Search

Acyl-CoA compounds are stable in adult Ascaris suum mitochondrial preparations. However, when incubated in the presence of 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), acetyl-CoA or propionyl-CoA are hydrolyzed to form free coenzyme A. This acetyl-CoA hydrolase activity has been partially purified and found to be specific for the above CoA derivatives. Gel filtration indicates an apparent molecular weight of 232,000. The hydrolase activity has been purified free from acyl-CoA transferase activities and appears not to be accounted for on the basis of a thiolase. Because Ascaris is an intestinal parasite that metabolizes primarily anaerobically and accumulates a large number of volatile fatty acids that are formed as the coenzyme A derivatives, the hydrolase would be expected to function in the regeneration of free CoA. However, how the hydrolase reaction would be pulled in the absence of the nonphysiologic DTNB is not known.

Succinyl-CoA is an intermediate in the formation of the fermentation product, propionate, by Fasciola hepatica adults. Acyl-CoA transferase activities are present in crude homogenates of Fasciola, which could account for the synthesis of succinyl-CoA from succinate by the transfer of CoA from either propionyl-CoA or acetyl-CoA. No transferase activity was apparent from 2-methylbutyryl-CoA or 2-methylvaleryl-CoA as was previously reported for the nematode, Ascaris suum. Heat denaturation experiments indicate that all of the Fasciola transferase activities may result from a single protein.

Drugs that act on calmodulin and protein kinase C (PKC) were investigated in the filariid Acanthocheilonema viteae. The filariid was slit open longitudinally and attached to an isotonic muscle transducer in a warmed (37 C) chamber containing physiologic solution bubbled with 95% N2-5% CO2. The calmodulin inhibitors, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), increased the spontaneous contractions of the parasite at low concentrations and induced a contraction followed by a flaccid paralysis at high concentrations. Trifluoperazine and W-7 also reduced the contractions from acetylcholine (ACh) and KCl in a concentration-dependent manner. The phorbol esters, phorbol 12-myristate 13-acetate and phorbol 12, 13-dibutyrate, which activate PKC, were either inactive or only weakly active at inducing contractions. Staurosporine (10(-6) M), a PKC inhibitor, enhanced and then blocked the spontaneous contractions of the filariid. Two other PKC inhibitors, H-7 (10(-4) M) and sphingosine (3 x 10(-5) M), induced much smaller increases in the spontaneous contractions and did not inhibit them. Staurosporine and sphingosine inhibited the ACh contractions; however, staurosporine only slightly reduced the maximal KCl contraction. These results support a role for calmodulin, but not for PKC, in filarial muscle contraction.

Studies of the muscle phospholipid metabolism of Ascaris suum suggest an effect of cholinergic drugs on the turnover of phosphatidylcholine and the generation of glycerophosphorylcholine (GPC). 31P-nuclear magnetic resonance (NMR) studies of helminths revealed the presence of a major peak that was assigned to GPC. The primary effect of the cholinergic drugs on the parasites' phosphate profile appeared to be on the level of GPC. In in vivo studies, decreases in internal GPC concentrations occurred prior to any decrease in the concentration of ATP. The importance of these studies relies on the correct identity of this major 31P-NMR resonance. More recently, the identity of this resonance as GPC was questioned by experimental data obtained from C. elegans dauer larvae using the NMR technique. Because studies from our laboratory suggested that phospholipid metabolism may be intimately connected with the parasite's responses to drugs, the identity of the assigned resonance in the 31P-NMR spectrum as GPC in Ascaris suum was reexamined and found to be correct. Similar studies with C. elegans indicate the presence of both GPC and GPE.

A branched-chain acyl-CoA transferase activity which transfers coenzyme A from either 2-methylbutyryl or 2-methylvaleryl-CoA to succinate is present in the muscle mitochondria from the intestinal nematode, Ascaris suum. Its physiological function is discussed. This activity appears to differ from the previously described acetyl-CoA: propionate and propionyl-CoA:succinate acyl-CoA transferases on the basis of heat stability, substrate specificity and the requirement of a "factor" from boiled Ascaris mitochondria for optimal activity of only the branched-chain acyl-CoA transferase. The "factor" has been recovered from HPLC and some of its properties examined. It could not be replaced by a crude soluble fraction from rat liver mitochondria, or by adenine, guanine or inosine di- or triphosphates. Activity was lost upon ashing, but was not affected by treatment with either pepsin or chymotrypsin.

