysomal and the cytosolic fractions.     

Cytochemical demonstration of malate synthase and glycolate oxidase in microbodies of cucumber cotyledons

The cytochemical localizations of malate synthase (glyoxysomal marker) and glycolate oxidase (peroxisomal marker) have been examined in cotyledon segments and sucrose-gradient fractions from germinated cucumber (Cucumis sativus L.) seedlings. The seedlings were grown in the dark for 4 days, transferred to 4 hours of continuous light, then returned to the dark for 24 hours. Under these conditions, high specific activities for both glyoxysomal and peroxisomal enzymes are maintained in cotyledon homogenates and microbody-enriched fractions. Electron cytochemistry of the marker enzymes reveals that all or virtually all the microbodies observed in cotyledonary cells and sucrose-gradient fractions contain both enzymes. The staining in gradient fractions was determined from scoring a minimum of 600 photographed microbodies for each enzyme. After correcting for the number of particles stained for catalase reactivity (representing true microbodies), 94 and 97% of the microbodies were found stained for malate synthase and glycolate oxidase activity, respectively.     

Presence of glyoxylate cycle enzymes in the mitochondria of Euglena gracilis

Isocitrate lyase and malate synthase are specific enzymes of the glyoxylate cycle, used here as glyoxysomal markers. Both enzymes were found in the mitochondrial fraction after organelle fractionation by isopycnic centrifugation. Electron microscopy of this fraction indicated that mitochondria were the only recognizable organelles. Using an immunogold labeling method with anti-(malate synthase) antiserum, the only organelles stained in cells were the mitochondria. These results show that the glyoxylate cycle is present in mitochondria in Euglena.     

Effect of gibberellin a(3) on the endoplasmic reticulum and on the formation of glyoxysomes in the endosperm of germinating castor bean

Seedlings of castor bean (Ricinus communis cv. Hale) were exposed to a range of concentrations of gibberellin A₃ (GA₃). Treatments for 20 hours with GA₃ concentrations of 0.5 μM or higher resulted in increased levels of NADH-cytochrome c reductase, phosphorylcholine glyceride transferase, and malate synthase in endoplasmic reticulum (ER) isolated from endosperm on linear sucrose gradients. GA₃ treatment also resulted in increased RNA associated with ER. Malate synthase and catalase in crude homogenates were enhanced by 1 to 100 μM GA₃ concentrations. Isocitrate lyase, citrate synthase, malate synthase, catalase, and glycolate oxidase in isolated glyoxysomes were enhanced by 60, 20, 18, 40, and 28%, respectively, over controls. Treatment with abscisic acid led to decreased levels of glyoxysomal enzymes and reduced glyoxysomal protein. The effect of GA₃ and abscisic acid on the specific activities of glyoxysomes of different densities suggests that GA₃ influences enzyme levels and glyoxysome assembly.     

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes in tissues and callus cultures ofCereus peruvianus (cactaceae)

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes were investigated in seeds and in seedlings and calli cultures ofC. peruvianus to determine if the changes in MDH isozyme banding patterns could be used as biochemical markers to identify the origin of regenerated plants from callus tissues. Four cytoplasmic MDH isozymes (sMDH), five mitochondrial MDH isozymes (mMDH), and one glyoxysomal MDH isozyme (gMDH) were detected and showed tissue- and stage-specific expression. A relationship of mMDH and gMDH isozyme patterns with callus tissues subcultured in three hormonal combinations and with the plants regenerated from these callus tissues was demonstrated. Furthermore, temperature and mechanical stress were found to be closely related to mMDH-1 activity in callus culture. Therefore, the different patterns of MDH isozymes in the various tissues ofC. peruvianus can be used as biochemical markers for the study of gene expression during development and as powerful tools in monitoring studies on callus cultures. This research was supported by the CNPq.