Fluorescence immunohistochemical localization of malate dehydrogenase isoenzymes in watermelon cotyledons : a developmental study of glyoxysomes and mitochondria

Monospecific antibodies to glyoxysomal, mitochondrial, and cytosolic I malate dehydrogenase were used for the fluorescence immunohistochemical localization of these isoenzymes in dark-grown watermelon (Citrullus vulgaris Schrad.) cotyledons. It was demonstrated that, with cell organelles isolated by sucrose density gradient centrifugation, antibodies to glyoxysomal malate dehydrogenase were specific markers for glyoxysomes, and similarly, antibodies to mitochondrial malate dehydrogenase were markers for mitochondria. The time course of the glyoxysomal malate dehydrogenase appearance and decline was not synchronous for the individual tissues and differed completely from that of the mitochondria. The cytosolic malate dehydrogenase I was confined to restricted regions of the lower epidermis. The activity which was definitively localized outside the cell organelles decreased during the first days of germination.     

Fate of glyoxysomal malate dehydrogenase from watermelon after heterologous in-vivo translation in xenopus oocytes

Total poly A⁺-mRNA from watermelon cotyledons was translated in Xenopus laevis oocytes. Watermelon glyoxysomal malate dehydrogenase was found as its higher molecular weight precursor (pre-gMDH) and accumulates over at least 48 hours of translation. Organelle separation and immunocytochemistry located the watermelon pre-gMDH in the cytosol of the oocyte. The heterologous translation product from oocytes can be imported into isolated glyoxysomes from endosperm of castor bean. This import was correct in terms of protection against proteolysis and cleavage of the presequence within the limits of accuracy. We conclude that watermelon pre-gMDH accumulates in mRNA-injected oocytes as an import competent cytosolic precursor.     

Sequence homologies between glyoxysomal and mitochondrial malate dehydrogenase

The comparison of mitochondrial and glyoxysomal malate dehydrogenase (EC 1.1.1.37) from cotyledons of germinating watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) by means of serological methods and peptide patterns revealed a high degree of homology. The N-terminal sequence analysis yielded a distinct presequence of eight or nine amino-acid residues, respectively, which is followed by an almost identical stretch of at least 20 amino-acid residues. A very similar domain has been recognized for mitochondrial malate dehydrogenase from porcine heart and yeast, and for Escherichia coli malate dehydrogenase.Abbreviations gMDH glyoxysomal malate dehydrogenase - mMDH mitochondrial malate dehydrogenase - SDS sodium dodecyl sulfate     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

Functional Analysis of the N-Terminal Prepeptides of Watermelon Mitochondrial and Glyoxysomal Malate Dehydrogenases*

Mitochondrial and glyoxysomal malate dehydrogenase (mMDH; gMDH; L-malate: NAD⁺ oxidoreductase; EC 1.1.1.37) of watermelon (Citrullus vulgaris) cotyledons are synthesized with N-terminal cleavable presequences which are shown to specify sorting of the two proteins. The two presequences differ in length (27 or 37 amino acids) and primary structure. Precursor proteins of the two isoenzymes with site-directed mutations in their presequences and hybrid precursor proteins with reciprocally exchanged presequences were analyzed for proper import using two approaches, namely in vitro using isolated watermelon organelles or in vivo after synthesis in the heterologous host, Hansenula polymorpha. The mitochondrial presequence is essential and sufficient to target the mature glyoxysomal isoenzyme into mitochondria (Gietl et al., 1994). As to the function of the mitochondrial presequence a substitution of ^?3R (considered important for one step precursor cleavage in yeast and mammals) with ^?3L permitted import into mitochondria but cleavage of the transit peptide and conversion into active mature enzyme was impeded. Substitution of ^?13R^?12S (in a sequence reminiscent of the octapeptide motif serving as a substrate for the mammalian and yeast intermediate peptidase) into ^?13L¹²F permitted mitochondrial import and processing like the wild type transit peptide. Purified rat mitochondrial processing protease, which can effect single step cleavage of mitochondrial protein precursors, cleaves in vitro translated watermelon mMDH precursor into its mature form. The glyoxysomal presequence is essential and sufficient to target the mature mitochondrial isoenzyme into peroxisomes of Hansenula polymorpha, but these peroxisomes lack a processing enzyme to cleave the presequence (Gietl et al., 1994). We here show that isolated watermelon organelles also import the hybrid proteins in vitro and process the glyoxysomal presequence. Site directed mutations within the conserved RI-X₅-HL-motif impede efficiency of import and cleavage by watermelon organelles.     

