High Concentrations Of Malate Research Articles

Volatile aroma components of Australian port wines

AbstractThe volatile aroma compositions of a blended port wine, having an equivalent age of 20 years, and a 100‐year‐old port wine were examined by gas chromatography and gas chromatography‐mass spectrometry. The compounds identified were products of yeast fermentation, acetalisation, esterification and oxidation reactions occurring during storage, carbohydrate degradation (of residual grape sugar and wood hemi‐celluloses) and wood extraction. High concentrations of ethyl lactate, diethyl succinate and diethyl malate were found relative to quantities present in young wine. 1‐Ethoxy‐1‐(3‐methylbutoxy)ethane, 1‐ethoxy‐1‐(2‐methylbutoxy)ethane, 1,1‐diethoxypropan‐2‐one, cis‐5‐hydroxy‐2‐methyl‐1,3‐dioxane and cis‐4‐hydroxymethyl‐2‐methyl‐1,3‐dioxolane were identified. The presence of these and other acetals demonstrated that carbonyl compounds were also present. Carbonyl compounds are likely to contribute to the ‘rancio’ flavour of these port wines. Carbohydrate‐derived compounds found included furfural, 2‐acetylfuran, 5‐methylfurfural, ethyl laevulinate, ethyl furoate and 5‐ethoxymethyl furfural. Oak lactone, which is a wood extractive, was also identified and is likely to contribute to the woody aroma distinguishable in these port wines.

Fasciola hepatica: The subcellular distribution and kinetic and electrophoretic properties of malate dehydrogenase

The optimal pH for oxalacetate reduction was 9.0 for the mitochondrial and supernatant enzyme. The optimal pH for malate oxidation was 10 for both fractions. Maximal activity during oxalacetate reduction was 3.26 ± 0.26 μmoles of NADH oxidized/mg of protein/min and 3.02 ± 0.27 for the supernatant and mitochondrial fractions, respectively. Maximal malate oxidation was 0.495 ± 0.08 and 0.72 ± 0.12 (not statistically different) for the supernatant and mitochondrial fractions, respectively. High concentrations of malate inhibited activity to a greater extent in the supernatant fraction while oxalacetate inhibited activity to a greater extent in the mitochondrial fraction. The supernatant malate dehydrogenase was more heat stable than the mitochrondrial enzyme. Electrophoresis showed three isoenzymes of malate dehydrogenase in whole homogenates of Fasciola hepatica and all three were represented in the supernatant while only one band was seen in the mitochondrial fraction. The presence of two types of malate dehydrogenase, one in the supernatant with three isoenzymes and one in the mitochondria with one isoenzyme, is proposed.

Malate Dehydrogenase and NAD Malic Enzyme in the Oxidation of Malate by Sweet Potato Mitochondria

Over a range of concentrations from less than 0.1 mm to more than 70 mm, sweet potato root mitochondria display a bimodal substrate saturation isotherm for malate. The high affinity portion of the isotherm has an apparent Km for malate of 0.85 mm and fits a rectangular hyperbolic function. The low affinity portion of the isotherm is sigmoid in character and gives an apparent S(0.5) of 40.6 mm and a Hill number of 3.7.Extracts of sweet potato mitochondria contain both malate dehydrogenase and NAD malic enzyme. The malate dehydrogenase, assayed in the forward direction at pH 7.2, shows typical Michaelis-Menten kinetics with a Km for malate of 0.38 mm. The NAD malic enzyme shows pronounced sigmoidicity in response to malate with a Hill number of 3.5 and an S(0.5) of 41.6 mm.On the basis of the normal kinetics, the Km, and the fact that oxaloacetate production from malate by mitochondria appears most active at low malate concentrations, the high affinity portion of the malate isotherm with mitochondria is attributed to malate dehydrogenase. The low affinity portion of the malate isotherm with mitochondria is thought, on the basis of the similarity of S(0.5) values, the Hill numbers, and the greater production of pyruvate from malate at high malate concentrations, to represent the activity of the NAD malic enzyme.

