Andrew Holowiecki. A Thesis

Transcription

1 Catalysis of Mitochondrial NADH NAD + Transhydrogenation in Adult Ascaris suum (Nematoda) Andrew Holowiecki A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE December 2009 Committee: Carmen F. Fioravanti, Advisor Daniel M. Pavuk Jill Zeilstra-Ryalls

2 2009 Andrew Holowiecki All Rights Reserved

3 iii ABSTRACT Carmen F. Fioravanti, Advisor Adult Ascaris suum inhabits the small intestine of its swine host where oxygen tension is low. Despite the lack of oxygen, A. suum generates mitochondrial ATP by an NADH-requiring, inner membrane-associated (IM), electron transport-coupled fumarate reductase, producing succinate. Compelling data suggests that the malic enzyme resides in the mitochondrial intermembrane space (IMS), forming the NADH required for anaerobic phosphorylation. Thus, the transfer of reducing power from IMS NADH across the IM to matrix NAD + would be needed to form the NADH required for anaerobic ATP generation. An IM-associated NADH NAD + transhydrogenation reaction has been implicated in this transfer and is thought to be a catalytic activity of lipoamide dehydrogenase in ascarid mitochondria. The purpose of this study was to ascertain whether the NADH NAD + transhydrogenation reaction in adult A. suum results from more than one catalytic activity, viz., lipoamide dehydrogenase and NADH dehydrogenase. Studies of the mitochondrial NADH NAD + transhydrogenation reaction, lipoamide dehydrogenase, and NADH dehydrogenase were performed using disrupted adult A. suum mitochondria as the source of enzymes. Based on studies evaluating the effects of ph on the ascarid activities, and the thermal labilities of these reactions, it appears that the lipoamide dehydrogenase and NADH dehydrogenase catalyze an NADH NAD + transhydrogenation reaction in adult A. suum mitochondria. These findings were supported further by intramitochondrial localizations of the three activities as well as the effects of inhibitors on these systems.

4 iv In light of these findings, it is concluded that the NADH NAD + transhydrogenation reaction in adult A. suum is the result of lipoamide dehydrogenase and NADH dehydrogenase systems. Presumably, these studies will aid in the ultimate development of specific chemotherapeutic strategies for development of anthelmintics.

5 v I would like to dedicate this thesis to my parents, Stanley and Katharina Holowiecki for encouraging me to go to school, and to not quit. You have succeeded in giving me all the opportunities that you never had. Thank you. I would also like to dedicate this thesis to Henry and Becky Beiro for helping me to keep things in perspective. A great man once said all models are wrong, some models are useful. The model you have created for life is useful. Thank you. Finally, and most importantly, this work is dedicated to my wife, Kristy. This achievement would have been impossible without you. Your love, support, tolerance, patience, respect, and understanding have allowed me to pursue my dreams. I love you, and I need you.

6 vi ACKNOWLEDGMENTS First, I would like to thank my advisor, Dr. Fioravanti, for his guidance and patience. Your ability to teach and inspire has undoubtedly helped me grow as a student, teacher, and person. You have set a great example of how to be a true professional, and a leader. You never taught me through intimidation, and I never lost my sense of dignity. Thank you. Additionally, I would like to thank the other members of the Fioravanti Lab, viz., Kurt Vandock, and Chris Drummond. Your assistance in helping me to learn and improve my lab techniques was essential to my success. Gratitude is expressed to Jeff Meyers at J.H. Routh Packing for allowing us to collect Ascaris suum. I would also like to thank my other committee members, Dr. Dan Pavuk and Dr. Jill Zeilstra-Ryalls for their support and willingness to contribute to my education. Finally, I would like to thank Dr. Kathryn Durham of Lorain County Community College. You made biology interesting, and you taught me how to learn. I would also like to thank Dr. James Beil of Lorain County Community College. Your patience teaching me as an undergraduate and your willingness to help me as a graduate student has helped me to achieve my goals. This work was supported in part by research grant AI from the National Institutes of Health, awarded to Dr. Carmen F. Fioravanti. This research was also supported by a Grant- In- Aid of Research from the National Academy of Sciences, administered by Sigma Xi, The Scientific Research Society.

7 vii TABLE OF CONTENTS Page INTRODUCTION... 1 CHAPTER I. A REVIEW OF THE LITERATURE... 3 CHAPTER II. MITOCHONDRIAL NADH NAD + TRANSHYDROGENATION IN ADULT ASCARIS SUUM (NEMATODA) Introduction.. 12 Materials and Methods.. 14 Results Discussion. 26 CONCLUSIONS AND FUTURE DIRECTIONS REFERENCES.. 33

8 viii LIST OF TABLES Table Page 1 Mitochondrial localizations of NADH NAD + transhydrogenation, lipoamide dehydrogenase, and NADH dehydrogenase activities of A. suum mitochondria 23 2 Effects of inhibitors on activities of Ascaris suum mitochondria assessed under acidic and basic conditions. 25

9 ix LIST OF FIGURES Figure Page 1.1 The life cycle of Ascaris lumbricoides Pathway of carbohydrate dissimilation in Ascaris lumbricoides muscle Pathway describing intramitochondrial malate oxidation and the potential role of IM-associated NADH NAD + transhydrogenation The effects of ph on NADH NAD + transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities of adult Ascaris suum mitochondria Thermal lability profiles of the NADH NAD + transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities of adult Ascaris suum mitochondria assessed under acidic conditions Thermal lability profiles of NADH NAD + transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities of adult A. suum mitochondria assayed under basic conditions Proposed roles of lipoamide dehydrogenase (LD) and NADH dehydrogenase (ND) as NADH NAD + transhydrogenation mechanisms in the mitochondrial energetics of adult Ascaris suum

