Regulation of malate dehydrogenases from neonatal,adolescent, and mature rat brain

Since the malate-aspartate shuttle in brain has been shown to be closely linked to brain energy metabolism and neurotransmitter synthesis, the activity of MDH, one of the enzymes of the malateaspartate shuttle, was studied in cortical non-synaptic mitochondria (mMDH) and cytosol (cMDH) in 1–4 day, 18–20 day and 7–8 week old rats. The mean mMDH activity (nmol/min/mg protein) was 10,517±734 (mean±SEM), 8,882±241 and 10,323±561 and cMDH activity was 2,453±99, 4,673±152 and 6,821±205 in 1–4 day, 18–20 day and 7–8 week old rats, respectively. While cMDH activity increased with age (p<0.0001), mMDH activity showed no change. This study also determined if endogenous compounds, previously shown to alter malate metabolism, affected MDH activities. Lactate inhibited only cMDH activity, by a competitive mechanism. Oxaloacetate inhibited mMDH by partial non-competitive inhibition and cMDH by competitive inhibition. Alpha-ketoglutarate competitively inhibited both enzymes; however, the inhibition of mMDH activity was more pronounced than that of cMDH activity. Citrate inhibited mMDH via an uncompetitive mechanism and cMDH via a noncompetitive mechanism. The mechanisms of inhibition of mMDH and cMDH by each of the effectors were the same over the three ages. The results suggest mMDH and cMDH activities show a dissimilar developmental pattern and may be regulated differently by endogenous effectors. The greater sensitivity of mMDH, compared to cMDH, to certain effectors may be related to the dual role of mMDH in the tricarboxylic acid cycle and the malate-aspartate shuttle.These data were presented in part at the meeting of the Federation of American Societies for Experimental Biology in Atlanta, Georgia, April 1991. This work was performed in partial fulfillment of the requirements for the M.S. Degree in Nutritional Sciences (P.M.)     

Phylogenetic Relationships and Biochemical Properties of the Duplicated Cytosolic and Mitochondrial Isoforms of Malate Dehydrogenase from a Teleost Fish, Sphyraena idiastes

Unlike birds and mammals, teleost fish express two paralogous isoforms (paralogues) of cytosolic malate dehydrogenase (cMDH; EC 1.1.1.37; NAD⁺: malate oxidoreductase) whose evolutionary relationships to the single cMDH of tetrapods are unknown. We sequenced complementary DNAs for both cMDHs and the mitochondrial isoform (mMDH) of the fish Sphyraena idiastes (south temperate barracuda) and compared the sequences, kinetic properties, and thermal stabilities of the three isoforms with those of mammalian orthologues. Both fish cMDHs comprise 333 residues and have subunit masses of approximately 36 kDa. One cytosolic isoform, cMDH-S, was significantly more heat-stable than either the other cMDH (cMDH-L) or mMDH. In contradiction to the generally accepted model of vertebrate cMDH evolution, our phylogenetic analysis indicates that the duplication of the fish cytosolic paralogues occurred after the divergence of the lineages leading to teleosts and tetrapods. cMDH-L and cMDH-S differed in optimal concentrations of substrates and cofactors and apparent Michaelis–Menten constants, suggesting that the two paralogues may play distinct physiological roles. Differences in intrinsic thermal stability among MDH paralogues may reflect different degrees of stabilization in vivo by extrinsic stabilizers, notably protein concentration in the case of mMDH. Thermal stabilities of porcine mMDH and cMDH-L, but not cMDH-S, were significantly increased when denaturation was measured at a high protein (bovine serum albumin; BSA) concentration, but the BSA-induced stabilization reduced the catalytic activity. Received: 5 April 2001 / Accepted: 28 June 2001     

An ancient transpecific polymorphism shows extreme divergence in a multitrait cline in an intertidal snail (Nucella lapillus (L.))

