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PubMed1983

Ornithine decarboxylation in rat liver nuclei.

1. Decarboxylation of ornithine in rat liver cytosol produces putrescine; on the contrary, no putrescine is formed in nuclei. 2. In nuclei, decarboxylation is not inhibited by alpha-difluoromethylornithine and depends on NADH reoxidation. This shows that decarboxylation occurs after transamination of ornithine. 3. The increase of ornithine transaminase activity may be partially responsible for the enhanced decarboxylation after 1-methyl-3-isobutylxanthine administration. Data obtained in the presence of Ca2+ suggest that modification of nuclear Ca2+ concentration may also be responsible.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Interactions between carbonic anhydrase and some decarboxylating enzymes as studied by a new bioelectrochemical approach.

This work presents the results of a study, carried out by recently developed amperometric bioelectrodes, on the interactions between carbonic anhydrase (CA) and the decarboxylating enzymes arginine decarboxylase (ADC), L-lysine decarboxylase (LDC), and L-ornithine decarboxylase (ODC). These are all pyridoxal-phosphate dependent enzymes and catalyze the decarboxylation reaction of the respective amino acids, to give carbon dioxide and the corresponding diamine (agmatine, cadaverine, and putrescine, respectively). The rate of each decarboxylase catalyzed reaction was measured by monitoring the production of the respective diamine by a plant tissue diamino oxidase (DAO) based bioelectrode. DAO is the enzyme which catalyzes the oxidation of agmatine, cadaverine, and putrescine with the production of NH and H2O2. DAO-based bioelectrodes consist of an amperometric H2O2 electrode, coupled to the biocatalytic membrane formed by a whole plant tissue (lentil cotyledon) containing the enzyme DAO, immobilized on a dialysis membrane by polyazetidine prepolymer (PAP). The bioelectrodes were calibrated and characterized in standard solutions of agmatine, cadaverine, and putrescine. Kinetic studies to measure decarboxylase activity were performed in the presence of different concentrations of ADC, LDC, and ODC, resulting in a lowest detection limit of 10, 25, and 10 U l(-1), respectively. The effect of bovine CA II (bCAII) was evaluated in the presence of 500 U l(-1) of each decarboxylase, showing a marked increase of the rate of the decarboxylation reaction. These results suggest that (i) CA can be used to enhance the performance of decarboxylase-based biosensors, and (ii) it possibly plays further physiological roles, acting synergistically, at specific cellular and subcellular sites, with low-activity decarboxylating enzymes.

Animals↗
PubMed1994

Secondary 18O and primary 13C isotope effects as a probe of transition-state structure for enzymatic decarboxylation of oxalacetate.

A new method for directly measuring 18O isotope effects on decarboxylation reactions has been developed. By running the reaction under high vacuum (10(-5) torr), CO2 leaves the solution before exchange with the oxygens of water to an extent greater than 2%. Thus, the method permits determination of 18O isotope effects with the precision of the isotope ratio mass spectrometer, and without the necessity of resorting to the remote label method and its attendant required syntheses. The method is used to determine 18O isotope effects for decarboxylation of oxalacetate (OAA) by Mg2+, and enzymatically by OAA decarboxylase from Pseudomonas putida; 13C isotope effects are also reported for this enzyme, as well as for decarboxylation of OAA by pyruvate kinase. Initial velocity patterns and pH profiles are reported for the P. putida enzyme, and all available data are used to discuss the kinetic and chemical mechanism of decarboxylation.

Carbon Isotopes↗
PubMed2005

On the mechanism of the palladium(II)-catalyzed decarboxylative olefination of arene carboxylic acids. Crystallographic characterization of non-phosphine palladium(II) intermediates and observation of their stepwise transformation in Heck-like processes.

