Assay of 3-hydroxybutyrate in the picomolar range

A radioisotopic procedure for the assay of 3-hydroxybutyrate is presented. It is based on the measurement of NADH, generated in the 3-hydroxybutyrate dehydrogenase reaction, through the conversion of 2-[U-14C]ketoglutarate to 14C-labeled L-glutamate in the presence of beef liver glutamate dehydrogenase. The assay is linear in the range of 2.5 to 20.0 pmole/sample and about 100-times more sensitive than previous methods. The procedure proved useful for the measurement of 3-hydroxybutyrate in liver samples not exceeding 25 micrograms wet weight.     

Synthesis of a new long-wavelength latent fluorimetric indicator for analytes determination in the DT-Diaphorase coupling dehydrogenase assay system

We synthesized a new long-wavelength latent fluorogenic probe BQC (1) to monitor DTD activity. The fluorogenic chemical transformation of BQC triggered by DTD in the presence of NADH is through a series of tandem reactions, DTD-catalyzed benzoquinone reduction, trimethyl-locks cyclization and intramolecular urea formation, which are spontaneous and irreversible at physiological temperature in aqueous media. The fluorescence signal revealed by this process is specific and exhibited in the near red spectrum region with emission maxima at 595 nm, and it could be competitively inhibited by menadione. The fluorescent response of BQC is insensitive to various biological thiol reductants. Furthermore, pro-fluorophore BQC is a sensitive fluorimetric indicator for analytes determination in the oxygen-insensitive DTD-coupled dehydrogenases assay by including NAD(+) which will convert to NADH by reaction in the presence of analytes. This novel oxygen-insensitive assay demonstrates a good relationship in detecting 3-hydroxybutyrate and glucose-1-phosphate in 1-10 microM range, which presents to the applicability for the construction of fiber-optic biosensors in the future clinical diagnostic.     

A sensitive radioisotopic method for the measurement of NAD(P)H: its application to the assay of metabolites and enzymatic activities

A radioisotopic method for the assay of NADH or NADPH is presented, which is based on the conversion of 2-[U-14C]ketoglutarate to 14C-labeled glutamate in the reaction catalyzed by glutamate dehydrogenase. The efficiency of the method is close to 75%, its precision (coefficient of variation) close to 5%, and its sensitivity close to 0.1 pmol/sample. This simple and rapid method can be applied to the measurement of several metabolites and enzymatic activities. In the present study, its application to the assay of sorbitol, 3-hydroxybutyrate, glutamate dehydrogenase, 3-hydroxybutyrate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase is documented.     

Quantum dots as nano plug-in's for efficient NADH resonance energy routing

The routing of fluorescent signals from NADH to quantum dots (QDs) has been a subject of extensive research for FRET based applications. In the present study, the spectral cross talk of NAD(+)/NADH with QDs was used to monitor the reaction of NAD(+)-dependent dehydrogenase enzyme. CdTe QD may undergo dipolar interaction with NADH as a result of broad spectral absorption due to multiple excitonic states resulting from quantum confinement effects. Thus, non-radiative energy transfer can take place from NADH to CdTe QD enhancing QDs fluorescence. Energy routing assay of NADH-QD was applied for detection of formaldehyde as a model analyte in the range 1000-0.01 ng/mL by the proposed technique. We observed proportionate quenching of CdTe QD fluorescence by NAD(+) and enhancement in the presence of NADH formed by various concentrations of enzyme (0.028-0.4 U). Hence, it was possible to detect formaldehyde in the range 1000-0.01 ng/mL with a limit of detection (LOD) at 0.01 ng/mL and regression coefficient R(2)=0.9982. Therefore, a unique optical sensor was developed for the detection of the formaldehyde in sensitive level based on the above mechanism. This method can be used to follow the activity of NAD(+)-dependent enzymes and detection of dehydrogenases in general.     

Evidence for the existence of enzymatic variants of beta-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria.

