ENERGETIC METABOLISM OF ADULT HYMENOLEPIS DIMINUTA

The adult intestinal helminth, Hymenolepis diminuta, continues to serve as a model for the study of cestode physiologically anaerobic energetics as well as the anaerobic energetics of parasitic helminths generally. It is now well established that carbohydrate dissimilation by adult H. diminuta results in acetate, lactate and succinate accumulation, with succinate being the major end product (Fairbairn et al. Reference Fairbairn, Wertheim, Harpur and Schiller1961; Scheibel and Saz, Reference Scheibel and Saz1966; Watts and Fairbairn, Reference Watts and Fairbairn1974). Equally convincing are the data demonstrating that succinate accumulation is the product of the anaerobic, mitochondrial, ATP-generating utilization of malate (Scheibel and Saz, Reference Scheibel and Saz1966; Bueding and Saz, Reference Bueding and Saz1968; Saz et al. Reference Saz, Berta and Kowalski1972).

In H. diminuta, malate arises in the cytosol via CO₂ fixation into phosphoenolpyruvate followed by reduction of the resulting oxalacetate (Prescott and Campbell, Reference Prescott and Campbell1965; Scheibel and Saz, Reference Scheibel and Saz1966; Bueding and Saz, Reference Bueding and Saz1968; Scheibel et al. Reference Scheibel, Saz and Bueding1968). Cytosolic malate then serves as the mitochondrial substrate. Upon entering the mitochondrial matrix, malate is oxidatively decarboxylated by the NADP-specific ‘malic’ enzyme, thereby yielding pyruvate, CO₂ and reducing power for electron transport in the form of NADPH (Prescott and Campbell, Reference Prescott and Campbell1965; Li et al. Reference Li, Gracy and Harris1972; Saz et al. Reference Saz, Berta and Kowalski1972; McKelvey and Fioravanti, Reference McKelvey and Fioravanti1984, Reference McKelvey and Fioravanti1985). Malate also is converted to fumarate by matrix fumarase (Read, Reference Read1953; McKelvey and Fioravanti, Reference McKelvey and Fioravanti1985). With the electron transport-coupled reduction of fumarate to succinate, as catalyzed by the fumarate reductase, the malate dismutation reaction is completed (Scheibel and Saz, Reference Scheibel and Saz1966; Saz et al. Reference Saz, Berta and Kowalski1972). Net, anaerobic ATP generation by isolated H. diminuta mitochondria is site I-dependent, as indicated by rotenone-sensitivity, and is significantly inhibited by oligomycin and protonophoric agents (including niclosamide, a known anticestodal agent (Saz et al. Reference Saz, Berta and Kowalski1972)), but not antimycin A. In addition, phosphorylation by H. diminuta mitochondria is inhibited by malonate in keeping with the involvement of the fumarate reductase system (Saz et al. Reference Saz, Berta and Kowalski1972).

Aside from the NADH-utilizing fumarate reductase (Scheibel et al. Reference Scheibel, Saz and Bueding1968), H. diminuta mitochondria display a less active, inner membrane-associated, rotenone-sensitive and peroxide-forming NADH oxidase that appears to require tightly bound manganous ion. Peroxide is formed by NADPH oxidation as well (Fioravanti and Saz, Reference Fioravanti, Saz and Arai1980; Fioravanti, Reference Fioravanti1981, Reference Fioravanti1982a; McKelvey and Fioravanti, Reference McKelvey and Fioravanti1985; Fioravanti and Reisig, Reference Fioravanti and Reisig1990). Both the NADH-utilizing fumarate reductase and oxidase exhibit a benzoquinone preference inasmuch as they employ rhodoquinone rather than ubiquinone as reflected in NADH oxidation, NADH-dependent oxygen consumption, hydrogen peroxide production, and succinate accumulation (Fioravanti and Kim, Reference Fioravanti and Kim1988). H. diminuta mitochondria also display a membrane-associated succinoxidase activity that results in oxygen consumption and peroxide formation (Fioravanti, Reference Fioravanti1982a; Fioravanti and Reisig, Reference Fioravanti and Reisig1990). NADH- and succinate-dependent peroxide formation accounts for about 50 and 40 percent, respectively, of the total oxygen consumed by isolated mitochondrial membranes (Fioravanti and Reisig, Reference Fioravanti and Reisig1990). Interestingly, the fumarate reductase inhibitor, malonate (Saz et al. Reference Saz, Berta and Kowalski1972), not only inhibits fumarate reduction and succinate oxidation, but also significantly inhibits H. diminuta mitochondrial NADH oxidase (Fioravanti, Reference Fioravanti1982a; Fioravanti and Reisig, Reference Fioravanti and Reisig1990). Consistent with the physiological role of fumarate as the terminal electron transport acceptor, NADH utilization by H. diminuta mitochondrial membranes increases precipitously in the presence of fumarate (despite the presence of oxygen) whereas NADH- and succinate-dependent peroxide accumulation is virtually abolished and oxygen consumption is minimal. Neither NADH- nor succinate-dependent oxygen utilization by H. diminuta mitochondrial membranes are appreciably sensitive to antimycin A, sodium azide or potassium cyanide (Fioravanti and Reisig, Reference Fioravanti and Reisig1990).

Taken together, the above findings support the model proposed by Fioravanti and Reisig (Reference Fioravanti and Reisig1990) of the H. diminuta mitochondrial electron transport system given in Fig. 1. Accordingly, both oxygen- and fumarate-dependent NADH oxidations employ a common site I-containing complex, viz., an NADH–rhodoquinone reductase. Branching occurs after this complex, at the benzoquinone level, as indicated by the rhodoquinone requirement of NADH-dependent oxygen utilization and peroxide formation. Because malonate inhibits NADH-/succinate-dependent peroxide formation/oxygen utilization as well as fumarate reduction, it appears that the NADH oxidase, succinoxidase, and fumarate reductase use a common malonate-sensitive, flavin-containing fumarate reductase that terminates the major branch of the system (Fig. 1). This is supported further since the presence of fumarate virtually abolishes NADH-dependent peroxide formation, and succinate oxidation. Succinate-dependent peroxide accumulation/oxygen consumption involves the above-mentioned flavin-containing branch and the lesser branch originating at the rhodoquinone. Thus, reducing power from NADH or succinate uses both branches of the electron transport system to reduce oxygen while fumarate reduction would need only the major physiological branch. The non-peroxide-forming branch is presumed to result in water formation (Fig. 1).