The effects of K+ on muscle contractility were explored in the filarial nematode Acanthocheilonema viteae (Dipetalonema viteae). The parasite was slit open longitudinally and mounted in a smooth muscle chamber that was filled with aerated (95% N2-5% CO2) physiological solution at 37 degrees C. KCl at concentrations ranging from 20 to 100 mM induced a rapid isotonic contraction of the filarial muscle. The maximal response from KCl was similar to the maximal response to acetylcholine chloride (ACh). When KCl was applied for several minutes, tolerance frequently occurred. Contractions were also induced by K2SO4 but not by NaCl, Na2SO4 or sucrose. Nifedipine was more than 10 times as potent in reducing the KCl-induced contraction as in reducing that caused by ACh. The KCl-induced contraction was abolished in a Ca-free physiological medium containing ethyleneglycol-bis-(beta-aminoethyl ether) N, N, N', N'-tetraacetic acid (EGTA, 10(-4) M). Low [Ca2+]/[Mg2+] solutions blocked the spontaneous activity, the KCl-induced contractions, and the ACh-induced contractions. KCl also induced contractions in denervated muscle strips, supporting the hypothesis that K+ acts directly on the muscle cells. These results indicate that K+ can depolarize the muscle membrane and induce a muscle contraction that is dependent on extracellular calcium ions.

The incorporation of [methyl-14C]choline into the choline-containing compounds of Ascaris suum muscle and the effects of acetylcholine and its agonists, carbachol and levamisole, on this incorporation were studied. Previous experiments reported a stimulation of phosphatidylcholine (lecithin) metabolism upon the administration of acetylcholine. Acetylcholine administered in vitro to A. suum muscle and body wall preparations resulted in a stimulation of phospholipase C activity that, in turn, produced an increased rate of hydrolysis of phosphatidylcholine to the corresponding diacylglyceride (DAG). The DAG, in turn, may act as a second messenger as it is required for the activation of an A. suum protein kinase C. Evidence presented here is in accordance with this hypothesis. The administration of cholinergics resulted in a stimulation of phosphatidylcholine turnover. Acetylcholine also stimulated isotope incorporation into glycerophosphorylcholine, presumably as a consequence of enhanced phospholipid turnover. These events appear to be associated with the ligand binding to the acetylcholine receptors of the A. suum muscle. Choline kinase activity is suggested in order to maintain the observed high ratio of phosphorylcholine to choline. Findings indicate that in the parasite's muscle phosphatidylcholine metabolism may be linked to receptor-dependent responses and subsequent signal transduction.

Quantitatively, the major phospholipid in the muscle of the nematode Ascaris suum was found to be phosphatidylcholine (lecithin). Stimulation of Ascaris muscle with acetylcholine or the agonists carbachol and levamisole increased the level of phosphorylcholine, 1,2-diacylglycerides and phosphatidic acid. Increased levels of these compounds, together with the demonstration of phospholipase C activity, suggest that phospholipid hydrolysis may be associated with the ACh response of the muscle via second messenger pathways. In other tissues, diacylglycerides and phosphatidic acid have been reported to regulate protein kinase C activity. Protein kinase C activity also was demonstrated in the muscle of Ascaris. For optimal activity the kinase was dependent upon Ca2+, unsaturated 1,2-diacylglyceride and phospholipid. All of the data are in accord with the possible involvement of a second messenger system being operative in the ACh-stimulated contraction of Ascaris muscle.

The propionyl-CoA condensing enzyme which catalyzes the first step in the biosynthesis of 2-methylbutyrate and 2-methylvalerate by Ascaris muscle appears to exist in at least three forms in the mitochondria of this parasitic nematode. Two forms, A and B, were separated by ion exchange chromatography on CM-Sephadex. Chromatography on phosphocellulose resulted in the recovery of one minor peak (I) and two major peaks with activity (II and III). A and B as well as I, II, and III differed in their specific activities. Forms B and III were the most retained by their resins, and were the most active forms of the enzyme in each case. Inhibition studies with metabolites from Ascaris mitochondria indicate that CoASH, a product of the condensation reaction, and acetyl-CoA are effective inhibitors of the condensing and thiolytic activities of the Ascaris enzyme, respectively. Incubation of the active enzyme form B for 2 h with 0.1 mM CoASH at room temperature under nitrogen caused the loss of 92% of the propionyl-CoA condensing activity and 67% of the thiolase activity when assayed in standard mixtures. The propionyl-CoA condensing enzyme exhibited a hyperbolic dependence of the condensation velocity to changes in substrate concentration. However, in the presence of CoASH the Michaelis-Menten kinetics was transformed into a sigmoidal kinetics indicating a deviation from a simple product inhibition. Inactivation of the most active forms of the enzyme with CoASH was accompanied by (a) a change in the chemical reactivity of the protein toward p-chloromurcuribenzoate, (b) a change in the uv-visible spectrum of the protein, and (c) a change in the elution patterns from both CM-Sephadex and phosphocellulose column chromatography, where-upon one, two, or more protein peaks were obtained. The several protein peaks resolved by rechromatography of the [14C]CoASH-inactivated enzyme III on phosphocellulose had different CoASH contents. The elution positions were correlated with the less active forms (I and II) having increased [14C]CoASH activities. Similarly, the two peaks isolated upon rechromatography of the CoASH-partially inactivated enzyme B on CM-Sephadex had different isotope contents and the elution position of enzyme A corresponded to the less active form. The results described indicate that the CoASH modification of Ascaris propionyl-CoA condensing enzyme may be responsible for the existence of several forms of the enzyme and might represent a mode of control by chemically modulating the amount of the active forms of the enzyme.

