Purification and properties of fructose diphosphate aldolase from Ascaris suum muscle

A fructose diphosphate aldolase has been isolated from ascarid muscle and crystallized by simple column chromatography and an ammonium sulfate fractionation procedure. It was found to be homogeneous on electrophoresis and Sephadex G-200 gel filtration. This enzyme has a fructose diphosphate/fructose 1-phosphate activity ratio close to 40 and specific activity for fructose diphosphate cleavage close to 11. K_m values of ascarid aldolase are 1 × 10⁻⁶m and 2 × 10⁻³m for fructose diphosphate and fructose 1-phosphate, respectively. The enzyme reveals a number of catalytic and molecular properties similar to those found for class I fructose diphosphate aldolases. It has C-terminal functional tyrosine residues, a molecular weight of 155,000, and is inactivated by NaBH₄ in presence of substrate. Data show the presence of two types of subunits in ascarid aldolase; the subunits have different electrophoretic mobilities but similar molecular weights of 40,000. Immunological studies indicate that the antibody-binding sites of the molecules of the rabbit muscle aldolase A or rabbit liver aldolase B are structurally different from those of ascarid aldolase. Hybridization studies show the formation of one middle hybrid form from a binary mixture of the subunits of ascarid and rabbit muscle aldolases. Hybridization between rabbit liver aldolase and ascarid aldolase was not observed. The results indicate that ascarid aldolase is structurally more related to the mammalian aldolase A than to the aldolase B.     

Purification and properties of an acyl CoA transferase from Ascaris suum muscle mitochondria

An acyl CoA transferase has been purified to electrophoretic homogeneity from the soluble compartment of Ascaris suum muscle mitochondria. From SDS-PAGE, isoelectric focusing and molecular exclusion chromatography, homogeneity was confirmed and the enzyme appears to be composed of two similar or identical subunits of apparent mol. wts of 50,000 resulting in an apparent mol. wt of 100,000 for the holoenzyme. The apparent isoelectric point was 5.6 +/- 0.1 by both chromatofocusing columns and slab gel isoelectric focusing. The transferase was relatively specific for the short, straight-chain acyl CoA donors as well as the CoA acceptors, being active on acetyl CoA, propionyl CoA, butyryl CoA, valeryl CoA and hexanoyl CoA as donors to acetate and propionate. Neither succinyl CoA nor succinate were appreciably active as CoA donor or acceptor, respectively. This enzyme cannot serve physiologically to activate succinate for decarboxylation to propionate, but may serve to ensure a supply of propionyl CoA which appears to be required in catalytic amounts for the decarboxylation of succinate.     

Purification of an anticoagulant from the body fluid of Ascaris suum

An anticoagulant has been purified from the body fluid of Ascaris suum by sequential passage through Sephadex G-50, CM-cellulose and Sephadex G-25 columns then treated with 2 M NaCl, passaged through a Sephadex G-25 column, separated from the phosphate buffer by precipitation of the latter with the CaCl2, then passaged through a Sephadex G-10 column in water. In the body fluid of the worm, the anticoagulant is ionically-bound to a carrier substance. The complex can be split by treatment with 2 M NaCl. The molecular weight of the anticoagulant is slightly less than 1400.     

Purification and characterization of phosphoenolpyruvate carboxykinase from the parasitic helminth Ascaris suum

