Purification of lipoamide dehydrogenase from Ascaris muscle mitochondria and its relationship to NADH:NAD+ transhydrogenase activity

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3) has been isolated from Ascaris suum muscle mitochondria. This activity has been purified to apparent homogeneity from both the pyruvate dehydrogenase complex and from 150,000g mitochondrial supernatants which were devoid of pyruvate dehydrogenase complex activity. The enzymes from both sources exhibited similar kinetic, catalytic, and regulatory properties and appear to be identical as judged by polyacrylamide gel electrophoresis. The native enzyme acts as a dimer, containing 2 mol of FAD, and has a subunit molecular weight of 54,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel chromatography. The enzyme also possesses substantial NADH:NAD⁺ transhydrogenase activity. Heat denaturation and differential solubilization experiments imply that the transhydrogenase activity previously reported is, in fact, associated with the lipoamide dehydrogenase moiety of the Ascaris pyruvate dehydrogenase complex. Whether or not this activity functions physiologically in hydride ion translocation, as previously suggested, remains to be demonstrated.     

Nitroreductase activity of heart lipoamide dehydrogenase.

A novel reaction catalysed by lipoamide dehydrogenase is described. In the presence of NADH, lipoamide dehydrogenase reduces the nitro group of 4-nitropyridine and 4-nitropyridine N-oxide. The elution profiles from a DEAE-cellulose column for the dehydrogenase and nitroreductase activities are identical. Chemical modifications of critical amino acid residues suggest that the two activities share a common catalytic domain. Nitro reduction catalysed by lipoamide dehydrogenase was monitored spectrophotometrically and chromatographically. The major product from the enzymic reduction of 4-nitropyridine was isolated and characterized structurally as NN-bis(pyridinyl)hydroxylamine, which is formed presumably via 4-hydroxyaminopyridine in a four-electron redox reaction.     

Effects of barium on gastric microsomal K+-stimulated para-nitrophenyl phosphatase activity

Ba2+ at low concentrations (1 mM or lower) stimulates the basal and K+-stimulated para-nitrophenyl phosphatase activity associated with the purified microsomal fractions from the oxyntic cells of bullfrog gastric mucosa. However, at higher concentrations Ba2+ acts as a competitive inhibitor of the K+-stimulated phosphatase. Ba2+ alone, in absence of any Mg2+, cannot maintain the enzyme activity; hence Ba2+ can only influence or modulate the Mg2+-dependent phosphatase. The effects are unique for Ba2+ because all other divalent cations such as Ca2+ and Zn2+ act as inhibitors of this enzyme under similar conditions. The data offer a possible biochemical basis for the known pharmacological effects of Ba2+ in gastric acid secretion.     

(Ca + Mg)-stimulated ATPase activity of a rat brain microsomal preparation.

ATPase activity of freshly prepared brain microsomes was stimulated 20% when 0.1 mm CaCl₂ was added in the presence of a “saturating” concentration of MgCl₂ (4 mm). This (Ca + Mg)-stimulated activity declined rapidly on storage. Treatment of the microsomes with 0.12% deoxycholate in 0.15 m KCl, followed by centrifugation and resuspension in sucrose, produced a preparation both stable on storage at ?15 °C and with an increased stimulation in the presence of CaCl₂. SrCl₂ was more effective than CaCl₂, but BaCl₂ was a poor activator. KCl and NaCl stimulated the (Ca + Mg)-ATPase activity by reducing substrate (ATP) inhibition. The K_m for ATP was 0.1 mm, a third that of the Mg-ATPase. CTP, ITP, and GTP could not substitute for ATP, although they were fair substrates for the Mg-ATPase. The energy of activation of the (Ca + Mg)-ATPase was 21 kcal, nearly twice that of the Mg-ATPase. After sucrose density-gradient centrifugation of the microsomal preparation, the (Ca + Mg)-ATPase activity was distributed with the (Na + K)-ATPase and not with the mitochondrial marker succinic dehydrogenase. Studies with ouabain, oligomycin, and azide distinguished the (Ca + Mg)-stimulated ATPase from (Na + K)- and mitochondrial ATPases. Sensitivity to ruthenium red suggested a link to Ca transport, although the microsomal ⁴⁵Ca accumulating system was much more sensitive to the inhibitor than was this ATPase activity.     

Correlation between Ca2+-stimulated adenosine triphosphatase activity and Ca2+ uptake in microsomal fraction of rat submandibular gland

Both the Ca2+-ATPase activity and the Ca2+ uptake in a microsomal fraction of rat submandibular gland were inhibited by the addition of indomethacin in vitro. The decrease of both the Ca2+-ATPase activity and the Ca2+ uptake caused by the drug closely paralleled each other (r = 0.97). The inhibitory manner of indomethacin on Ca2+-ATPase and Ca2+ uptake was noncompetitive for Ca2+. These results suggest that the Ca2+-ATPase in the microsomal fraction of rat submandibular gland is a Ca2+ pump in this tissue.     