The condensation of two propionyl-CoA units or a propionyl-CoA with acetyl-CoA is required for the synthesis of 2-methylvalerate or 2-methylbutyrate, respectively, two of the major fermentation products of Ascaris anaerobic muscle metabolism. An enzyme that preferentially catalyzes the condensation of propionyl-CoA rather than acetyl-CoA has been purified from the mitochondria of the parasitic intestinal nematode Ascaris lumbricoides var. suum. The purified enzyme is over 10 times more active with propionyl-CoA than with acetyl-CoA as substrate. It also catalyzes the coenzyme A-dependent hydrolysis of acetoacetyl-CoA at a rate four times higher than the propionyl-CoA condensation reaction. The purified Ascaris condensing enzyme preferentially forms the 2-methyl-branched-chain keto acids rather than the corresponding straight chain compounds. The native molecular weight of the purified enzyme was estimated to be 160,000 by gel filtration chromatography and 158,000 by high pressure liquid chromatography. The enzyme migrated as a single protein band with Mr 40,000 during sodium dodecyl sulfate-polyacrylamide electrophoresis, indicating that the enzyme is composed of four subunits of the same molecular weight. Chromatography on CM-sephadex resulted in the isolation of two separate peaks of activity, designated as A and B. Both A and B had the same molecular weight and subunit composition. However, they differed in their specific activities and isoelectric points. The pIs of condensing enzymes A and B were 7.6 and 8.4, respectively. Propionyl-CoA was the best substrate for the condensation reaction with both enzymes. However, the specific activity of enzyme B for both propionyl-CoA condensation (3.4 mumol/min/mg protein) and acetoacetyl-CoA thiolysis (13.8 mumol/min/mg protein) was 2.4 times higher than that obtained with enzyme A. Similarly, chromatography on phosphocellulose resolved the Ascaris condensing enzyme activity into one minor and two major peaks. All of these components had the same molecular weight and subunit composition, but differed in their specific activities. The two major phosphocellulose peaks cross-reacted immunologically when examined by the Ouchterlony double immunodiffusion technique. In addition, antiserum against the phosphocellulose most active form cross-reacted with forms A and B isolated by chromatography of the enzyme on CM-Sephadex, indicating that all forms were immunochemically related.

1. Isotonic contractions were recorded from the filarial nematode, Dipetalonema viteae (Acanthocheilonema viteae), in an isolated tissue chamber. 2. Nicotine (10(-6) M) and pilocarpine (10(-5) M) increased the spontaneous contractions in the intact filariid, but acetylcholine (ACh, 10(-4) M) and muscarine (10(-5) M) were inactive. 3. When ACh was applied to an opened D. viteae, it was 10,000 times more potent. This indicates that the cuticle is an effective barrier to the penetration of ACh to the muscle cells. 4. The effects of ACh on the opened D. viteae were not affected by hexamethonium (10(-3) M) or atropine (10(-5) M) and were only partially reduced by (+)-tubocurarine (10(-4) M). 5. gamma-Aminobutyric acid (GABA, 10(-3) M) reduced the spontaneous activity of the intact D. viteae; however, the effect of GABA had a slow onset and recovery. Muscimol (10(-5) M) was more potent than GABA and had a more rapid onset and recovery. 6. GABA was 1,000 times more potent on the opened D. viteae than on the intact D. viteae. Baclofen (10(-3) M) was inactive on both preparations. 7. The effect of GABA was not antagonized by bicuculline (10(-4) M), picrotoxin (10(-5) M or penicillin G (10(-3) M). 8. It is concluded that the filariid cuticle acts like a lipid structure and blocks the penetration of polar substances, such as ACh and GABA. Also, due to the lack of efficacy of the ACh and GABA antagonists, it was concluded that the nematode receptors are somewhat different from the mammalian ACh and GABA receptors.