Clines in intraspecific genetic variation are frequently associated with an environmental transition. Here, divergence among nucleotide sequences of two nuclear loci, cytosolic and mitochondrial malate dehydrogenase (cMDH and mMDH, respectively), is described, in a multitrait cline over a distance of ca. 3 km where shell phenotype, allozyme, mitochondrial DNA haplotype, and centric fusion (Robertsonian translocations) frequencies covary with temperature and humidity and change abruptly in a continuous population of the dog-whelk (Nucella lapillus), a common intertidal snail of the north temperate Atlantic. Protein electrophoresis has already shown two alleles of mMDH varying from fixation of one allele to near fixation of the other, whereas cMDH appears to be monomorphic. The results of this study show a striking disparity in nucleotide sequence divergence among alleles at the two loci, with extreme molecular differentiation in one of them. Four alleles of cMDH were found to have nucleotide and amino acid sequence divergences of 0.4% and 0.3%, respectively. In contrast, the two mMDH cDNA alleles differed by 23% and 20% at the nucleotide and amino acid levels, respectively. Analysis of a 91-bp partial nucleotide sequence of mMDH from Nucella freycineti, the closest relative of N. lapillus, revealed two similar alleles and indicated that the divergence in mMDH in N. lapillus represents an ancient transpecific polymorphism in these Nucella. Together with earlier studies on variation in N. lapillus, it is argued that the polymorphism in mMDH and the clines in N. lapillus represent the presence of two persistent coadapted gene complexes, multitrait coevolving genetic solutions to environmental variation, which may presently enable this snail to exploit a diverse environment successfully.     

Comparison of properties of malate dehydrogenase isoenzymes from hare and rabbit hearts

The hare heart mitochondrial malate dehydrogenase (mMDH) was established to have a much higher electrophoretic mobility than the corresponding enzyme from the rabbit heart. Differences of kinetic properties of both mMDH and cytoplasmic malate dehydrogenase (cMDH) from these two sources were shown. The hare heart mMDH and cMDH isoenzymes have a higher affinity to malate (in direct reaction) and oxaloacetate and NADH (in reverse reaction), i.e., they have lower K _M values in comparison with the isoenzymes from the rabbit heart. Malate dehydrogenase seems to operate more effectively in the hare heart, which might be important in adaptive and evolutionary aspects.     

High Polymorphism and Autotetraploid Origin of the Rare Endemic SpeciesOxytropis chankaensisJurtz. (Fabaceae) Inferred from Allozyme Data

Using starch electrophoresis, we examined 19 enzyme systems presumably controlled by 35 loci in the rare endemic tetraploid speciesOxytropis chankaensisJurtz. (Fabaceae). Electrophoretic patterns and their genetic interpretation are presented. The isozyme data suggest tetrasomic inheritance in O. chankaensis. Three or four alleles at a particular locus were found in a number of individual plants, which indicate the autotetraploid origin of this species. Seventeen loci were shown to be polymorphic. As reliable gene markers for population systems, we recommend highly active polymorphic systems showing good allozyme separation (Ce-2, Gpi-2, Gpt-2, Idh-2, Lap, Mdh-2, and Mdh-3). Parameters of allozyme variability proved to be very high for a rare species with a restricted range: P = 48.6%, A = 2, A _p = 3.06, H _ob = 0.173.     

Cloning and primary structure of putative cytosolic and mitochondrial malate dehydrogenase from the mollusc Nucella lapillus (L.)

The evolutionary history of the malate dehydrogenase (MDH) gene family [NAD-dependent MDH; EC 1.1.1.37 and NAD(P)-dependent MDH; EC 1.1.1.82] has received much attention. MDHs have also featured extensively as electrophoretic markers in population genetics and evolutionary ecology, and in many cases, intraspecific variation in MDH has been correlated with environmental variables. However, while the amino acid residues essential for MDH function are known, no studies have examined intraspecific nucleotide variation despite evidence indicating that natural selection may be operating on this locus. This study presents two sets of degenerate oligonucleotide PCR primers to facilitate the cloning of cytosolic MDH (cMDH) and mitochondrial MDH (mMDH) from a broad range of animals (cMDH) and animals and plants (mMDH). These primers were used to obtain putative cMDH and mMDH cDNAs from the mollusc Nucella lapillus. The N. lapillus cMDH cDNA was found to encode a putative cMDH protein of 334aa and 36kDa, while the mMDH cDNA encoded a putative mature mMDH protein of 315aa and 33kDa. The putative amino acid sequences of the two compartmentalised N. lapillus MDHs are presented and compared to other known MDH sequences.     