Import of glyoxysomal malate dehydrogenase precursor into glyoxysomes: A heterologous in-vitro system

A heterologous in-vitro system is described for the import of the precursor to glyoxysomal malate dehydrogenase from watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) cotyledons into glyoxysomes from castor-bean (Ricinus communis L.) endosperm. The 41-kDa precursor is posttranslationally sequestered and correctly processed to the mature 33-kDa subunit by a crude glyoxysomal fraction or by glyoxysomes purified on a sucrose gradient. The import and the cleavage of the extrasequence is not inhibited by metal chelators such as 1,10-phenanthroline and ethylenediaminetetraacetic acid. Uncouplers (carbonylcyanide m-chlorophenylhydrazone), ionophores (valinomycin), or inhibitors of oxidative phosphorylation (oligomycin) and ATP-ADP translocation (carboxyatractyloside) do not interfere, thus indicating the independence of the process of import by the organelle from the energization of the glyoxysomal membrane.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - EDTA ethylenediaminetetraacctic acid - gMDH glyoxysomal malate dehydrogenase - PMSF phenylmethylsulfonyl fluoride     

Cell-free synthesis of glyoxysomal malate dehydrogenase

Polyadenylated mRNA was isolated from germinating watermelon cotyledons and translated in a wheat germ protein synthesizing system. The synthesis of glyoxysomal malate dehydrogenase was detected by direct immunoprecipitation and electrophoretic analysis of the precipitate. In addition to a small amount of the authentic isoenzyme (subunit molecular weight = 33 000), the major part of the incorporated [35S] methionine was observed in a polypeptide with a molecular weight of 38 000. The possible role of the larger molecule as a precursor of glyoxysomal malate dehydrogenase is discussed.     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

Partitioning of malate dehydrogenase isoenzymes into glyoxysomes, mitochondria, and chloroplasts

Malate dehydrogenase isoenzymes catalyzing the oxidation of malate to oxaloacetate are highly active enzymes in mitochondria, in peroxisomes, in chloroplasts, and in the cytosol. Determination of the primary structure of the isoenzymes has disclosed that they are encoded in different nuclear genes. All three organelle-targeted malate dehydrogenases are synthesized with an amino terminal extension that is cleaved off in connection with the import of the enzyme precursor into the organelle. The sequence of the 27 amino acids of the mitochondrial transit peptide is unrelated to the 37-residue glyoxysomal transit peptide, which in turn is entirely different in sequence from the 57-residue chloroplastic transit peptide. With the exception of malate dehydrogenase and 3-ketoacyl thiolase, peroxisomal enzymes are synthesized without transit peptides and are frequently translocated into the organelle with a peroxisomal targeting signal consisting of a conserved tripeptide at the carboxy terminus of the protein. Based on the observation that this tripeptide (Ala-His-Leu) occurs in the transit peptides of glyoxysomal malate dehydrogenase and peroxisomal 3-ketoacyl thiolase, the possible significance of amino terminal transit peptides for peroxisome import is discussed.     

Regulation of Glyoxysomal Enzymes during Germination of Cucumber: 3. IN VITRO TRANSLATION AND CHARACTERIZATION OF FOUR GLYOXYSOMAL ENZYMES

Monospecific antibodies raised against four glyoxysomal enzymes (isocitrate lyase, catalase, malate synthase, and malate dehydrogenase) have been used to detect these proteins among the products of in vitro translation in a wheat germ system programmed with cotyledonary RNA from cucumber seedlings. In vitro immunoprecipitates were compared electrophoretically with the same enzymes labeled in vivo and also with the purified proteins. Isocitrate lyase yields two bands on sodium dodecyl sulfate-polyacrylamide gels, as synthesized both in vitro (61.5K and 60K products) and in vivo (63K and 61.5K polypeptides). Both the 63K and 61.5K subunits can also be demonstrated for the isolated enzyme. The two subunits are antigenically cross-reactive and yield similar electrophoretic profiles upon partial proteolytic digestion. A larger subunit is seen in vitro than in vivo for both malate dehydrogenase (38K versus 33K) and catalase (55K versus 54K); this suggests a need for processing which is often a characteristic of proteins that must be transported across or into membranes. Malate synthase has a molecular weight of 57K both in vitro and in vivo, but the isolated enzyme is a glycoprotein, containing N-acetyl glucosamine, mannose, and possibly also fucose and xylose. This indicates that the polypeptide portion of the isolated enzyme is smaller than the in vitro product and suggests processing of malate synthase also. None of the other three enzymes appears to be glycosylated. The implications of these size differences for the compartmentalization of matrix and membrane-bound glyoxysomal enzymes are discussed.     