Radioisotopically labeled glucose and pyruvate were employed to elucidate biochemical mechanisms utilized by the filariid Dipetalonema viteae during cultivation. Adults isolated from amicrofilaremic hamsters were incubated at 37 C in a mixture of NCTC135:IMDM (NI), with either D-[14C-(U)]glucose or [1-14C]pyruvate, under a gas phase of 5% CO2/N2 for 3 days. Labeled organic acids were separated and quantified by ion exchange chromatography. High performance liquid chromatography (HPLC) was used for separation and quantification of the 23 free amino acids in the NI medium. Ion exchange chromatography revealed that lactate was the major glycolytic end product, accounting for 90-97% of the original carbon utilized. Small amounts of radioactivity were recovered in succinate and variably in acetate fractions. HPLC analysis demonstrated that some amino acids increased, some decreased, and some remained at the initial concentration. Alanine exhibited the greatest change, consistently increasing from 2 to 4 times the original concentration. Analyses of purified amino acid peaks revealed radioactivity only in the alanine peak, accounting for 2-4% of the original carbon utilized.

The effects of the anthelmintic drug, levamisole, on glycogen metabolism in the adult filariid, Litomosoides carinii, were examined. Incubation of helminths for up to 6 hr in the presence of levamisole resulted in a fourfold increase in total worm glycogen levels. In accord with this observation, levamisole also produced an eightfold increase in the rate of incorporation of [14C]-glucose into [14C]-glycogen over a 6-hr incubation period. In contrast, levamisole had no significant effect on 14CO2 evolution or lactate formation from exogenous glucose indicating that glycolysis was not affected. Because levamisole stimulated the incorporation of [14C]-glucose into glycogen, its effects on glycogen synthase activity were determined. Glycogen synthase in L. carinii was found to exist in both glucose-6-phosphate dependent (D) and glucose-6-phosphate independent (I) forms. In vitro incubation of intact L. carinii for 2 hr in the presence of levamisole resulted in a significant increase in the activity ratio (activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P). This suggests that levamisole may act to stimulate the conversion of the less active (D) form of the glycogen synthase to the more active (I) form.

The trematode, Fasciola hepatica, and the cestode, Spirometra mansonoides have been shown to be similar to the nematode Ascaris lumbricoides in that all three decarboxylate succinate to propionate plus CO2. Associated with this decarboxylation is an incorporation of 32Pi into organic phosphate. Both the decarboxylation and phosphorylation are markedly stimulated by the addition of propionyl-CoA, are dependent on coenzyme B12 and are inhibited by avidin. The trematode and cestode exhibit propionyl-CoA carboxylase, methylmalonyl-CoA mutase and acyl-CoA transferase activities in sonicated mitochondrial preparations. Data are consistent with the occurrence of a mitochondrial substrate level site for ATP generation which is coupled with the decarboxylation of succinate. In Fasciola preparations, acetyl-CoA stimulates the decarboxylation and phosphorylation to a considerably larger extent than propionyl-CoA, indicating the possibility that acetyl-CoA may serve physiologically in these reactions by donating the CoA moiety to succinate.

The rate of 14CO2 evolution from 1-[14C]pyruvate by intact Ascaris mitochondria was very slow, but increased with increasing concentrations of pyruvate. At all concentrations of pyruvate, in an aerobic environment, pyruvate decarboxylation was stimulated greatly by the addition of fumarate, malate, or succinate. However, under anaerobic conditions, only malate and fumarate stimulated pyruvate decarboxylation; succinate had no effect. This implies that the aerobic metabolism of succinate, presumably to other dicarboxylic acids, may be required for the stimulation. Incubation of sonicated mitochondria with pyruvate plus fumarate, under rate-limiting concentrations of NAD+, resulted in approximately equal quantities of pyruvate utilized and succinate formed, suggesting that pyruvate oxidation and fumarate reduction may be linked. Branched-chain, volatile fatty acids were not formed during incubations with either malate or succinate, or succinate plus acetate. However, incubations of intact Ascaris mitochondria with pyruvate plus succinate yielded 2-methylbutyrate and 2-methylvalerate, whereas incubations with pyruvate plus propionate yielded almost exclusively 2-methylvalerate. Oxygen dramatically inhibited the synthesis of the branched-chain acids from succinate plus pyruvate, attesting to the apparent anaerobic nature of Ascaris mitochondrial metabolism. Significantly, the addition of glucose plus ADP stimulated the formation of all volatile fatty acids. Therefore, the synthesis of branched-chain acids may be related directly to increased energy generation. Alternatively, they may function in the regulatory role of maintaining the mitochondrial redox balance.

