Enzyme localization in the anaerobic mitochondria of Ascaris lumbricoides.

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.

Purification and properties of the Ascaris pyruvate dehydrogenase complex.

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.

Succinate decarboxylation to propionate and the associated phosphorylation in Fasciola hepatica and Spirometra mansonoides.

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.

Phospholipids and protein kinase C in acetylcholine-dependent signal transduction in Ascaris suum.

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.

Actions of acetylcholine and GABA on spontaneous contractions of the filariid, Dipetalonema viteae.

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.

Acetyl-CoA hydrolase activity and function in Ascaris suum muscle mitochondria.

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.

Effects of cholinergic agents on the metabolism of choline in muscle from Ascaris suum.

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.

The effects of stibophen on phosphofructokinases and aldolases of adult filariids.

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.

The effect of levamisole on glycogen synthase and the metabolism of Litomosoides carinii.

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.

Comparative utilization of pyruvate by Brugia pahangi, Dipetalonema viteae, and Litomosoides carinii.

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.

The carbohydrate metabolism of Brugia pahangi microfilariae.

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.

Relationships between pyruvate decarboxylation and branched-chain volatile acid synthesis in Ascaris mitochondria.

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.

Pathway of formation of branched-chain volatile fatty acids in Ascaris mitochondria.

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.

Acyl-CoA transferase activities in homogenates of Fasciola hepatica adults.

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.

Effects of calmodulin and protein kinase C antagonists on muscle in the filariid, Acanthocheilonema viteae.

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.

Glycerophosphorylcholine, a component of both Ascaris suum muscle and Caenorhabditis elegans.

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.

Aerobic and anaerobic fermentation of glucose by Echinostoma liei.

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.

Biochemical analyses of secretory and excretory products of adult Dipetalonema viteae in culture.

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 presence and possible function of methylmalonyl CoA mutase and propionyl CoA carboxylase in Spirometra mansonoides.

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.