Mechanistic studies of a palladium-mediated decarboxylative olefination of arene carboxylic acids are presented, providing spectroscopic and, in two instances, crystallographic evidence for intermediates in a proposed stepwise process. Sequentially, the proposed pathway involves carboxyl exchange between palladium(II) bis(trifluoroacetate) and an arene carboxylic acid substrate, rate-determining decarboxylation to form an arylpalladium(II) trifluoroacetate intermediate (containing two trans-disposed S-bound dimethyl sulfoxide ligands in a crystallographically characterized form), then olefin insertion and beta-hydride elimination. Because of the unique mode of generation of the arylpalladium(II) trifluoroacetate intermediate, a species believed to be substantially electron-deficient relative to phosphine-containing arylpalladium(II) complexes previously studied, it has been possible to gain new insights into those steps that are common to the Heck reaction, namely, olefin insertion and beta-hydride elimination. The present results show that there are notable differences in reactivity between arylpalladium(II) intermediates generated by decarboxylative palladation and those produced in conventional Heck reactions. Specifically, we have found that more electron-rich alkenes react preferentially with an arylpalladium(II) trifluoroacetate intermediate formed by decarboxylative palladation, whereas an opposite trend is found in conventional Heck reactions. In addition, we have found that the aralkylpalladium(II) trifluoroacetate intermediates that are formed upon olefin insertion in the present study are stabilized with respect to beta-hydride elimination as compared to the corresponding phosphine-ligated aralkylpalladium(II) complexes. We have also crystallographically characterized an aralkylpalladium(II) trifluoroacetate intermediate derived from arylpalladium(II) insertion into norbornene, and this structure, too, contains an S-bound dimethyl sulfoxide ligand; the ipso-carbon of the transferred aryl group and trifluoroacetate function as the third and fourth ligands in the observed distorted square-planar palladium(II) complex.

Journal Article↗
PubMed1996

Electronic Factors Influencing the Decarboxylation of beta-Keto Acids. A Model Enzyme Study.

A theoretical study of the mechanism of decarboxylation of beta-keto acids is described. A cyclic transition structure was found with essentially complete proton transfer from the carboxylic acid to the beta-carbonyl group. The activation barrier for decarboxylation of formylacetic acid is predicted to be 28.6 kcal/mol (MP4SDTQ/6-31G//MP2/6-31G) while loss of CO(2) from its anion exhibits a barrier of only 20.6 kcal/mol (MP4SDTQ/6-31+G//MP2/6-31+G). Barrier heights of decarboxylation of malonic acid and alpha,alpha-dimethylacetoacetic acid are predicted to be 33.2 and 26.7 kcal/mol, respectively. Model enzyme studies using a thio methyl ester of malonate anion suggests that the role of malonyl-CoA is to afford a polarizable sulfur atom to stabilize the developing enolate anion in the transition structure for decarboxylation. Adjacent positively charged ammonium ions are also observed to stabilize the loss of CO(2) from a carboxylate anion by through-bond Coulombic stabilization of the transition structure.

Journal Article↗
PubMed1997

The effect of surfactants on the rate of decarboxylation of p-aminosalicylic acid in acidic aqueous solutions.

p-Aminosalicylic acid, which exists in four ionic forms and whose decarboxylation is well known, was used as a model drug to investigate the effects of surfactants with different charges on its stability. The greatest reduction in the rate of decarboxylation at 50 degrees C occurred when the charge on the micelles was opposite to that of the charge on the drug or when both the drug and the micelle had a neutral charge. Thus, at the highest surfactant concentration, 3 or 5% (w/v), there was a 59% reduction in the rate at pH 2.68 in the presence of the nonionic surfactant polyoxyethylene 24 monocetyl ether, 69% reduction in the rate at pH 4.88 in the presence of the cationic surfactant hexadecyl trimethylammonium bromide, and a 43% reduction at pH 1.01 in the presence of the anionic surfactant sodium cetyl sulfate. The decrease in the rate of decarboxylation was attributed to the partitioning of the drug into the micelles, which provided a phase for improved stabilization. Partition coefficients and rate constants for decarboxylation of the drug inside the micelles were calculated.

Aminosalicylic Acid↗
PubMed2006

Decarboxylation of 6-nitrobenzisoxazole-3-carboxylate in mixed micelles of zwitterionic and positively charged surfactants.

