An experimental analysis of the relationships between fitness components and malic enzyme genotypes inTribolium confusum

The hypothesis that natural selection is capable of maintaining allozyme variation in natural populations was tested using a species of flour beetles,Tribolium confusum. We selected a polymorphic locus (a locus encoding variation for malic enzyme) in an experimental population ofT. confusum and scored the genotypes at this locus for a series of fitness components on different flour types. Measurements included survival rate, development time, fecundity, and rate of egg cannibalism. Flour type had significant effects on most traits. Significant differences among genotypes for fecundity and rates of egg cannibalism and the presence of genotype × flour type interactions for development time were demonstrated. Thus, changes in allele frequencies at the malic enzyme locus could in part be under the influence of natural selection. The existence of genotype × flour type interactions suggests that environmental heterogeneity could maintain allozyme variation at the malic enzyme locus.     

Enzyme changes associated with mitoichondrial malic enzyme deficiency in mice

A genetically determined absence of mitochondrial malic enzyme (EC 1.1.1.40) in c3H/c6H mice is accompanied by a four-fold increase in liver glucose-6-phosphate dehydrogenase and a two-fold increase for 6-phosphogluconate dehydrogenase activity. Smaller increases in the activity of serine dehydratase and glutamic oxaloacetic transaminase are observed while the level of glutamic pyruvate transaminase activity is reduced in the liver of deficient mice. Unexpectedly, the level of activity of total malic enzyme in the livers of mitochondrial malic enzyme-deficient mice is increased approximately 50% compared to littermate controls. No similar increase in soluble malic enzyme activity is observed in heart of kidney tissue of mutant mice and the levels of total malic enzyme in these tissues are in accord with expected levels of activity in mitochondrial malic enzyme-deficient mice. The divergence in levels of enzyme activity between mutant and wild-type mice begins at 19--21 days of age. Immunoinactivation experiments with monospecific antisera to the soluble malic enzyme and glucose-6-phosphate dehydrogenase demonstrate that the activity increases represent increases in the amount of enzyme protein. The alterations are not consistent with a single hormonal response.     

Genetic regulation of malic enzyme activity in the mouse

Cytosolic malic enzyme catalyzes the NADP(+)-dependent oxidative decarboxylation of malate to pyruvate and CO2. Additionally, this enzyme produces large amounts of reducing equivalents (NADPH) required for de novo fatty acid synthesis and provides a precursor for oxaloacetate replacement in the mitochondria. Malic enzyme is considered a key lipogenic enzyme and changes in enzyme activity parallel changes in the lipogenic rate. As would be expected, the activity of malic enzyme responds to a variety of dietary and hormonal factors acting mainly on the rate of enzyme synthesis. In the mouse, the structural locus for malic enzyme (Mod-1) is located on chromosome 9. Two alleles reflecting differences in electrophoretic mobility have been identified. This report demonstrates that the amount of hepatic malic enzyme activity is strain-dependent and is regulated by a malic enzyme regulator locus (Mod1r) located on the proximal end of chromosome 12. Two alleles have been identified: Mod1ra, conferring high enzyme activity (C57BL/6J), and Mod1rb, conferring low enzyme activity (C57BL/KsJ). Biochemical studies have demonstrated differences in the apparent Km and Vmax and in specific activity on purification and immunoprecipitation, features that suggest changes in enzyme structure even though no differences were observed by electrophoresis and isoelectric focusing. These combined data suggest that differences in both enzyme quantity and structure may be involved in the genetic regulation of malic enzyme activity in mice.     

Nutritional and hormonal regulation of malic enzyme synthesis in rat mammary gland.

Cytosolic malic enzyme was purified from rat mammary gland by L-malate affinity chromatography. The pure enzyme obtained was used to produce a specific antiserum in a rabbit. Relative synthesis of malic enzyme in the mammary gland of mid-lactating rats was 0.097%, measured by labelling the enzyme in isolated acini. When food was removed, malic enzyme synthesis decreased to 35% and 20% of the control value at 4 and 6 h respectively. Incorporation of [3H]leucine into soluble proteins was constant during the first 6 h of starvation. When lactating rats (maintained with their pups) were starved for 24 h and then re-fed, the relative rate of enzyme synthesis increased 2.5-, 4-, and 4.5-fold at 3 h, 6 h and 18 h respectively after initiation of re-feeding. The relative rate of malic enzyme synthesis was about 50% of normal at 15 h after weaning, whereas the rate of synthesis of soluble proteins did not change. Administration of bromocriptine or adrenalectomy of lactating rats decreased the relative rate of synthesis of malic enzyme by 40% or 30% respectively; these effects were counteracted by hormone supplementation. Hormone therapy also caused an increase in the rate of incorporation of [3H]leucine into soluble proteins and in malic enzyme activity.     