Genetic and cytogenetic studies of the malate dehydrogenases of Drosophila melanogaster

Genetic and cytogenetic locations of the structural genes for the NAD-dependent malate dehydrogenases have been studied. The mitochondrial form (mMDH) is coded for by a gene (mMdh) found at 62.6 on the third chromosome and included in Df(3R)R14, which includes 90C2-91A3 in the salivary gland chromosomes. Based on its inclusion within several J (Jammed; 2-41.0) deficiencies, the structural gene (cMdh) for the cytoplasmic form (cMDH) was determined to lie in region 31B-E, confirming the earlier finding of Grell. Flies lacking any cMDH activity (cMdhn-gamma 10069/Df(2L)J-der-27) were both viable and fertile.     

Inheritance and linkage relationships of ten isozyme genes in hazelnut

Summary The inheritance of 6-phosphogluconate dehydrogenase (6PGD), malate dehydrogenase (MHD), aconitase (ACO), phosphoglucomutase (PGM), phosphoglucoisomerase (PGI), and glutamate-oxalacetate transaminase (GOT) polymorphic isozymes was studied in leaf extracts of nine hazelnut progenies using horizontal starch gel electrophoresis. Evidence of Mendelian inheritance was obtained for ten loci: 6-Pgd-2, Mdh-1, Aco-1, Aco-2, Pgm-1, Pgm-2, Pgm-3, Pgi-2, Pgi-3, and Got-2, which permitted the analysis of 28 alleles (2.8 per locus). The presence of null alleles was detected in Pgm-1 and Pgm-3. Joint segregation analysis of pairs of isozymes revealed four linkages: Mdh-1-Pgi-2, Aco-2-Pgm-2, Pgm-1-Pgm-3, and 6Pdg-2-Pgm-2.     

Cloning and sequence analysis of cDNAs encoding mammalian cytosolic malate dehydrogenase. Comparison of the amino acid sequences of mammalian and bacterial malate dehydrogenase

A cDNA clone, named ppcMDH-1 and covering a part of the coding region for the porcine cytosolic malate dehydrogenase (cMDH) mRNA, was isolated from a porcine liver cDNA library. Subsequently, mouse cMDH cDNA clones were isolated from mouse liver and heart cDNA libraries, using the ppcMDH-1 cDNA as a probe. The longest clone, named pmcMDH-5, was sequenced and the primary structure of the mouse cMDH deduced from its cDNA sequence showed that the mouse cMDH consists of the 334-amino acid residues. When the amino acid sequence of the mouse cMDH was compared with that of the porcine cMDH, they shared a 93% homology. On the other hand, the amino acid sequences of mouse cMDH and mitochondrial MDH (mMDH) showed about 23% overall homology. Surprisingly, comparison of the amino acid sequences among the mammalian and bacterial MDHs revealed that the homology between the mouse cMDH and thermophilic bacterial MDH, as well as the homology between the mouse mMDH and Escherichia coli MDH, markedly exceeds the intraspecies sequence homology between mMDH and cMDH from mice.     