10 1 INTRODUCTION The adult intestinal helminth of swine, Ascaris suum (Nematoda), is predominately anaerobic (Scheibel and Saz, 1966; Scheibel et al.,1968; Saz and Lescure, 1969) and produces organic end products as a result of carbohydrate catabolism (Saz and Bueding, 1966). Malate is generated in the cytosol and enters the mitochondria where it serves as the mitochondrial substrate for a dismutation reaction (Bueding and Saz, 1968; Saz and Lescure, 1969). The oxidative branch of this reaction produces pyruvate and carbon dioxide, and subsequently yields reducing power in the form of NADH (Rew and Saz, 1974). The reductive branch of this dismutation reaction entails the reduction of fumarate to succinate resulting from the electron transport-dependent, NADH requiring, fumarate reductase (Fioravanti and Saz, 1980). Fumarate reduction is accompanied by the concomitant, site I-coupled, formation of ATP. Reducing power in the form of NADH accumulates in the mitochondrial IMS via the NAD + -linked malic enzyme (Rew and Saz, 1974). Subsequently, reducing power from NADH must cross the IM of the mitochondria to reduce matrix NAD + to drive the anaerobic, ATP generating, electron transport system. A membrane- associated NADH NAD + transhydrogenation reaction has been implicated in this transmembrane movement of hydride ions (Fioravanti and Saz, 1976; Kohler and Saz, 1976). Previous studies have implicated an association between this transhydrogenation and lipoamide dehydrogenase in adult Ascaris (Komuniecki and Saz, 1979). Conversely, in the intestinal parasite of rats, adult Hymenolepis diminuta (Cestoda), it has been reported that the NADH NAD + transhydrogenation reaction may be the result of both lipoamide dehydrogenase and NADH dehydrogenase (Walker and Fioravanti, 1995). Chapter 1 of this thesis entails a review of the literature relative to anaerobic energy generation in the helminths, with particular consideration given to the mitochondrial NADH NAD +

11 2 transhydrogenation reaction. Chapter 2 presents information addressing the question as to whether the mitochondrial NADH NAD + transhydrogenation reaction of adult A. suum is the sum of a catalytic entity other than an NADH NAD + transhydrogenase and/or lipoamide dehydrogenase system.

12 3 CHAPTER I. A REVIEW OF THE LITERATURE The Biology of Ascaris suum Ascaris suum, the adult intestinal roundworm (Nematode) of swine, is a member of the phylum Nematoda, the class Secernentea, the order Ascaridida, and the family Ascaridae (Bogitsh et al., 2005). Adult female ascarids are much larger than their male counterparts and male ascarids are easily distinguished from females by their ventrally curved tail. Adult A. suum is visually indistinguishable from the corresponding intestinal roundworm of humans, Ascaris lumbricoides, but differs from the former in that its eggs do not readily infect humans (Dailey, 1996). A. lumbricoides, the most common nematode parasitizing humans, is thought to infect over 1 billion people worldwide (Bogitsh et al., 2005). While the host specificity of A. suum and A. lumbricoides differs, their life cycles are alike. Both nematodes have a monoxenous life cycle, i.e., they are highly specific to only one host. Both A. suum and A. lumbricoides have four juvenile stages and undergo four molts in their development from egg to adult. Whereas the first two molts occur within the egg, the third and fourth molts occur within the host. The lifecycle of A. lumbricoides is presented in Figure Undeveloped eggs are passed in host feces and embryonate during incubation in an aerobic environment. Upon ingestion by a suitable host, the eggs hatch within the stomach, thereby releasing larvae which penetrate the intestines. From the intestines the larvae travel, via the venous blood stream to the liver and subsequently migrate to the lungs where they undergo a third molt. Thereafter the larvae move up the trachea where they are swallowed for a second time. The fourth and final molt occurs in the small intestine producing the adult egg-laying parasite.

13 4 FIGURE 1.1: The life cycle of Ascaris lumbricoides. Adult worms (1) live in the lumen of the small intestine. Female worms produce approximately 200,000 eggs per day, which are passed with the feces (2). Fertile eggs embryonate and become infective after approximately 18 days (3). Upon ingesting infective eggs (4), the larvae hatch (5), invade the intestinal mucosa, and are carried to the liver by the venous blood stream, and then, via systemic circulation, to the lungs (6). Further larval development occurs within the lungs (lasting 10 to 14 days) after which the larvae penetrate the alveolar walls, ascend the bronchial tree to the throat, and are swallowed (7). Inside the small intestine, the worms further develop into the adult egg laying parasite (1).

14 5 The ability of infectious parasites like A. suum and A. lumbricoides to not only survive, but to thrive in a variety of differing environments, is evident by their success in nature. Certainly their ability to go from one environment to another, regardless of changes in O 2 tension, host immune responses, and other environmental changes present difficulties in terms of creating specific chemotherapies (Barret, 1981). Nonetheless, biochemical studies of these ascarids, and other helminths, have contributed substantially to our understanding of aerobic and anaerobic respiration. For example, cytochromes, which have a key role in oxidative phosphorylation, were first observed by the parasitologist David Keilin when studying the intestinal horse parasite Gastrophilus intestinalis (Keilin, 1966; Slater, 2003). Additionally, Francesco Redi used A. lumbricoides to disprove the theory of spontaneous generation in the 17 th century (Read, 1972; Cox, 2002). A. lumbricoides also served as the first organism in which physiologically functional anaerobic mitochondria were described (Bueding, 1949; Saz and Weil, 1962). Ascaris suum Metabolism Adult A. suum is essentially anaerobic in terms of its energetics (Scheibel and Saz, 1966; Scheibel et al., 1968; Saz and Lescure, 1969) and produces succinate in addition to a variety of other fatty acids derived from succinate (Saz and Bueding, 1966). Although energy generation in adult A. suum is predominantly anaerobic, possibilities for the role of O 2 have been suggested. One such report implicates that cuticle formation in A. suum is dependent on the O 2 -utilizing enzyme proline hydroxylase (Fujimoto and Prockop, 1969; Cain and Fairbairn, 1971). The role of O 2 in other helminths has been questioned and it appears that O 2 may play a role separate from energy generation, such as egg development in Schistosoma mansoni (Schiller et al., 1975).

