Purification and characterization of aconitase isoforms from etiolated pumpkin cotyledons

Although aconitase (EC 4.2.1.3) is involved in the glyoxylate cycle, which is localized in glyoxysomes, we detected very low aconitase activity in glyoxysomal fractions after sucrose gradient centrifugation of extracts prepared from etiolated pumpkin ( Cucurbita sp.) colyledons. Two aconitase isoforms were purified to homogeneity, albeit in low yield, by hydrophobic interaction, hydroxylapatite and anion exchange chromatography. They were designated Aco I and Aco II; both were shown to be monomeric proteins of M₁ 100 000 or 98 000 by gel filtration and SDS-PAGE analysis, respectively; isoelectric points were 5.0 and 4.8, respectively. Kinetic studies revealed similarities between Aco I and Aco II. A third aconitase isoform (Aco III) was revealed but not purified to homogeneity.     

Cytosolic Aconitase Participates in the Glyoxylate Cycle in Etiolated Pumpkin Cotyledons

Two different aconitases are known to be expressed after thegermination of oil-seed plants. One is a mitochondrial aconitasethat is involved in the tricarboxylic acid cycle. The otherparticipates in the glyoxylate cycle, playing a role in gluconeogenesisfrom stored oil. We isolated and characterized a cDNA for anaconitase from etiolated pumpkin cotyledons. The cDNA was 3,145bp long and capable of encoding a protein of 98 kDa. N-terminaland C-terminal amino acid sequences deduced from the cDNA didnot contain mitochondrial or glyoxysomal targeting signals.A search of protein databases suggested that the cDNA encodeda cytosolic aconitase. Immuno blotting analysis with a specificantibody against the aconitase expressed in Escherichia colirevealed that developmental changes in the amount of the aconitasewere correlated with changes in levels of other enzymes of theglyoxylate cycle during growth of seedlings. Further analysisby subcellular fractionation and immunofluorescence microscopyrevealed that aconitase was present only in the cytosol andmitochondria. No glyoxysomal aconitase was found in etiolatedcotyledons even though all the other enzymes of the glyoxylatecycle are known to be localized in glyoxysomes. Taken together,the data suggest that the cytosolic aconitase participates inthe glyoxylate cycle with four glyoxysomal enzymes. (Received December 1, 1994; Accepted March 17, 1995)     

Activation of aconitase in mouse fast-twitch skeletal muscle during contraction-mediated oxidative stress

Aconitase is a mitochondrial enzyme that converts citrate to isocitrate in the tricarboxylic acid cycle and is inactivated by reactive oxygen species (ROS). We investigated the effect of exercise/contraction, which is associated with elevated ROS production, on aconitase activity in skeletal muscle. Humans cycled at 75% of maximal workload, followed by six 60-s bouts at 125% of maximum workload. Biopsies were taken from the thigh muscle at rest and after the submaximal and supramaximal workloads. Isolated mouse extensor digitorum longus (EDL; fast twitch) and soleus (slow twitch) muscles were stimulated to perform repeated contractions for 10 min. Muscles were analyzed for enzyme activities and glutathione status. Exercise did not affect aconitase activity in human muscle despite increased oxidative stress, as judged by elevated levels of oxidized glutathione. Similarly, repeated contractions did not alter aconitase activity in soleus muscle. In contrast, repeated contractions significantly increased aconitase activity in EDL muscle by 50%, despite increased ROS production. This increase was not associated with a change in the amount of immunoreactive aconitase (Western blot) but was markedly inhibited by cyclosporin A, an inhibitor of the protein phosphatase calcineurin. Immunoprecipitation experiments demonstrated that aconitase was phosphorylated on serine residues. Aconitase in cell-free extracts was inactivated by the addition of the ROS hydrogen peroxide. In conclusion, the results suggest that aconitase activity can be regulated by at least two mechanisms: oxidation/reduction and phosphorylation/dephosphorylation. During contraction, a ROS-mediated inactivation of aconitase can be overcome, possibly by dephosphorylation of the enzyme. The dual-control system may be important in maintaining aerobic ATP production during muscle contraction. glutathione; reactive oxygen species     

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.     

