Tight binding of oxaloacetate to succinate dehydrogenase

[¹⁴C]Oxaloacetate forms a stable complex with succinate dehydrogenase which withstands repeated Sephadex filtration. Oxidized glutathione, 2-thenoyltrifluoroacetone, KCN and ageing at +4° at neutral pH do not prevent the enzyme to bind oxaloacetate. The binding is prevented by succinate or malonate but the complex, once formed, can not be split by these compounds, although the enzyme activity can be restored; the reconstitutive property of succinate dehydrogenase is, however, irreversibly lost. Bound oxaloacetate does not exchange with added oxaloacetate, but can be released by perchloric acid. Sonic particles of beef heart mitochondria can also bind oxaloacetate. However, this complex can be split by succinate or malonate.     

Kinetics of inhibition of ethanol metabolism in rats and the rate-limiting role of alcohol dehydrogenase

If liver alcohol dehydrogenase were rate-limiting in ethanol metabolism, inhibitors of the enzyme should inhibit the metabolism with the same type of kinetics and the same kinetic constants in vitro and in vivo. Against varied concentrations of ethanol, 4-methylpyrazole is a competitive inhibitor of purified rat liver alcohol dehydrogenase (Kis = 0.11 microM, in 83 mM potassium phosphate and 40 mM KCl buffer, pH 7.3, 37 degrees C) and is competitive in rats (with Kis = 1.4 mumol/kg). Isobutyramide is essentially an uncompetitive inhibitor of purified enzyme (Kii = 0.33 mM) and of metabolism in vivo (Kii = 1.0 mmol/kg). Low concentrations of both inhibitors decreased the rate of metabolism as a direct function of their concentrations. Qualitatively, therefore, alcohol dehydrogenase activity appears to be a major rate-limiting factor in ethanol metabolism. Quantitatively, however, the constants may not agree because of distribution in the animal or metabolism of the inhibitors. At saturating concentrations of inhibitors, ethanol is eliminated by inhibitor-insensitive pathways, at about 10% of the total rate at a dose of ethanol of 10 mmol/kg. Uncompetitive inhibitors of alcohol dehydrogenase should be especially useful for inhibiting the metabolism of alcohols since they are effective even at saturating levels of alcohol, in contrast to competitive inhibitors, whose action is overcome by saturation with alcohol.     

Effect of ATP translocation on citrulline and oxaloacetate synthesis by isolated rat liver mitochondria.

The possibility that the availability of ATP may affect the rate of synthesis of carbamoyl phosphate (measured as citrulline) by carbamoyl phosphate synthase (ammonia) was studied using respiring isolated rat liver mitochondria incubated with added ADP, with hexokinase, glucose, and ATP, or with atractylate, in order to enhance or prevent the efflux of mitochondrial ATP. The effects of these agents were compared with those on oxaloacetate synthesis from pyruvate. Addition of hexokinase, glucose, and ATP to isolated mitochondria resulted in an inhibition of citrulline synthesis which was proportional to the amounts of glucose 6-phosphate formed; under these conditions, matrix ATP and ATP/ADP tended to decrease. The addition of increasing amounts of ADP also resulted in proportional inhibition of citrulline synthesis, but in this case the matrix content of ATP and ADP increased, and ATP/ADP decreased very slightly. In the presence of atractylate, citrulline synthesis was maximal despite a 30% decrease in matrix ATP and ATP/ADP. These effects were observed whether pyruvate, succinate, glutamate, or β-OH-butyrate was used as the respiratory substrate. ADP, the hexokinase system, and atractylate had qualitatively similar but much less pronounced effects on oxaloacetate synthesis from pyruvate. Within the limits of variation observed in these experiments, the rate of synthesis of citrulline appears not to be affected by the matrix content of total ATP, total ADP, or by ATP/ADP. It is affected, however, by the velocity of translocation of ATP into the extramitochondrial medium. These findings suggest that carbamoyl phosphate synthase (ammonia) may be loosely associated with the mitochondrial inner membrane, and may compete for ATP with the ATP-ADP translocator to an extent determined by the extramitochondrial demands for ATP.     

The effect of halides on the equilibrium and reactivity of aspartate aminotransferase.