Fitness of allozyme variants in Drosophila pseudoobscura III. Possible factors contributing to the maintenance of polymorphisms in nature

Experiments have been performed to investigate the mechanisms maintaining enzyme polymorphisms in natural populations. We have measured effects on fitness of genotypic variants at three loci, Est-5, Odh, and Mdh-2, in D. pseudoobscura. Significant differences exist among the genotypes in the rate of development from egg-to-adult; there is also indication of differences in larval survival. In a population segregating for allelic variants at all three loci, there is indication that segregation distortion at meiosis or some form of gametic selection might be involved. The relative fitnesses of alternative genotypes are reversed when either different fitness components are considered, or the genotypic frequencies are changed, or the larval density is increased. These fitness reversals may contribute to the maintenance of the polymorphisms, and may account for cyclical oscillations of allozyme frequencies observed in natural populations.Research supported by U.S. Public Health Service Research Fellowship (1F05 TWO 1991-01) to D.M. and by contract AT(04-3)34 with the U.S. Atomic Energy Commission. Adress reprint requests from Europe to D.M.; from elsewhere to F.J.A.     

Adaptation of Drosophila enzymes to temperature--V. Heat shock effect on the malate dehydrogenase of Drosophila melanogaster

Drosophila melanogaster cMdh allozymic variants (MdhF MdhF/S, MdhS) were subjected to heat shock (33 degrees C/30 min----40 degrees C/30 min). This stress increases differentially MDH specific activity, with the cMdhF strain showing greater response as compared with the cMdhS one; heterozygotes exhibited generally intermediate values. Correlative differences were also revealed for some catalytic properties (Vmax, Vmax/Km ratio, thermostability) of the cMDH; this is not true for the mMDH. The catalytic behavior of the enzyme is correlated with the differential survival of the cMdh variants, with the cMdhF showing again higher survival than the cMdhS one, a fact which seems to contribute to temperature adaptation of D. melanogaster.     

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.     

Characterization of the Kinetics of Cardiac Cytosolic Malate Dehydrogenase and Comparative Analysis of Cytosolic and Mitochondrial Isoforms

Because the mitochondrial inner membrane is impermeable to pyridine nucleotides, transport of reducing equivalents between the mitochondrial matrix and the cytoplasm relies on shuttle mechanisms, including the malate-aspartate shuttle and the glycerol-3-phosphate shuttle. These shuttles are needed for reducing equivalents generated by metabolic reactions in the cytosol to be oxidized via aerobic metabolism. Two isoenzymes of malate dehydrogenase (MDH) operate as components of the malate-aspartate shuttle, in which a reducing equivalent is transported via malate, which when oxidized to oxaloacetate, transfers an electron pair to reduce NAD to NADH. Several competing mechanisms have been proposed for the MDH-catalyzed reaction. This study aims to identify the pH-dependent kinetic mechanism for cytoplasmic MDH (cMDH) catalyzed oxidation/reduction of MAL/OAA. Experiments were conducted assaying the forward and reverse directions with products initially present, varying pH between 6.5 and 9.0. By fitting time-course data to various mechanisms, it is determined that an ordered bi-bi mechanism with coenzyme binding first followed by the binding of substrate is able to explain the kinetic data. The proposed mechanism is similar to, but not identical to, the mechanism recently determined for the mitochondrial isoform, mMDH. cMDH and mMDH mechanisms are also shown to both be reduced versions of a common, more complex mechanism that can explain the kinetic data for both isoforms. Comparing the simulated activity (ratio of initial velocity to the enzyme concentration) under physiological conditions, the mitochondrial MDH (mMDH) activity is predicted to be higher than cMDH activity under mitochondrial matrix conditions while the cMDH activity is higher than mMDH activity under cytoplasmic conditions, suggesting that the functions of the isoforms are kinetically tuned to their individual physiological roles.     

Genetic structure of Halotydeus destructor and Penthaleus major populations in Victoria (Acari: Penthaleidae)

