Isolation and properties of a 'malic' enzyme from cauliflower bud mitochondria.
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ABSTRACT:
SUBMITTER: Macrae AR
PROVIDER: S-EPMC1176806 | biostudies-other | 1971 May
REPOSITORIES: biostudies-other
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Project description:The metabolic program of osteoblasts, the chief bone-making cells, remains incompletely understood. Here in murine calvarial cells, we establish that osteoblast differentiation under aerobic conditions is coupled with a marked increase in glucose consumption and lactate production but reduced oxygen consumption. As a result, aerobic glycolysis accounts for approximately 80% of the ATP production in mature osteoblasts. In vivo tracing with 13C-labeled glucose in the mouse shows that glucose in bone is readily metabolized to lactate but not organic acids in the TCA cycle. Glucose tracing in osteoblast cultures reveals that pyruvate is carboxylated to form malate integral to the malate-aspartate shuttle. RNA sequencing (RNA-seq) identifies Me2, encoding the mitochondrial NAD-dependent isoform of malic enzyme, as being specifically upregulated during osteoblast differentiation. Knockdown of Me2 markedly reduces the glycolytic flux and impairs osteoblast proliferation and differentiation. Thus, the mitochondrial malic enzyme functionally couples the mitochondria with aerobic glycolysis in osteoblasts.
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Project description:Dobzhansky-Muller incompatibilities can result from the interactions of more than a single pair of interacting genes and there are several different models of how such complex interactions can be structured. Previous empirical work has identified complex conspecific epistasis as a form of complex interaction that has contributed to postzygotic reproductive isolation between taxa, but other forms of complexity are also possible. Here, I probe the genetic basis of reproductive isolation in crosses of the intertidal copepod Tigriopus californicus by looking at the impact of markers in genes encoding metabolic enzymes in F(2) hybrids. The region of the genome associated with the locus ME2 is shown to have strong, repeatable impacts on the fitness of hybrids in crosses and epistatic interactions with another chromosomal region marked by the GOT2 locus in one set of crosses. In a cross between one of these populations and a third population, these two regions do not appear to interact despite the continuation of a large effect of the ME2 region itself in both crosses. The combined results suggest that the ME2 chromosomal region is involved in incompatibilities with several unique partners. If these deleterious interactions all stem from the same factor in this region, that would suggest a different form of complexity from complex conspecific epistasis, namely, multiple independent deleterious interactions stemming from the same factor. Confirmation of this idea will require more fine-scale mapping of the interactions of the ME2 region of the genome.
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Project description:Using isolated amyloplasts from cauliflower buds, we have characterized the interaction and transport of various carbohydrates across the envelope membrane of a heterotrophic plastid. According to our results, glucose 6-phosphate (Glc6P) and glucose 1-phosphate (Glc1P) do not share the same transport protein for uptake into cauliflower-bud amyloplasts. Glc6P-dependent starch synthesis is strongly inhibited in the presence of dihydroxyacetone phosphate (DHAP) or 4,4'-di-isothiocyano-2,2'- stilbenedisulphonic acid (DIDS), whereas Glc1P-dependent starch synthesis is hardly affected by these compounds. Analysis of the Glc6P uptake into proteoliposomes reconstituted from the envelope proteins of cauliflower-bud amyloplasts indicate that Glc6P is taken up in a counter-exchange mode with Pi, DHAP or Glc6P, whereas Glc1P does not act as a counter-exchange substrate. Pi is a strong competitive inhibitor of Glc6P uptake (Ki 0.8 mM) into proteoliposomes, whereas Glc1P does not significantly inhibit Glc6P transport. Beside a hexose-phosphate translocator, these amyloplasts possess an envelope protein mediating the transport of glucose across the membrane. This translocator exhibits an apparent Km for glucose of 2.2 mM and is inhibited by low concentrations of phloretin, known to be a specific inhibitor of glucose-transport proteins. Maltose inhibits the uptake of glucose (Ki 2.3 mM), indicating that both carbohydrates share the same translocator.
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Project description:An NAD(P)+-dependent 'malic' enzyme is shown to be present in mitochondria from small-intestinal mucosa. The intracellular location, activity and regulatory kinetic properties of the enzyme suggest that it participates in the major energy-producing pathway for net oxidation of glutamine-derived tricarboxylic acid-cycle intermediates.