Enzyme changes associated with mitochondrial malic enzyme deficiency in mice

A genetically determined absence of mitochondrial malic enzyme (EC 1.1.1.40) in c^3H/c^6H mice is accompanied by a four-fold increase in liver glucose-6-phosphate dehydrogenase and a two-fold increase for 6-phosphogluconate dehydrogenase activity. Smaller increases in the activity of serine dehydratase and glutamic oxaloacetic transaminase are observed while the level of glutamic pyruvate transaminase activity is reduced in the liver of deficient mice. Unexpectedly, the level of activity of total malic enzyme in the livers of mitochondrial malic enzyme-deficient mice is increased approximately 50% compared to littermate controls. No similar increase in soluble malic enzyme activity is observed in heart of kidney tissue of mutant mice and the levels of total malic enzyme in these tissues are in accord with expected levels of activity in mitochondrial malic enzyme-deficient mice. The divergence in levels of enzyme activity between mutant and wild-type mice begins at 19–21 days of age. Immunoinactivation experiments with monospecific antisera to the soluble malic enzyme and glucose-6-phosphate dehydrogenase demonstrate that the activity increases represent increases in the amount of enzyme protein. The alterations are not consistent with a single hormonal response.     

Mitochondrial malate dehydrogenase and malic enzyme: Mendelian inherited electrophoretic variants in the mouse

Malate dehydrogenase and malic enzyme each possess supernatant and mitochondrial molecular forms which are structurally and genetically independent. We describe electrophoretic variants of the mitochondrial enzymes of malate dehydrogenase and malic enzyme in mice. Progeny testing from genetic crosses indicated that the genes which code for mitochondrial malate dehydrogenase and malic enzyme were not inherited maternally but as independent unlinked nuclear autosomal genes. The locus for mitochondrial malic enzyme was located on linkage group I. Linkage analysis with a third mitochondrial enzyme marker, glutamic oxaloacetic transaminase, showed that the nuclear genes which code for the three mitochondrial enzymes were not closely linked to each other. This evidence suggests that clusters of nuclear genes coding for mitochondrial function are unlikely in mice.Supported by U.S. Public Health Service grants 5F2 HD-35,531 and GM-09966.     

Cytogenetic localization of a putative regulatory gene affecting an NADP+-dependent enzyme in Drosophila melanogaster

The cytogenetic localization of a putative regulatory locus affecting an NADP+-dependent enzyme is described. This locus, Mex, is in region 8D10-12 to 9A1-2 on the X chromosome of Drosophila melanogaster. Hyperploidy for this region is associated with significant increases in NADP+-dependent malic enzyme specific activity and specific immunologically cross-reacting material. This is the first report of a specific locus, unlinked to the locus coding for the major polypeptide of the enzyme, which affects an NADP+-dependent enzyme in Drosophila melanogaster.     

Genetic linkage between the loci for phosphohexose isomerase (PHI) and a serum protein (Xk) in horses

Genetic linkage between the equine loci for phosphohexose isomerase (PHI) and serum Xk protein was demonstrated by means of segregation data from three sire families. The recombination frequency was estimated from pooled data to be 0.23 +/- 0.02; a significant heterogeneity between sires for estimates of the recombination frequency was observed. No indication of linkage was detected between Xk and 14 other blood marker loci. Linkage between the Xk locus and the locus for soluble malic enzyme (ME1) has recently been reported in horses. An equine linkage group designated LG IV comprising the three loci ME1, PHI, and Xk has thus been established. The possibility that the linkage between PHI and Xk is homologous to the linkage between the loci for PHI and a serum postalbumin (PO-2) in pigs was discussed.     

