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K F Sheu

Publications and source records attributed to K F Sheu.

At least 73 records · Page 4Linked to original sources
PubMed1986

The subcellular localization of glutamate dehydrogenase (GDH): is GDH a marker for mitochondria in brain?

Glutamate dehydrogenase (GDH, EC 1.4.1.2) has long been used as a marker for mitochondria in brain and other tissues, despite reports indicating that GDH is also present in nuclei of liver and dorsal root ganglia. To examine whether GDH can be used as a marker to differentiate between mitochondria and nuclei in the brain, we have measured GDH by enzymatic activity and on immunoblots in rat brain mitochondria and nuclei which were highly enriched by density-gradient centrifugation methods. The activity of GDH was enriched in the nuclear fraction as well as in the mitochondrial fraction, while the activities of other "mitochondrial" enzymes (fumarase, NAD-isocitrate dehydrogenase and pyruvate dehydrogenase complex) were enriched only in the mitochondrial fraction. Immunoblots using polyclonal antibodies against bovine liver GDH confirmed the presence of GDH in the rat brain nuclear and mitochondrial fractions. The GDH in these two subcellular fractions had a very similar molecular weight of 56,000 daltons. The mitochondrial and nuclear GDH differed, however, in their susceptibility to solubilization by detergents and salts. The mitochondrial GDH could be solubilized by extraction with low concentrations of detergents (0.1% Triton X-100 and 0.1% Lubrol PX), while the nuclear GDH could be solubilized only by elevated concentrations of detergents (0.3% each) plus KCl (greater than 150 mM). Our results indicate that GDH is present in both nuclei and mitochondria in rat brain. The notion that GDH may serve as a marker for mitochondria needs to be re-evaluated.

Animals↗
PubMed1985

Differences in some of the metabolic properties of mitochondria isolated from cerebral cortex and olfactory bulb of the rat.

The metabolic properties of mitochondria from rat cerebral cortex and olfactory bulb were investigated. The pyruvate-supported oxygen uptake rates by olfactory bulb mitochondria were significantly lower than those by cerebrocortical mitochondria. This is consistent with the differences in pyruvate dehydrogenase complex activities between these mitochondrial preparations. Pyruvate dehydrogenase kinase, NAD-linked isocitrate dehydrogenase, and hexokinase activities in olfactory bulb mitochondria were significantly lower than those in cerebrocortical mitochondria. However, NADP-linked isocitrate dehydrogenase, and NAD-linked and NADP-linked glutamate dehydrogenase activities in olfactory bulb mitochondria were significantly higher than those in cerebrocortical mitochondria. The differences between these two mitochondrial preparations in terms of the activities of these energy-metabolizing enzymes reflect the differences detected in the homogenates of these regions.

Animals↗
PubMed1985

An immunochemical study of the pyruvate dehydrogenase deficit in Alzheimer's disease brain.

The activity of the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) was reduced to about 30% of control values in histologically unaffected occipital cortex of the brains of patients with Alzheimer's disease, as well as in histologically affected frontal cortex. In contrast, activity of another mitochondrial enzyme, glutamate dehydrogenase, was normal. Neither age nor time until postmortem study correlated significantly with PDHC activity in either Alzheimer or control samples, and PDHC was not inactivated significantly on incubation with homogenates of either Alzheimer or control brain. Antibodies against the highly purified bovine PDHC inhibited Alzheimer and control PDHC equally per unit of enzyme activity. Immunoblots also indicated that the PDHC antigens were not different in normal and Alzheimer brains. This antibody, however, inhibited Alzheimer PDHC more effectively than it did control PDHC, based on milligrams of protein, suggesting a reduced amount of normal PDHC protein. Other data suggest that the PDHC deficiency is related to mitochondrial damage and to impaired calcium homeostasis in Alzheimer nerve cells, which may then mediate a variety of other cellular impairments.

Aged↗
PubMed1985

Immunochemical characterization of pyruvate dehydrogenase complex in rat brain.