Purification and characterization of a novel pumpkin short-chain acyl-coenzyme A oxidase with structural similarity to acyl-coenzyme A dehydrogenases

A novel pumpkin (Cucurbita pepo) short-chain acyl-coenzyme A (CoA) oxidase (ACOX) was purified to homogeneity by hydrophobic-interaction, hydroxyapatite, affinity, and anion-exchange chromatography. The purified enzyme is a tetrameric protein, consisting of apparently identical 47-kD subunits. The protein structure of this oxidase differs from other plant and mammalian ACOXs, but is similar to the protein structure of mammalian mitochondrial acyl-CoA dehydrogenase (ACDH) and the recently identified plant mitochondrial ACDH. Subcellular organelle separation by sucrose density gradient centrifugation revealed that the enzyme is localized in glyoxysomes, whereas no immunoreactive bands of similar molecular weight were detected in mitochondrial fractions. The enzyme selectively catalyzes the oxidation of CoA esters of fatty acids with 4 to 10 carbon atoms, and exhibits the highest activity on C-6 fatty acids. Apparently, the enzyme has no activity on CoA esters of branched-chain or dicarboxylic fatty acids. The enzyme is slightly inhibited by high concentrations of substrate and it is not inhibited by Triton X-100 at concentrations up to 0.5% (v/v). The characteristics of this novel ACOX enzyme are discussed in relation to other ACOXs and ACDHs.     

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.     

Studies on aconitase species from Saccharomyces cerevisiae, porcine and bovine heart, obtained by a modified isolation method

Aconitase (citrate (isocitrate) hydro-lyase, EC 4.2.1.3) was isolated from Saccharomyces cerevisiae, porcine and bovine heart by a simplified method including affinity chromatography on Blue Dextran-Sepharose. Partial characterisation reveals that the aconitase species are all similar due to molecule size, amino acid composition, isoelectric point and enzymatic activity. Aconitase appears as a single polypeptide chain with a small carbohydrate content. A molecular weight of 79000 +/- 2000 and a Svedberg constant of s20,w = 4.75 +/- 0.2 S indicate a compact structure of aconitase. Due to different properties among the yeast aconitase species concerning isoelectric point and enzymatic activity a coherence between net charge of the protein and redox state of the Fe-S cluster can be expected.     

Protein kinase C located in rat liver nuclei. Partial purification and biochemical and immunochemical characterization

In the rat liver homogenate, maximal protein kinase C activity was found at two calcium concentrations (1.75 and 3.5 mM). Subcellular fractionation of the liver homogenate revealed that the protein kinase C activity requiring 1.75 mM calcium was present only in the cytosolic and particulate subcellular fractions. The protein kinase C activity requiring 3.5 mM calcium concentration was mainly located in the rat liver nuclei preparation. About 19% of the liver homogenate protein kinase C activity requiring 3.5 mM calcium was present in the nuclei. Goat anti-rat brain protein kinase C antibodies revealed a single immunoreactive band at 80-82 kDa in the rat liver nuclear, particulate, or cytosolic fractions. Based on the ratio of plasma membrane marker enzyme activity determined in the nuclear preparation, the purity of the isolated nuclei was ascertained. Rat liver nuclear protein kinase C activity has been partially purified. The purification steps sequentially employed were Triton X-100 extraction of isolated nuclei, DEAE-cellulose chromatography, Phenyl-Superose, and Mono Q (fast protein liquid) chromatography. The final purification step revealed, by silver nitrate staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two protein bands at 80 and 66 kDa, respectively. These findings provide definitive data regarding the nuclear location of protein kinase C. The nuclear location of protein kinase C may lead to an understanding of the molecular pathway involved in signal transduction from the plasma membrane to the nucleus.     

High molecular weight forms of basic fibroblast growth factor recognized by a new anti-bFGF antibody

An antibody against basic fibroblasts growth factor (bFGF) was raised using purified bovine pituitary bFGF. Western blot analysis revealed immunoreactive bands at 18, 24, 30-33 and 46 kDa in immunoaffinity purified extracts of pituitary and adrenal gland using this antibody. A similar staining pattern was obtained with ovary extracts with the exception of the missing 18 kDa band. A second anti-bFGF antibody raised against a synthetic peptide comprising the 24 N-terminal amino acids of bFGF reacted with the 18 kDa and the 46 kDa band of immunoaffinity purified ovary and adrenal gland extracts.     