Genetic linkage between the loci for phosphohexose isomerase (PHI) and a serum protein (Xk) in horses

Genetic linkage between the equine loci for phosphohexose isomerase (PHI) and serum Xk protein was demonstrated by means of segregation data from three sire families. The recombination frequency was estimated from pooled data to be 0.23 ± 0.02; a significant heterogeneity between sires for estimates of the recombination frequency was observed. No indication of linkage was detected between Xk and 14 other blood marker loci. Linkage between the Xk locus and the locus for soluble malic enzyme ( ME1 ) has recently been reported in horses. An equine linkage group designated LG IV comprising the three loci ME1, PH1 , and Xk has thus been established. The possibility that the linkage between PH1 and Xk is homologous to the linkage between the loci for PHI and a serum postalbumin (PO-2) in pigs was discussed.     

Changes in NAD(P)+-dependent malic enzyme and malate dehydrogenase activities during fibroblast proliferation

A sensitive isotope exchange method was developed to assess the requirements for and compartmentation of pyruvate and oxalacetate production from malate in proliferating and nonproliferating human fibroblasts. Malatedependent pyruvate production (malic enzyme activity) in the particulate fraction containing the mitochondria was dependent on either NAD⁺ or NADP⁺. The production of pyruvate from malate in the soluble, cytosolic fraction was strictly dependent on NADP⁺. Oxalacetate production from malate (malate dehydrogenase, EC 1.1.1.37) in both the particulate and soluble fraction was strictly dependent on NAD⁺. Relative to nonproliferating cells, NAD⁺-linked malic enzyme activity was slightly reduced and the NADP⁺-linked activity was unchanged in the particulate fraction of serum-stimulated, exponentially proliferating cells. However, a reduced activity of particulate malate dehydrogenase resulted in a two-fold increase in the ratio of NAD(P)⁺-linked malic enzyme to NAD⁺-linked malate dehydrogenase activity in the particulate fraction of proliferating fibroblasts. An increase in soluble NADP⁺-dependent malic enzyme activity and a decrease in NAD⁺-linked malate dehydrogenase indictated an increase in the ratio of pyruvate-producing to oxalacetate-producing malate oxidase activity in the cytosol of proliterating cells. These coordinate changes may affect the relative amount of malate that is oxidized to oxalacetate and pyruvate in proliferating cells and, therefore, the efficient utilization of glutamine as a respiratory fuel during cell proliferation.     

Regulation of the activity and synthesis of malic enzyme in 3T3-L1 cells

Malic enzyme activity in differentiated 3T3-L1 cells was about 20-fold greater than activity in undifferentiated cells. A new steady-state level was achieved about 8 days after initiating differentiation of confluent cultures with a 2-day exposure to dexamethasone, isobutylmethylxanthine, and insulin. This increase in enzyme activity resulted from an increase in the mass of malic enzyme as detected by immunotitration of enzyme activity with goat antiserum directed against purified rat liver malic enzyme. Malic enzyme synthesis was undetectable in undifferentiated cells and increased to about 0.2% of soluble protein in differentiated cells, suggesting that the increase in enzyme mass was due primarily to an increase in enzyme synthesis. Thyroid hormone, a potent stimulator of malic enzyme activity in hepatocytes in culture and in liver and adipose tissue in intact animals, decreased or increased malic enzyme activity in differentiating 3T3-L1 cells by about 40% when it was removed or added to the medium, respectively. Insulin, another physiologically important regulator of malic enzyme activity in vivo, had no effect on the initial rate of accumulation of malic enzyme activity in the differentiating cells and caused a 30 to 40% decrease in the final level of enzyme activity in the fully differentiated cells. Cyclic AMP, a potent inhibitor of malic enzyme synthesis in hepatocytes in culture, inhibited this process in 3T3-L1 cells by 30%. Malic enzyme is like several other enzymes in that the large increase in its concentration which accompanies differentiation of 3T3-L1 cells is due to increased synthesis of enzyme protein. However, the hormonal modulation of malic enzyme characteristic of liver and adipose tissue in intact animals does not appear to occur in differentiated 3T3-L1 cells, suggesting that differentiated 3T3-L1 cells may not be an appropriate model system in which to study the hormonal modulation of malic enzyme that occurs in liver and adipose tissue of intact animals.     