15 6 The adult intestinal cestode, Hymenolepis diminuta, also generates energy anaerobically (Scheibel and Saz, 1966) and produces succinate, acetate and lactate as end products of glucose catabolism, with succinate being the main end product (Fairbairn et al., 1961). Both A. suum and H. diminuta lack a fully functioning tricarboxylic acid cycle (Kmetic and Bueding, 1961; Ward and Fairbairn, 1970) and serve as models for anaerobic helminth energetics. Carbohydrate dissimilation in adult Ascaris suum muscle is summarized in Figure 1.2. Malate is generated in the cytosol and enters the mitochondria where it serves as the substrate for a dismutation reaction (Bueding and Saz, 1968; Saz and Lescure, 1969). In contrast to reported enzyme distribution studies in mammalian systems, ascarid fumarase and the NAD + -linked "malic" enzyme are essentially located within the IMS (Rew and Saz, 1974), thus suggesting that NADH, pyruvate, and fumarate are formed in the IMS (Rew and Saz, 1974). Glycolysis occurs within the cytosol. Glucose is broken down to phosphoenolpyruvate (PEP) (Saz, 1981), and in the absence of significant amounts of pyruvate kinase (PK) in the cytoplasm, CO 2 is fixed to PEP producing oxalacetate (OAA) (Saz and Lescure, 1969). Cytoplasmic NAD + is formed as a result of the reduction of OAA to malate by malate dehydrogenase and cytoplasmic NADH (Saz, 1981). Cytoplasmic malate enters into the mitochondria and serves as the mitochondrial substrate where it undergoes a dismutation reaction (Kohler and Saz, 1976). In the oxidative branch of the reaction, the NAD + -linked malic enzyme catalyzes the oxidation of malate to pyruvate and CO 2 thus regenerating reducing power in the form of NADH within the mitochondria (Rew and Saz, 1974). The reductive branch of this dismutation reaction entails the reduction of fumarate to succinate resulting from the electron transport-dependent, NADH requiring, fumarate reductase (Fioravanti and Saz, 1980). Fumarate reduction is accompanied by the concomitant, site I coupled, formation of ATP.

16 7 Figure 1.2: Pathway of carbohydrate dissimilation in Ascaris lumbricoides muscle (after Rew and Saz, 1974). Malate, derived from glycolytic activity and CO 2 fixation in the cytosol, enters the mitochondrion. Within the mitochondrion, malate undergoes a dismutation reaction. The oxidative branch of this reaction, as catalyzed by the malic enzyme ( L-malate:NAD oxidoreductase [decarboxylating]), results in the formation of pyruvate and CO 2.The reductive branch of this dismutation reaction entails malate oxidation by fumarase with subsequent fumarate reduction by the NADH-dependent fumarate reductase. The resulting reduction results in electron transport-dependent ATP generation.

17 8 In order for the dismutation reaction to be complete, the fumarate reductase reaction must occur, which presumably happens on the matrix side of the mitochondrial membrane. Reducing power, in the form of NADH, is apparently accumulated in the mitochondrial IMS by the action of the NAD + -linked malic enzyme (Rew and Saz, 1974). Although NADH is required to catalyze the fumarate reductase reaction, due to its apparent origin within the IMS, a mechanism is needed to describe the means of reducing power translocation. A membrane- associated NADH NAD + transhydrogenation reaction has been implicated in this transmembrane movement of hydride ions (Kohler and Saz, 1976). Figure 1.3 depicts the proposed transmembrane transhydrogenation. Localization studies by Rew and Saz are in contrast to Kohler (1977), and have shown fumarase to be located within the IMS rather than the matrix in Ascaris (1974). Kohler (1977) has indicated that fumarate is unable to penetrate the mitochondrial IM. This presents a dilemma, as fumarate serves as the final electron acceptor in Ascaris (Kmetec and Bueding, 1961; Saz and Lescure, 1969; and Seidman and Entner, 1961) and other invertebrates (Saz and Bueding, 1966; Hochachka and Mustafa, 1972). Thus, the reduction of fumarate to succinate would be expected to occur on the matrix side of the inner mitochondrial membrane. The discrepancy between the proposed location(s) of fumarase in Ascaris notwithstanding, further characterization of the + proposed NADH NAD transhydrogenase was initiated.

18 9 Figure 1.3: Pathway describing intramitochondrial malate oxidation and the potential role of IMassociated NADH NAD + transhydrogenation (after Kohler and Saz, 1976). Abbreviations used are as follows: OM, outer membrane; IM, inner membrane. Fumarate reduction is indicated as a reaction occurring on the matrix surface of the IM. The pyruvate dehydrogenase complex (PDH) catalyzes the oxidative decarboxylation of pyruvate to acetyl-coa, CO 2, and concomitant NADH accumulation (Matuda and Saheki, 1985; Behal et al., 1993). This series of reactions is catalyzed by a 3 component enzyme: pyruvate dehydrogenase (E1), EC ; dihydrolipoamide transacetylase (E2), EC ; and dihydrolipoamide dehydrogenase (E3), EC (lipoamide dehydrogenase).

19 10 As noted, in adult A. suum, NADH is generated within the IMS and hydride ions are thought to cross the mitochondrial IM in order to begin the fumarate reduction reaction (Komuniecki and Saz, 1979). Studies by Kohler and Saz (1976), and Fioravanti and Saz (1976) implicate this transfer of reducing power from the IMS into the matrix by the membrane bound NADH NAD + transhydrogenase. This NADH NAD + transhydrogenase activity has been reported to be primarily associated with the lipoyl or lipoamide dehydrogenase in A. suum (Kommuniecki and Saz, 1979). Isolation of lipoamide dehydrogenase from pig heart has revealed the presence of two differing forms of this enzyme, viz., a soluble and a membrane bound form (Sakurai et al., 1970); however, studies with the ascarid system indicate that both forms of the lipoamide dehydrogenase are identical (Komuniecki and Saz, 1979). In the adult cestode Hymenolepis diminuta, a mitochondrial transhydrogenation reaction between NADPH and NAD + and between NADH and NAD + occurs (Saz et al. 1972; Fioravanti and Saz, 1976). The reversible NADPH NAD + reaction results from an IM-associated pyridine nucleotide transhydrogenase (Fioravanti and Saz, 1976; Fioravanti, 1981; Fioravanti and Kim, 1983; McKelvey and Fioravanti, 1985). The NADH NAD + transhydrogenation reaction has been found to be associated with both lipoamide dehydrogenase, and possibly NADH dehydrogenase (Walker and Fioravanti, 1995). The mitochondrial NADH dehydrogenase is the flavin-containing first component of the IM-associated anaerobic electron transport system as well as a component of the outer membrane rotenone-insensitive NADH cytochrome c reductase (as reviewed by Fioravanti and Vandock, 2009). However, the NADH NAD + transhydrogenation in H. diminuta appear to originate from a source(s) other than the NADPH NAD + transhydrogenase (Fioravanti and Saz, 1976).