Biosynthesis and intracellular transport of glyoxysomal malate dehydrogenase in germinating pumpkin cotyledons

Glyoxysomal malate dehydrogenase was synthesized as a larger molecular mass precursor in germinating pumpkin cotyledons. In pulse-chase experiments, the radioactive larger molecular mass precursor (38 kDa) disappeared and was converted to the mature form (33 kDa) of the enzyme. When the radiolabeled cotyledon was fractionated into cytosolic and organellar fractions, the larger molecular mass precursor was first recovered in the cytosolic fraction and then only after a 20 min chase the mature form was found in the organellar fraction. This indicates that the higher molecular mass precursor is synthesized in the cytosol and the processing of the transient precursor is coupled to the transport into glyoxysomes.     

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.     

Partial characterization of a major family of proteins in turions of Hydrilla verticillata

Electrophoretograms of turions of dioecious Hydrilla verticillata (L. f.) Royle, run under non-denaturing conditions, had a major complex protein band at R_f0.45 (7.5% acrylamide). Extracts of monoecious plants under similar conditions had major bands at R_f 0.43 and 0.45. The polypeptides which comprise these bands were partially purified and characterized. The major protein fraction in extracts of dioecious turions had a molecular mass of 58 kDa on gel permeation chromatography. Electrophoresis of this fraction under denaturing conditions in the presence of sodium dodecyl sulfate indicated principal bands with molecular masses of 58 and 57 kDa. Extracts from turions of the monoecious biotype had major bands at 59 and 55 kDa after electrophoresis under denaturing conditions. Antisera were raised against the proteins from the dioecious turion at R_f 0.45 after electrophoresis under non-denaturing conditions. When blots of gels run under non-denaturing conditions were probed with these antisera, a complex band was seen at R_f 0.45 for extracts of the dioecious biotype, while bands were observed at R_f 0.43 and 0.45 for the monoecious extracts. After electrophoresis under denaturing conditions, immunoreactive bands were noted at 58 and 57 kDa or 59 and 55 kDa in extracts of dioecious and monoecious turions, respectively. Extracts of leaves and stems of H. verticillata had detectable amounts of immunoreactive proteins, regardless of photoperiod, hence turion production. Related plants with the aquatic habit had immunoreactive proteins in their leaves and organs of perennation [Elodea canadensis Michx., Elodea nuttallii (Planch.) St. John, and Egeria densa Planch., Potamogeton nodosus Poir. and P. pectinatus L.], but the presence of these proteins was not noted in other plants (Zea mays L., Allium cepa L., Spinacia oleracea L., Lemna gibba L., or Solanum tuberosum L.).     

Purification of allantoinase from soybean seeds and production and characterization of anti-allantoinase antibodies.

Allantoinase catalyzes the hydrolysis of allantoin to allantoic acid, a reaction important in both biogenesis and degradation of ureides. Ureide production in cotyledons of germinating soybean (Glycine max L.) seeds has not been studied extensively but may be important in mobilizing nitrogen reserves. Allantoinase was purified approximately 2500-fold from a crude extract of soybean seeds by differential centrifugation, heat treatment, ammonium sulfate fractionation, ethanol fractionation, and fast protein liquid chromatography (Pharmacia) with Mono-Q and Superose columns. The purified enzyme had a subunit size of 30 kD. Polyclonal antibodies produced against the purified protein titrated allantoinase activity in a crude extract of seed proteins. Antibodies recognized the 30-kD band in western blot analysis of crude seed extracts, indicating that they were specific for allantoinase.     

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.