Mitochondrial malate dehydrogenase from corn : purification of multiple forms

A method to fractionate corn (Zea mays L. B73) mitochondria into soluble proteins, high molecular weight soluble proteins, and membrane proteins was developed. These fractions were analyzed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assays of mitochondrial enzyme activities. The Krebs cycle enzymes were enriched in the soluble fraction. Malate dehydrogenase has been purified from the soluble fraction by a two-step fast protein liquid chromatography method. Six different malate dehydrogenase peaks were obtained from the Mono Q column. These peaks were individually purified using a Phenyl Superose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks showed that three of the isoenzymes consisted of different homodimers (I, III, VI) and three were different heterodimers (II, IV, V). Apparent molecular masses of the three different monomer subunits were 37, 38, and 39 kilodaltons. Nondenaturing gel analysis of the malate dehydrogenase peaks showed that each Mono Q peak contained a band of malate dehydrogenase activity with different mobility. These observations are consistent with three nuclear genes encoding corn mitochondrial malate dehydrogenase. Polyclonal antibodies raised against purified malate dehydrogenase were used to identify the gene products using Western blots of two-dimensional gels.     

Nitrate-induced changes in protein synthesis and translation of RNA in maize roots

Nitrate regulation of protein synthesis and RNA translation in maize (Zea mays L. var B73) roots was examined, using in vivo labeling with [³⁵S]methionine and in vitro translation. Nitrate enhanced the synthesis of a 31 kilodalton membrane polypeptide which was localized in a fraction enriched in tonoplast and/or endoplasmic reticulum membrane vesicles. The nitrate-enhanced synthesis was correlated with an acceleration of net nitrate uptake by seedlings during initial exposure to nitrate. Nitrate did not consistently enhance protein synthesis in other membrane fractions. Synthesis of up to four soluble polypeptides (21, 40, 90, and 168 kilodaltons) was also enhanced by nitrate. The most consistent enhancement was that of the 40 kilodalton polypeptide. No consistent nitrate-induced changes were noted in the organellar fraction (14,000g pellet of root homogenates). When roots were treated with nitrate, the amount of [³⁵S]methionine increased in six in vitro translation products (21, 24, 41, 56, 66, and 90 kilodaltons). Nitrate treatment did not enhance accumulation of label in translation products with a molecular weight of 31,000 (corresponding to the identified nitrate-inducible membrane polypeptide). Incubation of in vitro translation products with root membranes caused changes in the SDS-PAGE profiles in the vicinity of 31 kilodaltons. The results suggest that the nitrate-inducible, 31 kilodalton polypeptide from a fraction enriched in tonoplast and/or endoplasmic reticulum may be involved in regulating nitrate accumulation by maize roots.     

A cysteine endopeptidase isolated from castor bean endosperm microbodies processes the glyoxysomal malate dehydrogenase precursor protein.

A plant cysteine endopeptidase with a molecular mass of 35 kD was purified from microbodies of germinating castor bean (Ricinus communis) endosperm by virtue of its capacity to specifically process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro. Processing of the glyoxysomal malate dehydrogenase precursor occurs sequentially in three steps, the first intermediate resulting from cleavage after arginine-13 within the presequence and the second from cleavage after arginine-33. The endopeptidase is unable to remove the presequences of prethiolases from rape (Brassica napus) glyoxysomes and rat peroxisomes at the expected cleavage site. Protein sequence analysis of N-terminal and internal peptides revealed high identity to the mature papain-type cysteine endopeptidases from cotyledons of germinating mung bean (Vigna mungo) and French bean (Phaseolus vulgaris) seeds. These endopeptidases are synthesized with an extended pre-/prosequence at the N terminus and have been considered to be processed in the endoplasmic reticulum and targeted to protein-storing vacuoles.     

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.     

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.     

In vitro synthesis of precursor forms of pig heart aspartate aminotransferase isozymes

Precursor forms of the isozymes of aspartate aminotransferase from pig heart were synthesized in vitro and purified by binding to specific antibodies. Analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis showed that the precursor of the cytosolic enzyme has a similar molecular weight to that of the mature protein whereas the precursor of the mitochondrial isozyme has a molecular weight greater than that of the corresponding mature protein (ΔMW ? 2500). Preliminary sequence studies seem to suggest that the precursor of the mitochondrial isozyme has an extra N-terminal peptide sequence while that of the cytosolic protein has only an extra N-terminal methionine residue.     

