Ben G. Harris

Studies on enzymes from parasitic helminths: I. Purification and physical properties of malic enzyme from the muscle tissue of Ascaris suum

Abstract 1. 1. A procedure has been developed for the isolation of the mitochondrial malic enzyme ( l -malate: NAD+ oxidoreductase (decarboxylating), EC 1.1.1.39) from muscle tissue of the roundworm Ascaris suum. 2. 2. The enzyme is adjudged to be homogeneous by the criteria of analytical ultracentrifugation, zone electrophoresis, isoelectric focusing and rechromatography. 3. 3. Concomitant with the homogeneity studies the following physical parameters were established: s020,w = 8.9·10−13s; D20,w = 3.6·10−7cm2·s−1; apparent isoelectric pH = 6.63. 4. 4. Sedimentation velocity, sedimentation equilibrium ultracentrifugation, as well as analytical gel filtration, yield a molecular weight of 250 000 for the native enzyme. The enzyme can be dissociated into four identical monomers of molecular weight 64 000. 5. 5. The isolated enzyme utilizes NAD+ more effectively than NADP+ in the decarboxylation of malate. Oxaloacetate is not decarboxylated in the presence of either coenzyme. 6. 6. The enzyme exhibits an absolute requirement for Mn2+ or Mg2+ and is inhibited by (NH4)2SO4. The inhibition is partially reversed by Mn2+.

Expression of Ascaris suum Malic Enzyme in a Mutant Escherichia coli Allows Production of Succinic Acid from Glucose

The malic enzyme gene of Ascaris suum, was cloned into the vector pTRC99a in two forms encoding alternative amino-termini. The resulting plasmids, pMEA1 and pMEA2, were introduced into Escherichia coli NZN111, a strain that is unable to grow fermentatively because of inactivation of the genes encoding pyruvate dissimilation. Induction of pMEA1, which encodes the native animoterminus, gave better overexpression of malic enzyme, approx 12-fold compared to uninduced cells. Under the appropriate culture conditions, expression of malic enzyme allowed the fermentative dissimilation of glucose by NZN111. The major fermentation product formed in induced cultures was succinic acid.

Serotonin (5-hydroxytryptamine): A possible regulator of glycogenolysis in perfused muscle segments of Ascarissuum

Summary When isolated muscle segments from the parasite Ascaris suum were perfused with varying concentrations of serotonin (5-hydroxytryptamine), the level of cyclic AMP increased three-fold. Maximal levels of cyclic AMP were produced by the muscle at serotonin concentrations of 50 μM. Perfusion of saturating levels of serotonin through isolated muscle segments for various time periods resulted in an inactivation of glycogen synthase and an activation of phosphorylase with a concomitant rise in cyclic AMP values. Collectively, the results support the concept that serotonin may be functioning as a regulatory hormone of carbohydrate metabolism in this worm.

Triosephosphate isomerases and aldolases from light- and dark-grown Euglena gracilis.

Abstract Euglena gracilis synthesizes two distinct types of triosephosphate isomerase which can be resolved by isoelectric focusing. The more acidic Type A isomerase (pI = 4.4) predominates when cells are grown photoautotrophically and is localized in the chloroplasts. The Type B isoenzyme exhibits a more basic isoelectric pH (pI = 4.8), predominates under heterotrophic growth conditions and is of cytoplasmic origin. The two isoenzymes exhibit similar molecular weights (56,000–60,000) and catalytic properties but can be distinguished by their pH activity profiles. The situation parallels that of fructose diphosphate aldolase where a chloroplastic Class I enzyme (pI = 4.6, M r 120,000) found in autotrophically grown cells can be resolved from the cytoplasmic Class II (pI = 5.7, M r 88,000) enzyme which predominates under heterotrophic conditions. Inhibition of chloroplastic 70S ribosomal synthesis by chloramphenicol blocks the formation of the Type A triosephosphate isomerase and the Class I aldolase.

Studies on enzymes from parasitic helminths: II. Purification and properties of malic enzyme from the tapeworm, Hymenolepis diminuta☆

Abstract A procedure for the isolation of malic enzyme (EC 1.1.1.40) from the rat tapeworm, Hymenolepis diminuta , is described. The 320-fold purified enzyme exhibits a specific activity of approximately 30 units/mg. Homogeneity of the preparation was adjudged by zone electrophoresis, analytical ultracentrifugation, and chromatography. Molecular weight determinations by sedimentation equilibrium ultracentrifugation and gel filtration yield a value of approximately 120,000. The purified enzyme utilizes NADP as a coenzyme, has the ability to decarboxylate oxalacetate at pH 7.5, and does not require addition of metal ions to the assay mixture. The enzyme is localized in the mitochondria and the physiological significance of this subcellular distribution is discussed.