Phosphoenolpyruvate carboxykinase has been purified from homogenates of Ascaris suum muscle strips to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification is a three-step procedure which yields pure enzyme in milligram quantities with good yield. The subunit molecular weight of the Ascaris enzyme is between 75,000 and 80,000. The native molecular weight is 83,000 as determined by gel filtration. The kinetic constants for substrates of the carboxylation reaction were determined and compared to those measured for the avian liver enzyme. From kinetic studies it appears likely that two separate roles for divalent metal ions exist in the catalytic process. Studies conducted with Mn2+ or with micromolar concentrations of Mn2+, in the presence of millimolar concentrations of Mg2+ suggest that Mn2+ but not Mg2+ binds directly to and activates the enzyme while either Mn2+ or Mg2+ may bind to the nucleotide resulting in the metal-nucleotide complex. The metal-nucleotide is the active form of the substrate for the reaction. In the presence of Mg2+, an increase in the Mn2+ concentration results in a decrease in the Km for P-enolpyruvate suggesting a direct role for Mn2+ stimulation and regulation of activity. The concentrations of Mn2+ and Mg2+ in Ascaris muscle strips were determined by atomic absorption spectroscopy and support the proposed hypothesis of a specific Mn2+ activation of the enzyme. The nucleotides ATP and ITP act as competitive inhibitors against GTP with KI values of 0.50 and 0.75 mM, respectively. ITP is a competitive inhibitor against both IDP and P-enolpyruvate, suggesting overlapping binding sites for the two substrates on the enzyme.     

Purification and properties of cyclic-3',5'-GMP-dependent protein kinase from the nematode Ascaris suum

A cyclic-3',5'-GMP-dependent protein kinase was purified 7400-fold from the reproductive tract of female ascarids to a specific activity of 718 nmol min-1 mg-1 (histone as substrate). The yield of the preparation was 25%. The enzyme protein obtained was homogeneous as judged by isoelectrofocusing and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme behaved as a dimer of two 82-kDa subunits in gel permeation chromatography on Superose 12. The protein kinase was inactive in the absence of cyclic purine nucleotides. Half-maximum velocity was obtained in the presence of 18 nM cGMP, whereas 400-fold higher concentrations of cAMP were required for the same activity. The enzyme underwent autophosphorylation in first-order kinetics (rate constant 0.054 min-1), leading to maximum incorporation of 0.96 phosphate per subunit. The autophosphorylation led to a 4-fold increase in Vmax, while the Km remained almost unchanged. In an extract from the reproductive tract, cGMP-stimulated phosphorylation was primarily observed in five proteins (molecular masses of 66, 60, 43, 30, and 25 kDa). These proteins also incorporated phosphate when isolated reproductive tracts were incubated in the presence of [32P]phosphate. The phosphate content in cellular proteins was enhanced when the incubation was performed in the presence of 10(-4) M of either octyl-cAMP or octyl-cGMP. In addition to the proteins mentioned above, however, six more electrophoretic bands containing radioactive phosphate were identified after in situ labeling of reproductive tracts with radioactive phosphate.     

Correlation between hysteresis and allosteric properties for phosphofructokinase from Ascaris suum

The Ascaris suum phosphofructokinase exhibits hysteretic transitions in the time course for fructose 6-phosphate (F6P) phosphorylation in addition to allosteric properties when assayed at pH values below 8. Conditions that enhance hysteretic changes also enhance cooperative interactions and thus there appears to be a link between hysteresis and cooperativity. Initiation of reaction with either F6P or phosphofructokinase results in a pronounced lag, while initiation of the reaction with MgATP results in a burst at pH values below 8. Under conditions in which a lag is evident, increasing the concentration of F6P in the assay decreases the lag, while under conditions where a burst is evident, increasing the concentration of MgATP in the assay decreases the burst. The lag is enzyme-dependent going to a limiting value at high enzyme concentration, while the burst is enzyme-independent. As the pH increases, the Hill coefficient for F6P decreases from a pH-independent value of 3 at low pH to a value of 1 above pH 8. Over the same pH range, the burst rate increases to a point that it is too fast to measure at pH 8 (that is, the time course is linear). Finally, at pH 6.9, the saturation curve for F6P becomes more cooperative with the Hill coefficient equal to 3 above 4 mM MgATP. Data are interpreted in terms of the model suggested for the rabbit skeletal muscle phosphofructokinase (Frieden, C., Gilbert, H. R., and Bock, P.E. (1976) J. Biol. Chem. 251, 5644-5647) in which MgATP binds preferably to an inactive tetrameric enzyme form in which a group with a pK of 6.8 is protonated and F6P binds preferably to the unprotonated active tetrameric form.     