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Project description:Malic enzymes have high cofactor selectivity. An isoform-specific distribution of residues 314, 346, 347 and 362 implies that they may play key roles in determining the cofactor specificity. Currently, Glu314, Ser346, Lys347 and Lys362 in human c-NADP-ME were changed to the corresponding residues of human m-NAD(P)-ME (Glu, Lys, Tyr and Gln, respectively) or Ascaris suum m-NAD-ME (Ala, Ile, Asp and His, respectively). Kinetic data demonstrated that the S346K/K347Y/K362Q c-NADP-ME was transformed into a debilitated NAD?-utilizing enzyme, as shown by a severe decrease in catalytic efficiency using NADP? as the cofactor without a significant increase in catalysis using NAD? as the cofactor. However, the S346K/K347Y/K362H enzyme displayed an enhanced value for k(cat,NAD), suggesting that His at residue 362 may be more beneficial than Gln for NAD? binding. Furthermore, the S346I/K347D/K362H mutant had a very large K(m,NADP) value compared to other mutants, suggesting that this mutant exclusively utilizes NAD? as its cofactor. Since the S346K/K347Y/K362Q, S346K/K347Y/K362H and S346I/K347D/K362H c-NADP-ME mutants did not show significant reductions in their K(m,NAD) values, the E314A mutation was then introduced into these triple mutants. Comparison of the kinetic parameters of each triple-quadruple mutant pair (for example, S346K/K347Y/K362Q versus E314A/S346K/K347Y/K362Q) revealed that all of the K(m) values for NAD? and NADP(+) of the quadruple mutants were significantly decreased, while either k(cat,NAD) or k(cat,NADP) was substantially increased. By adding the E314A mutation to these triple mutant enzymes, the E314A/S346K/K347Y/K362Q, E314A/S346K/K347Y/K362H and E314A/S346I/K347D/K362H c-NADP-ME variants are no longer debilitated but become mainly NAD?-utilizing enzymes by a considerable increase in catalysis using NAD? as the cofactor. These results suggest that abolishing the repulsive effect of Glu314 in these quadruple mutants increases the binding affinity of NAD?. Here, we demonstrate that a series of E314A-containing c-NADP-ME quadruple mutants have been changed to NAD?-utilizing enzymes by abrogating NADP? binding and increasing NAD? binding.
| S-EPMC3183043 | biostudies-literature
Project description:Cytosolic malic enzyme (Me1) was predicted as a causal gene for abdominal using a novel statistical method named LCMS (Schadt et al., 2005, Nature Genetics). In order to validate this prediction, we profiled the liver tissues of Me1 knockout mice (Me1-/-) and their littermate wild-type (wt) controls to examine the gene expression signature as well as pathways/networks resulting from the single gene perturbation.
2009-03-08 | GSE11992 | GEO
Project description:Cytosolic malic enzyme (Me1) was predicted as a causal gene for abdominal using a novel statistical method named LCMS (Schadt et al., 2005, Nature Genetics). In order to validate this prediction, we profiled the liver tissues of Me1 knockout mice (Me1-/-) and their littermate wild-type (wt) controls to examine the gene expression signature as well as pathways/networks resulting from the single gene perturbation. 3 Me1-/- mice and 5 wt controls were profiled. Reference pool included RNA extracted from the liver of 5 wt control mice. Dye-swap was involved in the profiling.
2009-03-07 | E-GEOD-11992 | biostudies-arrayexpress
Project description:1. Cell-free extracts from culture epimastigotes of Trypanosoma cruzi contained two forms of NADP+-linked 'malic' enzyme (EC 1.1.1.40), I and II, with the same molecular weight but different electrophoretic mobilities and kinetic and regulatory properties. 2. The apparent Km for L-malate was lower for 'malic' enzyme I, with hyperbolic kinetics, whereas the kinetic pattern for 'malic' enzyme II was slightly sigmoidal (h 1.4). The kinetics for NADPH were hyperbolic for 'malic' enzyme I, and very complex for 'malic' enzyme II, suggesting both positive and negative co-operativity. 3. 'Malic' enzyme II was markedly inhibited by adenine nucleotides; AMP was the the most effective, at least in the presence of an excess of MnCl2. 'Malic' enzyme I was much less affected by the nucleotides. Both enzyme forms were inhibited by oxaloacetate, competitively towards L-malate, but the apparent Ki for 'malic' enzyme I (9 microM) was 10-fold lower than the value for 'malic' enzyme II. 'Malic' enzyme II, but not 'malic' enzyme I, was activated by L-aspartate and succinate (apparent Ka of 0.12 and 0.5 mM respectively); the activators caused a decrease in the apparent Km for L-malate and, to a lesser extent, in the apparent Km for NADP+. L-Aspartate, but not succinate, increased the apparent Vmax. 4. The inhibition by AMP suggests regulation by energy charge, with the L-malate-decarboxylation reaction catalysed by 'malic' enzyme II fulfilling a biosynthetic role. The inhibition by oxaloacetate and the activation by succinate are probably involved in the regulation of the 'partial aerobic fermentation' of glucose which yields succinate as final product. The activation by L-aspartate would facilitate the catabolism of this amino acid, when present in excess in the growth medium.
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