A comparison of rat brain and liver β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has revealed that significant differences exist between the enzymes with regard to their kinetic and physical properties. In contrast to the liver enzyme, brain β-hydroxybutyrate dehydrogenase is rapidly inactivated at 46° and is unstable when stored at ?20°. The brain dehydrogenase was found to have a larger K_m (apparent) for the 3-acetylpyridine analog of NAD⁺, and a greater energy of activation in the direction of β-hydroxybutyrate oxidation than the liver enzyme. In the reverse direction, the brain and liver dehydrogenase exhibit substrate inhibition by NADH (0.22 mM and 0.36 mM, respectively). The brain and liver β-hydroxybutyrate dehydrogenase did not differ significantly with regard to the Michaelis-Menten constants measured for NAD⁺ and β-hydroxybutyrate. The K_m constants of brain β-hydroxybutyrate dehydrogenase for acetoacetate (0.39 mM) and NADH (0.05 mM) were lower than those determined for the liver enzyme, acetoacetate (0.73 mM) and NADH (0.35 mM) respectively. These results suggest that the β-hydroxybutyrate dehydrogenase from rat brain and liver are isozymic variants.     

Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)     

The iron-sulfur-cluster-containing L-serine dehydratase from Peptostreptococcus asaccharolyticus. Stereochemistry of the deamination of L-threonine.

The stereochemistry of the deamination of L-threonine to 2-oxobutyrate, catalyzed by purified L-serine dehydratase of Peptostreptococcus asaccharolyticus, was elucidated. For this purpose the enzyme reaction was carried out with unlabelled L-threonine in 2H2O and in 3HOH, as well as with L-[3-3H]threonine in unlabelled water. Isotopically labelled 2-oxobutyrate thus formed was directly reduced in a coupled reaction with L- or D-lactate dehydrogenase and NADH. The (2R)- or (2S)-2-hydroxybutyrate species obtained were then subjected to configurational analyses of their labelled methylene group. The results from 1H-NMR spectroscopy and, after degradation of 2-hydroxybutyrate to propionate, the transcarboxylase assay consistently indicated that the deamination of L-threonine catalyzed by L-serine dehydratase of P. asaccharolyticus proceeds with inversion and retention in a 2:1 ratio. This partial racemization is the first ever to be observed for a reaction catalyzed by serine dehydratase, therefore confirming the distinction of the L-serine dehydratase of P. asaccharolyticus as an iron-sulfur protein from those dehydratases dependent on pyridoxal phosphate. For the latter enzymes exclusively, retention has been reported.     

Biosensor for rapid determination of 3-hydroxybutyrate using bi-enzyme system

A bi-enzyme-based Clark electrode was developed for the determination of 3-hydroxybutyrate. This sensor is based on the specific dehydrogenation by 3-hydroxybutyrate dehydrogenase (HBDH, E.C. 1.1.1.30) in combination with salicylate hydroxylase (SHL E.C. 1.14.13.1). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, HBDH, catalyses the specific dehydrogenation of 3-hydroxybutyrate consuming NAD(+). The products, NADH, initiate the irreversible decarboxylation and the hydroxylation of salicylate by SHL in the presence of oxygen. SHL forces the equilibrium of dehydrogenation of 3-hydroxybutyrate by HBDH to the product side by consuming NADH. Dissolved oxygen acts as an essential material for SHL during its enzymatic reactions. This results in a detectable signal due to the SHL-enzymatic consumptions of dissolved oxygen in the measurement of 3-hydroxybutyrate. Interferences from different amino acids and electroactive substances were found to be minimal due to the specificity of HBDH and the application of a Teflon membrane. The sensor has a fast response (2s) and short recovery time (2 min) with a linear range between 8 and 800 microM 3-hydroxybutyrate and a detection limit of 3.9 microM. A good agreement (R(2)=0.9925) with theoretical calculation was obtained in spiked serum sample measurements.     

Direct imaging of dehydrogenase activity within living cells using enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP)

Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NADH generation capacity within living cardiac myocytes. Quantitative validation of this approach was performed using a dehydrogenase system, in vitro. In intact cells the NADH ED-FRAP was sensitive to temperature (Q(10) of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD(+)) fluorescence. These data, coupled with the cellular patterns of NADH ED-FRAP changes with dehydrogenase stimulation, suggest that NADH ED-FRAP is localized to the mitochondria. These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism.     

