Biochemical studies of supernatant malate dehydrogenase allozymes in Drosophila melanogaster.

1. A biochemical comparison was made among cytoplasmic malate dehydrogenase allozymic variants from Drosophila melanogaster. Experiments were carried out on enzyme extracted from six different genotypes: three homozygotes and their respective heterozygotes. 2. The allozyme forms (MDH A, MDH B, MDH C) were indistinguishable in terms of NAD and L-malate optima, while they are distinguishable in terms of NADH and OAA saturation conditions. Activities were inhibited at concentrations greater than 0.36 and 0.40 mM NADH for BB and AA, CC, respectively, while in relation to OAA inhibition was observed at concentrations higher than 3 or 6 mM for the AA, CC and BB, respectively. 3. differences among genotypes were also observed in thermal stability: Km values for OAA, L-malate, NADH and NAD: and pG optima. 4. A simple method is presented for the separation of the cytoplasmic from the mitochondrial malate dehydrogenase.     

Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Corynebacterium glutamicum

Like many other bacteria, Corynebacterium glutamicum possesses two types of L-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure.     

Malate dehydrogenases--structure and function

Malate dehydrogenases (MDH, L-malate:NAD oxidoreductase, EC 1.1.1.37), catalyze the NAD/NADH-dependent interconversion of the substrates malate and oxaloacetate. This reaction plays a key part in the malate/aspartate shuttle across the mitochondrial membrane, and in the tricarboxylic acid cycle within the mitochondrial matrix. They are homodimeric molecules in most organisms, including all eukaryots and the most bacterial species. The enzymes share a common catalytic mechanism and their kinetic properties are similar, which demonstrates a high degree of structural similarity. The three-dimensional structures and elements essential for catalysis are conserved between mitochondrial and cytoplasmic forms of MDH in eukaryotic cells even though these isoenzymes are only marginally related at the level of primary structure.     

Specific inhibition of Physarum polycephalum DNA-polymerase-alpha-primase by poly(L-malate) and related polyanions.

Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-alpha-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-alpha-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(vinyl sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (N) interspaced between them. Poly(L-malate) (N = 5) and poly(D,L-malate) (N = 5) are the most efficient inhibitors, followed by poly(L-aspartate) (N = 6), poly(acrylate) (N = 3), poly(L-glutamate) (N = 8), poly(vinyl sulfate) (N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-alpha-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-alpha-primase in the G2 phase and M phase of the plasmodial cell cycle.     

Oxidative phosphorylation in Propionibacterium]

Oxidative phosphorylation during electron transport in the respiratory chain was found in two propionic bacteria, P. shermanii and P. petersonii. Its effectiveness, with oxygen as the terminal acceptor of electrons, was higher in P. petersonii, a more aerobic culture, than in P. shermanii. Oxidative phosphorylation with the participation of the electron transport chain was not found in P. petersonii in the absence of oxygen. Oxidative phosphorylation can take place together with the reactions of propionic fermentation in P. shermanii upon a small rearrangement of the respiration chain (if fumarate reductase is substituted for cytochrome oxidase).     