     

Ovigerous-hair stripping substance (OHSS) in an estuarine crab: purification, preliminary characterization, and appearance of the activity in the developing embryos

Ovigerous-hair stripping substance (OHSS) is an active factor in crab hatch water (i.e., filtered medium into which zoea larvae have been released). This factor participates in stripping off the egg attachment structures (i.e., egg case, funiculus, and the coat investing ovigerous hairs) that remain attached to the female's ovigerous hairs after larval release. Thus this activity prepares the hairs for the next clutch of embryos. OHSS activity of an estuarine crab, Sesarma haematocheir, eluted as a single peak on molecular-sieve chromatography, but this peak still showed two protein bands at 32 kDa and 30 kDa on SDS-PAGE. The two protein bands stained with a polyclonal antiserum raised to the active fractions from molecular-sieve chromatography. Moreover, antibodies purified from this polyclonal OHSS antiserum also recognized both the 32-kDa and 30-kDa bands. OHSS immunoreactivity and biological activity were associated with the attachment structures that remained connected to the ovigerous hairs after hatching. In developing embryos, both protein bands could be stained immunochemically at least 10 days before hatching. But OHSS biological activity appeared only 3 days before hatching. The immunoreactive protein bands were not observed in the zoea, but OHSS bioreactivity was present, though greatly reduced. The 32-kDa protein, at least, is probably an active OHSS, and the 30-kDa protein band may also be OHSS-related. The OHSS appears to be produced and stored by the developing embryo. Upon hatching, most of the material may be trapped by the remnant structures, and the remainder is released into the ambient water.     

Identification and characterization of proteases involved in specific proteolysis of vitellogenin and yolk proteins in salmonids.

A pepstatin A-sensitive enzyme involved in yolk formation was purified from the masu salmon (Oncorhynchus masou) ovary using in vitro generation of yolk proteins from purified vitellogenin to assay enzymatic activity. Purification of the enzyme involved precipitation of ovarian extracts by water and ammonium sulfate followed by five steps of column chromatography. After SDS-PAGE and Western blotting, the purified enzyme appeared as a single approximately 42 kDa band that was immunoreactive to anti-human cathepsin D. The course of proteolytic cleavage of the three major yolk proteins (lipovitellin, beta'-component, and phosvitin) in fertilized masu salmon and Sakhalin taimen (Hucho perryi) eggs and embryos was visualized by SDS-PAGE and Western blotting using specific antisera. Major yolk protein bands appeared in positions corresponding to 92 kDa, 68 kDa, and 22 kDa (lipovitellin-derived peptides), as well as 17 kDa (beta'-component). During embryo development, the 92 kDa and 22 kDa bands gradually decreased in intensity, becoming undetectable in alevins. The 68 kDa band and a minor 24 kDa band became more intense after the eyed stage. Two additional peptides, corresponding to 40 and 28 kDa, newly appeared in alevins. During embryonic growth, the beta'-component band (17 kDa) persisted and phosvitin appeared to be progressively dephosphorylated. In vitro analysis of lipovitellin proteolysis indicated that the enzyme involved is a Pefabloc SC-sensitive serine protease. These results demonstrate, for the first time, that a cathepsin D-like protease and serine proteases play key roles in yolk formation and degradation, respectively, in salmonid fishes.     

Iron and aconitase activity

Aconitase activated with Fe(2+), cysteine and ascorbate incorporates 1 g-atom of Fe(2+)/mol. Loss of this Fe(2+) by transfer to ferrozine, a Fe(2+) chelator, results in loss of activity. Ascorbate increases the rate of transfer of the essential Fe(2+) whereas citrate retards the rate of transfer. Transfer of Fe(2+) from inactive aconitase, 2 g-atoms of Fe/mol, can be accomplished in the presence of urea and ascorbate. The correlation of activity with the presence of an added g-atom of Fe(2+)/mol leads to the conclusion that active aconitase has only one active site per mol.     

Purification and characterization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus

A hygromycin B phosphotransferase activity from Streptomyces hygroscopicus has been highly purified by ammonium sulphate fractionation followed by affinity column chromatography through Sepharose-6B-hygromycin-B. The combined active fractions showed a single protein band (41 kDa) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. When gel electrophoresis was performed under non-denaturing conditions, the single protein band promoted in situ phosphorylation of hygromycin B, indicating that this protein corresponded to the purified hygromycin B phosphotransferase. The enzyme has been purified 236-fold and approximate Km values of 0.56 microM for hygromycin B and ATP, respectively, were deduced.