Salts- induced oxidase activity of lipoamide dehydrogenase from pig heart.

A weak NADH oxidase activity of lipoamide dehydrogenase at neutral pH is increased as much as 15-fold by the addition of KI or (NH4)2SO4. The addition of NAD+ shifts the optimum pH for the KI-induced oxidase activity from 6.3 to 5.5 without changing the maximum activity. The optimum pH is similarly shifted to 5.6 when sulfhyldryl groups of the enzyme are oxidized in the presence of small amount of cupric ion. The NADH: lipoamide and NADH: p-benzoquinone reductase activities are strongly inhibited by KI but both are increased by the presence of (NH4)2SO4. The known intermediate having a charge-transfer band at 530 nm can be seen upon an addition of NADH to the enzyme in the presence of (NH4)2SO4 but not in the presence of KI. The enzyme flavin is reductase by a stoichiometric amount of NADH when KI is present.     

Effect of phospholipase A2 on Ca2+-stimulated adenosine triphosphatase activity in microsomal fraction of rat submandibular gland

The effect of phospholipase A2 on the Ca2+-ATPase (EC 3.6.1.3) activity in the microsomal fraction of rat submandibular gland was kinetically studied in vitro. The Ca2+-ATPase activity was significantly increased by the treatment with phospholipase A2 in the presence of bovine serum albumin as a scavenger for hydrolyzed products. When the microsomal fraction was incubated with phospholipase A2 in the absence of bovine serum albumin, the Ca2+-ATPase activity was not altered. The Vmax and Km values for both ATP and Ca2+ were increased by the phospholipase A2 treatment, respectively. These results indicated that the activation of Ca2+-ATPase by the phospholipase A2 treatment is due to the increase of Vmax.     

Isolation and characterization of lipoamide dehydrogenase from mackerel dark muscle

• 1.1. Lipoamide dehydrogenase was purified 1500-fold from mackerel dark muscle.
• 2.2. The enzyme was homogeneous as judged by acrylamide gel electrophoresis in the presence and absence of SDS.
• 3.3. Molecular weights of 102,000 and 55,000 were estimated for the native and denatured enzyme, respectively.
• 4.4. Optimal activity for the enzyme was obtained at around pH 5.7 and enhanced with citri acid.
• 5.5. Loss of activity was less than 5% by incubating the enzyme at 70°C for 20 min.
• 6.6. An apparent K_m of 3.1 × 10⁻³ M was obtained for dl-lipoic acid and 1.5 × 10⁻⁵ M for NADH.
• 7.7. The properties of lipoamide dehydrogenase from mackerel dark muscle observed in this investigation were very similar to those reported for the enzyme from other sources.

     

A Ca2+-stimulated ATPase activity in rabbit neutrophil membranes.

An ATPase activity specifically stimulated by micromolar Ca2+ concentrations has been identified in association with rabbit neurophil membranes. These studies provide the basis of further characterization of the Ca2+-ATPase activity with regard to neutrophil function.     

Ca2+-stimulated phosphorylation of muscle glycogen synthase by phosphorylase b kinase.

Phosphorylase b kinase from rabbit muscle phosphorylates glycogen synthase purified from the same tissue. The reaction is markedly stimulated by Ca2+ and results in a decrease in the synthase %I activity. Phosphorylase b kinase action leads to the incorporation of phosphate (0.6 to 0.8 mol/mol of subunit) preferentially into a single cyanogen bromide fragment of synthase (fragment III). Cyclic AMP-independent synthase kinase also shows a specificity for the site(s) contained in fragment III whereas the cyclic AMP-dependent protein kinase exerts a preference for the site(s) located in a distinct cyanogen bromide fragment (fragment II). A Ca2+-stimulated endogenous kinase also results in the phosphorylation of fragment III and can be attributed to the presence of phosphorylase b kinase. The finding of a Ca2+-stimulated phosphorylation of glycogen synthase has important implications for the regulation of glycogen metabolism and particularly those processes thought to be controlled by cytoplasmic Ca2+ concentration.     

Some properties of the Ca2+-stimulated ATPase of a rat liver microsomal fraction

1. The heavy microsomal fraction from rat liver apparently has very little Ca2+-stimulated ATPase activity, although it has an active, ATP-driven Ca2+ accumulation system. 2. The addition of ionophore A23187 to the ATPase assay, to allow continuous Ca2+ recycling during the assay time, reveals the presence of a substantial Ca2+-stimulated ATPase with Vmax. 160 nmol of Pi/10 min per mg of protein and Km for Ca2+ 0.19 microM. 3. The Ca2+-stimulated ATPase, but not the basal Mg2+-stimulated ATPase, is potently inhibited by orthovanadate. Both the Ca2+-stimulated ATPase and the vanadate inhibition are enhanced by the presence of Mg2+. 4. Ca2+-stimulated ATPase activity is not responsive to calmodulin or the calmodulin antagonist trifluoperazine.     