Fig. 1. Model of the Hymenolepis diminuta mitochondrial electron transport system. The physiological route for reducing equivalents is indicated by bold lines. Designations are as follows: RQ, rhodoquinone; Fp, flavin-containing component of the fumarate reductase.

Adult H. diminuta mitochondria display cytochrome c reducing and oxidizing activities, i.e. membrane-associated NAD(P)H- and succinate-dependent cytochrome c reductase, cytochrome c oxidase, and a cytochrome c peroxidase as assessed by exogenous oxidized or reduced cytochrome c utilization (Kim and Fioravanti, Reference Kim and Fioravanti1985; McKelvey and Fioravanti, Reference McKelvey and Fioravanti1986). Rotenone-sensitive NADH-dependent cytochrome c reductase and succinate-dependent cytochrome c reductase activities exhibit antimycin A sensitivity whereas cytochrome oxidase activity is inhibited by sodium azide and potassium cyanide. In contrast, these inhibitors are without appreciable effect on H. diminuta NADH oxidase, succinoxidase and fumarate reductase (Saz et al. Reference Saz, Berta and Kowalski1972; Fioravanti and Reisig, Reference Fioravanti and Reisig1990). While fumarate reduction involves the major branch of the cestode electron transport system, thereby explaining a lack of significant antimycin A, azide and cyanide effects, at this juncture it is assumed that membrane-associated, exogenous cytochrome c reduction/oxidation reflects activities of the lesser branch of the cestode electron transport system (Fig. 1). Even though further study is needed, the lack of appreciable inhibition of NADH oxidase and succinoxidase by antimycin A, azide and cyanide may reflect a basal level of cytochrome c-dependent activities, under the conditions of assay, that cannot be diminished further by the inhibitors. Nonetheless, these findings and others demonstrating rotenone-sensitive and -insensitive NADH-dependent cytochrome c reductase (McKelvey and Fioravanti, Reference McKelvey and Fioravanti1985), point to cytochrome c as an interesting component of H. diminuta mitochondria.

The H. diminuta electron transport system can form peroxide when exposed to oxygen (e.g. Kim and Fioravanti, Reference Kim and Fioravanti1985; Fioravanti and Reisig, Reference Fioravanti and Reisig1990). It is noteworthy, therefore, that adult H. diminuta lacks appreciable catalase, glutathione peroxidase or NAD(P)H peroxidase activities, but does display peroxide-forming superoxide dismutase activity (Paul and Barrett, Reference Paul and Barrett1980; Barrett and Beis, Reference Barrett and Beis1982). Although the subject of differing opinions, the occurrence and intramitochondrial localization of peroxidase activity (or peroxidase-like activity) in adult H. diminuta has been reported using histochemical and biochemical methods (Threadgold et al. Reference Threadgold, Arme and Read1968; Lumsden et al. Reference Lumsden, Oaks and Mills1969; Robinson and Bogitsh, Reference Robinson and Bogitsh1976, Reference Robinson and Bogitsh1978; Paul and Barrett, Reference Paul and Barrett1980; Kim and Fioravanti, Reference Kim and Fioravanti1985; McKelvey and Fioravanti, Reference McKelvey and Fioravanti1986). A mitochondrial membrane association for a H. diminuta cytochrome c peroxidase has been indicated (Threadgold et al. Reference Threadgold, Arme and Read1968; Robinson and Bogitsh, Reference Robinson and Bogitsh1976, Reference Robinson and Bogitsh1978; Paul and Barrett, Reference Paul and Barrett1980), but Kim and Fioravanti (Reference Kim and Fioravanti1985), using a biochemical assessment found that cytochrome c peroxidase activity is associated predominantly (95% of recovered activity) with the soluble mitochondrial fraction. Indeed, an evaluation of the intramitochondrial localization of the cestode cytochrome c peroxidase indicated a 55% matrix and 32% intermembrane space distribution, respectively (McKelvey and Fioravanti, Reference McKelvey and Fioravanti1986). Given the potential for peroxide formation by H. diminuta mitochondria and the apparent lack of a number of common peroxide-utilizing systems in the cestode, the occurrence of mitochondrial cytochrome c peroxidase activity merits additional study.

Further compelling data supporting the physiologically anaerobic character of H. diminuta energetics are evident in terms of in vitro cultivation. Schiller (Reference Schiller1965) demonstrated that H. diminuta can be cultivated from excysted cycticercoid to ovigerous adult in an atmosphere of 97% N₂-3% CO₂. Moreover, Schiller demonstrated that oncospheres, derived from these cultivated cestodes, develop into infective cysticercoids in the intermediate beetle host. Employing cultivation methods (modified by Schiller), Roberts and Mong (Reference Roberts and Mong1969) cultivated 6-day-old H. diminuta for 12 days in an atmosphere of 95% N₂-5% CO₂ with egg production; these eggs developed into infective cysticercoids when given to beetles. The addition of O₂ to the gas phase (1, 5, 20%) did not stimulate helminth growth. Voge et al. (Reference Voge, Jaffe, Bruckner and Meymarian1976) found that in vitro cultivation of hatched H. diminuta oncospheres to infective cysticercoids occurred more rapidly in 100% N₂ than in air. Of note were the findings that use of 95% N₂-5% CO₂ or 95% N₂-5% O₂ atmospheres gave results similar to those with 100% N₂, thereby indicating that CO₂, while needed for post-cycticercoid development, is not essential to cysticercoid development itself.

Saz et al. (Reference Saz, Berta and Kowalski1972) were the first to note that NADPH, formed by the action of the ‘malic’ enzyme, could serve as a source of reducing power for NADH-dependent, anaerobic phosphorylation in adult H.diminuta mitochondria. More importantly, their data indicated that the intermediary action of a membrane-associated NADPH→NAD⁺ transhydrogenase in H. diminuta, and presumably other helminth systems, plays a crucial role in anaerobic, electron transport-coupled succinate formation. Thus, for the first time in any of the invertebrates, a physiological role of the transhydrogenase as a metabolic connector was made apparent in the H. diminuta model.