For the pork heart, extramitochondrial aspartate aminotransferase (EC 2.6.1.1), the “half-reaction” equilibrium, amino acid + phosphopyridoxal enzyme ? keto acid + phosphopyridoxamine enzyme, is displaced in favor of the phosphopyridoxamine enzyme by the addition of halide ions. The order of effectiveness is I^? > Br^? > Cl^? > F^?. A kinetic analysis of this equilibrium with alanine and pyruvate as substrates showed that halide ions (0.01–0.1 m) both increase the rate of the forward reaction and decrease the rate of the reverse reaction. Chloride ions decrease the rate of the reverse reaction by competitively inhibiting the formation of an intermediate enzyme-pyruvate complex. The rate of the forward reaction is proportional to the alanine concentration up to 0.5 m alanine, indicating that the initial combination of alanine with the enzyme is the rate-limiting step in this direction. The activation by anions must therefore involve the initial binding of the substrates to the enzyme. Chloride ions also cause a marked activation of the enzyme in the presence of glutarate by displacing the inhibitory glutarate from the enzyme. These results indicate that some enzyme activations may be due to relieving a preexisting inhibition by ligand substitution reactions. The finding that aspartate aminotransferase has an anion-sensitive “half-reaction” equilibrium, or redox potential, suggests that transaminases may function in both active and passive transport of anions across membranes.     

Purification of the sodium transport enzyme oxaloacetate decarboxylase by affinity chromatography on avidin sepharose

Aspartic acid can be covalently linked to yeast aspartyl-tRNA synthetase and to other proteins, in the absence of tRNA, under conditions where the synthetase activates the amino acid into aspartyl-adenylate, i.e., in the presence of ATP and MgCl₂. The linkage between aspartic acid and the protein is acid and alkali resistant; thus it is likely a peptide-like amide bond formed between the activated carboxylate group of aspartic acid and the primary amine function of the side chain of lysine residues.     

Partial purification and reconstitution of the aspartate transport system from Halobacterium halobium

Membrane vesicles of Halobacterium halobium R1Wrm bind to an aspartic acid-agarose affinity column. After disruption of the bound vesicles by low ionic strength, a protein fraction is eluted from the column with 2.5% cholate in 3 M NaCl. When this fraction is reconstituted with soybean lipids to form proteoliposomes, the proteoliposomes exhibit active aspartate accumulation. Aspartate transport in the reconstituted system is driven by a chemical sodium gradient (out greater than in), exhibits sensitivity to an electrical potential, and is specific for L-aspartate. These characteristics are consistent with observations on aspartate transport in intact membrane vesicles of H. halobium. Initial aspartate transport rates in the reconstituted system are about ninefold enhanced over the native system. The system developed should be useful in future purification schemes and studies of the molecular details of membrane transport.     

Interactions between carbamyl phosphate synthase-I-mitochondrial aspartate aminotransferase and palmitoyl-CoA

Carbamyl phosphate synthase-I and glutamate dehydrogenase both form a complex with mitochondrial aspartate aminotransferase. Instead of these two enzymes competing for the aminotransferase, carbamyl phosphate synthase-I enhances glutamate dehydrogenase-aminotransferase interaction. This suggests that a complex can be formed between all three enzymes. Since this complex is stable in the presence of substrates and modifiers of the three enzymes, it could conceivably convert NH₄⁺ produced from aspartate into carbamyl phosphate. Furthermore, since carbamyl phosphate synthase-I is the predominant protein in liver mitochondria, it could play a major role in placing the aminotransferase and glutamate dehydrogenase in close proximity. Malate removes glutamate dehydrogenase from the tri-enzyme complex and thus could play a role in determining whether glutamate dehydrogenase interacts with carbamyl phosphate synthase-I or is available to participate in reactions with the Krebs cycle. Palmitoyl-CoA has a high affinity for both carbamyl phosphate synthase-I and glutamate dehydrogenase. ATP and malate which, respectively, decrease and enhance binding of palmitoyl-CoA to glutamate dehydrogenase, respectively decrease and enhance the ability of this enzyme to compete with carbamyl phosphate synthase-I for palmitoyl-CoA. Since carbamyl phosphate synthase-I is present in high levels in liver mitochondria and has a high affinity for palmitoyl-CoA, it could play a major role as a reservoir for palmitoyl-CoA.     

Inhibition of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase by glyoxylate

The overall reaction catalyzed by the pyruvate dehydrogenase complex from rat epididymal fat tissue is inhibited by glyoxylate at concentrations greater than 10 μm. The inhibition is competitive with respect to pyruvate; K_i was found to be 80 μm. Qualitatively similar results were observed using pyruvate dehydrogenase from rat liver, kidney, and heart. Glyoxylate also inhibits the pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat, with the inhibition being readily detectable using 50 μm glyoxylate. These effects of glyoxylate are largely reversed by millimolar concentrations of thiols (especially cysteine) because such compounds form relatively stable adducts with glyoxylate. Presumably these inhibitions by low levels of glyoxylate had not been previously observed, because others have used high concentrations of thiols in pyruvate dehydrogenase assays. Since the inhibitory effects are seen with suspected physiological concentrations, it seems likely that glyoxylate partially controls the activity of pyruvate dehydrogenase in vivo.     

The effect of sulfhydryl group reagents on the permeation of mitochondrial aspartate aminotransferase into mitochondria in vitro.