Larval stages of Oesophagostomum radiatum grown in vitro and adults grown in vivo were incubated in complex media or in a simple salt solution containing radioactive glucose. Glucose disappearance and end product accumulation of third-stage larvae in a simple salt solution indicated that they excreted CO2 and acetic, propionic, and lactic acids. Larvae in third molt, fourth stage, and adults all excreted CO2, acetic, propionic, and lactic acids at twice the rate of third-stage larvae plus an additional product, methylbutyric acid. Carbon dioxide arose primarily from the 3 or 4 carbons of glucose. An anaerobic atmosphere (95% N2:5% CO2) had no apparent effect on metabolism. When incubation was done in complex media, isobutyric and 3-methylbutyric acids were seen as major excretion products (10 and 24%, respectively). However, these acids were quantitatively minor when incubations took place in simple salts-glucose medium (1 and 0-3%, respectively).

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.

2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, by Ascaris lumbricoides muscle. These volatile acids are major fermentation products of Ascaris metabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold from Ascaris muscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+ agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4 degrees C. The molecular weight of the native protein (Mr = 64,000 by exclusion chromatography) and the size of the subunit (Mr = 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for the Ascaris enzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed.

Disrupted Ascaris mitochondria formed 2-methylbutyrate (2-MB) and 2-methylvalerate (2-MV) when incubated anaerobically with acetyl CoA, propionyl CoA and NADH. However, when mitochondrial membranes were removed by high speed centrifugation and the mitochondrial soluble fraction was incubated with the same substrates, 2-methylcrotonate (tiglate) and a compound tentatively identified as 2-methyl-2- pentenoate accumulated rather than 2-MB or 2-MV. These data suggest that the terminal reduction of the unsaturated intermediates to the saturated 2-MB and 2-MV was catalyzed by an enzyme system at least partially bound to membranes. This supposition was further supported by the findings that disrupted Ascaris mitochondria also formed 2-MB and lesser amounts of 2-MV when incubated with tiglyl CoA plus NADH, and both soluble and membrane-bound components appear to be involved in this reduction. The possibility that electron transport associated ATP synthesis may be coupled to these reductions remains to be examined.

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.

Trivalent organic antimonials, such as stibophen, have been employed for the chemotherapy of schistosome and filariid infections. The effects of stibophen on adult Litomosoides carinii, Dipetalonema witei (= viteae), and Brugia pahangi were examined. In vitro, lactate accumulation was markedly inhibited by the antimonials as was phosphofructokinase activities in homogenates. Incubation of filariids with stibophen and determination of internal concentrations of hexose phosphate also indicated a decreased phosphofructokinase activity. In addition, a second inhibitory effect of stibophen on aldolase has been observed which appears to be specific for stibophen and is not displayed by potassium antimony tartrate. Both inhibitory activities may contribute to the chemotherapeutic effect of stibophen. In addition to the schistosomes and filariids, stibophen also inhibits Ascaris and Hymenolepis diminuta phosphofructokinases at low concentrations, where no inhibition of the corresponding mammalian liver enzyme was demonstrable.

4-Isothiocyanato-4'-nitrodiphenylamine possessed anthelmintic activity against Nippostrongylus dubius and Hymenolepis nana in mice and H. diminuta in rats as determined by both egg counts and recovery of parasites at autopsy. No activity was detected against the cestode. Spirometra mansonoides, in cats.

Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides var. suum function anaerobically in electron transport-associated phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and intermembrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria. Succinate and pyruvate dehydrogenases as well as NADH oxidase, Mg(++)-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c reductases were determined to be distributed as in mammalian mitochondria. In contrast with the mammalian systems, fumarase and NAD-linked "malic" enzyme were isolated primarily from the intermembrane space fraction of the worm mitochondria. These enzymes are required for the anaerobic energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space. The need for and possible mechanism of a proton translocation system to obtain energy generation is suggested.