THE HYMENOLEPIS DIMINUTA MITOCHONDRIAL NADPH→NAD⁺ SYSTEM

The H. diminuta mitochondrial inner membrane-associated NADPH→NAD⁺ transhydrogenase (EC 1.6.1.1) catalyzes a reversible hydride ion transfer as given in the following equation:

The NADH- and NADPH-forming activities, denoted as the NADPH→NAD⁺ and NADH→NADP⁺ reactions, respectively, are readily assessed spectrophotometrically in the cestode using the appropriate acetylpyridine derivatives of NAD(P)⁺ (i.e. AcPyAD(P)) as hydride ion acceptors (Saz et al. Reference Saz, Berta and Kowalski1972; Fioravanti and Saz, Reference Fioravanti and Saz1976; McKelvey and Fioravanti, Reference McKelvey and Fioravanti1985). In support of the considerations of Saz et al. (Reference Saz, Berta and Kowalski1972), a reduced pyridine nucleotide preference of the H. diminuta electron transport system is apparent (Fioravanti, Reference Fioravanti1981) with NADH rather than NADPH being the preferred reductant (Table 1). Significantly, however, pyridine nucleotide specificity is circumvented by the transhydrogenase in its catalysis of NADPH→NAD⁺ hydride ion transfer. Whereas NADPH, formed by the ‘malic’ enzyme, does not appear to be an effective reductant for the oxidase or fumarate reductase, significant rotenone-sensitive NADPH oxidation occurs in the presence of NAD⁺. With respect to fumarate-dependent succinate formation, the coupling of the NADPH→NAD⁺ transhydrogenase with fumarate reduction by H. diminuta mitochondrial membranes is essentially unchanged whether assessed under aerobic conditions or conditions of reduced oxygen tension (Fioravanti, Reference Fioravanti1981) (Table 1). Additionally, coupling of malate utilization with the H. diminuta electron transport system, via the NADPH→NAD⁺ transhydrogenase, was demonstrated (McKelvey and Fioravanti, Reference McKelvey and Fioravanti1984).

Table 1. NADPH Utilization by Hymenolepis diminuta mitochondrial membranes under conditions of reduced oxygen tension.

Activity expressed as nmoles/min per mg protein; 0·35 mg of protein employed for assay.

An association of the H. diminuta transhydrogenase with the mitochondrial membrane fraction was reported by Saz et al. (Reference Saz, Berta and Kowalski1972) and, subsequently, the transhydrogenase was found to be a mitochondrial inner membrane (IM) component (Fioravanti and Saz, Reference Fioravanti and Saz1976; McKelvey and Fioravanti, Reference McKelvey and Fioravanti1985). This IM association was demonstrated using hypotonically shocked and sonicated cestode mitochondria, in conjunction with sucrose step-gradient centrifugation and appropriate marker enzymes (Table 2). The transhydrogenase activity was predominantly localized with the H. diminuta IM mitochondrial fraction as noted for the IM systems/markers (i.e. NADH oxidase, fumarate reductase, succinate dehydrogenase, and rotenone-sensitive NADH→cytochrome c reductase) (Table 2). The validity of marker enzyme localizations was established employing isolated rat liver mitochondria subjected to the same procedures. The findings with H. diminuta mitochondria were supported further by transhydrogenase and marker enzyme assessments following fractionation of incubated cestode mitochondria using increasing amounts of digititonin (McKelvey and Fioravanti, Reference McKelvey and Fioravanti1985). Assessments of enzyme activities of ‘intact’ vs sonically disrupted H. diminuta mitochondria and of isolated submitochondrial particles (SMP) were consistent with transhydrogenase-catalyzed hydride ion transfer, between reduced and oxidized pyridine nucleotides, occurring on the matrix surface of the IM (McKelvey and Fioravanti, Reference McKelvey and Fioravanti1985; Fioravanti et al. Reference Fioravanti, McKelvey and Reisig1992). Significantly, the ‘malic’ enzyme (as well as fumarase) is predominantly localized in the mitochondrial matrix of H. diminuta, in accord with the localizations of the intermembrane and matrix markers, i.e. adenylate kinase and citrate synthase (Table 2). Therefore, NADPH reducing power for electron transport would be readily available for transhydrogenation (McKelvey and Fioravanti, Reference McKelvey and Fioravanti1985).

Table 2. Enzyme distribution in Hymenolepis diminuta mitochondria

^a Units express total activity in μmol/min of mitochondria and mitochondrial fractions derived from 5·0 g of tissue.

^b Numbers in parentheses express percent distribution of recovered activity.

^c NADH→cytochrome c activity sensitive to 100 μm rotenone.

^d NADH→cytochrome c activity insensitive to 100 μm rotenone.

CHARACTERIZATION OF THE HYMENOLEPIS DIMINUTA MITOCHONDRIAL NADPH→NAD⁺ TRANSHYDROGENASE

The H. diminuta mitochondrial NADPH→NAD⁺ transhydrogenase is phospholipid-dependent (Fioravanti and Kim, Reference Fioravanti and Kim1983). When extracted with hexane, lyophilized cestode mitochondrial membranes display increased NADPH→NAD⁺ activity, but decreased NADH oxidase and fumarate reductase activities. However, with the addition of aqueous acetone to lyophilized and hexane-treated membranes, a diminishment in NADPH→NAD⁺ activity as well as NADH oxidase and fumarate reductase is noted (Fioravanti and Kim, Reference Fioravanti and Kim1983). These data indicate: (1) neutral lipid(s) are needed by the cestode oxidase and fumarate reductase; and (2) the cestode transhydrogenase, NADH oxidase, and fumarate reductase are phospholipid dependent. Consistent with a phospholipid dependency, phospholipase treatments of H. diminuta mitochondrial membranes significantly depress transhydrogenase activity with phospholipase A₂ being more effective in this regard than phospholipase C, thereby implying that conversion of membrane phospholipids to lysophosphatides was more inhibitory than conversion to diacylglycerols. Because transhydrogenase activity of either acetone-extracted lyophilized/hexane-treated membranes or phospholipase-treated membranes was unaffected by phospholipid supplementation, partially phospholipid(s)-depleted (~60%) membranes were prepared by detergent-treatment/ammonium sulfate precipitation, i.e. the 30–55 fraction (Table 3). The need for phospholipid by the transhydrogenase was demonstrable using the 30–55 fraction inasmuch as the addition of phosphatidylcholine to this fraction increases activity significantly. Neither phosphatidylethanolamine nor phoshphatidylserine effectively altered transhydrogenase activity (Table 4) (Fioravanti and Kim, Reference Fioravanti and Kim1983).