Ca-2+-stimulated membrane phosphorylation and ATPase activity of the human erythrocyte.

1. Human erythrocyte membranes were preincubated with ethyleneglycolbis-(beta-aminoethyl)-N,N' tetraacetate (EGTA) and subsequently labelled for short periods with micromolar concentrations of [8-3-H, gamma-32-P]ATP. Under these conditions, and at temperatures smaller than or equal to 22 degrees C, both ATP hydrolysis and membrane phosphorylation were stimulated by Ca-2+. 2. The properties of the Ca-2+-stimulated ATP hydrolysis and associated phosphorylation of a 150 000 molecular weight protein component, previously described (Knauf, P. A., Proverbio, F. and Hoffman, J. F. (1974) J. Gen. Physiol. 63, 324-336), have been studied. The behavior of the phosphorylated component, ECaP, has properties consistent with its role as a phosphorylated intermediate of Ca-2+-ATPase activity, including: (1) similar dependence of the steady-state level of ECaP and Ca-2+-ATPase on ATP concentration; (2) rapid turnover apparent upon the addition of excess non-radioactive ATP; and (3) good correlation between the steady-state levels of Ca-2+-dependent phosphorylation and Ca-2+-ATPase activity in separate preparations possessing variable specific activity. Addition of excess EGTA to ECaP caused only partial dephosphorylation. Sensitivity of Ca-2+-stimulated ATP hydrolysis and associated phosphorylation to micromolar concentrations of Ca-2+ implicates this activity in the "high-affinity" Ca-2+-pump system of the human erythrocyte (Schatzmann, H. J. (1973) J. Physiol. London 235, 551-569).     

Characterization and immunochemistry of pyruvate dehydrogenase complex of Ascaris muscle

Pyruvate dehydrogenase complex and lipoamide dehydrogenase were purified from muscle of Ascaris lumbricoides var. suum which contains relatively a large amount of the complex. Molecular weights of three constituent enzymes of Ascaris pyruvate dehydrogenase complex were as follows; alpha- and beta-subunits of pyruvate dehydrogenase were 42,000 and 37,000, respectively, lipoate acetyltransferase was 76,000 and lipoamide dehydrogenase was 56,000. Furthermore, two unknown polypeptides having molecular weight of 46,000 and 41,000 were detected. Anti-Ascaris lipoamide dehydrogenase antibody precipitated three constituent enzymes and two unknown polypeptides, suggesting that lipoamide dehydrogenase not only binds tightly to complex, but also two unknown polypeptides bind tightly to complex.     

pH dependent kinetic studies of lipoamide dehydrogenase catalysis.

1. Kinetic studies of lipoamide dehydrogenase and its modified enzymes catalyzing lipoamide oxidoreduction and ancillary reactions at various pH are compared. 2. The asymptotic kinetics of lipoamide oxidoreductions switch between the ping pong and ordered mechanisms by varying pH of the reactions. 3. pH-rate profiles of these reactions are bell-shaped suggesting the participation of 2 ionizable residues with pK values of 6.6 +/- 0.5 and above 8 respectively. 4. The unusually high pK value for the catalytic site histidine is attributed to its involvement in an ion-pair formation. 5. In the absence of the catalytic site histidine, the pH-rate profile for the lipoamide reduction of the photooxidized enzyme is no longer bell-shaped but it is similar to those of the transhydrogenation and NADH-oxidation of the native enzyme. 6. This implies the participation of a low-pK protonated group in these reactions.     

Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography.

1. Lipoamide dehydrogenase NADH: lipoamide oxidoreductase, (EC 1.6.4.3) from pig heart has been separated into two sets of isoenzymes by chromatography on lipoyl- and NAD+-derivatized Sepharose-4B matrices. The first fraction is eluted at 30 mM sodium phosphate buffer (pH 7.2), the other requires a higher ionic strength. The two groups originate from the alpha-ketoglutarate and the pyruvate dehydrogenase complex respectively. 2, Hydrophobic chromatography on a homologous series of alkyl-Sepharoses lead to similar results. The first fraction is eluted with 30 mM phosphate buffer in the case of propyl- and butyl-Sepharose but a high ionic strength is required in the case of an increased chain length (C5--C6). The second fraction is reversibly bound on Sepharose-NC3 and -NC4 but binding becomes irreversible at higher chain lengths. 3. Aminoalkyl-Sepharose behave qualitatively as the alkyl derivatives although elution, particularly in the case of the second fraction, can be realized at lower ionic strength. 4. Matrices which are negatively charged (Sepharose-NCnCOOH, n equal 3--7) have no affinity at pH 7.2. 5. The influence of a neutral polar substituent has been studied by comparing the following matrices: Sepharose-NC6OH, Sepharose-NC6NH2 and Sepharose NC6. Binding of the various isoenzymes is completely reversible in the case of a Sepharose-NC6OH matrix and the elution behaviour is identical to that on propyl- and butyl matrices.