When mitochondria are incubated with radioactively labeled mitochondrial aspartate aminotransferase (EC 2.6.1.1), the enzyme is taken up into the organelles. Mersalyl and p-hydroxymercuriphenyl sulfonic acid, but not N-ethylmaleimide or ethacrynic acid, decrease the extent of this uptake. Inhibition of the uptake by low concentrations of mercurial reagents is due to blockage of a single sulfhydryl group per monomer of the enzyme. Blockage of mitochondrial thiols does not inhibit uptake of the enzyme. A single sulfhydryl group out of a total of six per monomer of the native enzyme reacts with 5,5′-dithiobis-(2-nitrobenzoic acid). This is the same sulfhydryl group that reacts with low levels of mercurial reagents with consequent inhibition of uptake of the enzyme into mitochondria but without effect on the catalytic activity. N-Ethylmaleimide does not react with this group. N-Ethylmaleimide reacts with a different sulfhydryl group with concomitant decrease in enzymic activity but with no effect on uptake of the enzyme into mitochondria. High levels of mercurial reagents similarly decrease enzymic activity. Unlike the effect on uptake into mitochondria, the inhibition by mercurial reagents of enzymic activity is not reversed by treatment with cysteine. The significance of these observations with respect to the mechanism of uptake of aspartate aminotransferase into mitochondria is discussed, and comparisons are made between the reactivities of sulfhydryl groups in rat liver aspartate aminotransferase and in the enzymes from other animals.     

Sensitive ultraviolet monitoring of aldoses in automated borate complex anion-exchange chromatography with 2-cyanoacetamide

A convenient method for postcolumn carbohydrate labeling has been developed. Eluates of borate complex anion-exchange columns are mixed with a reagent solution prepared from an aqueous solution of 2-cyanoacetamide and a borate buffer (pH 10.5), and the mixture is heated in a 10-m reaction coil at 100°C. Measurement of the absorbance of the product at 276 nm permits high reproducibility determination of 5 to 500 nmol of aldoses. Some carbonyl compounds are positive to this reaction, but most do not interfere with the analysis because their peaks do not appear in the aldose region. Ascorbate gives a small peak between those of mannose and fucose, but interference is negligible for equimolar amounts of ascorbate and these aldoses. This method is applied to and gives satisfactory results in the analysis of monosaccharides from various types of glycoconjugates.     

Purification and properties of soybean nodule xanthine dehydrogenase

Xanthine dehydrogenase (EC 1.2.1.37), an essential enzyme for ureide metabolism was purified from the cytosol fraction of soybean nodules. The purified xanthine dehydrogenase was shown to be homogeneous by electrophoresis and a pI of 4.7 was determined by isoelectric focusing. The enzyme had a molecular weight of 285,000 and two subunits of molecular weight 141,000 each. The holoenzyme contained 1.7 (±0.7) mol Mo and 8.1 (±2.0) mol Fe/mol enzyme and the enzyme also contained FMN and is thus a molybdoironflavoprotein. Soybean xanthine dehydrogenase is the second enzyme in plants demonstrated to contain Mo and the first xanthine-oxidizing enzyme reported to contain FMN, rather than FAD as the flavin cofactor.     

New assay for enzymatic phosphate release: application to aspartate transcarbamylase and other enzymes

The colorimetric method for phosphate determination described in the preceding paper is adapted for the assay of orthophosphate liberated in the aspartate transcarbamylase reaction. The method provides for simple, accurate, and sensitive measurement of enzyme activity. The assay uses ammonium molybdate and zinc acetate to form a colored complex with the enzymatically released phosphate; mild conditions which minimize the nonenzymatic background degradation of the substrate, carbamoyl phosphate, are used. Since the assay procedure is relatively rapid, it is especially attractive in situations where results are desired immediately. The method can be used for the assay of any enzyme which releases inorganic phosphate, even in the presence of labile organophosphate compounds.     

Spectrophotometric assay of NADase-catalyzed reactions

A spectrophotometric method for the assay of NADase-catalyzed reactions was developed. The assay consisted of monitoring the decrease in absorbance at 275 nm accompanying the enzyme-catalyzed hydrolysis of ?-NAD. A millimolar extinction coefficient of 0.89 at 275 nm was determined for the hydrolysis of the nicotinamide-ribosidic bond of ?-NAD. Under assay conditions the assay was shown to be linear up to 50% completion. A linear relationship between the rate of ?-NAD hydrolyzed and the amount of NADase added was observed. The K_m and V_max values for Bungarus fasciatus venom NADase-catalyzed hydrolysis of ?-NAD were determined spectrophotometrically and were shown to be the same as those determined by other analytical methods.     