Table 3. Phosphorus content of mitochondrial membranes and the 30–55 fraction of Hymenolepis diminuta

* Partially lipid depleted preparation, derived from mitochondrial membranes following precipitation between 30 to 55% ammonium sulfate saturation.

Table 4. Stimulation of transhydrogenase activity by phospholipid addition to the 30–55 fraction of Hymenolepis diminuta

* Indicates μmol of phospholipid added to the 30–55 fraction, in the presence of 0·1% sodium cholate, and subjected to sonication.

† Activities are expressed per mg protein; 0·12 mg protein was employed for each assay.

‡ Material subjected to equivalent treatment without the addition of phospholipid.

Using isolated and everted IM vesicles, i.e. SMP, the cestode transhydrogenase was found to catalyze non energy-linked and energy-linked NADH→NADP⁺ reactions (Fioravanti et al. Reference Fioravanti, McKelvey and Reisig1992). As presented in Table 5, rotenone addition to H. diminuta SMP results in somewhat of an increase in the rate of the NADPH→NAD⁺ reaction, but significantly inhibits the NADH→NADP⁺ reaction as measured by reduction of the appropriate AcPyNAD(P) acceptor (Fioravanti et al. Reference Fioravanti, McKelvey and Reisig1992). These findings indicate that rotenone inhibits oxidation of NADH or AcPyADH and that an energization of the SMP, via electron transport- associated NADH oxidation, stimulates the NADH→NADP⁺ reaction. When, H. diminuta SMP were treated with Mg⁺⁺ plus ATP in the presence of rotenone, a significant increase in NADH→NADP⁺ transhydrogenation also was noted as compared to samples containing rotenone alone (Table 6). This increased activity was not matched by the individual addition of either Mg⁺⁺ or ATP. Furthermore, the Mg⁺⁺ plus ATP-dependent increase in NADH →NADP⁺ activity was lowered to the level observed with rotenone alone when either ethylenediaminetetraacetate (EDTA) or oligomycin were added to the reaction vessels, in keeping with the involvement of Mg⁺⁺-dependent ATPase. Thus, an energy-linkage was apparent wherein ATP hydrolysis is energizing the mitochondrial membranes, thereby driving NADPH formation. Collectively, these data made evident, for the first time, the occurrence of non-energy-linked and energy-linked NADH→NADP⁺ mitochondrial transhydrogenations in the parasitic helminths (Fioravanti et al. Reference Fioravanti, McKelvey and Reisig1992).

Table 5. Reduced pyridine nucleotide utilization by Hymenolepis diminuta submitochondrial particles

Values are mean±s.e.; the number of observations is presented inside the parentheses, 0·03 mg of protein was used for the assays.

† Rotenone concentration was 100 μm.

Table 6. Energy-linked NADH→NADP⁺ transhydrogenation catalyzed by Hymenolepis diminuta submitochondrial particles

* All assays contained 100 μm rotenone.

† Values are mean±S.E.; the number of observations is presented inside the parentheses; numbers in brackets are observed values; 0·03 mg of protein was used for the assays.

‡ Where indicated MgCl₂ and ATP were present at 3·0 mm and 2·0 mm, respectively.

In other eukaryotes, e.g. mammalian (Danielson and Earnster, Reference Danielson and Ernster1963; Lee and Earnster, Reference Lee and Ernster1989) and insect (Mayer et al. Reference Mayer, Svoboda and Weirich1978; Vandock et al. Reference Vandock, Smith and Fioravanti2008) systems, mitochondrial succinate oxidation energizes the reversible NADPH→NAD⁺ transhydrogenase, resulting in an increased NADH→NADP⁺ (energy-linked) activity. As indicated above, H. diminuta mitochondria engage in an inner membrane-associated oxidation of succinate (McKelvey and Fioravanti, Reference McKelvey and Fioravanti1985; Fioravanti and Reisig, Reference Fioravanti and Reisig1990). However, the addition of succinate to H. diminuta SMP was without appreciable effect on the NADH→NADP⁺ activity (Table 6). While the data of Fioravanti et al. (Reference Fioravanti, McKelvey and Reisig1992) reflect an energy-linked transhydrogenation driven by either NADH oxidation or ATP hydrolysis, the lack of succinate-dependent energization supports the view that the H. diminuta electron transport system does not engage in appreciable site II or site III activity and thus, appreciable site II or site III phosphorylation.

Certainly, the findings of Fioravanti et al. (Reference Fioravanti, McKelvey and Reisig1992) demonstrate that non-energy- and energy-linked NADH→NADP⁺ transhydrogenations are catalyzed by H. diminuta mitochondria. Employing cestode SMP, Park and Fioravanti (Reference Park and Fioravanti2006) characterized these reactions in greater detail. For these studies, and subsequent considerations, the following designations pertain. The NADH→NADP⁺ reaction measured in the presence of rotenone, is deemed to be essentially non energy-linked and is so designated. The energy-linked transhydrogenations are denoted as follows: (1) the NADH→NADP⁺ reaction driven by electron transport-dependent NADH oxidation is refered to as ETD; (2) the reaction driven by ATP hydrolysis is designated ATPD. All assessments of energy-linked NADH→NADP⁺ reactions were corrected for non-energy-linked activity (Park and Fioravanti, Reference Park and Fioravanti2006).

The data of Table 7 indicate that the H. diminuta SMP non-energy-linked transhydrogenation is unaffected by N,N′-dicyclohexylcarbodiimide (DCCD), a known inhibitor of proton translocating systems, as well as the protonophores carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and niclosamide (Heytler, Reference Heytler, Fleischer and Packer1979; Fisher and Earle, Reference Fisher, Earle, Everse, Anderson and You1982; Hassinen and Vuokila, Reference Hassinen and Vuokila1993). All of these inhibitors markedly depressed the cestode ETD and ATPD reactions (Table 7). Although these data do not differentiate the effects of inhibitors on the transhydrogenase or the NADH oxidase and ATPase systems, they are demonstrative of a role of transmembrane proton translocation in the cestode energy-linked transhydrogenations.