The rate of decarboxylation of 6-nitrobenzisoxazole-3-carboxylate, NBOC, was determined in micelles of N-hexadecyl-N,N,N-trimethylammonium bromide or chloride (CTAB or CTAC), N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS), N-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (DPS), N-dodecyl-N,N,N-trimethylammonium bromide (DTAB), hexadecylphosphocholine (HPC), and their mixtures. Quantitative analysis of the effect on micelles on the velocity of NBOC decarboxylation allowed the estimation of the rate constants in the micellar pseudophase, k(m), for the pure surfactants and their mixtures. The extent of micellar catalysis for NBOC decarboxylation, expressed as the ratio k(m)/k(w), where k(w) is the rate constant in water, varied from 240 for HPS to 62 for HPC. With HPS or DPS, k(m) decreased linearly with CTAB(C) mole fraction, suggesting ideal mixing. With HPC, k(m) increased to a maximum at a CTAB(C) mole fraction of ca. 0.5 and then decreased at higher CTAB(C). Addition of CTAB(C) to HPC, where the negative charge of the surfactant is close to the hydrophobic core, produces tight ion pairs at the interface and, consequently, decreases interfacial water contents. Interfacial dehydration at the surface in equimolar HPC/CTAB(C) mixtures, and interfacial solubilization site of the substrate, can explain the observed catalytic synergy, since the rate of NBOC decarboxylation increases markedly with the decrease in hydrogen bonding to the carboxylate group.

Journal Article↗
PubMed1987

Role of activation of myocardial branched-chain 2-oxo acid dehydrogenase complex in the regulation of leucine decarboxylation during cardiac work in vitro.

The oxidative decarboxylation of L-[1-14C]leucine was measured in the isolated perfused rat heart under both working and non-working conditions. Stimulation of decarboxylation of the labelled substrate was observed in working hearts as cardiac work was increased, and in Langendorff hearts upon increasing the coronary flow rate. The rate of L-[1-14C]leucine decarboxylation was significantly higher (P less than 0.05) in hearts working against moderate afterload pressure when compared to Langendorff hearts perfused at a matching coronary flow rate. The rate of release of 4-methyl-2-oxo[1-14C]pentanoate to the perfusate was high in Langendorff hearts, and was unaffected by changes in coronary flow. In contrast, perfusate levels of 14C-labelled 4-methyl-2-oxopentanoate decreased significantly upon the establishment of the working condition (P less than 0.05). These findings suggested an enhancement in the efficiency of the decarboxylation of the 2-oxo acid in response to cardiac work. The amount of branched-chain 2-oxo acid dehydrogenase complex present in the active form was measured in freeze-clamped hearts. Cardiac work resulted in a rapid activation of the complex (P less than 0.02) within 5 min of work when compared to control Langendorff hearts perfused at matching coronary flow rates. To a lesser extent, increasing the coronary flow rate in Langendorff-perfused hearts also led to activation of the enzyme complex. These studies suggest the following: a) L-leucine oxidation in myocardial tissue can be accelerated by exercise as it is in other tissues; b) this regulatory response can be evoked by the contractile activity of the heart itself, independent of contributions by circulating factors or nervous stimuli; and c) regulation of the activity state of the branched-chain 2-oxo acid dehydrogenase complex is involved in the mechanism by which metabolic flux through this pathway is controlled during cardiac work.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1978

Prolyl hydroxylase half reaction: peptidyl prolyl-independent decarboxylation of alpha-ketoglutarate.

Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) is a mixed-function oxygenase that hydroxylates peptidyl proline with the simultaneous and stoichiometric decarboxylation of alpha-ketoglutarate to succinate and CO2. It has been found that highly purified preparations of the enzyme can decarboxylate alpha-ketoglutarate in the absence of a peptidyl proline substrate. The uncoupled decarboxylation proceeds at only a fraction of the rate of the whole reaction and for study requires substrate quantities of the pure enzyme, as well as oxygen, ferrous ion, and ascorbate. No hydroxyproline is formed under these conditions. Immobilized antiserum to prolyl hydroxylase was found to remove both activities from enzyme preparations. However, addition of free antiserum during incubation inhibits only the complete reaction. Poly(L-proline), a specific inhibitor of prolyl hydroxylation, enhances the uncoupled decarboxylation of alpha-ketoglutarate without itself being hydroxylated. All of these findings prove that alpha-ketoglutarate can serve as substrate in the absence of peptidyl proline and is most likely the initial site of attack by oxygen. In the coupled reaction an oxidized form of the keto acid, perhaps a peroxy acid, then attacks prolyl residues in the unhydroxylated substrate.

Animals↗
PubMed1967

CO(2) Metabolism in Corn Roots. I. Kinetics of Carboxylation and Decarboxylation.

