Spectrophotometric measurement of pyruvate dehydrogenase complex activity in cultured human fibroblasts

A spectrophotometric assay for the pyruvate dehydrogenase complex (PDHC) has been adapted for use with cultured human firbroblasts. It is a coupled enzyme assay utilizing pigeon liver arylamine acetyltransferase to measure the acetyl-CoA produced by PDHC. Activity is proportional to fibroblasts protein and to tine and depends completely on added pyruvate, CoA and NAD. In extracts in which PDHC had been activated (dephosphorylated) by the method of Sheu et al. (Sheu, R.K.-F., Hu, C.C. and Utter, M.F. (1981) J. Clin. Invest. 67, 1463–1471), activities in control cell lines are 5–50 fold higher than in earlier reports. Low activity has been demonstrated in a line previously eported to be PDHC-deficient.     

A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase.

A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.     

A method for the determination of pyruvate carboxylase activity during the glutamic acid fermentation with Corynebacterium glutamicum

A discontinuous lactate dehydrogenase coupled assay is described for the evaluation of the pyruvate carboxylase activity (Pc, EC 6.4.1.1) in a glutamate overproducing strain of Corynebacterium glutamicum. After an initial permeabilisation period of the cells, the method consisted of the fluorometric determination of the remaining pyruvate level after transformation into oxaloacetate by the endogenous Pc. The assay was demonstrated to be powerful and enabled the determination of the C. glutamicum Pc activity grown on different carbon sources. Besides, this method was used to assay Pc activity in C. glutamicum 2262 during a temperature triggered glutamate producing process with biotin excess or limitation.     

Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response

Glucose-stimulated insulin secretion is a multistep process dependent on beta-cell metabolic flux. Our previous studies on intact pancreatic islets used two-photon NAD(P)H imaging as a quantitative measure of the combined redox signal from NADH and NADPH (referred to as NAD(P)H). These studies showed that pyruvate, a non-secretagogue, enters beta-cells and causes a transient rise in NAD(P)H. To further characterize the metabolic fate of pyruvate, we have now developed one-photon flavoprotein microscopy as a simultaneous assay of lipoamide dehydrogenase (LipDH) autofluorescence. This flavoprotein is in direct equilibrium with mitochondrial NADH. Hence, a comparison of LipDH and NAD(P)H autofluorescence provides a method to distinguish the production of NADH, NADPH, or both. Using this method, the glucose dose response is consistent with an increase in both NADH and NADPH. In contrast, the transient rise in NAD(P)H observed with pyruvate stimulation is not accompanied by a significant change in LipDH, which indicates that pyruvate raises cellular NADPH without raising NADH. In comparison, methyl pyruvate stimulated a robust NADH and NADPH response. These data provide new evidence that exogenous pyruvate does not induce a significant rise in mitochondrial NADH. This inability likely results in its failure to produce the ATP necessary for stimulated secretion of insulin. Overall, these data are consistent with either a restricted pyruvate dehydrogenase-dependent metabolism or a buffering of the NADH response by other metabolic mechanisms.     

A spectrophotometric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase activity

We report an assay for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, the enzyme which catalyzes the fourth reaction step of the 2-C-methyl-D-erythritol 4-phosphate pathway for the synthesis of isoprenoids, which is based on the spectrophotometrical determination of adenosine 5'-diphosphate using pyruvate kinase and L-lactate dehydrogenase as auxiliary enzymes. This method can be adapted to microtiter plates, can be automated, and because of its simplicity and speed can be useful for the functional characterization of the enzyme and for the screening of inhibitors with potential antibiotic or antimalarial action.     

A non-isotopic in vitro assay for histone acetylation

We describe a simple, robust, and relatively inexpensive non-radioactive in vitro assay for measuring histone acetyl-transferase activity. The assay takes advantage of easy to purify recombinant E. coli-derived fusion proteins containing the NH(2)-terminal tails of histones H3 and H4 linked to epitope-tagged maltose-binding protein (MBP), and immunoblotting with antibodies specific to acetylated H3 and H4. Here we show the specificity and dynamic range of this assay for the histone acetyl-transferases, p300 and PCAF. This assay may be adapted readily for other substrates by simply generating new fusion proteins and for other acetyl-transferases by modifying reaction conditions.     