Table 7. NADH→NADP⁺ transhydrogenation catalyzed by Hymenolepis diminuta submitochondrial particles

Values are mean±s.e.; the number of observations is presented inside the parentheses. DCCD, N,N′-dicyclohexylcarbodiimide; CCCP, 3-chlorophenylhydrazone; FCCP, 4-(trifluoromethoxy) phenylhydrazone. 0·03 mg of protein was used for the assays.

The data of Park and Fioravanti (Reference Park and Fioravanti2006) presented in Table 7 reflect a coupling of ATP hydrolysis with the ATPD transhydrogenation. This consideration is made even more convincing by the data given in Table 8. Measurements of ATP hydrolysis, assessed as phosphorus liberated, versus AcPyADP reduction indicate an ATP/NADPH ratio of 1·39 in H. diminuta. Interestingly, addition of the multivalent protein, bovine serum albumin, increased the rate of phosphorus formation and the accumulation of AcPyADPH yielding an ATP/NADPH ratio of 1·08. These data form a basis for further studies. Nevertheless, they indicate a stoichiometric relationship of ATP hydrolysis to NADPH formation of about 1:1 (Table 8).

Table 8. Relationship of ATP hydrolysis and energy-linked NADH→NADP⁺ transhydrogenation catalyzed by Hymenolepis diminuta submitochondrial particles

Where indicated, BSA (bovine serum albumin) was present at 0·5% wt vol⁻¹. Reactions were measured in the presence of 25 μm rotenone. Values are mean±s.e.; the number of observations is presented inside the parentheses, whereas the numbers inside the square brackets are observed values. 0·15 mg of protein was used for the assays.

An evaluation of the effects of pH on the non-energy-linked, ETD, and ATPD reactions proved of interest (Fig. 2). Whereas the ETD reaction was unaffected over a pH range of 5·0–8·0, the non-energy-linked and ATPD reactions displayed significant peaks at pH 5·5 and 6·5, respectively. Presumably, the ATPD peak also suggests a more optimal pH for the cestode ATPase. Although these data may well reflect more optimal pH ranges, it is significant that with acidification, the non-energy-linked reaction simulates ATPD activity. Thus, the intriguing notion that the non energy-linked reaction assumes the characteristics of the ATPD activity, via a pH-dependent change in the transhydrogenase (conformational, protonation (Galante et al. Reference Galante, Lee and Hatefi1980)), is presented (Park and Fioravanti, Reference Park and Fioravanti2006).

Fig. 2. The effects of pH on the NADH→NADP⁺ transhydrogenation reactions of adult Hymenolepis diminuta submitochondrial particles. Symbols used are: –○–, non-energy-linked; –•–, electron transport–driven; –□–, ATP–driven. Error bars represent s.e. Activities were measured in the absence of BSA. 25 μm rotenone was employed for assessments of the electron transport–(ETD) and ATP–driven (ATPD) reactions. The mean value for the non-energy-linked reaction at pH 8·0 was 11·3±s.e. 1·1 whereas that for the ATPD reaction was 10·9±3·2. Values for the ETD reaction at pH 7·5 and 8·0 were 14·8±2·1 and 10·4±1·3, respectively. Numbers of observations were: non-energy-linked – pH 5·0, 8; pH 5·5, 6; pH 6·0–6·5, 5; pH 7·0–8·0, 7. ETD-pH 5·0–6·5, 5; pH 7·0, 7; pH 7·5, 4; pH 8·0, 8. ATPD- pH 5·0, 7; pH 5·5, 8; pH 6·0, 7; pH 6·5, 5; pH 7·0, 7; pH 7·5, 6; pH 8·0, 7. An amount of 0·03 mg protein was employed for assays.

The above data imply that a proton gradient, established either by the electron transport-dependent utilization of NADH or ATP hydrolysis by the Mg⁺⁺-dependent ATPase, drives the H. diminuta energy-linked, transhydrogenase-catalyzed reduction of NADP⁺. The possibility that the H. diminuta transhydrogenase can act in transmembrane proton translocation was first evaluated by Mercer et al. (Reference Mercer, McKelvey and Fioravanti1999). They proposed that the helminth transhydrogenase, in catalyzing NAD⁺ reduction, could act in the simultaneous movement of protons from the matrix to the intermembrane space compartment as reported for the corresponding mammalian transhydrogenase (Danielson and Ernster, Reference Danielson and Ernster1963; Rydström et al. Reference Rydström, Kanner and Racker1975). To this end, H. diminuta SMP were evaluated with respect to transhydrogenase-catalyzed proton translocation.

Presented in Table 9 are the effects of protonophores or DCCD on the catalysis of the NADPH→NAD⁺ activity of H. diminuta SMP. In the presence of protonophores, a marked increase in AcPyAD reduction is apparent while DCCD significantly inhibits the activity, thereby indicating that with hydride ion transfer between NADPH and NAD⁺, the enzyme concomitantly catalyzes transmembrane proton translocation into the SMP intravesicular space. The increased activity noted in the presence of the protonophores indicates that the protonophores relieved an inhibition of the NADPH→NAD⁺ system created by the enzyme-dependent intravesicular accumulation of protons (Mercer et al. Reference Mercer, McKelvey and Fioravanti1999).

Table 9. Effects of protonophores and DCCD on the NADPH→NAD⁺ transhydrogenase activity of Hymenolepis diminuta submitochondrial particles

Values are means±s.e.; the number of observations is presented inside the parentheses, 0·02 mg of protein was used for the assays. Mean value for each treatment differed significantly when compared to the untreated control.