1. A body wall preparation is described which permits intracellular recording from the somatic muscle cells of the small filarial nematode, Dipetalonema viteae. Using this preparation, resting membrane potentials were measured and spontaneous muscle depolarizations described. 2. Stimulatory effects noted upon the addition of acetylcholine, or the cholinergic agonists suggest the hypothesis that acetylcholine is the excitatory neurotransmitter. However, in contrast with vertebrate tissues, the cholinergic antagonists, d-tubocurarine, hexamethonium and pentolinium do not inhibit somatic muscle activity of the worm. 3. GABA inhibited somatic muscle depolarizations, suggesting the possibility that it may serve as an inhibitory neurotransmitter. 4. The anthelmintic drug, levamisole, produced a depolarizing block. Effects of other pharmacological agents are described, discussed and compared with effects on vertebrate muscles.

Mitochondria from the muscle of Ascaris lumbricoides var. suis function anaerobically. NADH is generated in the intermembrane space as a consequence of the "malic" enzyme reaction. It has been suggested that this reducing equivalent in the form of hydride ion, would be translocated across the inner membrane in order to mediate ATP generation via the fumarate reductase reaction. In accord with this suggestion, intact Ascaris mitochondria showed appreciable NADH oxidase activity. Sonication resulted in an approximately 2-fold increase in NADH oxidase activity, whereas "malic" enzyme, fumarase, and NADH:NAD+ transhydrogenase activities increased approximately 7- to 14-fold, respectively. Phosphorylation capabilities and permeability toward pyridine nucleotides also indicated the intactness of the mitochondria. Ascaris mitochondria incubated anaerobically in the presence of fumarate, and [14C]NADH catalyzed a rapid reduction of the fumarate to succinate with the concomitant formation of equivalent quantities of extramitochondrial NAD+. However, very little isotope was recovered from the washed mitochondria, indicating the possibility of hydride ion translocation in the absence of nucleotide translocation. NADH:NAD+ transhydrogenase has been isolated from the muscle mitochondria of the intestinal nematode, Ascaris lumbricoides var. suis. The enzyme seems to have been solubilized from the mitochondrial membrane fraction by treatment with sodium deoxycholate followed by dialysis and subsequent adsorption by and elution from alumina C gamma. No NADPH:NAD+ transhydrogenase activity was detectable, making the Ascaris system unique over others reported. Activity was protected by L-cysteine, reduced glutathione and dithioerythritol, but strongly inhibited by low concentrations of p-chloromercuribenzoate or silver nitrate. The thionicotinamide derivative of NAD+ (thioNAD+) was employed to accept hydride ions from NADH in order to assay spectrophotometrically at 398 nm. Apparent Km values for thioNAD+ and NADH were 1 X 10(-4) M and 8 X 10(-6) M, respectively. That the physiological nucleotide, could act as hydride ion acceptor from NADH was indicated by the findings that NAD+ competitively inhibited the reduction of thioNAD+ when assayed at 398 nm. The additional finding of a noncompetitive inhibition between NAD+ and NADH suggested at least two binding sites on the enzyme, one for NADH and another common site for NAD+ and thioNAD+. More conclusive evidence indicating the participation of NAD+ as acceptor was obtained by incubation of the enzyme with NADH and [14C]NAD+ and demonstrating a rapid formation of [14C]NADH. These findings, in conjunction with those discussed above, suggest a physiological function of this enzyme in hydride ion translocation.

The metabolism of pyruvate by the adult filarial parasites Brugia pahangi, Dipetalonema viteae, and Litomosoides carinii has been compared. Istopic carbon-balance studies indicate the presence of significant pyruvate dehydrogenase activity in L. carinii but little or no activity in either B. pahangi or D. viteae. In all 3 helminths, the quantities of pyruvate that were completely oxidized to CO2 and water were very small. The activities of some of the tricarboxylic acid cycle enzymes of B. pahangi also were determined. In particular, a relatively low level of isocitrate dehydrogenase was noted in the mitochondria of B. pahangi. It is suggested that the tricarboxylic acid energy generating pathway is of doubtful importance as an energy yielding pathway in any of these parasites.