NAD+-malic enzyme. Regulatory properties of the enzyme from Ascaris suum.

The regulatory properties of the NAD-dependent malic enzyme from the mitochondria of Ascaris suum have been studied. The malate saturation curve exhibits sigmoidicity and the degree of this sigmoidicity increases as the pH is increased. Fumarate was the only compound tested that stimulated the enzyme activity, whereas oxalacetate was the most powerful inhibitor. Activation by low levels of fumarate was found to be competitive with malate. It is proposed that this stimulation has physiological significance in controlling the dismutation reaction in the parasite. The branched-chain volatile fatty acid excretion products, tiglate, 2-methylbutanoate, and 2-methylpentanoate, inhibited the enzyme activity and this inhibition was competitive with malate. The Ki values for these compounds are in the physiological range of their concentrations; therefore, it is suggested that they may aid in controlling the malic enzyme activity in vivo. Oxalacetate inhibition of malic enzyme activity was competitive with malate, and the Ki values decreased with an increase in pH. Two alternatives are proposed which could account for the lack of oxalacetate decarboxylation by the ascarid malic enzyme.     

Surface properties of membrane vesicles prepared from muscle cells of Ascaris suum.

To facilitate biochemical, pharmacological, and biophysical studies on the membrane of the body muscle of Ascaris suum, a method for preparing intact vesicles was developed. Vesicles were prepared by incubating a muscle flap preparation with 1 mg/ml collagenase in a saline solution and then washing in saline without enzyme. The vesicles then formed gradually over the next hour as outgrowths of the original surface membrane from the bag region of the muscle. The vesicles were harvested readily by suction using a Pasteur pipette. The structure of the vesicles was examined with the transmission electron microscope. The whole-cell patch-clamp technique showed that the vesicles had a high input resistance and that the membrane was complete. The vesicle membrane was shown to contain Ca-activated Cl channels and gamma-aminobutyric acid-activated Cl channels. The vesicles also were shown to be suitable for fluorescence recovery after photobleaching studies designed to examine lateral and vertical movement of a lipid probe (5-N [octadecanoyl]-aminofluorescein) in the membrane. This probe had a mean lateral diffusion coefficient (DL) of 8.1 x 10(-9) cm2/sec, but only a proportion (68.4%) of the probe was mobile. The latter observation illustrated the nonuniform nature of the membrane. Ivermectin (10(-7) M) had no effect on DL or percent recovery. Trypan blue quenching experiments showed that the lipid probe remained in the outer monolayer of the membrane. These observations illustrate the experimental value of the vesicles; they are potentially useful in discerning anthelmintic mode of action and in drug screening.     

Ascaris suum actin: properties and similarities to rabbit actin.

Unlike other enzymes of the aromatic multienzyme system, chorismate synthase and the aromatic complex of $\text{Neurospora crassa}$ were found to bind to a column of cellulose phosphate and to elute at a relatively high concentration of phosphate (～ 0.2 $\text{M}$). The fact that other enzymes with similar ionic properties failed to bind to phosphocellulose suggests that the binding of the former two enzyme systems is due to a specific affinity for phosphate. This conclusion is not only supported by the fact that these same enzymes did not bind to a column of carboxymethyl cellulose, but also is consistent with the nature of the catalytic reactions of the enzymes. Both the shikimate kinase enzyme of the aromatic complex and chorismate synthase would be expected to have active sites which accomodate a phosphate moiety. We anticipate that other enzymes which involve phospho-substrates will also be amendable to this procedure.     

Purification and characterization of acid trehalase from muscle of Ascaris suum (Nematoda)

Acid trehalase (EC 3.2.1.28) was isolated from muscle of Ascaris suum by fractionating with ammonium sulfate, acetone and column chromatography on DEAE-cellulose and phenyl sepharose CL-4B. The purified homogeneous preparation of native acid trehalase exhibited a molecular mass of 76 kDa and of 38 kDa on SDS-PAGE. The enzyme has the optimum pH 4.9, pI 4.3, Km of 6.6 mM and Vmax=34.5 nM min(-1) x mg(-1). Besides trehalose, it hydrolyses sucrose, isomaltose and maltose and, to a lesser degree melezitose, and it does not act on cellobiose and lactose. Acid trehalase was activated by MgCl2, KNO3, NaCl, CaCl2, CH2ICOOH and p-chloromercuribenzoate and inhibited by EDTA, ZnSO4 and FeCl3.     