Characterization of ligand-induced states of maize homoserine dehydrogenase

The threonine-sensitive homoserine dehydrogenase (L-homoserine: NAD(P)+ oxido-reductase), isolated from seedlings of Zea mays L., is characterized by variable kinetic and regulatory properties. Previous analysis of this enzyme suggested that it is capable of ligand-mediated interconversions among four kinetically distinct states (S. Krishnaswamy and J. K. Bryan (1983) Arch. Biochem. Biophys. 222, 449-463). These forms of the enzyme have been identified and found to differ in oligomeric configuration and conformation. In the presence of KCl and threonine a rapid equilibrium among three species of the enzyme (B, T, and K) is established. Each of these species can undergo a unique slow transition to a steady-state form under assay conditions. Results obtained from gel-filtration chromatography and sucrose density centrifugation indicate that the B and steady-state forms are tetramers and the T and K states are dimers. Evidence is presented to indicate that the rapid conversion from one dimeric species to the other can only occur via formation of the tetrameric B state. Chromatography under reacting-enzyme conditions provides direct support for the slow formation of a common steady-state species from any one of the other forms of the enzyme. The rate of transition is influenced by threonine, homoserine, NAD+, and, for transitions involving association reactions, by enzyme concentration. Small, reproducible differences in the apparent size of the T and K forms, and the B and steady-state species, are attributed to changes in conformation. This conclusion is supported by differential susceptibility of the enzymic states to proteolytic inactivation, by different rates of inactivation by dithio-bis-nitrobenzoate, and by alterations in their thermal stability. In addition, the B, T, and K states of the enzyme exhibit unique intrinsic fluorescence spectra. Spectral changes are shown to closely parallel changes in kinetic and hysteretic properties of the enzyme. The results of diverse methods of analysis are internally consistent, and provide considerable support for the conclusion that this pleiotropic regulatory enzyme can exist in any of several physically distinct states.     

Differences in the mechanism of NADPH- and cumene hydroperoxide-supported reactions of cytochrome P-450

A study has been carried out on the association of aldolase with the human erythrocyte membrane. It has been shown that the conditions employed during hypotonic hemolysis affect the amount of aldolase that remains bound to the cell membrane. Thus, the in vivo nature of this binding cannot be ascertained by this technique. Therefore, a method has been developed in which aldolase is crosslinked with glutaraldehyde to the inner surface of the membrane in intact red blood cells. Under the specified conditions, over 90% of the intracellular aldolase can be crosslinked to the membrane with less than 10% of the hemoglobin becoming bound. These results suggest that the localization of aldolase in situ is on or near the inner surface of the membrane. The amount of aldolase bound to the membrane following crosslinking can be decreased by preincubating the cells with cytoskeletal agents such as cytochalasin B, colchicine, and vinblastine sulfate. The in vitro binding of aldolase to the purified spectrin-actin and F-actin complexes was studied. Aldolase bound both complexes very tightly (K_D ? 10^?9m) and this binding could be inhibited by cytochalasin B, but not by colchicine. A competition binding study was carried out to determine if the binding of aldolase to F-actin involved specific interactions. Neither bovine serum albumin nor cytochrome c significantly inhibited the binding of aldolase to F-actin when each was present at equimolar concentrations with aldolase. However, glyceraldehyde 3-phosphate dehydrogenase inhibited aldolase binding to F-actin and when present at equimolar concentrations with aldolase completely blocked the association. The association of aldolase and other glycolytic enzymes with the erythrocyte membrane is discussed and it is postulated that aldolase could be localized in vivo on the inner surface of the membrane by attachment to actin or a spectrin-actin complex.     

Regulation of glutamate dehydrogenase by palmitoyl-coenzyme A

Glutamate dehydrogenase is inhibited more by palmitoyl-CoA when the reduced form of triphosphopyridine nucleotide instead of the reduced form of diphosphopyridine nucleotide is the coenzyme. Inhibition is further enhanced by α-ketoglutarate and malate. Thus, for example, in the presence of TPNH plus malate, the amount of palmitoyl-CoA required for 50% inhibition is 10-fold lower (0.03 μm) than previously reported values obtained with reduced diphosphopyridine nucleotide as a coenzyme. Allosteric modifiers such as ATP, GTP, and leucine decrease inhibition of glutamate dehydrogenase by palmitoyl-CoA. Palmitoyl-CoA and ADP are competitive. Thus, the palmitoyl-CoA binding site is apparently in the vicinity of the site of these allosteric modifiers and is probably at the ADP site. The fact that ADP (which has only one site per polypeptide chain) can completely prevent inhibition by palmitoyl-CoA suggests that there is only one kinetically significant palmitoyl-CoA binding site per polypeptide chain. This is consistent with the fact that adding one equivalent of palmitoyl-CoA per polypeptide chain inhibits about 80%. The high affinity of glutamate dehydrogenase for palmitoyl-CoA enables it to successfully compete with other mitochondrial proteins for palmitoyl-CoA.