Cellular metabolite distribution and the control of gluconeogenesis in the perfused isolated rat liver

Glucose production was measured in isolated rat livers perfused with 100 ml of blood-free recirculating medium. The gluconeogenic rate using L-alanine as substrate was only 55% of that obtained with L-lactate. The steady-state concentration of gluconeogenic and tricarboxylic acid cycle intermediates were measured in freeze clamped biopsies. Livers perfused with L-lactate displayed higher concentrations of malate, alpha-glycerophosphate and beta-hydroxybutyrate probably as a result of a higher state of reduction of the nicotinamide system. Hexose-phosphate intermediates were also increased when L-lactate was the substrate. Phosphoenolpyruvate and 3-phosphoglycerate were considerably elevated when L-alanine was the glucose precursor. Livers perfused with L-lactate displayed higher cytosolic concentration of all the tricarboxylic acid cycle intermediates except oxaloacetate while glutamate was slightly and aspartate considerably higher when alanine was the substrate. In the mitochondrial compartment the pattern of distribution tended to be the opposite; that is, livers perfused with L-lactate showed lower concentrations of all the intermediates except alpha-ketoglutarate. The mitochondrial: cytosolic metabolite gradients of all the intermediates whose distribution was studied were higher in livers perfused with L-alanine. The relevance of these findings to the observed differences in the gluconeogenic fluxes are discussed.

Pathways for the oxidation of malate and reduced pyridine nucleotide by wheat mitochondria.

Piericidin A and oxaloacetate have been used as inhibitors to investigate the operation and localisation of NAD+‐linked dehydrogenases and the possible compartmentation of these enzymes within the matrix of the mitochondrion. Mitochondria isolated from etiolated shoots of wheat (Triticum vulgare) were shown to oxidise a variety of NAD+‐linked substrates. Oxaloacetate, when added to rapidly respiring preparations (i.e. state 3) resulted in a large but transient inhibition of the oxidation of both pyruvate and citrate, but had little effect on the rate of oxidation of malate. The products of oxidation of malate, in the absence of added NAD+, were found to be a large quantity of pyruvate together with smaller quantities of oxaloacetate and citrate. Using potassium ferricyanide as a non‐penetrating electron acceptor it was possible to show that the malate oxidation occurred in the matrix. This is consistent with the operation of both the malic enzyme and a low level of malate dehydrogenase within the matrix of the mitochondrion. The low level of inhibition of malate oxidation by oxaloacetate was a function of the concentration of malate and appeared to be of a competitive nature. Measurement of the redox state of the endogenous NAD+ pool showed that under phosphorylatig conditions both malate and citrate reduce approximately 15% of the total coenzyme. The addition of 0.2 mM oxaloacetate to phosphorylating aerobic mitochondria failed to oxidise any of the NAD+ reduced by malate but resulted in a partial oxidation of NAD+ reduced by citrate. The transition from the aerobic to anaerobic state in the presence of malate resulted in a biphasic reduction of the endogenous NAD+, whereas in the presence of citrate it was monophasic. The oxidation of NAD+‐linked substrates, including malate, was found to be only partially inhibited by piercidin A. The piericidin‐A‐resistant pathway was found to be located internally on the basis of experiments using ferricyanide as a non‐penetrating electron acceptor. The first site of phosphorylation was not coupled to oxidation mediated by the piericidin‐A‐resistant pathway. The presence of piericidin A altered the shape of the malate saturation curve. In the absence of the inhibitor it was found to be biphasic; saturating at 60 mM and in its presence to be monophasic saturing at 15 mM. At high concentrations of malate (60 mM) piericidin A caused approximately a 30% inhibition, similar to that found with other NAD+‐linked substrates, while at lower concentrations of malate (15 mM) piericidin A caused a much higher inhibition (80%), which subsequently recovered to the much higher piericidin‐A‐resistant rate observed with higher concentrations of malate. In the presence of piericidin A oxaloacetate caused severe but transient inhibition of malate oxidation, similar in magnitude to that observed during pyruvate oxidation in the absence of piericidin A. It was concluded that the data were consistent with the spatial separation of both the internal malic enzyme and malate dehydrogenase together with the internally located piericidin‐A‐sensitive and resistent electron‐transport pathways.

Purification and properties of cytoplasmic and mitochondrial malate dehydrogenases of Physarum polycephalum.