Purification and properties of the cyclic 3',5'-AMP-binding protein from the muscle of the Nematode Ascaris suum

The cyclic 3',5'-AMP-binding protein was isolated from the muscle of Ascaris suum and purified to apparent homogeneity. It migrated as a protein with a relative Mr 54,000 on electrophoresis under denaturing conditions. On gel filtration columns it was eluted at a volume corresponding to a protein of Mr greater than 200,000 under conditions which kept the cyclic 3',5'-AMP-binding property intact. The purified catalytic subunit of protein kinase from Ascaris and the C subunit of cyclic 3',5'-AMP-dependent protein kinase from bovine heart were inhibited by the cyclic 3',5'-AMP-binding protein. Gel filtration studies indicated the formation of a stable protein complex between the protein kinase and the cyclic 3',5'-AMP-binding protein from Ascaris.     

Molecular and catalytic properties of an arginine kinase from the nematode Ascaris suum

We amplified the cDNA coding for arginine kinase (AK) from the parasitic nematode Ascaris suum, cloned it in pMAL plasmid and expressed the enzyme as a fusion protein with the maltose-binding protein. The whole cDNA was 1260 bp, encoding 400 amino acids, and the recombinant protein had a molecular mass of 45,341 Da. Ascaris suum recombinant AK showed significant activity and strong affinity ( K(m)(Arg) = 0.126 mM) for the substrate L-arginine. It also exhibited high catalytic efficiency ( k(ca)/K(m)(Arg) = 352) comparable with AKs from other organisms. Sequence analysis revealed high amino acid sequence identity between A. suum AK and other nematode AKs, all of which cluster in a phylogenetic tree. However, comparison of gene structures showed that A. suum AK gene intron/exon organization is quite distinct from that of other nematode AKs. Phosphagen kinases (PKs) from certain parasites have been shown to be potential novel drug targets or tools for detection of infection. The characterization of A. suum AK will be useful in the development of strategies for control not only of A. suum but also of related species infecting humans.     

Isoenzymatic pattern and structure of glutamate dehydrogenase from Ascaris suum.

The isoenzymatic pattern of glutamate dehydrogenase (GDH) has been described for Ascaris suum a parasite of Sus scrofa domestica. Only one band of activity has been revealed, suggesting a monomorphic condition for this enzyme. Also, the structure of GDH has been assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Only one subunit was present with a molecular weight of about 55,000. A hexameric structure for GDH of A. suum is suggested.     

Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. II. Purification and characterization of b-type cytochrome and NADH-ferricyanide reductase from Ascaris muscle microsomes.

A b-type cytochrome and NADH-ferricyanide (FC) reductase were solubilized from Ascaris muscle microsomes by detergents and purified by column chromatography. The purified b-type cytochrome displayed absorption bands at 560 (alpha-peak), 525 (beta-peak), and 424 nm (gamma-peak), with a marked shoulder at 555 nm in the reduced from, 415 nm (gamma-peak) in the oxidized form. This absorption spectrum was different from that of rat liver microsomal cytochrome b5. The molecular weight was estimated to be about 100,000 by SDS-polyacrylamide gel electrophoresis, and the absorption spectrum of alkaline pyridine ferrohemochrome suggested that the prosthetic group of this cytochrome is protoheme. The molecular weight of the purified NADH-FC reductase was estimated to be about 55,000 by SDS-polyacrylamide gel electrophoresis. The purified reductase required NADH as a specific electron donor. The reductase efficiently reduced some redox dyes with NADH, but the reduction of cytochrome c was much slower. The purified reductase, like the membrane-bound reductase, was not inhibited by thiol reagents.