The kinetics of (14)CO(2) carboxylation and decarboxylation in corn root tips were determined to ascertain the sequence of product formation and subsequent utilization, and to obtain further evidence to predict the enzymes mediating the carboxylation and decarboxylations. The carboxylation data indicated that the first product was oxaloacetate followed by malate and aspartate. Malate was the first stable product which could be detected. Decarboxylation data indicated that a large fraction of the (14)CO(2) release and turnover of (14)C was accountable for by a decrease in malate: however, essentially all labeled amino acids turned over rapidly and at a greater rate than organic acids. The data generally support the hypothesis that CO(2) fixation in corn root tips is via P-enolpyruvate carboxylase and malic dehydrogenase and that subsequent malate metabolism is for the most part by direct decarboxylation, possibly by the malic enzyme.

Journal Article↗
PubMed1974

Structure and Biosynthesis of Cuticular Lipids: Hydroxylation of Palmitic Acid and Decarboxylation of C(28), C(30), and C(32) Acids in Vicia faba Flowers.

The structure and composition of the cutin monomers from the flower petals of Vicia faba were determined by hydrogenolysis (LiAlH(4)) or deuterolysis (LiAlD(4)) followed by thin layer chromatography and combined gas-liquid chromatography and mass spectrometry. The major components were 10, 16-dihydroxyhexadecanoic acid (79.8%), 9, 16-dihydroxyhexadecanoic acid (4.2%), 16-hydroxyhexadecanoic acid (4.2%), 18-hydroxyoctadecanoic acid (1.6%), and hexadecanoic acid (2.4%). These results show that flower petal cutin is very similar to leaf cutin of V. faba. Developing petals readily incorporated exogenous [1-(14)C]palmitic acid into cutin. Direct conversion of the exogeneous acid into 16-hydroxyhexadecanoic acid, 10, 16-dihydroxy-, and 9, 16-dihydroxyhexadecanoic acid was demonstrated by radio gas-liquid chromatography of their chemical degradation products. About 1% of the exogenous [1-(14)C]palmitic acid was incorporated into C(27), C(29), and C(31)n-alkanes, which were identified by combined gas-liquid chromatography and mass spectrometry as the major components of the hydrocarbons of V. faba flowers. The radioactivity distribution among these three alkanes (C(27), 15%; C(29), 48%; C(31), 38%) was similar to the per cent composition of the alkanes (C(27), 12%; C(29), 43%; C(31), 44%). [1-(14)C]Stearic acid was also incorporated into C(27), C(29), and C(31)n-alkanes in good yield (3%). Trichloroacetate, which has been postulated to be an inhibitor of fatty acid elongation, inhibited the conversion of [1-(14)C]stearic acid to alkanes, and the inhibition was greatest for the longer alkanes. Developing flower petals also incorporated exogenous C(28), C(30), and C(32) acids into alkanes in 0.5% to 5% yields. [G-(3)H]n-octacosanoic acid (C(28)) was incorporated into C(27), C(29), and C(31)n-alkanes. [G-(3)H]n-triacontanoic acid (C(30)) was incorporated mainly into C(29) and C(31) alkanes, whereas [9, 10, 11-(3)H]n-dotriacontanoic acid (C(32)) was converted mainly to C(31) alkane. Trichloroacetate inhibited the conversion of the exogenous acids into alkanes with carbon chains longer than the exogenous acid, and at the same time increased the amount of the direct decarboxylation product formed. These results clearly demonstrate direct decarboxylation as well as elongation and decarboxylation of exogenous fatty acids, and thus constitute the most direct evidence thus far obtained for an elongation-decarboxylation mechanism for the biosynthesis of alkanes.

Journal Article↗
PubMed1980

Malate Decarboxylation by Kalanchoë daigremontiana Mitochondria and Its Role in Crassulacean Acid Metabolism.

Mitochondria isolated from Kalanchoë daigremontiana, a Crassulacean acid metabolism plant, decarboxylate added malate to pyruvate at rates of up to 100 micromoles per hour per milligram original chlorophyll in the presence of ADP. Omitting ADP reduces this rate by approximately 50%. Antimycin A inhibits malate decarboxylation and this inhibition could be relieved by addition of aspartate and alpha-ketoglutarate to the mitochondria. Increasing the pH of the external medium inhibited malate decarboxylation; a dramatic decrease in pyruvate production was observed between pH 7.2 and pH 7.4. It is suggested that cytoplasmic pH changes may regulate the contribution of mitochondria to malate decarboxylation in the light in vivo.