20 11 Purpose of Study The purpose of the present study is to determine if lipoamide dehydrogenase is the sole enzymatic entity catalyzing NADH NAD + transhydrogenation in A. suum, or if another system, viz., NADH dehydrogenase contributes to this activity? Published reports have suggested an association of NADH NAD + transhydrogenase activity with lipoamide dehydrogenase, and NADH dehydrogenase in adult H. diminuta (Walker and Fioravanti, 1995). In mammalian systems, it has been proposed that the NADH dehydrogenase contains two closely related active sites (Hatafi and Galante, 1977). One site would accommodate the dehydrogenation of NAD(P)H while the other site would allow for a second nucleotide to bind for transhydrogenation (Hatafi and Galante, 1977). This study was undertaken to assess the possibility that adult ascarid mitochondrial NADH NAD + transhydrogenation activity is the result of a catalytic activity or activities in addition to that of the lipoamide dehydrogenase. Using disrupted Ascaris mitochondria as the source of the enzymes, three NADH utilizing activities were evaluated as indicated by the following reactions and designations: + 1. NADH + NAD NADH (NADH NAD + transhydrogenation) + 2. NADH + H + ferricyanide NAD + + ferrocyanide (NADH dehydrogenase) 3. Lipoamide + NADH + H + Dihydrolipoamide + NAD + (lipoamide dehydrogenase). These evaluations consisted of comparative thermal lability studies, ph optima, insoluble and soluble mitochondrial localization, and the effects of some inhibitors. The results of this study are presented here.

21 12 CHAPTER II. MITOCHONDRIAL NADH NAD + TRANSHYDROGENATION IN ADULT ASCARIS SUUM (NEMATODA) Introduction As an adult, Ascaris suum, the intestinal nematode of swine, is essentially anaerobic in terms of its energy generation (Bueding, 1949; Fairbairn, 1957, 1970; Saz and Bueding, 1966; Saz and Lescure, 1969). Like a number of parasitic helminths (Saz, 1971), this nematode produces succinate (derived from malate) and a variety of fatty acid-end products as the result of carbohydrate catabolism (Saz and Bueding, 1966). Malate, formed in the cytosol by CO 2 fixation into glycolytically formed oxalacetate, serves as the anaerobic mitochondrial substrate in A. suum muscle (Bueding and Saz, 1968; Saz and Lescure, 1969). Upon entering the mitochondrion, malate undergoes a dismutation reaction (Bueding and Saz, 1968; Saz and Lescure, 1969). One arm of the dismutation is catalyzed by the malic enzyme resulting in the formation of pyruvate, CO 2 and NADH. Malate is also converted to fumarate by fumarase that, in turn, is reduced to succinate by the NADH requiring, electron transport coupled, fumarate reductase (Fioravanti and Saz, 1980). Studies by Rew and Saz (1974) indicated that both the malic enzyme and fumarase are localized in the mitochondrial IMS of A. suum. Thus, upon entering the mitochondrion, the oxidative decarboxylation of malate, by the NAD + -utilizing malic enzyme, would result in NADH formation within this space. Conversion of malate to fumarate also would occur in the IMS. Since, it is expected that the reduction of fumarate to succinate occurs on the matrix side of the mitochondrial IM, a translocation of reducing equivalents from NADH in the IMS to matrix

22 13 NAD +, producing the required NADH for electron transport, would be needed inasmuch as NADH is impermeable to the eukaryotic mitochondrial IM (Lehninger, 1951). Consistent with the considerations of Rew and Saz (1974), Kohler and Saz (1976) presented evidence demonstrating that A. suum mitochondria are capable of translocating reducing equivalents from NADH in the IMS to matrix NAD +. These data, therefore, support the occurrence of an IMassociated, hydride-translocating mechanism in the adult A. suum system. Both Fioravanti and Saz (1976) and Kohler and Saz (1976) suggested that this hydride transfer is physiologically accomplished by an IM-associated NADH NAD + transhydrogenation mechanism. Subsequently, Kohler (1977) reported that fumarate does not penetrate the mitochondrial IM of A. suum. If fumarate does not penetrate the IM of Ascaris, the IM localization of fumarase reported by Rew and Saz (1974) versus the data of Kohler (1977) presents somewhat of a dilemma. Nevertheless, the data concerning the ascarid IM-associated NADH NAD transhydrogenation (Rew and Saz, 1974; Fioravanti and Saz, 1976; Kohler and Saz, 1976) remain consistent with the notion that a mechanism(s) exists whereby reducing equivalents arising in the IM are translocated across the IM to matrix NAD +. + Komuniecki and Saz (1979) presented convincing data that the mitochondrial NADH + NAD transhydrogenation in adult A. suum mitochondria appears to be associated predominately with the lipoamide dehydrogenase system. For another parasitic helminth, viz., the adult cestode Hymenolepis diminuta, Walker and Fioravanti (1995) presented evidence that the mitochondrial NADH NAD + transhydrogenation reaction can result from the catalytic activities of both lipoamide dehydrogenase and NADH dehydrogenase. Given these latter findings, the potential association of mitochondrial NADH NAD + transhydrogenation activity resulting from a catalytic activity associated with NADH dehydrogenase as well as lipoamide dehydrogenase

