Howard J. Saz

Anaerobic phosphorylation in Ascaris mitochondria and the effects of anthelmintics

Abstract 1. 1. A rapid anaerobic, malate dependent, net esterification of inorganic radioactive phosphate (32Pi) has been demonstrated in muscle mitochondria obtained from the intestinal nematode, Ascaris lumbricoides var. suum. Of a number of substrates tested, the system was essentially specific for malate or fumarate. 2. 2. 32Pi/malate ratios obtained approximated 0.5. These findings and the observed inhibitory effect of malonate on 32Pi esterification are in accord with the previously postulated mitochondrial dismutation of malate to pyruvate and succinate. 3. 3. Oligomycin, rotenone and other uncouplers of oxidative phosphorylation in mammalian mitochondria also inhibit the anaerobic incorporation of 32Pi in Ascaris mitochondria, providing additional evidence for the involvement of an electron transport associated phosphorylation. Antimycin, however, has little inhibitory effect. 4. 4. Low concentrations of a number of anticestodal chemotherapeutic agents and the antinematodal agent, dithiazanine, markedly inhibit this system.

Transhydrogenase and anaerobic phosphorylation in Hymenolepis diminuta mitchondria

1. l. It has been confirmed that the malic enzyme of the cestode Hymenolepis diminuta has a specific requirement for NADP. 2. 2. A non-energy linked mitochondrial NADPH-NAD transhydrogenase is present in this tapeworm which should allow for the generation of intramitochondrial NADH. 3. 3. An aerobic, malate dependent, electron transport associated net phosphorylation system which is sensitive to uncouplers of oxidative phosphorylation and a number of anticestodal agents has been demonstrated in H. diminuta mitochondria.

PROPIONYL-COA CONDENSING ENZYME FROM ASCARIS MUSCLE MITOCHONDRIA. I, ISOLATION AND CHARACTERIZATION OF MULTIPLE FORMS

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.

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.

Propionyl-CoA condensing enzyme from Ascaris muscle mitochondria. II. Coenzyme A modulation.

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.

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.

Pyridine nucleotide transhydrogenases of parasitic helminths

Abstract Mitochondria from the parasitic helminth, Hymenolepis diminuta , catalyzed both NADPH:NAD + and NADH:NADP + transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD + would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD + system was approximately five times more active than the NADH:NADP + system. The NADH:NADP + reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD + reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD + transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta . Saturation kinetics indicated that the NADH:NAD + reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides , catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD + activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD + transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides , where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD + transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system.

Phosphorylation associated with succinate decarboxylation to propionate in Ascaris mitochondria

Abstract In mammalian tissues, propionyl CoA carboxylase and methylmalonyl CoA mutase act physiologically primarily in the ATP requiring direction of succinate formation. Therefore, propionate is glycogenic. However, many invertebrates and bacteria accumulate propionate from succinate. Employing acyl CoA transferase, propionyl CoA carboxylase, and methylmalonyl CoA mutase in the direction of propionate formation, substrate level ATP should be generated. The intestinal worm, Ascaris lumbricoides possesses mitochondria which function anaerobically and accumulate propionate and volatile fatty acids derived from propionate. Results of the present study indicate that a site of mitochondrial phosporylation may be present at the substrate level during the decarboxylation of succinate to propionate and C02. Mitochondrial preparations from Ascaris muscle exhibit propionyl CoA carboxylase, methylmalonyl CoA mutase, and acyl CoA transferase activities. Inorganic 32P is esterified during succinate decarboxylation. In accord with the proposed reactions, both succinate decarboxylation and 32P esterification are stimulated six- to eightfold upon the addition of propionyl CoA, and both stimulations are inhibited by avidin.

31P-NMR studies of the metabolisms of the parasitic helminths Ascaris suum and Fasciola hepatica

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 organisms 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.

Biochemical aspects of filarial parasites

Abstract Filarial parasites infect approximately 300 million people yet there is no chemotherapy which is both adequate and safe, and almost nothing is known of the biochemistry of these worms. Circumstantial evidence indicates that the adult filariids which infect man may be homolactate fermenting anaerobes, and the development of chemotherapeutic agents is likely to depend on our finding differences between parasite and host metabolisms.