New allozyme variability in Italian honey bees

Adult workers of the honey bee, Apis mellifera ligustica, from Italy were assayed for enzyme polymorphism using a variety of electrophoretic conditions. Three polymorphic enzyme systems are described, two of which, malic enzyme and an esterase, were previously unknown in indigenous A. m. ligustica. In addition, a new allozyme for the Mdh locus is reported.     

CO(2) Metabolism in Corn Roots. II. Intracellular Distribution of Enzymes

Three enzymes assumed to mediate CO₂ metabolism in corn root tips, P-enolpyruvate carboxylase, malic dehydrogenase, and the malic enzyme, were extracted to determine their relative specific activities and their partitioning between soluble and particulate fractions. The data indicated that the intracellular location of these 3 enzymes is nonparticulate and thus these enzymatic reactions of CO₂ metabolism are apparently nonparticulate. The soluble malic dehydrogenase fraction differed from the particulate fraction in several kinetic properties, viz., response to the thionicotinamide analog of nicotinamide-adenine dinucleotide, oxaloacetate substrate inhibition at pH 8.3, and Km's for nicotinamide-adenine dinucleotide and l-malate. It was concluded that the soluble-malic dehydrogenase differed from the particulate forms in both structure and function. The soluble malic dehydrogenase is apparently involved in CO₂ metabolism.     

Turnover of rat liver malic enzyme during induction by protein deprivation.

The half-lives of hepatic malic enzyme and total liver soluble proteins were determined in protein-sufficient and protein-deficient rats after injection of tracer doses of radioactively labeled leucine. The results obtained in these experiments have demonstrated that the increased levels of malic enzyme obtained under conditions of severe protein restriction are due to elevated rates of synthesis of the enzyme protein, with no apparent change in the rate of its degradation.     

A null mutation of cytoplasmic malic enzyme in mice

In the course of conducting a biochemical screening program for mutant enzymes in mice, individuals with an apparent nonfunctional allele at the locus (Mod-1) responsible for cytoplasmic malic enzyme were observed. The variant, later attributed to a germinal mutation, was identified by starch gel electrophoresis and by enzyme activity measurements. A series of matings were made, and mice homozygous for the nonfunctional, null, allele (Mod-1) were produced. In liver, kidney, and testis homogenates, the homozygous mutant exhibited less than 10% of the enzyme activity of the control mice. By an enzyme immuno-inactivation study, the residual enzyme activity was shown to be mitochondrial malic enzyme in all of the tissues examined. By double immuno-diffusion experiments, the kidney homogenate of the mutant formed no precipitin lines with the antiserum to cytoplasmic malic enzyme. Thus, the null mutants express no proteins that crossreact with the antiserum to cytoplasmic malic enzyme (CRM negative). Tissue enzyme assays revealed no significant differences between the normal and the mutant mice in activities of other enzymes in the related metabolic pathways. Because malic acid and malic enzyme together are reported to serve as a pump for NADPH generation in cytoplasm, total cellular NADP⁺ and NADPH concentrations in liver were determined for the control and the mutant mice. In liver from two individual mutant mice, lower NADPH/NADP⁺ ratio was detected in comparison to the level in liver from control mice. In spite of the lower levels of NADPH in the mutant mice, their body weight and lipid content were not significantly altered. Mice without cytoplasmic malic enzyme exhibited no striking deficiencies in metabolism or viability.     

Genetic variation of the cytoplasmic and mitochondrial malic enzymes in the monkey Macaca nemestrina

Electrophoretic variation of both the cytoplasmic and mitochondrial forms of the malic enzyme is described in Macaca nemestrina. Pedigree analysis of the observed phenotypes demonstrates that the two subcellular forms of the malic enzyme are genetically independent. The identity of the electrophoretic phenotypes in brain, heart muscle, liver, kidney, adrenal, and spleen from any given individual shows that each subcellular form is determined by the same genetic locus in a wide variety of tissues. After separation by ion exchange chromatography, the cytoplasmic and mitochondrial malic enzymes were shown to be distinct in their heat stability and K _m for malate, but no significant differences were found among the variants of the cytoplasmic enzyme or among the variants of the mitochondrial enzyme. It is possible that the polymorphism of the mitochondrial malic enzyme is selectively neutral.This study was supported by grant GM-15253 from the National Institutes of Health. One of us (G.S.O.) was a Special Fellow, U.S. Public Health Service (5F3 HD 43, 122-02); Fellow, National Genetics Foundation.     