Pyruvate dehydrogenase complex (PDHC) in rat brain was studied immunochemically, using antibodies against the bovine kidney PDHC, by immunoblotting, immunoprecipitation, inhibition of enzyme activity, and enzyme-linked immunoabsorbent assay (ELISA). The immunoblots showed that the antibodies bound strongly to the alpha peptide of the pyruvate dehydrogenase (E1) component, and to the dihydrolipoyl transacetylase (E2) and the dihydrolipoyl dehydrogenase (E3) components of PDHC. A similar immunoblotting pattern was observed in all eight brain regions examined. On immunoblotting of the subcellular fractions, these PDHC peptides were observed in mitochondria and synaptosomes but not in the postmitochondrial supernatants. This agrees with other evidence that brain PDHC is localized in the mitochondria. These results, together with those from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitin, also showed that the alpha E1, beta E1, and E3 peptides of rat brain PDHC are very similar in sizes to those of the bovine kidney PDHC, being 42, 36, and 58 kD, respectively. The size of the E2 peptide, 66 kD, is different from that of bovine kidney E2, 73 kD. The relative abundance of PDHC protein in nonsynaptic mitochondria was compared by enzyme activity titration and ELISA. Both methods demonstrated that the amount of PDHC antigen in the mitochondria from cerebral cortex is greater than that in the olfactory bulb mitochondria. This is consistent with the results of the activity measurement. The ELISA also showed that the PDHCs in both mitochondrial populations are antigenically similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Relationship between activation state of pyruvate dehydrogenase complex and rate of pyruvate oxidation in isolated cerebro-cortical mitochondria: effects of potassium ions and adenine nucleotides.

The relation between the activation (phosphorylation) state of pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) and the rate of pyruvate oxidation has been examined in isolated, metabolically active, and tightly coupled mitochondria from rat cerebral cortex. With pyruvate and malate as the substrates, the activation state of PDHC decreased on addition of ADP, while the rates of oxygen uptake and 14CO2 formation from [1-14C]pyruvate increased. The lack of correlation between the activation state of PDHC and rate of pyruvate oxidation was seen in media containing 5, 30, or 100 mM KCl. Both the activation state of PDHC and pyruvate oxidation increased, however, when KCl was increased from 5 to 100 mM. Although the PDHC is inactivated by an ATP-dependent kinase (EC 2.7.1.99), direct measurement of ATP and ADP failed to show a consistent relationship between the activation state of PDHC and either ATP levels or ATP/ADP ratios. Comparison of the activation state of PDHC in uncoupled or oligomycin-treated mitochondria also failed to correlate PDHC activation state to adenine nucleotides. In brain mitochondria, unlike those from other tissues, the activation state of PDHC does not seem to be related clearly to the rate of pyruvate oxidation, or to the mitochondrial adenylate energy charge.

Acetyltransferases↗
PubMed1984

Stereochemical course of thiophosphoryl group transfer catalyzed by mitochondrial phosphoenolpyruvate carboxykinase.

Guinea pig liver mitochondrial phosphoenolpyruvate carboxykinase catalyzes the conversion of (Rp)-guanosine 5'-(3-thio[3-18O]triphosphate) and oxalacetate to (Sp)-[18O] thiophosphoenolpyruvate , GDP, and CO2 by a mechanism that involves overall inversion in the configuration of the chiral [18O]thiophosphate group. This result is most consistent with a single displacement mechanism in which the [18O]thiophosphoryl group is transferred from (Rp)-guanosine 5'-(3-thio[3-18O]triphosphate) bound at the active site directly to enolpyruvate generated at the active site by the decarboxylation of oxalacetate. In particular, this result does not indicate the involvement of a covalent thiophosphoryl-enzyme on the reaction pathway.

Animals↗
PubMed1984

Correlation of enzymatic, metabolic, and behavioral deficits in thiamin deficiency and its reversal.

To clarify the enzymatic mechanisms of brain damage in thiamin deficiency, glucose oxidation, acetylcholine synthesis, and the activities of the three major thiamin pyrophosphate (TPP) dependent brain enzymes were compared in untreated controls, in symptomatic pyrithiamin-induced thiamin-deficient rats, and in animals in which the symptoms had been reversed by treatment with thiamin. Although brain slices from symptomatic animals produced 14CO2 and 14C-acetylcholine from [U-14C]glucose at rates similar to controls under resting conditions, their K+-induced-increase declined by 50 and 75%, respectively. In brain homogenates from these same animals, the activities of two TPP-dependent enzymes transketolase (EC 2.2.1.1) and 2-oxoglutarate dehydrogenase complex (EC 1.2.4.2, EC 2.3.1.61, EC 1.6.4.3) decreased 60-65% and 36%, respectively. The activity of the third TPP-dependent enzyme, pyruvate dehydrogenase complex (EC 1.2.4.1, EC 2.3.1.12, EC 1.6.4.3) did not change nor did the activity of its activator pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43). Although treatment with thiamin for seven days reversed the neurological symptoms and restored glucose oxidation, acetylcholine synthesis and 2-oxoglutarate dehydrogenase activity to normal, transketolase activity remained 30-32% lower than controls. The activities of other TPP-independent enzymes (hexokinase, phosphofructokinase, and glutamate dehydrogenase) were normal in both deficient and reversed animals.