Pyruvate Dehydrogenase Activity in Osmotically Shocked Rat Brain Mitochondria: Stimulation by Oxaloacetate

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.     

A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue.

A radioactive assay for the determination of pyruvate dehydrogenase complex activity in muscle tissue has been developed. The assay measures the rate of acetyl-CoA formation from pyruvate in a reaction mixture containing NAD+ and CoASH. The acetyl-CoA is determined as [14C]citrate after condensation with [14C]-oxaloacetate by citrate synthase. The method is specific and sensitive to the picomole range of acetyl-CoA formed. In eleven normal subjects, the active form of pyruvate dehydrogenase (PDCa) in resting human skeletal muscle samples obtained using the needle biopsy technique was 0.44 +/- 0.16 (SD) mumol acetyl-CoA.min-1.g-1 wet wt. Total pyruvate dehydrogenase complex (PDCt) activity was determined after activation by pretreating the muscle homogenate with Ca2+, Mg2+, dichloroacetate, glucose, and hexokinase. The mean value for PDCt was 1.69 +/- 0.32 mumol acetyl-CoA.min-1.g-1 wet wt, n = 11. The precision of the method was determined by analyzing 4-5 samples of the same muscle piece. The coefficient of variation for PDCa was 8% and for PDCt 5%.     

A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay

In order to determine the activity of O-linked GlcNAc transferase (OGT), a modified coupled enzyme method was proposed. This method was based on the measurement of uridine 5′-(trihydrogen diphosphate) (UDP), a product generated in transglycosylation reaction. In the assay, UDP was coupled to the conversion of phosphoenolpyruvate to pyruvate using pyruvate kinase. Using a commercial pyruvate assay kit, the pyruvate was converted to a red terminal product, which could be photometrically measured at 570 nm or fluorometrically measured at 587 nm (E _m = 535 nm) on a microplate reader. Kinetic study of a truncated recombinant mOGT and quantitative analysis of OGT in two biological samples indicated that this method was practical and competitive for quantitative analysis of OGT.     

An immunochemical assay model system for the sensitive detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit pyruvate dehydrogenase (E1).

An immunochemical enzyme immunoassay model system was developed and compared for maximum sensitivity with a radioimmunoassay method and the classic enzyme activity method for the detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit, pyruvate dehydrogenase (E1), isolated from Escherichia coli. Cross-linked large molecular weight antibody-enzyme conjugate systems are compared with heterobifunctional singular antibody conjugates substituted with high levels of horseradish peroxidase. Both polyclonal and monoclonal antibodies generated to the Escherichia coli PDHc and E1 antigens were used to develop a double-antibody sandwich microtiter plate enzyme-linked immunosorbent assay. It is demonstrated that a double sandwich immunochemical assay system can be quantitative for PDHc, can detect PDHc in crude cell lysates and has levels of sensitivity of 2.0.10(-16) mol for the detection of PDHc. This assay model system provides specific antibody selection criteria and coupling methods needed to select specific antisera that cross-react with human PDHc. This rapid and sensitive immunochemical assay method clearly demonstrates that sensitive mass assay systems can be developed for the detection of PDHc. Different from Western blot, this methodology could be used to generate mass assays which could be applied to the rapid detection of mammalian antigens (employing the corresponding antibodies) implicated in a number of pyruvate dehydrogenase deficiencies associated with human disorders.     

A new oxidase method for analyzing L-lactate

A new oxidase-coupled colorimetric method for analysis of L-lactate in biological fluids has been developed without use of peroxidase. The method is based on lactate oxidase-catalysed transformation of lactate to pyruvate which is determined photometrically in the next dye-producing reaction of 3-methyl-2-benzothiazolinone hydrazone (MBTH) in the presence of ferric ions. Sensitivity of the method is estimated as 0.1 micromole of analyte in 4-ml of reaction mixture. Linearity is observed in the range 0.1-1.0 micromole of L-lactate in sample (r = 0.99943; p < 0.0001). The developed method has been adapted for assay of L-lactic acid in kefirs and yogurts.     