Malic enzyme: its purification and characterization fromMucor circinelloides and occurrence in other oleaginous fungi

Malic enzyme was purified 43-fold from Mucor circinelloides. The enzyme was dependent on Mg²⁺ or Mn²⁺ for activity, was not active with Dmalate and had a pH optimum at 7.8. The apparent K_m values for malate and NADP⁺ were 488 ΜM and 41 Μm respectively. The M_r of the native enzyme was 160 kDa. Five metabolic analogues of malate: oxaloacetate, tartronic acid, 1-methylenecyclopropane trans-2,3-dicarboxyIic acid, malonic acid and glutaric acid, were found to inhibit malic enzyme activity at 10 mM. Four oleaginous fungi, Mucor circinelloides, Mortierella alpina, Mortierella elongata and Pythium ultimum, were also examined, all possessed a soluble malic enzyme, two also possessed a microsomal malic enzyme.     

Equine marker genes: Polymorphism for soluble erythrocyte malic enzyme

Polymorphism for equine erythrocyte malic enzyme is detectable on starch gel electrophoresis. The frequency of MEI^S was 0.06 in 667 Standardbred and 0.09 in 85 Thoroughbred horses. No genetically determined electrophoretic variation in soluble malate dehydrogenase was detected.     

Light-stimulated synthesis of NADP malic enzyme in leaves of maize

Illumination of etiolated maize plants for 80 h brings about a 15-20-fold increase in activity of NADP malic enzyme (EC 1.1.1.40). Increases in NADP malic enzyme protein and in the level of translatable mRNA for this protein occur simultaneously with the activity increase. Radiolabeled amino acids are also incorporated into NADP malic enzyme during this time. These results are consistent with the conclusion that an increase in NADP malic enzyme activity during greening results from de novo synthesis of NADP malic enzyme protein. Polyadenylated RNA extracted from greening maize leaves directs the synthesis in vitro of a protein 12,000 daltons larger than NADP malic enzyme purified from corn leaves. This protein is a precursor of NADP malic enzyme because 1) both the precursor and mature NADP malic enzyme are immunoprecipitated by antibody made against NADP malic enzyme purified from corn leaves, 2) both NADP malic enzyme protein and the level of mRNA for the precursor increase during greening, and 3) peptide maps of the precursor and of mature NADP malic enzyme are very similar. Mature NADP malic enzyme and its precursor (synthesized in vitro) both migrate on sodium dodecyl sulfate-polyacrylamide gradient gels as doublet bands. Peptide analyses show all bands to be structurally related.     

Regulation of carbon flux from amino acids into sugar phosphates in Xenopus embryos

Xenopus laevis oocytes and embryos are glycogenic cells, metabolizing sugar phosphates into glycogen. These cells have very low pyruvate kinase activity in vivo and, consequently, make little pyruvate and lactate through glycolysis. Nevertheless, oocytes and embryos do contain significant pyruvate and lactate levels. To determine the source of carbon for sugar phosphates and pyruvate, 14C-labeled intermediary metabolites were injected into fertilized eggs and their metabolism examined by thin-layer chromatography. Alanine, pyruvate, and lactate form a pool of carbon that fluxes into sugar phosphates. Cytosolic (nonmitochondrial) aspartate, oxaloacetate, and malate form a pool of carbon which is largely blocked in the short-term from entering the smaller alanine/pyruvate/lactate pool. The data indicate that the major source of carbon for sugar phosphates in fertilized eggs and rapidly cleaving embryos is the alanine/pyruvate/lactate pool. Pyruvate from this pool is converted in the mitochondria to phosphoenolpyruvate, which in turn is metabolized outside the mitochondria to sugar phosphates. A key enzyme in regulating flux from amino acid carbon to pyruvate is malic enzyme. Three malic enzyme isozymes, one soluble and two mitochondrial, were partially isolated and kinetically characterized from total ovarian tissue. Full-grown oocytes and eggs, however, have very low soluble malic enzyme activity, which results in the separation of the cytosolic aspartate/oxaloacetate/malate and alanine/pyruvate/lactate pools.