The redlegged earth mite (Halotydeus destructor) and the blue oat mite (Penthaleus major) are major pests of pastures and crops in southern Australia. Reproductive modes, migration rates and levels of differentiation between populations were investigated using allozyme electrophoresis. Collections were made throughout Victoria and a sample was also obtained from Western Australia. Three enzyme loci were polymorphic in H. destructor (Mdh-1, Mdh-2 and Idh). Genotype frequencies of these loci did not differ between phenotypic males and females, providing no evidence for haplodiploidy. Allele frequencies were in Hardy-Weinberg equilibrium, indicating that H. destructor is diploid and sexual. This was confirmed via crosses between males and females. Allele frequencies differed between Victorian sites, although F statistics indicated little differentiation over all loci. A sample from Western Australia did not differ in allele frequencies from the Victorian sites. Four polymorphic loci were found in P. major (Mdh-1, Mdh-2, Idh and Gpi). Only a few multilocus genotypes occurred in a sample, indicating that P. major is parthenogenic. No male P. major were found in this study. A number of colour morphs were also identified and a genetic association between genital plate colour and clonal type was found in one population of P. major. Two different body colour morphs were associated with different clonal types.     

Cloning,expression and biochemical characterization of mitochondrial and cytosolic malate dehydrogenase from Phytophthora infestans

The genes of the mitochondrial and cytosolic malate dehydrogenase (mMDH and cMDH) of Phytophthora infestans were cloned and overexpressed in Escherichia coli as active enzymes. The catalytic properties of these proteins were determined: both enzymes have a similar specific activity. In addition, the natural mitochondrial isoenzyme was semi-purified from mycelia and its catalytic properties determined: the recombinant mitochondrial isoform behaved as the natural enzyme. A phylogenetic analysis indicated that mMDH, present in all stramenopiles studied, can be useful to study the relationships between these organisms. MDH with the conserved domain MDH_cytoplasmic_cytosolic is absent in some stramenopiles as well as in fungi. This enzyme seems to be less related within the stramenopile group. The Phytophthora cMDHs have an insertion of six amino acids that is also present in the stramenopile cMDHs studied, with the exception of Thalassiosira pseudonana cMDH, and is absent in other known eukaryotic cMDHs.     

Peroxidase and leucine-aminopeptidase in diploidMedicago species closely related to alfalfa: Multiple gene loci,multiple allelism,and linkage

Summary The genetics of peroxidase and leucine-aminopeptidase isozymes was studied utilizing starch gel electrophoresis in the diploidsMedicago sativa L. (M. coerulea Less.) andM. falcata L. Three anodal and one cathodal sets of peroxidase isozymes identify four linked loci. In addition, two anodal sets of leucine-aminopeptidase isozymes identify two loci that may be linked. The allozymes at each of the loci segregated as expected for monomeric enzymes. However in several crosses there were deficiencies in the number of progeny in particular genotypic classes. This could result from the segregation of recessive deleterious genes linked to some of the allozyme alleles. This is the first report of multiple loci and multiple alleles determining isozymes inMedicago. Supported by grants from the Alberta Research Council (No. D1B02 to R.C. von Borstel) and the Computer Use and Policy Committee, University of Alberta     

Genetic analysis of the two groups of duplicated genes coding for mitochondrial malate dehydrogenase in Zea mays: Possible origin of Mdh genes by chromosome segment duplication

Summary The results presented in this paper demonstrate that there are four mitochondrial malate dehydrogenase genes (mMdh1, mMdh2, mMdh3, and mMdh4) as was proposed by Yang et al. (1977), but with the following modification. Yang et al. (1977) postulated that mMdh1 and mMdh2 were closely linked as were mMdh3 and mMdh4. They also proposed that mMdh3 and mMdh4 arose by gene duplication from mMdh1 and mMdh2 respectively. In the modified model mMdh3 and mMdh4 are linked, but the other two genes mMdh1 and mMdh2 are unlinked. In addition, we demonstrate using reversible denaturation techniques, which mMDH isozymes are heterodimeric in structure. Hence, the origin of all the mMDH isozymes has been elucidated.Since the number of genes in the modified mMDH model remains the same as the Yang et al. model (1977), the original nomenclature is used. We illustrate further that the three gene model proposed by Goodman et al. (1980) and accepted by their collaborators, Newton and Schwartz (1980) is not consistent with data from our genetic crosses. However, the modified version of the Yang et al. (1977) model will account for all MDH data published to date. The psossible role of chromosome segment duplication in the evolution of the MDH genes in Zea mays is discussed.This research was supported, in part, by NIH Grants T501-GM296, GM22733, and by the N.C. Agricultural FoundationPaper No. 6833 of the Journal Series of the North Carolina Agricultural Research Service     