Polynucleotide phosphorylase-based photometric assay for inorganic phosphate

Polynucleotide phosphorylase is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of nucleotide diphosphates. By taking advantage of this property, we developed a photometric assay for inorganic phosphate. In the presence of polyadenylic acid, phosphate is converted into adenosine 5'-diphosphate (ADP) by this enzyme. ADP then reacts with phosphoenolpyruvate in a pyruvate kinase-catalyzed reaction, thus giving rise to adenosine 5'-triphosphate and pyruvate. Finally, pyruvate oxidizes reduced nicotinamide adenine dinucleotide (NADH) through the action of L-lactate dehydrogenase, with concomitant decrease in absorbance at 340 nm. As expected, in this detection system 1 mol of NADH was oxidized per mole of phosphate. The assay showed an excellent reproducibility, as the standard deviations never exceeded 5%. It also was shown to be unaffected by several compounds that are regarded as major interferents of the traditional colorimetric assays. Absence of interference was also demonstrated when determining phosphate content in different biological samples, such as human serum and perchloric acid extracts from Escherichia coli, yeast, and bovine liver. An E. coli strain overexpressing His-tagged polynucleotide phosphorylase developed in our laboratories allowed quick and straightforward purification of enzyme, making the assay feasible and convenient. Since all other reagents required are inexpensive, the assay represents a cheaper alternative to commercially available phosphate assay kits.     

A spectrophotometric, enzymatic assay for D-3-hydroxybutyrate that is not dependent on hydrazine

Because of the potential carcinogenic properties of hydrazine and because of other health hazards associated with its use in the laboratory, an enzymatic assay has been developed for D-3-hydroxybutyrate that is not dependent on hydrazine to drive the reaction toward completion. The use of a high concentration of NAD+ and a buffer at pH 9.5 resulted in a favorable conversion of D-3-hydroxybutyrate to acetoacetate by D-3-hydroxybutyrate dehydrogenase even though the reaction favors D-3-hydroxybutyrate formation under physiological conditions. The assay was also completed faster than previous assays using hydrazine so that the amount of enzyme used for the assay could be reduced. The recovery of D-3-hydroxybutyrate added to liver samples was 98 +/- 1% (mean +/- SEM, n = 6). The assay was found to be suitable for the measurement of D-3-hydroxybutyrate in samples such as perchloric acid extracts of isolated hepatocytes even when the acetoacetate to D-3-hydroxybutyrate ratio was 4 to 1. This assay presents a reliable alternative to the use of hydrazine and may be used for the assay of D-3-hydroxybutyrate in a variety of physiological and experimental samples.     

Decreased rate of ketone-body oxidation and decreased activity of D-3-hydroxybutyrate dehydrogenase and succinyl-CoA:3-oxo-acid CoA-transferase in heart mitochondria of diabetic rats.

Heart mitochondria from chronically diabetic rats ('diabetic mitochondria'), in metabolic State 3, oxidized 3-hydroxybutyrate and acetoacetate at a relatively slow rate, as compared with mitochondria from normal rats ('normal mitochondria'). No significant differences were observed, however, with pyruvate or L-glutamate plus L-malate as substrates. Diabetic mitochondria also showed decreased 3-hydroxybutyrate dehydrogenase and succinyl-CoA: 3-oxoacid CoA-transferase activities, but cytochrome content and NADH-dehydrogenase, succinate dehydrogenase, cytochrome oxidase and acetoacetyl-CoA thiolase activities proved normal. The decrease of 3-hydroxybutyrate dehydrogenase activity was observed in diabetic mitochondria subjected to different disruption procedures, namely freeze-thawing, sonication or hypoosmotic treatment, between pH 7.5 and 8.5, at temperatures in the range 6-36 degrees C, and in the presence of L-cysteine. Determination of the kinetic parameters of the enzyme reaction in diabetic mitochondria revealed diminution of maximal velocity (Vmax) as its outstanding feature. The decrease in 3-hydroxybutyrate dehydrogenase in diabetic mitochondria was a slow-developing effect, which reached full expression 2-3 months after the onset of diabetes; 1 week after onset, no significant difference between enzyme activity in diabetic and normal mitochondria could be established. Insulin administration to chronically diabetic rats for 2 weeks resulted in limited recovery of enzyme activity. G.l.c. analysis of fatty acid composition and measurement of diphenylhexatriene fluorescence anisotropy failed to reveal significant differences between diabetic and normal mitochondria. The Arrhenius-plot characteristics for 3-hydroxybutyrate dehydrogenase in membranes of diabetic and normal mitochondria were similar. It is assumed that the variation of the assayed enzymes in diabetic mitochondria results from a slow adaptation to the metabolic conditions resulting from diabetes, rather than to insulin deficiency itself.     

A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with alpha-ketoglutarate results in formation of alpha-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between alpha-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (alpha-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure.     