Animals↗
PubMed1984

Studies on the bovine brain pyruvate dehydrogenase complex using the antibodies against kidney enzyme complex.

Pyruvate dehydrogenase complex (PDHC) was purified from bovine kidney with a specific activity of 12-16 mumol of NADH or acetyl-CoA formed/min/mg protein. The four peptides comprising its three catalytic components were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Rabbit antibodies against this highly purified PDHC (anti-PDHC) exhibited similar binding affinity to the phospho-PDHC as it did to the PDHC antigen. To test whether there exist brain isozymes of PDHC differing from kidney enzyme, which has been extensively characterized, the PDHCs in bovine brain and kidney were compared using this anti-PDHC. The PDHC activities in the brain and kidney mitochondrial extracts were inhibited to the same degree by varying amounts of anti-PDHC. Brain PDHC was precipitated with the anti-PDHC and resolved by SDS-PAGE. The four brain PDHC peptides isolated immunochemically with anti-PDHC had the same sizes as the kidney PDHC peptides. These PDHC peptides from kidney and brain were further compared by their peptide fragment patterns, which were generated by partial proteolysis with Staphylococcus aureus V8 protease or by CNBr and resolved by SDS-PAGE. The peptide patterns generated with the former method indicated that the alpha and beta peptides of the pyruvate dehydrogenase (E1) component and the peptide of dihydrolipoyl transacetylase (E2) component of kidney PDHC were very similar to the corresponding peptides immunologically isolated from brain. The peptide patterns generated with CNBr further confirmed that the beta E1 and E2 peptides of kidney PDHC were similar to the corresponding peptides from brain.

Animals↗
PubMed1984

Properties and regional distribution of pyruvate dehydrogenase kinase in rat brain.

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.

Adenosine Triphosphate↗
PubMed1984

Pyruvate dehydrogenase phosphate (PDHb) phosphatase activity in fibroblasts from Leigh's disease.

The defective activation of pyruvate dehydrogenase complex (PDHC) in Leigh's disease (subacute necrotizing encephalomyelopathy) could be due to deficiency of pyruvate dehydrogenase phosphate (PDHb) phosphatase (EC 3.1.3.43). This enzyme catalyzes the dephosphorylation and activation of phospho-PDHC. In cultured skin fibroblasts, we assayed this enzyme by measuring the rate of activation of the exogenously added, purified phospho-PDHC (bovine kidney). PDHb phosphatase activity did not differ significantly among normal control cells, Leigh's lines, spinocerebellar ataxias, or other neurologic disorders. The results do not support the idea that PDHb phosphatase is deficient in Leigh's disease.

Animals↗
PubMed1984

Central hypoventilation syndrome in pyruvate dehydrogenase complex deficiency.

The presentation and treatment of a central hypoventilation syndrome in a boy with pyruvate dehydrogenase complex (PDHC) deficiency are reported. Dephosphorylated PDHC was assayed in disrupted fibroblasts after pretreatment with dichloroacetate, a pyruvate dehydrogenase kinase inhibitor. Maximal specific activity of activated patient PDHC was 10% to 30% of control values. Patient PDHC activity was not increased by alterations in concentrations of pyruvate or cofactors (thiamine pyrophosphate [TPP], coenzyme A [CoA], oxidized form of nicotinamide adenine dinucleotide [NAD+]). Clinically, normalization of plasma lactate by a high-lipid diet did not prevent slowly progressive neurologic decline. The patient manifested intermittent ataxia, episodic profound weakness, moderate psychomotor retardation, ophthalmoplegia, and retinal pigment epithelial changes. A true central hypoventilation syndrome was documented on the basis of rigorous radiologic, electrophysiologic, and pulmonary function criteria. Theophylline, progesterone, and ritalin neither altered ventilatory response to CO2 nor permitted weaning from the ventilator. In contrast, peripheral chemoreceptor stimulants (intravenous doxapram; oral almitrine) effected an acute doubling of minute ventilation with appropriate decreases in PaCO2. However, a positive response to long-term therapy with almitrine could not be unequivocally shown. It was concluded that measurement of disrupted fibroblast PDHC following dichloroacetate activation constitutes an accurate assay for PDHC deficiency. PDHC deficiency must be considered in the differential diagnosis of the central hypoventilation syndrome; this appears to be the first report of such an association. Finally, a therapeutic trial of a peripheral chemoreceptor agonist is warranted in the management of central hypoventilation syndrome.