To assess further the potential of the NADPH→NAD⁺ transhydrogenase to act as a proton pump, fluorometric assessments of the H. diminuta SMP NADPH→NAD⁺ transhydrogenation were performed using as a probe, 8-anilino-1-napthalene-sulfonic acid (ANS). Under the conditions employed, the added ANS would be in its anionic form and, thus, attracted to an intravesicular accumulation of a positive charge. In its attraction to this intravesicular environment, ANS fluorescence would be enhanced by its binding to the SMP membrane (Azzi et al. Reference Azzi, Chance, Radda and Lee1969). The assay system contained NADPH and NAD, with appropriate substrate generating systems, as well as rotenone and potassium cyanide. As presented in Fig. 3A, with the addition of NAD⁺ to the assay, a time-dependent increase in ANS fluorescence was noted. In contrast, with additions of the protonophores, i.e. CCCP (Fig. 3B) or niclosamide (Fig. 3C), a rapid collapse in the increasing fluorescence observed in the absence of the inhibitors was apparent. Moreover, with the addition of DCCD to the reaction medium, prior to the start of the reaction with NAD⁺, increased fluorescence was essentially abolished (Fig. 3D). Accordingly, these data support the concept that the H. diminuta transhydrogenase system is a transmembrane component that catalyzes hydride ion transfer with concomitant proton translocation from the mitochondrial matrix to the intermembrane space (Mercer et al. Reference Mercer, McKelvey and Fioravanti1999).

Fig. 3. Enhancement of ANS fluorescence by Hymenolepis diminuta SMP-catalyzed NADPH→NAD⁺ transhydrogenation. For assays 2·0–3·0 mg protein was employed. (A) Fluorescence enhancement following the start of transhydrogenation with NAD. (B) Quenching of transhydrogenase-dependent fluorescence by 1 mm CCCP. (C) Quenching of fluorescence by 3 mm niclosamide. (D) Inhibition of transhydrogenase-dependent enhanced fluorescence by the addition of 600 mm DCCD prior to the start of the reaction with NAD.

The findings supporting a concomitant proton translocation with NADPH→NAD⁺ transhydrogenation in H. diminuta mitochondria, in conjunction with those supporting a proton gradient as the driving force for the energy-linked NADH→NADP⁺ reactions (Fioravanti et al. Reference Fioravanti, McKelvey and Reisig1992; Park and Fioravanti, Reference Park and Fioravanti2006), suggested that the helminth transhydrogenase, in catalyzing NADH formation, serves as an additional site for anaerobic phosphorylation. In this context, it is noteworthy that bovine heart SMP engage in transhydrogenase-dependent phosphorylation when catalyzing NADPH→NAD⁺ transhydrogenation in the virtual absence of electron transport activity (Van de Stadt et al. Reference Van de Stadt, Nieuwenhuis and Van Dam1971; Donstov et al. Reference Donstov, Grinius, Jasaitis, Severina and Skulachev1972). Mercer-Haines and Fioravanti (Reference Mercer-Haines and Fioravanti2008) evaluated the possibility that the H. diminuta transhydrogenase can serve as an additional phosphorylation site using SMP as the enzyme source. Net phosphorylation, measured by ³²P incorporation into ATP, was assayed by a modification of the procedure of Saz et al. (Reference Saz, Berta and Kowalski1972) and employed substrate generating systems in the absence or presence of rotenone and the additives indicated in Tables 10 and 11.

Table 10. Reduced pyridine nucleotide-dependent phosphorylation by Hymenolepis diminuta submitochondrial particles

Values are mean incorporations after 5 min±s.e. Numbers of observations are in parentheses. 0·4–0·5 mg protein was employed for assays. ND indicates not detected.

^a Exogenous NADH-generating system consisted of glutamate (3·0 mm) and glutamate dehydrogenase (5 U).

^b Significantly different when compared to non-rotenone containing counterparts.

^c Significantly different when compared to counterparts without fumarate.

^d Exogenous NADPH-generating system consisted of glucose-6-phosphate (3·0 mm) and glucose-6-phosphate dehydrogenase (5 U).

^e No exogenous NAD-generating system.

Table 11. NADPH→NAD⁺ transhydrogenase as an energy-coupling site in Hymenolepis diminuta submitochondrial particles

Values are mean incorporations after 5 min±s.e. Numbers of observations are in parentheses. 0·4–0·5 mg protein was employed for assays. ND indicates not detected.

^a Exogenous-generating systems employed were as follows: NADPH, glucose-6-phosphate (3·0 mm) and glucose-6-phosphate dehydrogenase (5 U); NAD, pyruvate (15 mm) and lactate dehydrogenase (5 U).

^b DCCD, N,N′-dicyclohexylcarbodiimide.

^c No exogenous NAD-generating system.

^d Significantly different when compared to counterparts containing an NAD-generating system.

Given in Table 10, are the data obtained wherein reduced pyridine nucleotide-dependent net phosphorylation by H. diminuta SMP was assessed. For these studies, exogenous NAD(P)H-generating systems were employed as indicated. Obviously, NADH-dependent phosphorylation was enhanced when fumarate was added to the system and in both the absence and presence of fumarate, rotenone significantly inhibited net ATP generation (Table 10). Significantly, when NADPH was substituted for NADH in measurements of net phosphorylation, either in the absence or presence of fumarate, no phosphorylation was measurable. However, when NADPH served as the source of reducing power, in the presence of NAD⁺, marked net ATP generation was noted and the degree of this net phosphorylation increased with fumarate supplementation. Collectively, the data of Table 10 support prior findings and demonstrate, based on net ³²P incorporation, that: (1) the H. diminuta electron transport system indeed displays a preference for NADH; and (2) via the transhydrogenase system NADPH can serve as a source of reducing equivalents for electron transport (Mercer-Haines and Fioravanti, Reference Mercer-Haines and Fioravanti2008).