Tolerance to, and uptake and degradation of 2,4,6-trinitrotoluene (TNT) are enhanced by the expression of a bacterial nitroreductase gene in Arabidopsis thaliana

Arabidopsis thaliana was transformed with a gene encoding a nitroreductase (NTR, E.C.1.6.99.7) with activity against a wide range of nitroaromatic compounds. The gene was transferred from Escherichia coli by an Agrobacterium-mediated in planta method. The obtained seeds were sowed to produce T1 plants, and they were assayed for the integration of the transgene in the plant genome. Transgenic plants that were positive with the PCR analysis were self-pollinated to produce T2 generation plants. Seven lines obtained were assayed for the NTR activity. While the non-transformed wild-type plants showed no detectable NTR activity, the enzyme activity of the transgenic plant lines was approx. 20 times higher. Using the line with the highest NTR activity, the phytoremediation characteristics of plants against 2,4,6-trinitrotoluene (TNT) was investigated. While the wild-type plants did not grow in the presence of 0.1 mM TNT, the transgenic plants grew almost normally in this condition. The uptake of TNT by seedlings of transgenic plants increased by 7 to 8 times when theywere floated on TNT solution. HPLC analysis showed that the peak due to TNT taken upinto plant body was much smaller in the transgenic plants as compared with that of the wild type, and that a number of peaks attributable to the degradation products of TNT, including 4-amino-2,6-dinitrotoluene, were detected in the extract from the transgenic plants. This indicates that the expression of bacterial NTR improved the capability of plants to degrade TNT.     

Investigations into the pathway of electron transport to the nitrogenase from nodule bacteroids

Summary A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N₂ fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H₂ to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein. Evidence is available indicating that nodule bacteroids contain flavoproteins analogous to Azotobacter, azotoflavin, and spinach ferredoxin-NADP reductase. It is concluded that physiologically important systems involved in transport of electrons from dehydrogenases to nitrogenase in bacteroids very likely will include relatively specific electron transport proteins such as bacteroid non-heme iron protein and a flavoprotein from bacteroids that is analogous to azotoflavin.     

Inhibition of lipogenesis by halothane in isolated rat liver cells.

1. Halothane at clinically effective concentrations [2.5 and 4% (v/v) of the gas phase of the incubation flask] was found to inhibit significantly lipogenesis from endogenous substrates, e.g., glycogen, or from added lactate plus pyruvate. This was accompanied by a decrease in the ratio of the free [NAD+]/[NADH] of the mitochondrion and the cytoplasm, as shown by the [3-hydroxybutyrate]/[acetoacetate] ratio and the [lactate]/[pyruvate] ratio. 2. Acetoacetate or pyruvate decreased the inhibitory effect of halothane and restored lipogenesis to control rates. They were reduced rapidly by 3-hydroxybutyrate dehydrogenase or lactate dehydrogenase respectively, with the concomitant oxidation of NADH and the generation of NAD+. 3. These results suggest that the mechanism by which halothane inhibits lipogenesis from glycogen or lactate is by inhibition of the oxidation of NADH; this results in inhibition of flux of carbon through pyruvate dehydrogenase and a shortage of acetyl-CoA for fatty acid synthesis. Thus when NADH acceptors are added in the presence of halothane, the concentration of mitochondrial NAD+ is raised so that the flux of carbon through pyruvate dehydrogenase increases and lipogenesis is restored.     

A modification of isocitrate and malate dehydrogenase assays for use in crude cell free extracts

A modification of the assays for isocitrate and malate dehydrogenase, using phenazine methosulphate and 2,6-dichlorophenolindophenol, permits measurements on cell-free extracts. Phenazine methosulfate at concentrations higher than 30 nmoles/3 ml prevents the accumulation of NADPH or NADH and thus reduces errors due to endogenous oxidation of these compounds. The use of 2,6-dichlorophenolindophenol rather than a tetrazolium salt as the terminal electron acceptor allows continuous spectrophotometric measurement of enzyme activities.Assay for NADP-specific isocitrate dehydrogenase can be performed in aerobic or anaerobic conditions. Assays for malate dehydrogenase should be run under anaerobic conditions because of the interference by oxygen on the phenazine methosulfate mediated reduction of 2,6-dichlorophenolindophenol by NADH. Under anaerobic conditions, where NADH oxidase is inoperative, the phenazine methosulfate/dichlorophenolindophenol assay is more sensitive than the assay using direct measurement of NADH at 340 nm.     

The effects of Mg2+ on certain steps in the mechanisms of the dehydrogenase and esterase reactions catalysed by sheep liver aldehyde dehydrogenase. Support for the view that dehydrogenase and esterase activities occur at the same site on the enzyme.