Journal Article↗
PubMed1984

Ethanolamine glycerophospholipid formation by decarboxylation of serine glycerophospholipids in myelinating organ cultures of cerebellum.

Serine decarboxylation as a source of glycerophospholipid ethanolamine is known to occur in mammals. However, early investigators failed to demonstrate the pathway in brain. In the present study serine is shown to be decarboxylated to glycerophospholipid ethanolamine in myelinating organ cultures of rat cerebellum up to 32 days in vitro. The pattern of incorporation of L-[3-14C]serine into culture phospholipids strongly suggests a precursor-product relationship between serine glycerophospholipids (SGP) and ethanolamine glycerophospholipids (EGP), with serine label appearing in the ethanolamine moiety of EGP. The time course of labelling was similar for both acid-stable and acid-labile EGP. In contrast DL-[1-14C]serine failed to label EGP significantly due to the loss of serine carbon C1 on decarboxylation. Through the systematic hydrolysis of phospholipids from cerebellar cultures incubated with L-[3-14C], it was clear that in SGP, acid-stable EGP, and acid-labile EGP greater than to 70% of radiolabel resides in the base moiety of each of these molecular species. It is proposed that serine decarboxylation as a source of EGP ethanolamine may be important in the early stages of brain development.

Animals↗
PubMed1996

Antagonistic actions of renal dopamine and 5-hydroxytryptamine: effects of amine precursors on the cell inward transfer and decarboxylation.

1. The present work was designed to examine the interference of L-3,4-dihydroxyphenylalanine (L-DOPA) on the cell inward transport of L-5-hydroxytryptophan (L-5-HTP) and on its decarboxylation by aromatic L-amino acid decarboxylase (AAAD) in rat isolated renal tubules. 2. The accumulation of both L-5-HTP and L-DOPA in renal tubules was found to occur through non-saturable and saturable mechanisms. The kinetics of the saturable component L-5-HTP and L-DOPA uptake in renal tubules were as follows: L-5-HTP, Vmax = 24.9 +/- 4.5 nmol mg-1 protein h-1 and Km = 121 (95% confidence limits: 75, 193) microM (n = 5); L-DOPA, Vmax = 58.0 +/- 4.3 nmol mg-1 protein h-1 and Km = 135 (97, 188) microM (n = 5). When the saturation curve of L-5-HTP tubular uptake was performed in the presence of L-DOPA (250 microM), the maximal rate of accumulation of L-5-HTP in renal tubules was found to be markedly (P < 0.01) reduced (Vmax = 10.5 +/- 1.7 nmol mg-1 protein h-1, n = 4); this was accompanied by a significant (P < 0.05) increase in Km values (325 [199, 531] microM, n = 4). 3. L-DOPA (50 to 2000 microM) was found to produce a concentration-dependent decrease (38% to 91% reduction) in the tubular uptake of 5-HTP; the Ki value (in microM) of L-DOPA for inhibition of L-5-HTP uptake was found to be 29.1 (13.8, 61.5) (n = 6). 4. At the highest concentration tested the organic anion inhibitor, probenecid (10 microM) produced no significant (P = 0.09) changes in L-5-HTP and L-DOPA uptake (18% and 22% reduction, respectively). The organic cation inhibitor, cyanine 863 (1-ethyl-2-[1,4-dimethyl-2-phenyl-6-pyrimidinylidene)methyl]-quino linium) produced a potent inhibitory effect on the tubular uptake of L-5-HTP (Ki = 212 [35, 1289] nM, n = 8), being slightly less effective against L-DOPA uptake (Ki = 903 [584, 1396] nM, n = 5). The cyanine derivatives 1,1-diethyl-2,4-cyanine (decynium 24) and 1,1-diethyl-2,2-cyanine (decynium 22) potently inhibited the tubular uptake of both L-5-HTP (Ki = 100 [49, 204] and 120 [26, 561] nM, n = 4-6, respectively) and L-DOPA (Ki = 100 [40, 290] and 415 [157, 1094] nM, n = 5, respectively). 5. The Vmax and Km values for AAAD using L-DOPA as the substrate (Vmax = 479.9 +/- 74.0 nmol mg-1 protein h-1; Km = 2380 [1630, 3476] microM; n = 4) were both found to be significantly (P < 0.01) higher than those observed when using L-5-HTP (Vmax = 81.4 +/- 5.2 nmol mg-1 protein h-1, Km = 97 [87, 107] microM, n = 10). The addition of 5 mM L-DOPA to the incubation medium reduced by 30% (P < 0.02) the maximal rate of decarboxylation of L-5-HTP (Vmax = 56.7 +/- 3.1 nmol mg-1 protein h-1, n = 10) and resulted in a significant (P < 0.05) increase in Km values (249 [228, 270] microM, n = 10). 6. The results presented suggest that L-5-HTP and L-DOPA are using the same transporter (most probably, the organic cation transporter) in order to be taken up into renal tubular cells; L-DOPA exerts a competitive type of inhibition upon the tubular uptake and decarboxylation of L-5-HTP. The decrease in the formation of 5-HT as induced by L-DOPA may also depend on a decrease in the rate of its decarboxylation by AAAD.