GLYCOGEN METABOLIZING ENZYMES DURING STARVATION AND FEEDING OF ASCARIS SUUM MAINTAINED IN A PERFUSION CHAMBER

The glycogen content of muscle was correlated with the activity of glycogen synthase and glycogen phosphorylase from the parasitic roundworm Ascaris suum maintained in vitro. Adult female worms were maintained in the laboratory in a perfusion system during periods of starvation and feeding. During starvation, the levels of glucogen decreased at a rate of 0.1 to 0.2 mumoles/min/g wet weight of muscle-cuticle. During this time, 95% of the glycogen synthase (E.C. 2.4.1.11) was in the active D-form, and 48% of the phosphorylase (E.C. 2.4.1.1) was in the active a-form. Upon feeding, the rate of incorporation of glycosyl residues into glycogen proceeded at a rate of 0.75 to 1.0 mumoles/min/g muscle-cuticle. Glycogen synthase was 22% in the active I-form and phosphorylase a-levels remained virtually unchanged at 41% as compared with the starved worm. Total levels of both enzymes remained constant over the starvation-feeding period with 3.9 units/g phosphorylase and 0.4 units/g glycogen synthase. The apparent Km value for the substrate UDPG for glycogen synthase was 0.22 +/- 0.02 mM. For glycogen phosphorylase the Km value for G-1-P was 1.76 +/- 0.38 mM.

Reversible activation and inactivation of phosphofructokinase from Ascaris suum by the action of tissue-homologous protein phosphorylating and dephosphorylating enzymes

In the presence of ATP-Mg2+, purified phosphofructokinase from Ascaris suum muscle was effectively phosphorylated and activated in vitro by a protein kinase purified from the same tissue. Both effects were reversed by the action of a purified protein phosphatase from the same tissue. The findings suggest the presence of a highly potent interconversion mechanism for phosphofructokinase in the muscle of the parasitic nematode.

Crystallographic Studies on Ascaris suum NAD-Malic Enzyme Bound to Reduced Cofactor and Identification of an Effector Site

The crystal structure of the mitochondrial NAD-malic enzyme from Ascaris suum, in a quaternary complex with NADH, tartronate, and magnesium has been determined to 2.0-Å resolution. The structure closely resembles the previously determined structure of the same enzyme in binary complex with NAD. However, a significant difference is observed within the coenzyme-binding pocket of the active site with the nicotinamide ring of NADH molecule rotating by 198° over the C-1–N-1 bond into the active site without causing significant movement of the other catalytic residues. The implications of this conformational change in the nicotinamide ring to the catalytic mechanism are discussed. The structure also reveals a binding pocket for the divalent metal ion in the active site and a binding site for tartronate located in a highly positively charged environment within the subunit interface that is distinct from the active site. The tartronate binding site, presumably an allosteric site for the activator fumarate, shows striking similarities and differences with the activator site of the human NAD-malic enzyme that has been reported recently. Thus, the structure provides additional insights into the catalytic as well as the allosteric mechanisms of the enzyme.

Purification of malic enzyme from Ascaris suum using NAD+-agarose

Malic enzyme has been purified from Ascaris suum by polyethylene glycol precipitation, ion-exchange chromatography, ammonium sulfate precipitation, and NAD-agarose affinity chromatography to a specific activity of 80 units/mg (V/[E]t = 350 s-1). The preparation was shown to be homogeneous by SDS polyacrylamide disc gel electrophoresis. The procedure can be accomplished in a maximum of four days with a 74% yield.

Studies on enzymes from parasitic helminths—III. Purification and properties of lactate dehydrogenase from the tapeworm,Hymenolepis diminuta

Abstract 1. A procedure for the purification of lactate dehydrogenase (E.C. 1.1.1.27) from the tapeworm,Hymenolepis diminuta, is described. 2. The 128-fold purified enzyme exhibits a specific activity of 106 units/mg of protein and was adjudged to be homogeneous by rechromatography, sedimentation velocity and sedimentation equilibrium ultracentrifugation, and a variety of electrophoretic techniques. 3. Only one form of the enzyme is present in the tapeworm as demonstrated by electrophoresis on cellulose acetate, polyacrylamide-gel and isoelectric focusing. 4. The molecular weight of the native enzyme is 141,000 and exhibits a sedimentation coefficient of 6·89 × 10-13 sec. The enzyme is a tetramer composed of subunits of molecular weight 36,000. However, under low protein concentrations (0·1 mg/ml) on analytical gel filtration, the enzyme dissociates to the dimeric state and exhibits a molecular weight of 75,000. 5. Inhibitor studies are consistent with an active center which may be catalytically and structurally similar to the enzyme from vertebrates. 6. The enzyme is kinetically similar to vertebrate heart LDH. The Km for pyruvate was 0·17 mM and for lactate was 6·3 mM. TheH. diminuta lactate dehydrogenase also forms an abortive ternary complex with coenzyme and substrate. 7. The data are discussed in regard to the physiology of the parasite and the host.