Insulin stimulation of hepatic malic enzyme activity in normal and diabetic rats controlled by different regulatory processes

A comparison of the processes controlling the increase in hepatic malic enzyme activity in insulin-treated normal and diabetic rats indicated the existence of two distinct regulatory mechanisms. Livers were removed at 12, 36, and 60 h after insulin treatment of normal and alloxan-diabetic rats, and the activity, quantity, and specific activity (units/nmol), of malic enzyme was determined. In normal rats, a significant increase in activity occurred 12 h after insulin, whereas 36 h of insulin treatment was required for diabetic rats to show an increase in enzyme activity. This suggested that the return of malic enzyme activity from the depleted levels measured in diabetic rats probably involved a different sequence of events. A malic enzyme specific radioimmunoassay confirmed this. The increase in activity in insulin-treated normal rats was due to an increase in the quantity of malic enzyme. In insulin-treated diabetic rats, the increase in activity resulted from increases in both enzyme quantity and the specific activity of the enzyme, which returned to levels observed in normal rats.     

Induction of rat liver malic enzyme messenger RNA activity by insulin and by fructose

Several human normal and neoplastic cell lines were screened for production of PDGF receptor competing activity. Conditioned medium from two sarcomas and one glioma blocked 125I-PDGF binding to human foreskin fibroblasts in a dose-dependent manner. In each case this effect was abolished when the conditioned medium was pretreated with PDGF-antiserum, indicating that the receptor competing activity was immunologically related to PDGF. Direct evidence for de novo synthesis of a PDGF-like component in the cultures was afforded by 35S-cysteine labeling of the three cell lines, followed by immunoprecipitation with PDGF antiserum. This resulted in the specific precipitation of a 31,000 molecular weight labeled protein, which upon reduction was split into two polypeptides of molecular weights 17,000 and 16,500. The significance of these findings in view of the recently discovered structure homology between PDGF and the transforming gene product of simian sarcoma virus, p28sis, is discussed.     

High malic enzyme activity in tumor cells and its cross-reaction with antipigeon liver malic enzyme serum

Summary Rabbit antibodies against pigeon liver malic enzyme (EC 1.1.1.40) were prepared. The antiserum gave single precipitation line with crude pigeon liver extract. Cross reaction was observed with partially purified malic enzyme or crude extract from chicken liver. Positive cross reaction was also observed with the concentrated cytosolic fraction of two human carcinoma cell lines which were demonstrated to contain high malic enzyme activity. All other proteins examined did not react with the antibodies. When purified pigeon liver malic enzyme was mixed with the antiserumin vitro, a time-dependent inactivation of the enzyme activity was observed. Protection of the enzyme activity against antiserum inactivation was afforded by NADP⁺ orL-malate. Metal Mn²⁺ gave little protection.     

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.     

A possible plasma membrane particle containing malic enzyme activity

A definite membrane fraction from Cucurbita hypocotyls, maize coleoptiles, and other plant tissues contains a NADP-dependent malic enzyme activity, up to 10% of overall tissue activity, and probably other soluble proteins. This malic enzyme particle is identified as plasmalemma on the basis of sedimentation behavior, density distribution in sucrose gradients, in comparison with enzyme markers, and sluggish penetration by the sugar Metrizamide. Enzyme binding to the plasma membrane is stable and scarcely sensitive to salts and EDTA, although all activity is released to the supernatant in the presence of Triton-X-100 or under hypotonic conditions. The properties of bound enzyme are similar to those of free enzyme in cell extracts. It is proposed that osmotically sensitive plasma membrane vesicles, containing cytoplasm fragments, are formed during homogenization. Low malic enzyme activities are also associated with Cucurbita proplastids.Abbreviations Tris tris(hydroxymethyl)amino methane - NPA naphthylphtalamic acid - malic enzyme L-malate - NAD(P) oxidoreductase, decarboxylating (EC 1.1.1.40) - ER endoplasmic reticulum     

Metabolic regulation of malic enzyme activity from Paracoccus denitrificans by glyoxylate and acetylCoA.

$\text{Paracoccus denitrificans}$ contains both NAD⁺- and NADP⁺-linked malic enzyme activities when grown on malate/nitrate. The enzyme is inactive in the absence of NH₄⁺. AcetylCoA inhibits both activities competitively with respect to L-malate. Glyoxylate (0.5 mM) causes 60% inhibition of the NADP⁺-linked activity but has little effect on the NAD⁺-linked activity. Citrate, aspartate, AMP, ADP, and ATP, at 0.5mM, have little effect on either of the two activities. The results are discussed with regards to the control of malic enzyme activity within the cell.     

Mitochondrial malic enzyme polymorphism among different ethnic groups in Singapore

Mitochondrial malic enzyme (EC 1.1.1.40; MEM) was examined by starch-gel electrophoresis on post-mortem brain samples from 453 unrelated subjects of either sex comprising 161 Chinese, 150 Indians and 113 Malays and 29 from other racial groups. The estimated gene frequencies of MEM1 were found to be 0.7111, 0.6100 and 0.6769 in Chinese, Indians and Malays, respectively. No significant deviation from the Hardy-Weinberg equilibrium was observed in Chinese and Malays. However, there was a significant deviation with a deficiency of heterozygotes among Indians. MES did not show any polymorphism.     

Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose: roles of malic enzyme and fructose-1,6-bisphosphatase

In the biotechnological production of L-lysine and L-glutamate by Corynebacterium glutamicum media based on glucose, fructose or sucrose are typically used. Glutamate production by C. glutamicum was very similar on glucose, fructose, glucose plus fructose and sucrose. In contrast, lysine production of genetically defined C. glutamicum strains was significantly higher on glucose than on the other carbon sources. To test whether malic enzyme or fructose-1,6-bisphosphatase might limit growth and lysine on fructose, glucose plus fructose or sucrose, strains overexpressing either malE which encodes the NADPH-dependent malic enzyme or the fructose-1,6-bisphosphatase gene fbp were generated. Overexpression of malE did not improve lysine production on any of the tested carbon sources. Upon overexpression of fbp lysine yields on glucose and/or fructose were unchanged, but the lysine yield on sucrose increased twofold. Thus, fructose-1,6-bisphosphatase was identified as a limiting factor for lysine production by C. glutamicum with sucrose as the carbon source.     

Organ specific regulation of malic enzyme and hexosemonophosphate shunt dehydrogenases activity by high carbohydrate diet

The effect of starvation-refeeding transitions on the activity of malic enzyme and hexosemonophosphate shunt dehydrogenases in lipogenic and non-lipogenic tissues from rats was investigated. Starvation of the rats caused a decrease of malic enzyme activity in the liver, white and brown adipose tissue. Refeeding of the animals with high carbohydrate diet caused a several fold increase of malic enzyme activity in these tissues. Substitution of high fat for high carbohydrate diet resulted in only a slight increase of malic enzyme activity in the liver, white and brown adipose tissues. In the same rats, no significant effect of starvation-refeeding transition on malic enzyme activity in the kidney cortex, brain, heart, skeletal muscle and spleen was observed. The changes of the activity of hexosemonophosphate shunt dehydrogenases during starvation-refeeding transition essentially paralleled those of malic enzyme in all the tissues examined.     

Arl2 and Arl3 regulate different microtubule-dependent processes

Arl2 and Arl3 are closely related members of the Arf family of regulatory GTPases that arose from a common ancestor early in eukaryotic evolution yet retain extensive structural, biochemical, and functional features. The presence of Arl3 in centrosomes, mitotic spindles, midzones, midbodies, and cilia are all supportive of roles in microtubule-dependent processes. Knockdown of Arl3 by siRNA resulted in changes in cell morphology, increased acetylation of alpha-tubulin, failure of cytokinesis, and increased number of binucleated cells. We conclude that Arl3 binds microtubules in a regulated manner to alter specific aspects of cytokinesis. In contrast, an excess of Arl2 activity, achieved by expression of the [Q70L]Arl2 mutant, caused the loss of microtubules and cell cycle arrest in M phase. Initial characterization of the underlying defects suggests a defect in the ability to polymerize tubulin in the presence of excess Arl2 activity. We also show that Arl2 is present in centrosomes and propose that its action in regulating tubulin polymerization is mediated at centrosomes. Somewhat paradoxically, no phenotypes were observed Arl2 expression was knocked down or Arl3 activity was increased in HeLa cells. We conclude that Arl2 and Arl3 have related but distinct roles at centrosomes and in regulating microtubule-dependent processes.     

Regulation of hepatic malic enzyme by perfluorodecanoic acid

Perfluorodecanoic acid (PFDA) administration to adult male rats increased both the activity of hepatic malic enzyme and liver weight in a dose-dependent manner. Hepatomegaly and augmented activity of malic enzyme in liver were apparent within one day following PFDA administration and reached a plateau by three days posttreatment. Malic enzyme quantity per liver in PFDA-treated rats was elevated within one day following dosing and increased continually throughout five days posttreatment. Administration of PFDA to rats in the fed state also led to an increase in the specific activity of hepatic malic enzyme that peaked at three days following dosing. When compared to the fed condition, rats fasted for 48 hours had a decrease in both relative liver weight and the quantity of supernatant protein per liver. The total activity (U/liver) and specific activity of malic enzyme in the liver were also reduced in the fasted state. During the 24 hours after treatment in rats fasted for 48 hours, the body weight as well as the absolute and relative liver weight of animals receiving vehicle declined continuously in the absence of feed. Following the administration of PFDA to fasted rats, body weight was maintained until eight hours posttreatment but then declined at a rate similar to that found with the vehicle-treated group. Absolute and relative liver weight in PFDA-treated rats were increased significantly at eight hours posttreatment when compared to those receiving vehicle, and this increment was maintained throughout the rest of the 24 hours following dosing. While the activity and enzyme content of hepatic malic enzyme decreased in the vehicle-treated group, administration of PFDA to rats fasted for 48 hours prevented their decline. The specific activity of hepatic malic enzyme in 48 hours fasted rats receiving PFDA was also elevated significantly at 16 hours posttreatment. Thus, the administration of PFDA to the adult male rat in both the fed and fasted nutritional states was found to regulate hepatic malic enzyme by not only increasing enzyme quantity but also by augmenting the specific activity, (ie, catalytic state) of the enzyme.