23 14 became apparent. In the present study, data are presented indicating that an NADH NAD + transhydrogenation reaction in adult A. suum mitochondria is catalyzed by not only lipoamide dehydrogenase, but by NADH dehydrogenase as well. Materials and Methods Mitochondrial preparation Adult Ascaris suum were obtained from J.H. Routh Packing in Sandusky, Ohio. Muscle was dissected from female ascarids and mitochondria were extracted essentially as described by Fioravanti and Saz (1976). Muscle tissue was minced in mitochondrial medium (10 ml/g tissue) consisting of 250 mm sucrose, 15 mm ethylenediaminetetracetate (EDTA), and 10 mm Trishydroxymethylaminomethane-HCl (ph 7.5). Thereafter minced muscle was homogenized using a motorized Potter-Elvehjem homogenizer equipped with a Teflon pestle. Removal of cellular debris was accomplished via centrifugation of the homogenates at 482 x g for 10 min. Mitochondria were obtained from the resulting supernatant fraction by centrifugation at 9770 x g for 30 min. Extractions were performed at 4⁰ C. Isolated mitochondria were suspended in mitochondrial medium (~ 1 ml) and stored frozen. Enzyme Assays For enzymatic assays, mitochondria were thawed and diluted (1:5 v/v) with mitochondrial medium. Thereafter, the organelles were sonically disrupted (3-5 sec bursts with 30 sec cooling intervals) using a Branson sonifier, equipped with a microtip, at a power setting of 20W unless otherwise noted. When needed, mitochondria were separated into insoluble and soluble fractions by centrifugation of disrupted organelles at 257,320 x g for 60 min. Isolated fractions were suspended in 0.4 ml of mitochondrial medium and stored frozen.

24 15 NADH NAD + transhydrogenation was assessed by measuring acetylpyridine (AcPyAD) reduction at 375 nm (Fioravanti, 1981). In addition to enzyme, the 1.0 ml assay volume contained the following in µmoles: NADH, 0.24; AcPyAD, 0.6; and either sodium acetate, sodium phosphate, or Tris-HCl, 100 at the ph indicated. NADH dehydrogenase was evaluated by measuring the rate of ferricyanide reduction at 410 nm (Walker and Fioravanti, 1995). In addition to enzyme, the 1.0 ml assay volume contained the following in µmoles: NADH, 0.24; potassium ferricyanide, 0.6; and either sodium acetate, sodium phosphate, or Tris-HCl, 100 at the ph indicated. Lipoamide dehydrogenase was assessed by measuring NADH oxidation at 340 nm as described by Komuniecki and Saz (1979). In addition to enzyme and 3.8 % ethanol the 1.0 ml assay volume contained the following in µmoles: NAD, 0.1; NADH, 0.24; lipoamide, 2.5; EDTA, 2.0; and either sodium acetate, sodium phosphate, or Tris-HCl, 100 at the ph indicated. Thermal Lability Thermal lability profiles were performed using disrupted A. suum mitochondria as the source of enzyme activities. Mitochondria were suspended in approximately μl of mitochondrial medium and were heated in 5 min. intervals from 25⁰ - 95⁰ C. Termination of heat treatment was accomplished by quenching the samples at 4⁰ C. Protein determinations were performed according to the method of Bradford (1976) using crystalline bovine serum albumin as the standard. Spectrophotometric assays were performed at 25⁰ C using a Shimadzu UV-1700 series spectrophotometer.

25 16 Statistical evaluations were performed using JMP 8 software. Significant differences (P < 0.05) in the comparisons of enzyme activities were established by one-way ANOVA. Differences in the mean were established by a student s t test. Results An evaluation of the effects of ph on the catalysis of the NADH NAD + transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities by disrupted A. suum mitochondria was performed and the data obtained are presented in Figure 2.1. Although differences in the activities of the NADH NAD + transhydrogenation versus NADH dehydrogenase were noted at ph 9.0 and 8.5, with a decrease in ph from 8.0 to 7.0 both the NADH NAD + transhydrogenation and NADH dehydrogenase activities were essentially unchanged and virtually equivalent. Lipoamide dehydrogenase activity was relatively low at ph 8.5 and 8.0, but gradually increased, rising to the levels of the other reactions at ph 7.0 (Fig. 2.1). With further medium acidification, lipoamide dehydrogenase activity increased and peaked at ph 6.0. Thereafter, a decline in this activity was noted with nearly a complete loss of activity at ph 4.5. Interestingly, between ph 7.0 and ph 6.0, NADH dehydrogenase activity decreased, whereas NADH NAD + transhydrogenation activity essentially fell at a midpoint between NADH and lipoamide dehydrogenase. All three reactions displayed similar activity levels at ph 5.5. Subsequently, transhydrogenation activity decreased in a fashion similar to the lipoamide dehydrogenase, but not to the same extent, while NADH dehydrogenase activity increased immensely, exhibiting a ph 4.5 peak before decreasing at ph 4.0 (Fig. 2.1).

26 Activity (μmol/min/mg protein) ph Figure 2.1. The effects of ph on NADH NAD + transhydrogenation (NT), NADH dehydrogenase (ND), and lipoamide dehydrogenase (LD) activities of adult Ascaris suum mitochondria. Symbols used are:- - NADH NAD + transhydrogenation; - - NADH dehydrogenase; - - lipoamide dehydrogenase. Activity assessments at each ph were performed in triplicate and the means are presented here. A mean of 0.08 mg protein was employed for assays. Values designated for points in insert at given ph are as follows: ph 6.0, NT 0.85 ± 0.058; ND 0.53 ± 0.103; LD 1.20 ± 0.052; ph 6.5, NT 0.76 ± 0.045; ND 0.45 ± 0.108; LD 1.02 ± Values in insert are significantly different from each other ± SE; N=3 for each point.