Two isoenzymes of malate dehydrogenase (MDH) were demonstrated in plasmodia of Physarum polycephalum by polyacrylamide-gel electrophoresis. The more "cathodal" form was uniquely associated with mitochondria (M-MDH) and the other form was found in the soluble cytoplasm (S-MDH). The isoenzymes were separated by acetone fractionation of soluble plasmodial homogenates acidified to pH 5.0. The M-MDH was purified 201-fold by cetylpyridinium chloride treatment, fractionation with ammonium sulfate, gradient elution from sulfoethyl cellulose at pH 6.0, and Sephadex G-100 chromatography. The S-MDH was purified 155-fold by ammonium sulfate fractionation, diethylaminoethyl cellulose chromatography, gradient elution from sulfoethyl cellulose at pH 5.5, and Sephadex G-100 chromatography. The optimal cis-oxalacetate concentrations were 0.35 mM for M-MDH and 0.25 mM for S-MDH, and the optimal pH for both isoenzymes was 7.6 for oxalacetate reduction. The optimal l-malate concentrations were 5 mM for S-MDH and 6 mM for M-MDH, and both isoenzymes exhibited an optimal pH of 10.0 for L-malate oxidation. The Michaelis constants of S-MDH and M-MDH served to discriminate between the isoenzymes. The S-MDH was more heat-stable than the M-MDH. High concentrations of oxalacetate and malate inhibited S-MDH more than M-MDH. The isoenzymes were further distinguished by their utilization of analogues of nicotinamide adenine dinucleotide. Many properties of the Physarum isoenzymes were similar to those of more complex organisms, especially vertebrates.

Histochemical Localization of Some Dehydrogenase Enzymes in the Bull Testis and Epididymis

The distribution and relative activities of succinate, lactate, glucose-6-phosphate, malate, glutamate, and β-hydroxybutyrate dehydrogenases have been histochemically evaluated in the normal adult bull testis and caput epididymis. In the seminiferous tubule, the spermatogonial cells appear to display fairly high levels of activity in most of the enzymes investigated but, except for glutamate and β-hydroxybutyrate dehydrogenases, there was a marked decrease of activity in the older spermatocytes and spermatids. Sertoli cells showed a virtual absence of enzymatic activity. Leydig cells showed very high concentrations of enzymes, particularly glucose-6-phosphate, malate, and lactate dehydrogenase. Other enzymes were not very reactive in these cells, and could not be demonstrated in the remainder of the interstitium. Lactate, glucose-6-phosphate, and succinate dehydrogenase showed fairly strong activity in the epithelial cells of a portion of the caput epididymis. The surrounding muscle cells and connective tissue gave very weak reactions for all enzyme substrates tested. Spermatozoa in both tubular and epididymal lumina showed the presence of some dehydrogenases, all activity being confined to the mid-piece section of these cells.

PURIFICATION AND PROPERTIES OF MALATE DEHYDROGENASE (DECARBOXYLATING) FROM MYCOBACTERIUM 607.

Abstract Malate dehydrogenase (decarboxylating) ( l -malate: NADP oxidoreductase (decarboxylating), EC 1.1.1.40) has been purified 100-fold from Mycobacterium 607, and its properties for oxidative decarboxylation reaction have been studied. The pH optimum was 7.8. Below pH 5.5 the enzyme was unstable. Reaction with NAD was slow. K 3 for NADP was 4·10 −5 M. Bivalent cations such as Mn 2+ , Mg 2+ , Co 2+ or Ni 2+ were essential for activity. The order and extent of effectiveness of these cations depended on the concentration of malate and Ks + . K + activated the reaction, but higher substrate concentrations reduced or inhibitory. This inhibition was reversed completely by GSH and partially by increasing the activity bivalent cation concentration. The involvement of a sulfhydryl group in the enzymic reactions is also suggested by other inhibition studies. The apparent K s for malate at pH 7.4 was 1☆10 −3 M and at 8.2 it was 2.5·10 −3 M. Higher malate concentrations were inhibitory. Raising the pH lowered, and increasing the Mg 2+ concentration abolished, this effect. The cause of substrate inhibition is concluded to be its chelatioon of bivalent cation. Anions also affected the activity. Activity with Cl- was more than that with SO 4 2− and this effect was more marled at higher pH.