5-Hydroxytryptophan↗
PubMed1967

Decarboxylation of alpha-keto acids by Streptococcus lactis var. maltigenes.

Decarboxylation rates for a series of C-3 to C-6 alpha-keto acids were determined in the presence of resting cells and cell-free extracts of Streptococcus lactis var. maltigenes. The C-5 and C-6 acids branched at the penultimate carbon atom were converted most rapidly to the respective aldehydes in the manner described for alpha-carboxylases. Pyruvate and alpha-ketobutyrate did not behave as alpha-carboxylase substrates, in that O(2) was absorbed when they were reacted with resting cells. The same effect with pyruvate was noted in a nonmalty S. lactis, accounting for CO(2) produced by some "homofermentative" streptococci. Mixed substrate reactions indicated that the same enzyme was responsible for decarboxylation of alpha-ketoisocaproate and alpha-ketoisovalerate, but it appeared unlikely that this enzyme was responsible for the decarboxylation of pyruvate. Ultrasonic disruption of cells of the malty culture resulted in an extract inactive for decarboxylation of pyruvate in the absence of ferricyanide. Dialyzed cell-free extracts were inactive against all keto acids and could not be reactivated.

Butyrates↗
PubMed1983

Purification and characterization of a bifunctional L-(+)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) from Rhodopseudomonas sphaeroides Y.

A bifunctional enzyme, L-(+)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and EC 1.1.1. . . , respectively), was discovered in cells of Rhodopseudomonas sphaeroides Y, which accounts for the ability of this organism to grow on L-(+)-malate. The enzyme was purified 110-fold to homogeneity with a yield of 51%. During the course of purification, including ion-exchange chromatography and preparative gel electrophoresis, both enzyme activities appeared to be in association. The ratio of their activities remained almost constant [1:10, L-(+)-tartrate dehydrogenase/D-(+)-malate dehydrogenase (decarboxylating)] throughout all steps of purification. Analysis by polyacrylamide gel electrophoresis revealed the presence of a single protein band, the position of which was coincident with both L-(+)-tartrate dehydrogenase and D-(+)-malate dehydrogenase (decarboxylating) activities. The apparent molecular weight of the enzyme was determined to be 158,000 by gel filtration and 162,000 by ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single polypeptide chain with an estimated molecular weight of 38,500, indicating that the enzyme consisted of four subunits of identical size. The isoelectric point of the enzyme was between pH 5.0 and 5.2. The enzyme catalyzed the NAD-linked oxidation of L-(+)-tartrate as well as the oxidative decarboxylation of D-(+)-malate. For both reactions, the optimal pH was in a range from 8.4 to 9.0. The activation energy of the reaction (delta Ho) was 71.8 kJ/mol for L-(+)-tartrate and 54.6 kJ/mol for D-(+)-malate. NAD was required as a cosubstrate, and optimal activity depended on the presence of both Mn2+ and NH4+ ions. The reactions followed Michaelis-Menten kinetics, and the apparent Km values of the individual reactants were determined to be: L-(+)-tartrate, 2.3 X 10(-3) M; NAD, 2.8 X 10(-4) M; and Mn2+, 1.6 X 10(-5) M with respect to L-(+)-tartrate; and D-(+)-malate, 1.7 X 10(-4) M; NAD, 1.3 X 10(-4); and Mn2+, 1.6 X 10(-5) M with respect to D-(+)-malate. Of a variety of compounds tested, only meso-tartrate, oxaloacetate, and dihydroxyfumarate were effective inhibitors. meso-Tartrate and oxaloacetate caused competitive inhibition, whereas dihydroxyfumarate caused noncompetitive inhibition. The Ki values determined for the inhibitors were, in the above sequence, 1.0, 0.014, and 0.06 mM with respect to L-(+)-tartrate and 0.28, 0.012, and 0.027 mM with respect to D-(+)-malate.