27 18 The comparative thermal labilities of the NADH NAD + transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities of disrupted A. suum mitochondria were evaluated under acidic and basic assay conditions. Presented in Figure 2.2 are the data obtained employing acidic conditions of assay. For these assessments the acidic conditions employed were those yielding apparent maximal activities for the NADH NAD + transhydrogenation and lipoamide dehydrogenase, i.e., ph 5.5 and 6.0, respectively. Because NADH dehydrogenase activity proved somewhat difficult for routine measurements, activity was assessed at ph 5.0. At this ph linear measurements of activity were more amenable. As presented in Figure 2.2, with some variation all three activities were essentially similar in their lack of thermal lability up to a temperature of 55⁰ C when assessed under acidic conditions. The most notable lability at 65⁰ C was that of the NADH NAD + transhydrogenation and reflected a decline in activity that was made more apparent at 75⁰ C and then 85⁰ C, with complete inactivation at 95⁰ C. Conversely a mild increase in the lipoamide dehydrogenase activity was observed at 65⁰ C with a peak of activity at 75⁰ C. However, NADH dehydrogenase activity began to decline at 75⁰ C, but differed from the other activities when compared to controls. A greater degree of NADH dehydrogenase degradation under acidic conditions was noted at 85⁰ C (Fig. 2.2). The latter degree of degradation was intermediate between that noted for lipoamide dehydrogenase and the NADH NAD + transhydrogenation. As with the NADH NAD + transhydrogenation both the NADH dehydrogenase and lipoamide dehydrogenase activities were completely inactivated at 95⁰ C (Fig. 2.2). The comparative thermal labilities of the NADH NAD + transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities of disrupted A. suum mitochondria also

28 19 were evaluated under basic conditions of assay, i.e., at ph 8.0 and the data are presented in Figure 2.3. The NADH dehydrogenase and lipoamide dehydrogenase both displayed a degree of lability that was apparent up to 45⁰ C. Thereafter, a dramatic increase in lipoamide dehydrogenase activity was noted with a peak occurring at 65⁰ C. In contrast, NADH dehydrogenase activity markedly declined at 55⁰ C, and continued with even a greater activity loss from 65⁰ C - 85⁰ C before an almost complete loss of activity at 95⁰ C (Fig. 2.3). Whereas the NADH NAD + transhydrogenation reaction displayed essentially no lability up to 45⁰ C, this activity declined between 55⁰ C and 75⁰ C in a fashion that was intermediate between lipoamide dehydrogenase and NADH dehydrogenase activities. Thereafter, the transhydrogenation activity declined similarly to the decline noted for lipoamide dehydrogenase before a complete loss of activity at 95⁰ C (Fig. 2.3).

29 20 Figure 2.2. Thermal lability profiles of the NADH NAD + transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities of adult Ascaris suum mitochondria assessed under acidic conditions. Symbols used and the ph of the assay medium were: - - NADH NAD + transhydrogenation, ph 5.5; - - NADH dehydrogenase, ph 5.0; - - lipoamide dehydrogenase, ph 6.0. Control (100%) activities in μmol/min/mg protein were: NADH NAD + transhydrogenation, 1.5; NADH dehydrogenase, 2.9; and lipoamide dehydrogenase, 1.4. Activity assessments at each temperature were performed in triplicate and the means are presented here. A mean of 0.09 mg protein was employed for assays. Values designated for lipoamide dehydrogenase and NADH dehydrogenase differ significantly at 65⁰ C, 75⁰ C, and 85⁰C. Values at these temperatures are significantly different from each other ± SE; N=4 for lipoamide dehydrogenase, N= 3 for NADH NAD + transhydrogenation and NADH dehydrogenase.

30 21 Figure 2.3. Thermal lability profiles of NADH NAD + transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities of adult A. suum mitochondria assayed under basic conditions, i.e., ph 8.0. Symbols used were: - - NADH NAD + transhydrogenation; - - NADH dehydrogenase; - - lipoamide dehydrogenase. Control (100%) activities in μmol/min/mg protein were: - - NADH NAD + transhydrogenation, 0.81; - - NADH dehydrogenase, 0.77; - - lipoamide dehydrogenase, For all assessments, N= 2-3 and the means are presented here. A mean of 0.12 mg protein was employed for assays.

31 22 Adult A. suum mitochondria were separated into insoluble (membranes) and soluble fractions via differential centrifugation. Thereafter, assessments of the NADH NAD + transhydrogenation, lipoamide dehydrogenase, and NADH dehydrogenase activities were made using these fractions. The data obtained are presented in Table 1. All three activities were assessed under acidic and basic conditions. As given in Table 1, all three activities displayed a predominate insoluble association regardless of the ph of assessment. Under either condition tested (i.e., ph 5.5 or 7.5), the NADH NAD + transhydrogenation as well as the NADH dehydrogenase activities were clearly more pronounced in the insoluble fraction, being in excess of 70% of the recovered activities. However, the distribution of lipoamide dehydrogenase activity differed from the distributions of the other two activities when assessments were performed either under acidic or basic conditions with insoluble and soluble localizations being 64% and 36%, respectively under acidic conditions, and 67% and 33%, respectively under basic conditions (Table 1).

32 Table 1. Mitochondrial localizations of NADH NAD + transhydrogenation, lipoamide dehydrogenase, and NADH dehydrogenase activities of A. suum mitochondria. Total Units* % Recovered Activity Reaction ph Insoluble fraction Soluble fraction Insoluble fraction Soluble fraction NADH NAD + transhydrogenation ± ± % 24 % ± ± % 25 % Lipoamide dehydrogenase ± ± % 36% ± ± % 33% NADH dehydrogenase ± ± % 24 % ± ± % 21% * Units express total activity in μmol/min. Values are means ± SE. Lipoamide dehydrogenase values reflect the means of five assessments at ph 5.5 and four assessments at ph 7.5 while all other values are the means of six assessments mg protein was used for membrane fraction assessments while mg protein was used for soluble fraction assessments.