Location of genes coding isozyme markers on Aegilops umbellulata chromosomes adds data on homoeology among Triticeae chromosomes

Summary Zymogram analysis was used to identify the Aegilops umbellulata chromosomes that carry the structural genes for particular isozymes. Wheat, Aegilops and wheat-Aegilops hybrid derivative lines (which contained identified Aegilops chromosomes) were tested by gel electrophoresis for isozymes of particular enzymes. It was found that Aegilops chromosome A (nomenclature according to G. Kimber 1967) carries a structural gene for 6-phosphogluconate dehydrogenase, Aegilops chromosome B carries structural genes for glucose phosphate isomerase and phosphoglucose mutase, Aegilops chromosome D carries genes for leaf peroxidases, Aegilops chromosome E carries structural genes for endosperm peroxidases, acid phosphatases and leaf esterases, Aegilops chromosome F carries a gene for embryo plus scutellum peroxidases and Aegilops chromosome G carries structural genes for endosperm alkaline phosphatases, leaf alkaline phosphatases and leaf esterases. The results obtained indicate that chromosome B is partially homoeologous of the wheat chromosomes of group 1 and 4, and chromosome E is partially homoeologous of wheat chromosomes of groups 7 and 4. Circumstantial evidence is also provided about the possible association between chromosomes C, D and A of A. umbellulata respectively with chromosomes 5, 2 and 1 of wheat.     

Dissociation of mitochondrial malate dehydrogenase into active soluble subunits

Gel exclusion chromatographic studies demonstrate that cytosolic and mitochondrial malate dehydrogenases (cMDH and mMDH) dissociate into subunits in the presence of 0.1% of the non-ionic detergent Triton X-100 (TX-100). The presence of cofactor and catalytically competent cofactor-substrate pairs does not protect mMDH against this dissociation. In contrast, cMDH dimers resist dissociation in the presence of either addition. Since steady state kinetic studies indicate both enzymes are fully active in the presence of 0.1% TX-100, we conclude that quaternary structure is not a kinetically important feature of mMDH structure and cooperativity does not account for mMDH kinetic anomalies. In contrast, cooperativity is a reasonable explanation for cMDH kinetic properties even in the presence of 0.1% TX-100, since this enzyme's subunits associate in the presence of active site ligands. The existence of fully active mMDH subunits raises the possibility that this species rather than the dimer may be a constituent of proposed multi-enzyme complexes of the mitochondrion. Preliminary chromatographic experiments involving gently disrupted mitochondria have found MDH activity in differently sized complexes, all with molecular weights larger than the mMDH dimer but smaller than complexes anticipated for multi-enzyme complexes involving other enzymes and the mMDH dimer.     

Genetic control and developmental expression of malate dehydrogenase in Apis mellifera

Starch gel electrophoresis of extracts of Apis mellifera indicates that genetic variability exists for the enzyme cytoplasmic malate dehydrogenase (E.C. 1.1.1.37). Analysis of individuals throughout development indicates that the isozyme patterns are identical for larvae and adults and suggests a dimeric structure for the molecule. The isozyme pattern observed in pupae is more complex than that of larvae and adults may be due to an additional pupal-specific MDH gene being expressed or to an epigenetic modification of the isozymes. Forty-three colonies with artificially inseminated queens were used to study the Mendelian pattern of inheritance. The data revealed that the MDH isozymes are encoded by three alleles, Mdh-1A, Mdh-1B, and Mdh-1C. The frequency of the Mdh-1 alleles is different in two analyzed subspecies, A. m. adansonii (African bees) and A. m. ligustica (Italian bees), with Mdh-1A and Mdh-1B in the African bees being 0-768 and 0.202, respectively. For the Italian bees, these frequencies are 0.136 and 0:154, respectively.