Indeed, because the H. diminuta NADH→NADP⁺ reaction is enhanced significantly by mitochondrial ATP hydrolysis and the NADPH→NAD⁺ reaction results in a concomitant movement of protons from the mitochondrial matrix to the intermembrane space, evaluations were undertaken to determine if the cestode NADPH→NAD⁺ transhydrogenase, in catalyzing NAD⁺ reduction, could serve as an additional site for net ATP generation (Mercer-Haines and Fioravanti, Reference Mercer-Haines and Fioravanti2008). Employing H. diminuta SMP and both NADPH and NAD⁺ generating systems as indicated, assessments of the transhydrogenase system serving in the potential generation of ATP were made and these data are given in Table 11. As noted, in the presence of rotenone the catalysis of the NADPH→NAD⁺ reaction by the cestode transhydrogenase resulted in an appreciable net ATP generation. Conversely, when these assessments were made in the presence of rotenone with either DCCD or niclosamide supplementation, no ATP generation was detected in keeping with a transhydrogenase-dependent formation of a proton gradient by transmembrane pumping of protons. Within this context, it also is of note that when ³²P incorporation was assessed, in the presence of an NADPH generating system, rotenone and NAD⁺, in the absence of NAD⁺ generation, incorporation was markedly less than that observed in corresponding samples in which rotenone was absent (Table 10) or in which both the NADH generating system and rotenone were present (Table 11). Thus, the need for an ongoing generation of NADPH and NAD⁺ when the transhydrogenase serves as a phosphorylation site is apparent. Moreover, if these assessments were made in the presence of rotenone, but without an exogenous NAD⁺ generating system, only a low level of phosphorylation (~14% of that noted in the presence of the NAD⁺ generating system) was observed. The latter would likely reflect the ‘leakiness’ of the rotenone inhibitor (Table 11).

Based on the data obtained relating to the H. diminuta mitochondrial IM-associated and reversible NADPH→NAD⁺ transhydrogenase, the model for the involvement of this enzyme in the energetics of the cestode is presented in Fig. 4. With the oxidative decarboxylation of malate by the ‘malic’ enzyme, reducing equivalents for electron transport are accumulated in the matrix as NADPH. With NADPH, in the presence of NAD⁺, the IM-associated transhydrogenase catalyzes the formation of NADH. This scalar reaction, occurring on the matrix surface of the transhydrogenase, fosters a concomitant translocation of protons from the matrix to the intermembrane space compartment via the proton-pumping ability of the transhydrogenase. The proton gradient so established serves in driving ATP synthesis by the ATP synthase system. Likewise, the oxidation of transhydrogenase-formed NADH by the cestode electron transport system results not only in fumarate reduction but the establishment of a proton gradient, promoting net phosphorylation. With active malate oxidation, NADPH is formed allowing for NADH accumulation. Coupled to this is the oxidation of NADH via the electron transport-dependent fumarate reductase. With respect to the energy-linked NADPH-forming reactions (i.e. NADH→NADP⁺), a reversal of the role of the transhydrogenase occurs. The hydrolysis of ATP or NADH oxidation establishes a proton gradient, that with the transhydrogenase catalyzed transmembrane movement of protons, drives NADPH formation (Fig. 4). Therefore, in H. diminuta and presumably other helminths, the mitochondrial NADPH→NAD⁺ transhydrogenase can serve not only as a metabolic connector, but also as a site for anaerobic phosphorylation.

Fig. 4. Model for the role of Hymenolepis diminuta inner mitochondrial membrane-associated transhydrogenase. Designations are as follows: T, transhydrogenase; A, ATP synthase/ATPase; ETS, electron transport system; ME, ‘malic’ enzyme.

NADH→NAD⁺ MITOCHONDRIAL TRANSHYDROGENATION IN HYMENOPEPIS DIMINUTA

Fioravanti and Saz (Reference Fioravanti and Saz1976) first demonstrated the occurrence of a transhydrogenation reaction between NADH and NAD⁺ (AcPyAD) in H. diminuta mitochondria that is independent of the reversible NADPH→NAD⁺ transhydrogenase. Initial evaluations of this NADH→NAD⁺ transhydrogenation indicated a predominant IM localization. Furthermore, in adult Ascaris suum mitochondria, that apparently lack an NADPH→NAD⁺ transhydrogenase, an IM-associated NADH→NAD⁺ transhydrogenation was noted (Fioravanti and Saz, Reference Fioravanti and Saz1976). Two lines of evidence concerning the ascarid NADH→NAD⁺ transhydrogenation were presented. Firstly, the findings of Rew and Saz (Reference Rew and Saz1974) coupled with those of Köhler and Saz (Reference Köhler and Saz1976) indicated that the nematode, NAD⁺-preferring ‘malic’ enzyme was essentially localized in the mitochondrial intermembrane space and that the NADH→NAD⁺ transhydrogenation could serve in the transmembrane movement of reducing equivalents from the intermembrane space to the matrix compartment. Secondly, the work of Komuniecki and Saz (Reference Komuniecki and Saz1979) indicated that the ascarid NADH→NAD⁺ transhydrogenation predominantly reflected an activity associated with the nematode lipoamide dehydrogenase. Considered collectively, the above noted studies fostered a further evaluation of the mitochondrial NADH→NAD⁺ transhydrogenation of adult H. diminuta.

Employing sonically disrupted H. diminuta mitochondria, a comparison was made of the intramitochondrial localizations of the NADH→NAD⁺ reaction, the NADPH→NAD⁺ transhydrogenase, the NADH dehydrogenase, and the lipoamide dehydrogenase activities (Walker and Fioravanti, Reference Walker and Fioravanti1995). As expected, the data of Table 12 reveal that both the NADPH→NAD⁺ transhydrogenase and NADH dehydrogenase systems are primarily membrane associated. With respect to lipoamide dehydrogenase and the NADH→NAD⁺ transhydrogenation, however, the former was predominantly recovered in the mitochondrial soluble fraction (62%) whereas the latter was predominantly membrane-associated (66%). Given the data of Table 12, a further comparison of the four mitochondrial activities was made based on thermal lability.

Table 12. Intramitochondrial localization of NADPH→NAD⁺ transhydrogenase, NADH dehydrogenase, lipoamide dehydrogenase, and the NADH→NAD⁺ transhydrogenation in Hymenolepis diminuta

* Units express total activity in μmol/min of fractions from a 3·5 ml preparation of mitochondria equivalent to 28·3 mg protein. Each value is the mean of duplicate assays. Duplicates for each value did not vary by more than 8·5%.

† Numbers in parentheses express activity in μmol min⁻¹ (mg protein)⁻¹; 6·0 μg to 0·12 mg mitochondrial, 8·0 μg to 0·1 mg membrane, and 12·0 to 37·0 μg soluble protein were employed for assay.