β-Galactosidase of Propionibacterium shermanii

Ten strains of Propionibacterium shermanii were tested for beta-galactosidase (beta-gal) activity. Of these ten strains, five yielded enhanced enzyme activity when cell suspensions were treated with toluene-acetone; on solvent treatment, the remaining five lost a considerable portion of the activity found in whole-cell suspensions. By using a strain yielding decreased activity upon solvent treatment, explanations for the loss in activity were sought through assays for possible alternative beta-galactoside utilization mechanisms. When this strain was assayed for beta-D-phosphogalactoside galactohydrolase by using orthonitrophenyl-beta-D-galactopyranoside-6-P04 as a substrate, the activity was wither lower or indiffernt as compared with beta-gal activity determined simultaneously. Cell suspensions of P. shermanii 7 and 22 (strains chosen for further work) grown separately on the individual substrates (lactose, glucose, galactose, and sodium lactate) did not show significant differences in beta-gal activity. Optimal temperature for beta-gal activity in untreated and toluene-acetone-treated cell suspensions of strain 7 was 52 C. With strain 22, of the temperatures tested, maximal activity in untreated cell suspensions was noted at 58 C and with solvent-treated cells at 32 C. In the cell-free extract (CFE) system, both strains exhibited maximal activity at 52 C. Optimal pH for untreated and solvent-treated cell suspensions of both strains was around 7.5. In the P. shermanii 22 CFE system, maximal activity occurred at pH 7.0; pH had very little effect on enzyme activity in P. shermanii 7 CFE. Sodium or potassium phosphate buffers in the assay system yielded the best activity. In the CFE system of these two strains, Mn2+ was definitely stimulatory, but in untreated and solvent-treated cell systems of these strains presence or absence of Mn2+ in the assay system had variable effects on enzyme activity. Maximal beta-gal activity was noted in P. shermanii 7 cells harvested after 28 h of growth at 32 C in sodium lactate broth. Sulfhydryl-group blocking agents inhibited enzyme activity in P. shermanii 22 CFE; the inhibition was partly reversed by dithiothreitol.     

Cloning and structural characterization of the genes coding for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii.

The structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been cloned, with the use of synthetic oligonucleotides as primary hybridization probes. The genes are closely linked and are transcribed in the same direction. Nucleotide sequence analysis of 4.5 kb of DNA encompassing both genes allowed us to infer the complete amino acid sequence of the two subunits: the beta-subunit is the product of the upstream gene, and consists of 638 amino acid residues (Mr 69465) and the alpha-subunit consists of 728 amino acid residues (Mr 80,147). There is a very close structural homology between the two subunits, reflecting the probable duplication of a common ancestral gene. A sequence present only in the alpha-subunit is significantly homologous to a portion of the sequence of the methylmalonyl-CoA-binding subunit of transcarboxylase from P. shermanii [Samols, Thornton, Murtif, Kumar, Haase & Wood (1988) J. Biol. Chem. 263, 6461-6464], and this homologous region may form part of the CoA ester-binding site in both enzymes.     

Isolation of intact chains of polyphosphate from "Propionibacterium shermanii" grown on glucose or lactate

A procedure is presented for the isolation of intact polyphosphate (poly P) from "Propionibacterium shermanii." It is demonstrated, by including [32P]poly P during the extraction, that this procedure does not hydrolyze the poly P, and it is shown that two other widely used procedures do cause breakdown of the poly P. The procedure presented allows isolation of three fractions, short-chain poly P which is soluble in trichloroacetic acid, long-chain poly P which is soluble at neutral pH, and long-chain poly P which is present in volutin granules. Cells which had been grown on lactate did not contain short-chain poly P but did contain a high amount of long-chain poly P, which accumulated to 3% of the cell dry weight. At least 70% of this poly P was present in volutin granules. The poly P ranged in length from 250 to 725 phosphate residues and was the same average size as that synthesized in vitro by the poly P kinase from "P. shermanii". This indicates that the poly P kinase is responsible for catalyzing the synthesis of the poly P. In contrast to cells grown on lactate, those which had been grown on glucose did not contain volutin granules, did contain short-chain poly P and had 100-fold less long-chain poly P than lactate-grown cells. We propose that during the fermentation of glucose, the amount of poly P is lower than during growth on lactate because it is continuously utilized as a substrate in the phosphorylation of glucose.     

Utilization of lactose and production of corrinoids in selected strains of propionic acid bacteria in cheese-whey and casein media.

Comparative studies were carried out with 23 strains (14 species) of propionibacteria in two media-cheese-whey and casein. The degree of lactose fementation and the efficiency of the corrinoids synthesis were studied. Lactose fermentation showed great differences even within one species (e.g. 13.3% and 66.1% for various strains of P. shermanii). The differences were particularly sharp in casein medium (0% or 100%). The highest capacity for utilizing cheese-whey lactose (70--80%) was found in two strains of P. shermanii and P. petersonii and P. arabinosum. No definite correlation, however, was found either in the cheese-whey or in the casein medium, between the capability of lactose fermentation and the efficiency of the corrinoids. As the most technologically effective strains have been recognized P. shermanii 1, P. shermanii 566 and P. petersonii J.     