The effect of phenylpyruvate on pyruvate metabolism in rat brain

1. The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by isolated mitochondria from rat brain was investigated. 2. Phenylpyruvate inhibited the fixation of H(14)CO(3) (-) in the presence of pyruvate by intact rat brain mitochondria, whereas phenylalanine and other metabolites of this amino acid had no inhibitory effect on this process. 3. Pyruvate carboxylase activity in freeze-dried rat brain mitochondrial preparations was also inhibited only by phenylpyruvate, and a ;mixed type' inhibition was observed. 4. The K(m) for pyruvate of rat brain pyruvate carboxylase was about 0.2mm. 5. The concentration of phenylpyruvate required for a 50% inhibition of H(14)CO(3) (-) fixation by the intact mitochondria and of pyruvate carboxylase activity was dependent on the concentration of pyruvate used in the incubation medium. 6. The possible significance of inhibition of pyruvate carboxylase activity by phenylpyruvate in the brains of phenylketonuric patients is discussed.     

Control of glyceroneogenic activity in rat brown adipose tissue

Brown adipose tissue (BAT) glyceroneogenesis was evaluated in rats either fasted for 48 h or with streptozotocin-diabetes induced 3 days previously or adapted for 20 days to a high-protein, carbohydrate-free (HP) diet, conditions in which BAT glucose utilization is reduced. The three treatments induced an increase in BAT glyceroneogenic activity, evidenced by increased rates of incorporation of [1-14C]pyruvate into triacylglycerol (TAG)-glycerol in vitro and a marked, threefold increase in the activity of BAT phosphoenolpyruvate carboxykinase (PEPCK). BAT glycerokinase activity was not significantly affected by fasting or diabetes. After unilateral BAT denervation of rats fed either the HP or a balanced diet, glyceroneogenesis activity increased in denervated pads, evidenced by increased rates of nonglucose carbon incorporation into TAG-glycerol in vivo (difference between 3H2O and [14C]glucose incorporations) and of [1-14C]pyruvate in vitro. PEPCK activity was not significantly affected by denervation. The data suggest that BAT glyceroneogenesis is not under sympathetic control but is sensitive to hormonal/metabolic factors. In situations of reduced glucose use there is an increase in BAT glyceroneogenesis that may compensate the decreased generation of glycerol-3-phosphate from the hexose.     

Measurement of Rat Brain Kynurenine Aminotransferase at Physiological Kynurenine Concentrations

The production of the neuroinhibitory and neuroprotective metabolite kynurenic acid (KYNA) was investigated in rat brain by examining its biosynthetic enzyme, kynurenine aminotransferase (KAT). By using physiological (low micromolar) concentrations of the substrate L-kynurenine (KYN) and by determining the irreversible conversion of [3H]KYN to [3H]KYNA as a measure of KAT activity, a novel, simple, and sensitive assay was developed which permitted the detailed characterization of the enzyme. Only a single protein, which under routine assay conditions showed approximately equal activity with 2-oxoglutarate and pyruvate as the aminoacceptor, was found in rat brain. The enzyme was distributed heterogeneously between the nine brain regions studied, with the KAT-rich olfactory bulb displaying approximately five times higher activity than the cerebellum, the area with lowest KAT activity. In subcellular fractionation studies, the majority of KAT was recovered in mitochondria. In contrast to many known aminotransferases, partially purified KAT was shown to be highly substrate-specific. Thus, of the amino acids tested, only alpha-aminoadipate and tryptophan displayed moderate competition with KYN. Notably, 3-hydroxykynurenine, reportedly a very good substrate of KAT, competed rather poorly with KYN as well. Aminooxyacetic acid, a nonspecific transaminase inhibitor, blocked KAT activity with an apparent Ki of 5 microM. Kinetic analyses with partially purified rat brain KAT revealed a Km of 17 microM for KYN with 1 mM 2-oxoglutarate, but a much higher Km (910 microM) with 1 mM pyruvate. Km values for 2-oxoglutarate and pyruvate were 150 and 160 microM, respectively. The cellular localization of KAT was examined in striatal homogenates obtained from rats 7 days after an intrastriatal injection of quinolinate.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H NMR observation of redox potential in liver.

1H NMR spectral editing techniques can select the distinct signals of lactate, pyruvate, beta-hydroxybutyrate, and acetoacetate and provide a unique way to monitor the biochemical processes in vivo. These metabolite levels reflect the near-equilibrium dehydrogenase activity and therefore the cellular redox state. The quantitative comparison between the 1H NMR and biochemical assay data is in excellent agreement. Lactate/pyruvate and beta-hydroxybutyrate/acetoacetate ratios, obtained from normalized 1H NMR spectra, respond directly to changes in the cytosolic and mitochondrial redox states. Because NMR is noninvasive, our results set the groundwork for implementing these techniques to observe tissue redox states in vivo.     