33 24 Both copper chloride (CuCl 2 ) and cadmium chloride (CdCl 2 ) are known inhibitors of A. suum mitochondrial lipoamide dehydrogenase activity (Komuniecki and Saz, 1979). Within this context, assessments of the effects of these inhibitors on the NADH NAD + transhydrogenation and lipoamide dehydrogenase activities of disrupted A. suum mitochondria were performed and these data are given in Table 2. Under either acidic or basic conditions of assessment, CuCl 2 markedly inhibited both activities, when compared to corresponding controls, with the greater inhibitions for both noted under acidic conditions. Under basic conditions of assay, CuCl 2 exerted a greater inhibition on the lipoamide dehydrogenase activity, i.e., 96% versus 62% inhibition. With the addition of EDTA to the assays, CuCl 2 inhibition was clearly relieved for both reactions with the greater relief being noted with the transhydrogenation reaction under basic conditions (Table 2). Similarly, CdCl 2 inhibited both activities under acidic and basic conditions of assay with the greater inhibition noted in terms of the lipoamide dehydrogenase activity. As with CuCl 2, CdCl 2 inhibition was relieved by inclusion of EDTA in the assay system with the greater relief being observed under basic conditions (Table 2). Attempts to evaluate the effects of these inhibitors on the mitochondrial NADH dehydrogenase activity, as assessed by ferricyanide reduction, were unsuccessful, inasmuch as inhibitor additions resulted in a significant precipitation of ferricyanide.

34 Table 2. Effects of inhibitors on activities of Ascaris suum mitochondria assessed under acidic and basic conditions Activity (µmol/min/mg) NADH NAD + transhydrogenation Lipoamide dehydrogenase Addition(s) a b a b None ± 0.062* (13) ± (8) ± (14) ± (7) CuCl ± (5) [97] ± (5) [62] ±0.006 (5) [99] (1) [96] CuCl 2 plus EDTA ± (3) [23] ±0.029 (4) [7] ± (3) [14] ± (3) [+11] CdCl ± (8) [63] (2) [63] ± (5) [97] ±0.003 (4) [94] CdCl 2 plus EDTA ± (6) [14] 0.65 (1) [2] ±0.117(5) [22] ± (3) [22] *Values are means ± SE Number of observations is in parentheses. Percent inhibition compared to untreated sample is in brackets. a- designates acidic assay conditions, ph 5.5. b-designates basic assay conditions, ph 7.5. A mean protein concentration of 0.10 mg protein was used for assessments. Inhibitors were added such that the assay concentration was 0.4 mm while EDTA was 2.0 mm

35 26 Discussion An evaluation of the effects of ph on the NADH NAD + transhydrogenation, lipoamide dehydrogenase, and NADH dehydrogenase activities of disrupted A. suum mitochondria is presented here. Significant differences in activities were observed for all three reactions at ph 6.0 and ph 6.5. Interestingly, at ph 6.0 and ph 6.5, the NADH NAD + transhydrogenation reaction displayed an activity level in between that noted for NADH dehydrogenase and lipoamide dehydrogenase. These differences in activity under acidic assay conditions suggest that the NADH NAD + transhydrogenation reflects an activity that is catalyzed by both the lipoamide dehydrogenase and NADH dehydrogenase systems. It was also noted that the NADH dehydrogenase activity displays a marked increase in activity at ph 4.5 while the other two activities significantly decline in activity at this ph. Rew and Saz (1974) demonstrated the occurrence of two NADH cytochrome c reductase activities; one is associated with the IM and is rotenone-sensitive while the other is associated with the OM and is rotenone-insensitive. In our preliminary studies with the A. suum system, ph evaluations of both the rotenone-insensitive and rotenone-sensitive NADH cytochrome c reductase activities indicated that the rotenoneinsensitive activity simulates the peak of activity noted for the NADH dehydrogenase activity. Accordingly, it is suspected that the large peak in NADH dehydrogenase activity noted under acidic conditions may reflect, at least in part, an NADH dehydrogenase component of the rotenone-insensitive NADH cytochrome c reductase activity and warrants further investigation. The thermal profiles of the NADH NAD + transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase reactions were determined. Because of the effects of ph observed on these reactions, thermal lability assessments entailed evaluations under both acidic and basic conditions. All three reactions ceased after prolonged exposure to heat, in keeping with an

36 27 enzymatic catalysis of these reactions. While under acidic conditions differences in activities were not apparent until samples were incubated at 65⁰ C and 75⁰ C. At these latter temperatures, lipoamide dehydrogenase and NADH dehydrogenase activities differed from each other significantly. At 85⁰ C, lipoamide dehydrogenase activity differed significantly from both the NADH dehydrogenase and NADH NAD + transhydrogenation activities. Furthermore, the corresponding experiments performed under basic conditions indicate marked differences in activities when samples were heated at 55⁰ C and above. Taken together, these data also support the notion that the NADH NAD + transhydrogenation is the product of two catalytic entities; viz., NADH dehydrogenase and lipoamide dehydrogenase. Walker and Fioravanti (1995) observed that both the mitochondrial lipoamide dehydrogenase and NADH dehydrogenase systems of the adult, anaerobic cestode, Hymenolepis diminuta, are responsible for the catalysis of the NADH NAD + transhydrogenation. In view of the present findings, and those reported by Walker and Fioravanti (1995), it would appear that the catalysis of a mitochondrial NADH NAD + transhydrogenation in the mitochondria of parasitic helminths may be a more wide spread phenomenon. Employing both insoluble and soluble fractions derived from isolated A. suum mitochondria, assessments of the NADH NAD + transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase were performed. The data obtained demonstrated that all three activities have a predominant association with the mitochondrial insoluble fraction. However, despite this predominant association, and regardless of the ph of assessment, there was less lipoamide dehydrogenase activity associated with the insoluble fraction, and more of this activity in the soluble fraction, than noted for the other two activities. Indeed, the distributions of the other two activities, i.e., NADH dehydrogenase and NADH NAD + transhydrogenation, were