Functional difference of malate-aspartate shuttle system in liver between plateau zokor (Myospalax baileyi) and plateau pika (Ochotona curzoniae)

To explore the adaptive mechanisms of plateau zokor (Myospalax baileyi) to the enduring digging activity in the hypoxic environment and of plateau pika (Ochotona curzoniae) to the sprint running activity, the functional differences of malate-aspartate shuttle system (MA) in liver of plateau zokor and plateau pika were studied. The ratio of liver weight to body weight, the parameters of mitochondria in hepatocyte and the contents of lactic acid in serum were measured; the open reading frame of cytoplasmic malate dehydrogenase (MDH1), mitochondrial malate dehydrogenase (MDH2), and the partial sequence of aspartate glutamate carrier (AGC) and oxoglutarate malate carrier (OMC) genes were cloned and sequenced; MDH1, MDH2, AGC and OMC mRNA levels were determined by real-time PCR; the specific activities of MDH1 and MDH2 in liver of plateau zokor and plateau pika were measured using enzymatic methods. The results showed that, (1) the ratio of liver weight to body weight, the number and the specific surface of mitochondria in hepatocyte of plateau zokor were markedly higher than those of plateau pika (P < 0.01 or P < 0.05), but the content of lactic acid in serum of plateau pika was significantly higher than that of plateau zokor (P < 0.01); (2) MDH1 and MDH2 mRNA levels as well as their enzymatic activities in liver of plateau zokor were significantly higher than those of plateau pika (P < 0.01 or 0.05), AGC mRNA level of the zokor was significantly higher than that of the pika (P < 0.01), while no difference was found at OMC mRNA level between them (P > 0.05); (3) mRNA level and enzymatic activity of MDH1 was significantly lower than those of MDH2 in the pika liver (P < 0.01), MDH1 mRNA level of plateau zokor was markedly higher than that of MDH2 (P < 0.01), but the activities had no difference between MDH1 and MDH2 in liver of the zokor (P > 0.05). These results indicate that the plateau zokor obtains ATP in the enduring digging activity by enhancing the function of MA, while plateau pika gets glycogen for their sprint running activity by increasing the process of gluconeogenesis. As a result, plateau pika converts the lactic acid quickly produced in their skeletal muscle by anaerobic glycolysis and reduces dependence on the oxygen.     

Conditions for porphyrin formation by propionic acid bacteria

Porphyrin production by seven species of propionic acid bacteria (Propionibacterium shermanii, its mutant P. shermanii M-82, P. technicum, P. vannielii, P. rubrum, P. thoenii and P. jensenii) was being studied. All the bacteria were cultivated on a glucose-peptone medium. A positive correlation between the amount of the produced porphyrins and the vitamin B12-synthetizing activity was observed for the most of species. Exogenous delta-aminolevulinic acid stimulated the porphyrin accumulation, but the degree of its utilisation decreased as its content in the culture medium increased from 5 to 200 mg/l. A maximum synthesis of porphyrins by P. shermanii M-82 (mainly of coproporpyrin III) was observed at definite concentrations of glucose and cobalt salts.     

Primary structure of the monomer of the 12S subunit of transcarboxylase as deduced from DNA and characterization of the product expressed in Escherichia coli.

Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types: a hexameric central 12S subunit to which 6 outer 5S subunits are attached through 12 1.3S biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl coenzyme A and pyruvate serve as substrates to form propionyl coenzyme A (propionyl-CoA) and oxalacetate, the 12S subunit specifically catalyzing one of the two reactions. We report here the cloning, sequencing, and expression of the 12S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 12S peptides with the deduced sequence of an open reading frame present in a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3S biotinyl subunit. The cloned 12S gene encodes a protein of 604 amino acids and of M(r) 65,545. The deduced sequence shows regions of extensive homology with the beta subunit of mammalian propionyl-CoA carboxylase as well as regions of homology with acetyl-CoA carboxylase from several species. Two genomic fragments were subcloned into pUC19 in an orientation such that the 12S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots (immunoblots) which comigrated with authentic 12S. The Escherichia coli-expressed 12S was purified to apparent homogeneity by a three-step procedure and compared with authentic 12S from P. shermanii. Their quaternary structures were identical by electron microscopy, and the E. coli 12S preparation was fully active in the reactions catalyzed by this subunit. We conclude that we have cloned, sequenced, and expressed the 12S subunit which exists in a hexameric active form in E.coli.     

Citric-acid cycle key enzyme activities during in vitro growth and metacyclogenesis of Leishmania infantum promastigotes.

The activities of 5 key regulatory enzymes in most energetic systems, namely citrate synthase (EC 4.1.3.7, CS), NADP-specific isocitrate dehydrogenase (EC 1.1.1.42, ICDH), succinate dehydrogenase (EC 1.3.99.1, SDH), L-malate dehydrogenase (EC 1.1.1.37, MDH), and decarboxylating malic enzyme (EC 1.1.1.40, ME), were measured during the growth and metacyclogenesis of a cutaneous (CL) and a visceral (VL) strain of Leishmania infantum. As occurs with other Leishmania species, infective promastigotes were present along all phases of growth, but their percentages were higher at the early stationary phase for VL and the end of the same phase for CL. High CS and SDH activities were detected in both strains, as compared with other trypanosomatids, bringing more evidence for an actively functional citric-acid cycle in L. infantum. Both strains showed higher levels of CS, ICDH, and MDH and lower SDH and ME activities when more metacyclic promastigotes were present, but in VL these changes paralleled an increase in glucose consumption, whereas in CL these changes coincided with an NH3 hyperproduction. This suggests that the energy metabolism during L. infantum growth and metacyclogenesis is affected by regulated enzymes that probably respond to changes in the culture medium in the levels of glucose and amino acids.     

Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy.

The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated. In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generated by the metabolism of a glycolytic substrate. Malolactic fermentation results in the synthesis of ATP which is inhibited by the ionophore nigericin and the F0F1-ATPase inhibitor N,N-dicyclohexylcarbodiimide. Since substrate-level phosphorylation does not occur during malolactic fermentation, the generation of metabolic energy must originate from the uptake of L-malate and/or excretion of L-lactate. The initiation of malolactic fermentation is stimulated by the presence of L-lactate intracellularly, suggesting that L-malate is exchanged for L-lactate. Direct evidence for heterologous L-malate/L-lactate (and homologous L-malate/L-malate) antiport has been obtained with membrane vesicles of an L. lactis mutant deficient in malolactic enzyme. In membrane vesicles fused with liposomes, L-malate efflux and L-malate/L-lactate antiport are stimulated by a membrane potential (inside negative), indicating that net negative charge is moved to the outside in the efflux and antiport reaction. In membrane vesicles fused with liposomes in which cytochrome c oxidase was incorporated as a proton motive force-generating mechanism, transport of L-malate can be driven by a pH gradient alone, i.e., in the absence of L-lactate as countersubstrate. A membrane potential (inside negative) inhibits uptake of L-malate, indicating that L-malate is transported an an electronegative monoanionic species (or dianionic species together with a proton). The experiments described suggest that the generation of metabolic energy during malolactic fermentation arises from electrogenic malate/lactate antiport and electrogenic malate uptake (in combination with outward diffusion of lactic acid), together with proton consumption as result of decarboxylation of L-malate. The net energy gain would be equivalent to one proton translocated form the inside to the outside per L-malate metabolized.     