A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures

beta-Galactosidase (beta-Gal) activity is a widely accepted biomarker to detect senescence both in situ and in vitro. A cytochemical assay based on production of a blue-dyed precipitate that results from the cleavage of the chromogenic substrate X-Gal is commonly used. Blue and nonblue cells are counted under the microscope and a semiquantitative percentage of senescent cells can be obtained. Here, we present a quantitative, fast, and easy to use chemiluminescent assay to detect senescence. The Galacton chemiluminescent method used to detect the prokaryotic beta-Gal reporter enzyme in transfection studies was adapted to assay mammalian beta-Gal. The assay showed linear production of luminescence in a time- and cell-number-dependent manner. The chemiluminescent assay showed significant correlation with the cytochemical assay in detecting replicative senescence (Pearson r=0.8486, p<0.005). Moreover, the chemiluminescent method (Galacton) also detected stress-induced senescence in cells treated with H2O2 similar to the cytochemical assay (X-Gal) (Galacton: control 25,207.3+/-6548.6, H2O2 52,487.4+/-16,284.9, p<0.05; X-Gal: control 41.31+/-7.0%, H2O2 92.97+/-2.8%, p<0.01). Thus, our method is well suited to the detection of replicative and stress-induced senescence in cell culture.     

A novel assay for monoacylglycerol hydrolysis suitable for high-throughput screening

A simple assay for monoacylglycerol hydrolysis suitable for high-throughput screening is described. The assay uses [(3)H]2-oleoylglycerol as substrate, with the tritium label in the glycerol part of the molecule and the use of phenyl sepharose gel to separate the hydrolyzed product ([(3)H]glycerol) from substrate. Using cytosolic fractions derived from rat cerebella as a source of hydrolytic activity, the assay gives the appropriate pH profile and sensitivity to inhibition with compounds known to inhibit hydrolysis of this substrate. The assay could also be adapted to a 96-well plate format, using C6 cells as the source of hydrolytic activity. Thus the assay is simple and appropriate for high-throughput screening of inhibitors of monoacylglycerol hydrolysis.     

A 14CO2 ratios method for detecting pyruvate carboxylation

The pattern of oxidative metabolism of pyruvate may be assessed by comparing the steady-state 14CO2 production from four isotopes in identical samples. The assay requires measuring the ratios of steady-state 14CO2 production from two isotope pairs, [2-14C]pyruvate:[3-14C]pyruvate and [1-14C]acetate:[2-14C]acetate. These ratios are defined as the "pyruvate 14CO2 ratio" and the "acetate 14CO2 ratio," respectively. If pyruvate is metabolized exclusively via pyruvate dehydrogenase (PDH), the two ratios will be identical. Alternatively, if any pyruvate enters the tricarboxylic acid (TCA) cycle via pyruvate carboxylation (PC), the pyruvate 14CO2 ratio will be less than the acetate 14CO2 ratio. If pyruvate enters the TCA cycle only through PC (with oxaloacetate and fumarate in equilibrium) the pyruvate 14CO2 ratio will approach a value of 1.0. An equation is presented for the quantitative evaluation of pyruvate oxidation by these two pathways. We have used this method to detect relative changes in the pattern of pyruvate metabolism in rat liver mitochondria produced by exposure to 1 mM octanoyl carnitine, a compound known to alter the PC:PDH activity ratio. The major advantages of the method are (i) that it provides a sensitive method for detecting pyruvate carboxylation at physiological pyruvate concentrations and (ii) that it provides a method for distinguishing between effects on pyruvate transport and effects on pyruvate oxidation.     

Determination of kinase activity by using an enzyme electrode

A method for determining the enzymatic activity of hexokinases, acetate kinase and pyruvate kinase using an enzyme electrode was developed. The assay time is 2-3 min. The lower limit of the activity determining is 0,054 U/ml. The proposed method was applied to investigation of pyruvate kinase and acetate kinase reactivation under the action of mercaptoethanol.     

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria

The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 [mu]M in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion. We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intra-mitochondrial generation of pyruvate via malic enzyme.