Isolated H. diminuta mitochondrial membranes were exposed to increasing temperatures for five minute periods and these data are given in Fig. 5. NADPH→NAD⁺ activity increased up to 35°C and at 45°C, activity decreased with complete inactivation at 55°C. NADH dehydrogenase activity gradually declined at lower temperatures but dramatically decreased at 45°C with total inactivation at 65°C (Fig. 5). Whereas lipoamide dehydrogenase activity was stimulated at 25°C, the activity declined thereafter to about the control level at 45°C and above 45°C an increased activity was noted that peaked at 75°C. Temperatures in excess of 75°C diminished activity with total inactivation at 100°C. NADH→NAD⁺ activity appeared intermediate to that of both dehydrogenases. While lower temperatures increased activity, inactivation was apparent at 45°C and 55°C. With increasing temperature an increase in NADH→NAD⁺ activity was noted that exceeded that of untreated membranes at 75°C and thereafter a diminishment in activity was evident with complete inactivation at 100°C (Fig. 5). Based on these thermal profiles, in conjunction with the data of Table 12, it appears that the H. diminuta mitochondrial NADH→NAD⁺ transhydrogenation reaction is catalyzed by lipoamide dehydrogenase and possibly by NADH dehydrogenase rather than by an independent transhydrogenase system.

Fig. 5. Thermal profiles of NADPH→NAD⁺ transhydrogenase, NADH dehydrogenase, lipoamide dehydrogenase, and the NADH→NAD⁺ transhydrogenation reaction catalyzed by Hymenolepis diminuta mitochondrial membranes. Symbols used are: –□–, Lipoamide dehydrogenase; –▪–, NADPH→NAD⁺; –○–, NADH→NAD⁺; –•–, NADH dehydrogenase.

Further studies of the H. diminuta NADH→NAD⁺ transhydrogenation, associated with lipoamide dehydrogenase, were pursued (Walker et al. Reference Walker, Burkhart and Fioravanti1997). The occurrence of NADH→NAD⁺ and lipoamide dehydrogenase activities were assessed for H. diminuta cysticercoids, the mitochondria of 6-, 10-, and 14-day-old cestodes, and the mitochondria of pre-gravid/gravid regions of adults (Table 13). All of these preparations, catalyzed an NADH→NAD⁺ transhydrogenation and lipoamide reduction, with the latter being more prominent. A developmentally related increase in NADH→NAD⁺ activity is suggested when comparisons of mitochondria from 6-, 10-, and 14-day cestodes are made. In contrast, lipoamide dehydrogenase activity is essentially unchanged, thereby indicating that a system(s) other than lipoamide dehydrogenase contribute to the increased NADH→NAD⁺ activity as suggested for adult mitochondria (Walker and Fioravanti, Reference Walker and Fioravanti1995). Assessments of NADH→NAD⁺ and lipoamide dehydrogenase activities for adult H. diminuta segments indicate that the immature and mature segments have the highest levels of transhydrogenation while the highest level of lipoamide dehydrogenase is with the immature segment (Table 13). The levels of both mitochondrial activities in the immature segment are consistent with it being the most metabolically active region of the adult cestode (Roberts, Reference Roberts1961).

Table 13. NADH→NAD⁺ Transhydrogenation and lipoamide dehydrogenase activities of Hymenolepis diminuta

Values are mean±s.e.; the number of observations is presented inside the parentheses. 2·5–170 μg protein was used for assessments. For cysticercoids, the supernatant fraction obtained after disruption served as the source of activities, whereas isolated and disrupted mitochondria were used for all other assessments.

Isolation of the H. diminuta mitochondrial lipoamide dehydrogenase was undertaken and a representative purification is presented in Table 14. Utilizing the H. diminuta soluble mitochondrial fraction as starting material, coupled with heat treatment and column chromatographies, ~149 fold purification of the enzyme was obtained. As given in Fig. 6, the purified enzyme has a monomeric, M_r of 47 kDa. Via Sephadex G-200 chromatography, the native M_r of the enzyme was estimated to be 93 kDa, consistent with a homodimeric enzyme, and the enzyme's absorption spectra revealed the occurrence of flavin (Walker et al. Reference Walker, Burkhart and Fioravanti1997).

Fig. 6. SDS–PAGE of the purification of Hymenolepis diminuta lipoamide dehydrogenase. Lane 1, mitochondrial supernatant; Lane 2, heat-treated; Lane 3, concentrated; Lane 4, DEAE-Sepharose; Lane 5, Sephadex G-100; Lane 6, hydroxylapatite; Lane 7, molecular mass markers. Each lane contained 5 mg protein.

Table 14. Purification of the Hymenolepis diminuta lipoamide dehydrogenase

A listing of the reactions catalyzed by purified H. diminuta lipoamide dehydrogenase and the pH optimum of each of these activities is presented in Table 15. Aside from lipoamide dehydrogenase and NADH→NAD⁺ transhydrogenation activities, both NADH-dependent ferricyanide reduction (pH 6·5) and a low level of diaphorase (pH 6·0) were detected. Interestingly, the purified enzyme preparation also catalyzed a low level of NADPH→NAD⁺ transhydrogenation at acidic pH (4·5). An initial comparison of a partial amino acid sequence of the H. diminuta lipoamide dehydrogenase indicated that the cestode enzyme was most similar to the corresponding enzymes of other parasitic helminths.

Table 15. Reactions catalyzed by purified Hymenolepis diminuta lipoamide dehydrogenase

Buffers employed were as follows: lipoamide dehydrogenase, NADH diaphorase and NADH→ferricyanide, 100 mm potassium phosphate; NADH→NAD⁺, 100 mm Tris-HCl; and NADPH→NAD⁺, 100 mm acetate.

Employing purified H. diminuta lipoamide dehydrogenase as the antigen, polyclonal rabbit antibodies, directed against this enzyme, were prepared in conjunction with David Upite (unpublished observations). IgG was purified from the rabbit anti-lipoamide dehydrogenase serum and was employed to assess antigen retention in electron micrographs of thin sections of mouse liver mitochondria using the ‘HACH’ polymer as the embedding material (Olesen et al. Reference Olesen, Heckman, Lukinius, Schwab, Upite and Fioravanti1997). The effective use of the IgG prepared using H. diminuta purified lipoamide dehydrogenase made evident an appreciable degree of similarity of the H. diminuta enzyme with the corresponding mammalian mitochondrial system.