Malic Dehydrogenase Heterogeneity in Malaria (Plasmodium lophurae and P. berghei)*

SYNOPSIS. Molecular heterogeneity of malic dehydrogenase (MDH) in malaria was shown by zone electrophoresis in potato starch, starch gel, and by enzymatic activity with analogs of the coenzyme diphosphopyridine nucletide. A single anodal peak of MDH characterized the normal duck red blood cell whereas P. lophurae free of the host cell had a cathodal form of the enzyme. Infected duck erythrocytes had a combination of these electrophoretic forms. The isolated enzymes had different pH optima with oxaloacetate as substrate: pH 7.4 for the duck red cell and 6.4 for the plasmodial enzyme. The Km of each enzyme for oxaloacetate varied with the pH. The Km at pH 7.4 was 4.1 and 4.4 × 10⁵ M for parasite and host, respectively, whereas at 6.4 it was 2.0 × 10⁵ M for P. lophurae and 6.3 × 10⁵M for the duck erythrocyte. At pH 7.4 both enzymes were inhibited by oxaloacetate concentrations greater than 10^?4 M. P. berghei MDH also had a different electrophoretic character from that of the mouse red blood cell. Quantitatively, MDH activity increased with parasitization, and erythrocyte-free P. lophurae contained approximately twice the activity found in the uninfected duck erythrocyte. The quantity of MDH activity of the infected cell was ca. 50% less than the sum of the activities of the parasite and the uninfected cell. It is suggested that these properties of the parasite MDH may give it a physiologic advantage over the red cell under the conditions which prevail intraerythrocytically.     

In silico exploration of the fructose-6-phosphate phosphorylation step in glycolysis: genomic evidence of the coexistence of an atypical ATP-dependent along with a PPi-dependent phosphofructokinase in Propionibacterium freudenreichii subsp. shermanii

We performed a detailed bioinformatic study of the catalytic step of fructose-6-phosphate phosphorylation in glycolysis based on the raw genomic draft of Propionibacterium freudenreichii subsp. shermanii (P. shermanii) ATCC9614 [Meurice et al., 2004]. Our results provide the first in silico evidence of the coexistence of genes coding for an ATP-dependent phosphofructokinase (ATP-PFK) and a PPi-dependent phosphofructokinase (PPi-PFK), whereas the fructose-1,6-bisphosphatase (FBP) and ADP-dependent phosphofructokinase (ADP-PFK) are absent. The deduced amino acid sequence corresponding to the PPi-PFK (AJ508922) shares 100% similarity with the already characterised propionibacterial protein (P29495; Ladror et al., 1991]. The unexpected ATP-PFK gene (AJ509827) encodes a protein of 373 aa which is highly similar (50% positive residues) along at least 95% of its sequence length to different well-characterised ATP-PFKs. The characteristic PROSITE pattern important for the enzyme function of ATP-PFKs (PS00433) was conserved in the putative ATP-PFK sequence: 8 out of 9 amino acid residues. According to the recent evolutionary study of PFK proteins with different phosphate donors [Bapteste et al., 2003], the propionibacterial ATP-PFK harbours a G104-K124 residue combination, which strongly suggested that this enzyme belongs to the group of atypical ATP-PFKs. According to our phylogenetic analyses the amino acid sequence of the ATP-PFK is clustered with the atypical ATP-PFKs from group III of the Siebers classification [Siebers et al., 1998], whereas the expected PPi-PFK protein is closer to the PPi-PFKs from clade P [Müller et al., 2001]. The possible significance of the co-existence of these two PFKs and their importance for the regulation of glycolytic pathway flux in P. shermanii is discussed.     

Diauxic Growth of Propionibacterium shermanii

Propionibacterium shermanii has been anaerobically propagated in batch and continuous culture with glucose and/or lactate as energy source. Specific growth rate on lactate was observed to be the same as that on glucose. In terms of cell density, the yield on glucose is higher than the yield on lactate. But the molar ratio of yield on glucose to that on lactate, 8.35, is in good agreement with the theoretical value of 8. In a mixture of glucose and lactate, P. shermanii showed diauxic growth. It used lactate before glucose utilization began. Neither temporary growth cessation nor two distinct growth phases were observed. A mathematical model is proposed to describe the diauxic growth.     