In vitro synthesis and processing of tomato fruit polygalacturonase

The in vitro processing of tomato fruit polygalacturonase (PG) (poly[1,4-α-d-galacturonide]glucanohydrolase, EC 3.2.1.15) was studied. Complete chemical deglycosylation of a mixture of mature, purified PG 2A and PG 2B isozymes (45 and 46 kilodaltons; respectively) with trifluoromethane sulfonic acid yielded a single polypeptide of 42 kilodaltons. Similarly, N-terminal amino acid sequencing of the PG 2A/2B isozyme mixture yielded a single 21 amino acid N-terminal sequence, suggesting that the two isozymes result from differential post-translational processing of a single polypeptide. Translation of PG mRNA in vitro results in the synthesis of a single polypeptide with an apparent molecular weight of 54 kilodaltons. Nucleotide sequence analysis of a full-length PG cDNA clone indicates that the large size difference between the PG in vitro translation product and the mature isozymes is due to the presence of a 71 amino acid (8.2 kilodaltons) domain at the N-terminus of in vitro translated PG, consisting of a hydrophobic signal sequence followed by a highly charged prosequence. To determine the precise cleavage site of the signal sequence, PG mRNA was translated in vitro in the presence of canine pancreas microsomal membranes. This resulted in the production of two glycosylated PG processing intermediates with apparent molecular weights of 58 and 61 kilodaltons. The PG processing intermediates were shown to be sequestered within the lumen of the microsomal membranes by protease protection and centrifugational analysis. Deglycosylation of the PG processing intermediates with endoglycosidase H yielded a single polypeptide with an apparent molecular weight of 54 kilodaltons. The production of two distinct, glycosylated processing intermediates from the single in vitro translated PG polypeptide suggests a mechanism by which the differential glycosylation observed for the mature PG 2A and PG 2B isozymes may occur. Edman degradation of ³H-labeled 58 and 61 kilodalton PG processing intermediates indicates that the site of signal sequence cleavage is after amino acid 24 (serine). These results suggest that the proteolytic processing of PG occurs in at least two steps, the first being the co-translational removal of the 24 amino acid signal sequence and the second being the presumed post-translational removal of the remaining highly charged 47 amino acid prosequence.     

Malate dehydrogenase isoenzymes from cotton leaves: molecular weights

Malate dehydrogenase isolated from leaves of the cotton plant (Gossypium hirsutum L.) appears in the form of several isoenzymes. Four of the isoenzymes found in cotton leaf extracts appear to be charge isomers with a molecular weight of approximately 60,000. A fifth malate dehydrogenase isoenzyme found in leaf extracts has a molecular weight of approximately 500,000. Under appropriate conditions it is possible to form this high molecular weight isoenzyme from at least one of the smaller isoenzymes. In addition, malate dehydrogenase isoenzymes of approximately 700,000 and 130,000 molecular weight have been observed under some conditions, although these isoenzymes do not appear in the crude cotton leaf preparations. The relationship of this heterogeneity with respect to size and to the discrepancies in the number and size of malate dehydrogenase isoenzymes reported from plant tissues may be significant.     

Schistosoma mansoni: Purification and characterization of malate dehydrogenases

Isoelectric focusing of a homogenate of Schistosoma mansoni, followed by malate dehydrogenase-specific staining, showed the presence of two major and five minor malate dehydrogenase isoenzymes (EC 1.1.1.37), with isoelectric points ranging from 7.3 to 9.5. The malate dehydrogenase isoenzymes were purified by gel filtration, followed by ion-exchange chromatography on DEAE- and CM-cellulose. The isoenzymes could be differentiated by their susceptibility to substrate inhibition. No differences in the Michaelis-Menten constants for substrate were found. One of the isoenzymes is inhibited by 5′-AMP. Further purification of this particular isoenzyme was achieved by affinity chromatography on 5′-AMP-Sepharose 4B. Analysis after subcellular fractionation indicated a mitochondrial origin for this isoenzyme. The mitochondrial isoenzyme (at a recovery of 80%) was purified 218-fold compared to the crude soluble extract, and contained about 40% of the total malate dehydrogenase activity. The enzyme has a molecular weight of 65,500 and showed absolute specificity for l-malic acid, NAD, and NADH. The final preparation has a specific activity of 451 U/mg protein. Physicochemical studies, including binding constants, substrate inhibition, thermostability, and pH optima, demonstrated differences between the mitochondrial and cytoplasmic enzymes. A role for malate dehydrogenase in Schistosoma mansoni metabolism is discussed.