Stopped-flow experiments in spectrophotometric and fluorescence modes reveal different aspects of the aldehyde dehydrogenase mechanism. Spectrophotometric experiments show a rapid burst of NADH production whose course is not affected by Mg2+. The slower burst seen in the fluorescence mode is markedly accelerated by Mg2+. It is argued that the fluorescence burst accompanies acyl-enzyme hydrolysis and, therefore, that Mg2+ increases the rate of this process. Experiments on the hydrolysis of p-nitrophenyl propionate indicate that acyl-enzyme hydrolysis is indeed accelerated by Mg2+ and a combination of Mg2+ and NADH. Vmax. values for p-nitrophenyl propionate hydrolysis in the presence of NADH and NADH and Mg2+ agree closely with the specific rates of acyl hydrolysis from the E . NADH . acyl and E . NADH . acyl . Mg2+ complexes seen in the dehydrogenase reaction with propionaldehyde. These observations support the view that esterase and dehydrogenase activities occur at the same site on the enzyme. Other evidence is presented to support this conclusion.     

High-performance liquid chromatography-based methods of enzymatic analysis: electron transport chain activity in mitochondria from human skeletal muscle

This study addresses an application of pyridine nucleotide enzymatic analyses to evaluate the activity of the mitochondrial electron transport chain (reduced nicotinamide adenine dinucleotide (NADH) oxidase) and Complexes I and II in samples of human muscle as small as approximately 10 mg wet weight. Key aspects in this adaptation are the use of high-performance liquid chromatography with fluorescence detection of NADH and use of alamethicin, a channel-forming antibiotic that enables an unrestricted access of substrates into the mitochondrial matrix. The procedure includes disintegration of tissue by Polytron homogenizer, extraction of myosin from myofibrillar fragments by KCl/pyrophosphate to facilitate release of mitochondria, and preparation of fractions of subsarcolemmal and intermyofibrillar mitochondria. Oxidation of NADH or succinate is assayed in the presence of 40 microg/ml alamethicin and the reaction is terminated by H(2)SO(4), which also destroys the remaining NADH. Nicotinamide adenine dinucleotide (NAD) or fumarate concentrations are measured using alcohol dehydrogenase or fumarase plus malic dehydrogenase reactions, respectively. Generation of NADH, assessed in auxiliary reactions in the presence of hydrazine, is strictly proportional to NAD or fumarate content across a concentration range of 1-20 microM. NADH is quantitatively analyzed with a detection limit of 3-5 pmol by HPLC using a reverse-phase Hypersil ODS column connected to a fluorescence detector.     

Studies on the metabolism of glycolyl-CoA

Glycolyl-CoA can be formed during the course of the beta-oxidation by rat liver mitochondria of 4-hydroxybutyrate. The existence of this beta-oxidation has been previously supported by the occurrence of 4-hydroxybutyrate and its beta-oxidation catabolites in urine from patients with 4-hydroxybutyric aciduria, an inborn error of gamma-aminobutyric acid metabolism due to the deficiency of succinic semialdehyde dehydrogenase. The characteristics of the mitochondrial beta-oxidation of 4-hydroxybutyrate were, in rat liver, compared with those of the mitochondrial beta-oxidation of butyrate. The inhibition by malonate of the oxidation of 4-hydroxybutyrate was about twofold weaker than that of oxidation of butyrate, whereas both oxidations were abolished by preincubating the mitochondria with 1 mM valproic acid, a known inhibitor of mitochondrial beta-oxidation. Mitochondria from rat kidney cortex were demonstrated to catalyse, as previously shown for hepatic mitochondria, the carnitine-dependent oxidation of 12-hydroxylauroyl-CoA-omega-Hydroxymonocarboxylyl-CoAs are thus concluded to be precursors of glycolyl-CoA also in rat kidney cortex. In addition, 3-hydroxypyruvate was found to be a precursor of glycolyl-CoA, since it was oxidized by bovine heart pyruvate dehydrogenase with a cofactor requirement similar to that of pyruvate oxidation. Glycolyl-CoA was a substrate of carnitine acetyltransferase (pigeon breast muscle). Pig heart citrate synthase was capable of catalyzing the condensation of glycolyl-CoA with oxaloacetate. The product of this reaction induced low NADH production rates dependent on the addition of porcine heart aconitase and isocitrate dehydrogenase.