Many invertebrates accumulate propionate, or products derived from propionate, as products of fermentation. Evidence has been reported that the nematode, Ascaris suum, the cestode, Spirometra mansonoides, and the trematode, Fasciola hepatica, accumulate propionate by means of an adenosine triphosphate (ATP)-generating decarboxylation of succinate. To generate energy, an acyl coenzyme A (CoA) transferase that would transfer CoA to succinate is required as one component of the sequence of reactions. Recently, an acyl CoA transferase was isolated from Ascaris mitochondria and purified to electrophoretic homogeneity. However, upon examination of the substrate specificities of this enzyme, it was found essentially to lack the ability to use succinate or succinyl CoA as an acceptor or donor of CoA, respectively. Therefore, this transferase could not serve to activate succinate. This article describes the isolation of an additional acyl CoA transferase from Ascaris mitochondria that appears to be unique in its substrate specificity and that could easily account not only for the activation of succinate but also for the regulation of succinate metabolism primarily in the direction of decarboxylation to propionate. This is in contrast with mammalian tissues, which act in the opposite direction by catalyzing the fixation of CO2 into propionate, thereby forming succinate and accounting for the glycogenic nature of dietary propionate. Possible functions of the two acyl CoA transferases are discussed.

Many metazoans, and particularly the parasitic forms, have adapted well to their environments. This is particularly obvious in organisms that reside in anoxic surroundings, such as the large lumen-dwelling intestinal parasites. However, anaerobic energy metabolisms are not confined to parasites in microaerophilic environments. Some that reside in highly aerobic surroundings (e.g. blood or lungs) also have lost much or all of their aerobic capabilities for energy generation. Adaptations toward anaerobiosis are many and varied: homolactate fermentation (schistosomes and filarial worms), heterolacate fermentations (H. diminuta), and succinate and heterosuccinate fermentations (Ascaris). Even helminths that are obligate aerobes are not complete oxidizers. All accumulate aerobic fermentation products, indicating at best a limited terminal respiration. Some worms, particularly some of the larval stages, retain their ability to survive anaerobically but require the presence of oxygen for motility. Regardless of the type of oxygen requirement, all parasitic helminths examined are dramatically different from their mammalian hosts in regard to their energy metabolisms. Many similar adaptations have been shown to occur in nonparasitic organisms ranging from metazoans, through fish and mammals. There is much room for additional studies of these biochemical adaptations.

Amoscanate possesses chemotherapeutic activity against schistosomes, and in higher doses against many other helminths including filariids and Hymenolepis diminuta. The primary mode of action of this compound is unknown. Effects of the drug on the carbohydrate metabolism as well as on the tegumental and nephridial epithelia of H. diminuta were examined. At various time intervals after administration of the drug to rats infected with H. diminuta, the parasites were recovered and incubated in glucose-salts medium for 90 min. Chemotherapy resulted in decreases in succinate, lactate, and acetate recoveries, while ATP levels dropped. In addition, glycogen levels were depressed in drug-treated worms which were homogenized immediately upon isolation. Glycogen synthase I activity was inhibited 16-61% in cestodes obtained from Amoscanate-treated animals and homogenized immediately, but returned to normal levels after incubation for 90 min in glucose-salts medium prior to homogenization and assay. Phosphorylase a activity was found to be 25-30% higher in preparations of worms from drug-treated rats, which correlates with the rapid depletion of glycogen in parasites exposed to the drug. However, in contrast with glycogen synthase activity, the elevation of phosphorylase a activity in H. diminuta exposed to the drug was not readily reversible. Attempts to demonstrate activity of the drug in vitro by incubating intact cestodes directly with Amoscanate were unsuccessful. Thin sections of parasites obtained from Amoscanate-treated rats and examined by transmission electron microscopy revealed surface alterations of the tegument and nephridial canals. Alterations included bleb formation and erosion of microtriches from the tegument, as well as disappearance of microvilli from nephridial canals. However, these effects became manifest only after 4 or more hr exposure of the rat to the drug. Biochemical effects, on the other hand, were significant after 3 hr exposure.

With [U-14C]glucose we confirmed that essentially all of the glucose carbon used aerobically by intact Brugia pahangi adults was accounted for in the recovered lactate, in spite of the presence of cristate mitochondria in these parasites. Approximately 0.2% of the glucose carbon that disappeared was recovered in CO2. However, aerobiosis was required for the incorporation of alanine, proline, and glutamate into both respiratory CO2 and protein, but the glucose was used on the order of 1000 times faster than the amino acids. Homogenate fractions were examined for amino acid use, and all except the 100,000 g soluble fraction showed activity. However, all of the active fractions also contained living microfilariae and other developmental stages that arise from the adult uterus, since the females give birth to living larvae. Sonicates of the microfilariae formed CO2 from proline 30 times more rapidly than crude homogenates of the adult. Similarly, CO2 was formed from glucose 14 times more rapidly by intact microfilariae than by intact adults. The possibility arises that the low rates of CO2 formation from both the amino acids and glucose by intact adults may arise from the metabolism of the aerobic microfilariae and other developmental stages present in utero.