Alcohol Oxidoreductases↗
PubMed1975

Rat brain regional uptake and decarboxylation of L-DOPA following carotid injection.

Using the carotid injection technique, the regional uptake and decarboxylation of L-DOPA at the blood-brain barrier in the rat was studied. After a single intracarotid injection in the rat followed by decapitation 15 S later, the uptake of L-DOPA was measured relative to tritiated water injected simultaneously as a diffusible internal standard. Decarboxylation was investigated with an injection mixture of L-[carboxy-14C]DOPA and L-[2,3-3H]DOPA. Uptake of L-DOPA studied over the range of 15-5,076 nmol/ml appeared to be a composite of two separate mechanisms. The saturable component had a half-maximal velocity transport value, K-t, of 336 muG. A diffusional, nonsaturable component had a diffusion constant of 0.018. A regional study showed that uptake operated at approximately the same rate in the various brain areas despite marked regional variation in catecholamine concentration. The decarboxylation of L-DOPA also occurred at a similar rate in all regions. Even when L-DOPA was injected at a concentration of 3 mM, 33-51% was decarboxylated within the 15-S period. These results support the hypothesis that L-DOPA movement into the brain occures via a neutral amino acid transport mechanism common to cerebral capillaries of different regions of the brain.

Animals↗
PubMed1986

Branched chain alpha-keto acid oxidative decarboxylation in skeletal muscle mitochondria. Effect of isolation procedure and mitochondrial delta pH.

In order to study branched chain alpha-keto acid oxidative decarboxylation in skeletal muscle mitochondria, an improved procedure was developed for isolating muscle mitochondria. The procedure uses the protease Nagase in mannitol sucrose media (Procedure A). These mitochondria exhibited high rates of oxygen consumption, good respiratory control ratios, and improved rates of branched chain alpha-keto acid oxidation. At 20 microM [1-14C]alpha-ketoisovalerate (KIV), rates were 1.99 +/- 0.09 nmol/mg of mitochondrial protein/min versus 0.85 +/- 0.02 in mitochondria prepared in electrolyte media without Nagase treatment (Procedure B). The apparent kinetic constants for KIV and alpha-ketoisocaproate (KIC) oxidation were determined. In the presence of ATP, the Vmax and K0.5 for KIV were 17.7 +/- 2.5 nmol/mg of mitochondrial protein/min and 82 microM, respectively. The K0.5 for KIV was at least 2-fold higher than for KIC as were apparent Vmax values. Branched chain alpha-keto acid oxidative decarboxylation in skeletal muscle mitochondria was compared to the activity in mitochondria isolated from liver, heart, and kidney. Rates of KIV and KIC oxidative decarboxylation were highest in heart mitochondria and quite similar in skeletal muscle, liver, and kidney mitochondria. It is the low mitochondrial content of mixed skeletal muscle, not the specific activity of the branched chain alpha-keto acid dehydrogenase, that limits muscle oxidative capacity. The data also indicate that the total activity in muscle has been routinely underestimated. Addition of ATP which increased the matrix pH (increases delta pH) stimulated the rate of oxidative decarboxylation of branched chain alpha-keto acids. On the other hand, addition of uncoupler which decreased the delta pH inhibited the rate of oxidation. Nigericin in low K+ medium inhibited oxidation to about the same degree as uncoupler, while addition of valinomycin in high K+ medium, which will decrease the electrical potential, had little effect on oxidation rates. Transport of branched chain alpha-keto acids should be sensitive to the mitochondrial pH gradient. Hence, the effects of ATP and the mitochondrial inhibitors on rates of branched chain alpha-keto acid oxidation suggest that mitochondrial transport may be partially rate-controlling for oxidation at physiological concentrations of the branched chain alpha-keto acids.

Animals↗