Almitrine↗
PubMed1983

Pyruvate dehydrogenase phosphate (PDHb) phosphatase in brain: activity, properties, and subcellular localization.

The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg . ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho-PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K-phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K-phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.

Animals↗
PubMed1981

Pyruvate dehydrogenase complex activity in normal and deficient fibroblasts.

Pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts appears to be regulated by a phosphorylation-dephosphorylation mechanism, as is the case with other animal cells. The enzyme can be activated by pretreating the cells with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, before they are disrupted for measurement of PDC activity. With such treatment, the activity reaches 5-6 nmol/min per mg of protein at 37 degrees C with fibroblasts from infants. Such values represent an activation of about 5-20-fold over those observed with untreated cells. That this assay, based on [1-(14)C]pyruvate decarboxylation, represents a valid measurement of the overall PDC reaction is shown by the dependence of (14)CO(2) production on the presence of thiamin-PP, coenzyme A (CoA), Mg(++), and NAD(+). Also, it has been shown that acetyl-CoA and (14)CO(2) are formed in a 1:1 ratio. A similar degree of activation of PDC can also be achieved by adding purified pyruvate dehydrogenase phosphatase and high concentrations of Mg(++) and Ca(++), or in some cases by adding the metal ions alone to the cell homogenate after disruption. These results strongly suggest that activation is due to dephosphorylation. Addition of NaF, which inhibits dephosphorylation, leads to almost complete loss of PDC activity. Assays of completely activated PDC were performed on two cell lines originating from patients reported to be deficient in this enzyme (Blass, J. P., J. Avigan, and B. W. Ublendorf. 1970. J. Clin. Invest. 49: 423-432; Blass, J. P., J. D. Schuman, D. S. Young, and E. Ham. 1972. J. Clin. Invest. 51: 1545-1551). Even after activation with DCA, fibroblasts from the patients showed values of only 0.1 and 0.3 nmol/min per mg of protein. A familial study of one of these patients showed that both parents exhibited activity in fully activated cells about half that of normal values, whereas cells from a sibling appeared normal. These results demonstrate the inheritance nature of PDC deficiency, and that the present assay is sufficient to detect the heterozygous carriers of the deficiency. Application of the same procedures to fibroblasts obtained from 16 individuals who were believed to have normal PDC activities showed a range from about 2-2.5 nmol/min per mg protein for adults to 5-6 nmol/min per mg protein for cells from infants.

Acetyl Coenzyme A↗
PubMed1978

UDP-glucose pyrophosphorylase. Stereochemical course of the reaction of glucose 1-phosphate with uridine-5'[1-thiotriphosphate].

Of the two diastereoisomers of uridine-5'[1-thiotriphosphate] differing in configuration at Palpha, UDP-glucose pyrophosphorylase catalyzes the reaction of the one designated isomer B (Eckstein, F. (1975) Angew. Chem, Int. Ed. Engl. 14, 160-166) with glucose-1-P to produce uridine-5'[1-thiodiphosphate]glucose. Mild acid hydrolysis of the latter, under conditions known to be minimal for the hydrolysis of aldose phosphates, produces diastereoisomer A of uridine-5'[1-thiodiphosphate]. Therefore, the reaction of uridine-5'[1-thiotriphosphate](B) with glucose-1-P catalyzed by UDP-glucose pyrophosphorylase occurs with stereochemical inversion at Palpha to produce uridine-5'[1-thiodiphosphate]glucose(A). The 31P NMR spectra of these nucleotides are presented and used to elucidate the stereochemical course of the reaction. The apparent Km exhibited by this enzyme for uridine-5'[1-thiodiphosphate]glucose isomer A at 2 mM pyrophosphate and pH 7.6 is 75 micron, which is only 2.2 times that for UDP-glucose, 35 micron. The maximal velocity for UDP-glucose is 11 times that for uridine-5'[1-thiodiphosphate]glucose isomer A under these conditions.

Glucosephosphates↗