31P-NMR has been applied to the study of the metabolisms of the intact parasitic helminths Ascaris suum (the intestinal roundworm) and Fasciola hepatica (the liver fluke). After calibration of the chemical shift of Pi in muscle extracts the internal pH of adult Ascaris worms and the effect of the pH of the external medium on the organism's internal pH were measured. Assignments of nearly all of the observable 31P resonances could be made. A large resonance from glycerophosphorylcholine whose function is unclear was observed but no signals from energy storage compounds such as creatine phosphate were detected. The profiles of the phosphorus-containing metabolites in both organisms were monitored as a function of time. Changes in sugar phosphate distributions but not ATP/ADP were observed. Studies of the drug closantel on Fasciola hepatica were performed. Initial effects of the drug were a decrease in glucose 6-phosphate and an increase in Pi with no substantial change in ATP levels as observed by 31P-NMR. Studies involving treatment with closantel followed by rapid freezing, extraction, and analytical determination of glycolytic intermediates confirmed NMR observations. This NMR method can serve as a simple noninvasive procedure to study parasite metabolism and drug effects on metabolism.

The pyruvate dehyhdrogenase complex (pyruvate:lipoate oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1) has been isolated from Ascaris muscle mitochondria and purified to near homogeneity by differential centrifugation, (NH4)2SO4 fractionation and calcium phosphate gel-cellulose chromatography. It is similar in shape, size and physical characteristics to pyruvate dehydrogenase complexes isolated from mammalian sources. It has an absolute dependence on CoA, NAD+ and pyruvate for activity and is competitively inhibited by acetyl-CoA and NADH. However, much higher NADH/NAD+ ratios are necessary to inhibit activity, suggesting regulation by the more reduced state of the pyridine nucleotide pool in Ascaris mitochondria.

Evidence is presented that the microfilariae of Litomosoides carinii, Dipetalonema viteae and Brugia pahangi have an aerobic requirement for motility, but possibly not for survival. In addition, the data suggest that in an in vitro anaerobic environment, B. pahangi microfilariae ferment glucose only as far as lactate. In an aerobic environment, however, the data are consistent with a portion of glucose being dissimilated via a one step oxidative decarboxylation of pyruvate formed from glycolysis to acetate and CO2. In addition, a low level of complete oxidation, possibly via a tricarboxylic acid cycle pathway, may be occurring. Finally, if B. pahangi microfilariae are immobilized with levamisole in an aerobic atmosphere, the drug appears to alter the aerobic glucose metabolism of the parasite both qualitatively and quantitatively. A decreased glucose utilization occurs, together with a shift to a more nearly homolactate fermentation. It is suggested that the effects of levamisole on the metabolism of the microfilariid are secondary to the observed paralysis.

Both spargana and adult forms of Spirometra mansonoides were shown to accumulate lactate, succinate, acetate, and propionate upon in vitro incubation. Adults differed markedly from the spargana in that quantitatively the most significant products of the former were acetate and propionate while the latter formed primarily acetate and lactate. The adults accumulated approximately 32 times more propionate than the spargana per gram of tissue. In accord with this propionate formation, propionyl CoA carboxylase and methylmalonyl CoA mutase have been found to be present in both stages of the parasite. As might be predicted, however, the activities of the carboxylase and mutase were 100-fold and 10-fold higher, respectively, in the adults as compared to the larvae. A possible physiological relationship between propionate formation and energy generation is suggested. Accordingly, inorganic 32P was incorporated into ATP upon incubation of methylmalonyl CoA with a homogenate obtained from adult S. mansonoides. Since methylmalonyl CoA mutase requires vitamin B12 coenzyme, a relationship between vitamin B12 content and propionate formation in helminths is suggested.

Echinostoma liei was incubated in vitro aerobically and anaerobically with various species of 14C glucose. From the recovery of isotope in the respired CO2, it appeared that glucose was incompletely oxidized. The major portion of the CO2 arose from the 3 and 4 carbons of glucose. In addition to CO2, a number of volatile fatty acids accumulated as end-products both aerobically and anaerobically. Lactate and succinate were also isolated. Of these, n-valerate was recovered in the highest concentrations. Qualitatively, the same products were demonstrated after both aerobic and anaerobic incubations, but quantitatively considerably more fatty acids accumulated anaerobically.