37 28 essentially the same. These data again suggest that the NADH NAD + transhydrogenation is an enzymatic activity that results from both the lipoamide dehydrogenase and NADH dehydrogenase systems. It has been reported that the lipoamide dehydrogenase and NADH NAD transhydrogenation catalyzed by A. suum mitochondria are significantly inhibited by the presence of CuCl 2 and CdCl 2 (Komuniecki and Saz, 1979). In agreement with their findings, both these salts were also found to be significantly inhibitory for the A. suum mitochondrial NADH NAD + transhydrogenation and lipoamide dehydrogenase activities. Furthermore, the inhibitions exerted by these salts were relieved by addition of the chelating agent EDTA to the + assay system as observed by Komuniecki and Saz (1979). However, in the present study, CuCl 2 seemed to be a less effective inhibitor on the NADH NAD + transhydrogenation reaction than the lipoamide dehydrogenase reaction under basic conditions. Similarly, CdCl 2 was a less effective inhibitor of the NADH NAD + transhydrogenation reaction in comparison to lipoamide dehydrogenase under both acidic and basic assay conditions. Interestingly, under acidic conditions of assessment, CdCl 2 did not inhibit the NADH NAD + transhydrogenation reaction as potently as CuCl 2. These data suggest that there are differences between the transhydrogenation and lipoamide dehydrogenase reactions in regards to the degree of inhibition exerted by these salts. These differences are of note, inasmuch as one would expect that if the NADH NAD + transhydrogenation reaction is a result of lipoamide dehydrogenase, that both activities would be inhibited to the same degree. Thus, there would appear to be another entity responsible for the catalysis of the mitochondrial NADH NAD + transhydrogenation reaction in A. suum.

38 29 Based upon the collective findings obtained in the present study, a model is proposed denoting the mitochondrial energetics of adult A. suum, and this model is given in Figure 2.4. As indicated, Rew and Saz (1974) presented compelling data indicating that both the malic enzyme and fumarase of adult A. suum mitochondria are predominantly localized in the mitochondrial IMS. Figure 2.4. Proposed roles of lipoamide dehydrogenase (LD) and NADH dehydrogenase (ND) as NADH NAD + transhydrogenation mechanisms in the mitochondrial energetics of adult Ascaris suum. Abbreviations used are as follows: OM, outer membrane; IM, inner membrane; IMS, intermembrane space; ME, malic enzyme; F, fumarase; FR, fumarate reductase.

39 30 These findings were supported both by fractionation of isolated A. suum mitochondria coupled to sucrose gradient centrifugation, as well as by evaluating the release of enzyme activities noted when increasing digitonin concentrations were applied to the isolated nematode organelles (Rew and Saz, 1974). The findings of Rew and Saz (1974) were made emphatic by assessments of biochemical markers for the OM, IMS, IM, and matrix fractions. Furthermore, the efficacy of fractionation by the techniques used here for the A. suum organelles was verified using mammalian (rat liver) mitochondria subjected to the same techniques and marker enzyme assessments (Rew and Saz, 1974). In contrast to these findings, Kohler et al. (1983), using digitonin treatment of isolated A. suum mitochondria and marker enzymes (although without a mammalian organelle control), reported that fumarase was chiefly localized in the mitochondrial matrix and that malic enzyme was equally distributed between the IMS and matrix. Furthermore, Kohler (1977) indicated that fumarate did not traverse the ascarid IM. Regardless of these differences noted in a comparison of the Rew and Saz (1974) and Kohler et al. (1983) studies, the question of usage of malic enzyme dependent NADH formation by the ascarid system remains. The physiological utilization of NADH accumulated in the IMS by the ascarid electron transport system still necessitates a transmembrane transfer of reducing equivalents. Thus, the need for a transhydrogenation(s) reaction remains, and our data suggest that this is accomplished via NADH dehydrogenase and lipoamide dehydrogenase activities as given in Figure 2.4. In this regard it has been noted that IM associated, but externally oriented NADH dehydrogenase systems have been found in plants, animals, fungi, and protists, and also with respect to bacterial cellular membranes (Sotthibandhu and Palmer, 1975; Day et al., 1976; Brailovskaya et al., 2003; Rasmusson et al., 2008). A number of anaerobic, succinate-forming parasitic helminths are known to display a mitochondrial NADH NAD + transhydrogenation

40 31 reaction; examples include, Spirometra mansoides (Cestoda), Hymenolepis microstoma (Cestoda), Taenia crassiceps (Cestoda), and Setaria digitata (Nematoda), as reviewed by Fioravanti and Vandock (2009). Thus, it would be of interest to determine the impact of lipoamide dehydrogenase and NADH dehydrogenase on transmembrane hydride translocation in these organisms. Conclusions and Future Directions The adult intestinal nematode of swine, Ascaris suum, exhibits a mitochondrial NADH NAD + transhydrogenation reaction, which transfers reducing power in the form of hydride ions across the IM to matrix NAD +, thus regenerating the NADH needed to drive the NADH requiring, electron transport coupled, fumarate reductase (Fioravanti and Saz, 1976; Kohler and Saz, 1976; Fioravanti and Saz, 1980). This transhydrogenation reaction has been demonstrated to be the result of lipoamide dehydrogenase in A. suum (Komuniecki and Saz, 1979). The mitochondrial NADH NAD + transhydrogenation reaction is also present in the intestinal helminth of rats, Hymenolepis diminuta (Cestoda). However, the reaction in H. diminuta has been shown to result from both the lipoamide and NADH dehydrogenase activities (Walker and Fioravanti, 1995). Based on the findings in H. diminuta, studies to further assess the origin(s) of the NADH NAD + transhydrogenation reaction in adult A. suum were undertaken. The purpose of this study was to assess if the mitochondrial NADH NAD transhydrogenation reaction in adult A. suum results from lipoamide dehydrogenase and NADH dehydrogenase, as it does in H. diminuta. Comparative evaluations of the NADH NAD + transhydrogenation, lipoamide dehydrogenase, and NADH dehydrogenase reactions were carried out using disrupted A. suum mitochondria as the source of enzymes. These three activities were +