Mechanism of the ring contraction process in vitamin B12 biosynthesis by the anaerobe Propionibacterium shermanii under aerobic conditions

The mechanism of the ring contraction process during vitamin B(12) biosynthesis by the anaerobe Propionibacterium shermanii was investigated under both aerobic and anaerobic conditions by means of feeding experiments with delta-amino[1-(13)C]levulinic acid (a biosynthetic intermediate of tetrapyrrole) and delta-amino[1-(13)C,1,1,4-(18)O(3)]levulinic acid in combination with (13)C-NMR spectroscopy. We showed that the characteristic mechanism of the ring contraction process (the generation of precorrin-3x from formation of the gamma-lactone from the ring A acetate group at C1 and hydroxylation at C20 by molecular oxygen catalyzed by CobG, and the migration of ring D by cleavage of the carbon-oxygen bond at C1 of precorrin-3x) in the aerobe Pseudomonas denitrificans was not seen in P. shermanii under aerobic conditions, and the mechanism of the ring contraction process in P. shermanii was the same irrespective of the presence or absence of oxygen.     

Expression of novel cytosolic malate dehydrogenases (cMDH) in Lupinus angustifolius nodules during phosphorus starvation

During P deficiency, the increased activity of malate dehydrogenase (MDH, EC 1.1.1.37) can lead to malate accumulation. Cytosolic- and nodule-enhanced MDH (cMDH and neMDH, respectively) are known isoforms, which contribute to MDH activity in root nodules. The aim of this study was to investigate the role of the cMDH isoforms in nodule malate supply under P deficiency. Nodulated lupins (Lupinus angustifolius var. Tanjil) were hydroponically grown at adequate P (+P) or low P (−P). Total P concentration in nodules decreased under P deficiency, which coincided with an increase in total MDH activity. A consequence of higher MDH activity was the enhanced accumulation of malate derived from dark CO₂ fixation via PEPC and not from pyruvate. Although no measurable neMDH presence could be detected via PCR, gene-specific primers detected two 1 kb amplicons of cMDH, designated LangMDH1 (corresponding to +P, HQ690186) and LangMDH2 (corresponding to −P, HQ690187), respectively. Sequencing analyses of these cMDH amplicons showed them to be 96% identical on an amino acid level. There was a high degree of diversification between proteins detected in this study and other known MDH proteins, particularly those from other leguminous plants. Enhanced malate synthesis in P-deficient nodules was achieved via increased anaplerotic CO₂ fixation and subsequent higher MDH activities. Novel isoforms of cytosolic MDH may be involved, as shown by gene expression of specific genes under P deficiency.     

Intracellular mechanisms of the formation of homo- and heteropolymeric isozymes. I. Kinetics of the formation of the heteropolymeric isozyme of malate dehydrogenase in parasexual hybrids of two Acetabularia species

The isozyme pattern of malate dehydrogenase (MDH) of Acetabularia crenulata and A. mediterranea is characterized by heterogeneity in different regions of the cytoplasm of both algae, as well as by species specificity. The formation of the heteropolymeric MDH isozyme is restricted to a definite region of the cytoplasm of heterokaryons and nuclear-cytoplasmic A.crenulata-A.mediteranea hybrids at different stages of their development. The data obtained suggest that the concentrations of the free subunits of MDH, coded for by homologous genes, are unevenly distributed in the cytoplasm of hybrid cells. The heteropolymeric MDH isozyme in these cells is presumably the result of the de novo synthesis of isozyme subunits. This seems plausible inasmuch as no exchange occurs between the homopolymeric MDH isozymes of both parental types in the cytoplasm. The formation of the heteropolymeric MDH isozyme is tentatively related to the spatial compartmentalization of the mRNAs of homologous genes coding for the MDH subunits.