Carmen F. Fioravanti

COUPLING OF MITOCHONDRIAL NADPH: NAD TRANSHYDROGENASE WITH ELECTRON TRANSPORT IN ADULT HYMENOLEPIS DIMINUTA

The mitochondrial electron transport system of adult Hymenolepis diminuta exhibited an apparent specificity in terms of reduced pyridine nucleotide utilization. The preferred substrate for both the minor oxidase and the physiologically required fumarate reductase system was NADH. Intramitochondrial reducing equivalents, needed for phosphorylation via the anaerobic, electron transport-dependent, fumarate reductase, were generated as NADPH by the action of the cestodes NADP-specific malic enzyme. However, H. diminuta mitochondria catalyzed an NADPH : NAD transhydrogenation which would serve in hydride ion transfer from NADPH to NAD, thereby producing NADH required for the anaerobic, electron transport mechanism. Accordingly, NADPH utilization was increased when NAD was added to the mitochondrial system. The most significant increase occurred in the presence of both NAD and fumarate. These data indicate a coupling of the NADPH : NAD transhydrogenase with mitochondrial electron transport. This coupling of the transhydrogenase with electron transport was demonstrated using disrupted mitochondria and mitochondrial membrane preparations. Under conditions of reduced oxygen tension, the coupling of the transhydrogenase to fumarate reduction was apparent. In adult Ascaris suum, where the malic enzyme physiologically utilizes NAD, the mitochondria differ from those of H. diminuta because NADPH : NAD transhydrogenase activity was minimal under the conditions of assay. The rate of NADPH utilization by the nematode mitochondrial system is not increased appreciably in the presence of NAD when either oxygen or fumarate serves as the acceptor.

Mitochondrial malate dehydrogenase, decarboxylating ("malic" enzyme) and transhydrogenase activities of adult Hymenolepis microstoma (Cestoda).

Adult H. microstoma mitochondria catalyzed a malate dehydrogenase, decarboxylating (malic enzyme) activity. This malic enzyme was found as a soluble component of the mitochondrion, was specific for NADP, and required a divalent cation with Mn++ ion yielding the greatest activity. The H. microstoma malic enzyme could fulfill the need for generating intramitochondrial reducing equivalents required for electron transport. The H. microstoma mitochondria also exhibited an NADPH:NAD transhydrogenation reaction. The electron transport system of this cestode was apparently specific for NADH both in terms of the rotenone-sensitive oxidase and fumarate reductase systems. Electron transport-associated NADPH oxidation was increased markedly with the addition of NAD to the system. Coupling of NADPH utilization to fumarate reduction, in the presence of NAD, was apparent under conditions of reduced oxygen tension. This was consistent with the presence of the NADPH:NAD transhydrogenase which catalyzed a transfer of reducing equivalents from NADPH to NAD, producing NADH for electron transport function. The data presented suggest that H. microstoma mitochondria can engage in an anaerobic, electron transport-associated production of succinate, and presumably concomitant phosphorylation. Malate may serve as the mitochondrial substrate supplying reducing equivalents for electron transport via the activity of the malic enzyme coupled to the NADPH:NAD transhydrogenase. In addition to the NADPH:NAD transhydrogenase activity, H. microstoma mitochondria catalyzed an NADH:NAD transhydrogenation.

Energy-linked mitochondrial pyridine nucleotide transhydrogenase of adult Hymenolepis diminuta

Employing phosphorylating submitochondrial particles as the source of pyridine nucleotide transhydrogenase, the occurrence of an energy-linked NADH----NADP+ transhydrogenation in the adult cestode Hymenolepis diminuta was demonstrated. The isolated particles displayed rotenone-sensitive NADH utilization and the reversible transhydrogenase, with the NADPH----NAD+ transhydrogenation being more prominent. Although not inhibiting the NADPH----NAD+ reaction, rotenone, but not oligomycin, inhibited the catalysis of NADH----NADP+ transhydrogenation. In the presence of rotenone, Mg2+ plus ATP stimulated by more than 3-fold NADH----NADP+ transhydrogenation. This stimulation was ATP specific and was abolished by EDTA or oligomycin. Succinate was essentially without effect on the NADH----NADP+ reaction. These data demonstrate the occurrence of an energy-linked transhydrogenation between NADH and NADP+ with energization resulting from either electron transport-dependent NADH oxidation or ATP utilization via the phosphorylating mechanism in accord with the preparation of phosphorylating particles. This is the first demonstration of an energy-linked transhydrogenation in the parasitic helminths and apparently in the invertebrates generally.

Mitochondrial hydrogen peroxide formation and the fumarate reductase of Hymenolepis diminuta.

The catalysis of hydrogen peroxide accumulation by the mitochondrial, membrane-associated NADH oxidase and less active succinoxidase of adult Hymenolepis diminuta was confirmed. NADH-dependent peroxide formation by isolated mitochondrial membranes occurred at about half the coincident rates of NADH and oxygen utilization, whereas succinate-dependent peroxide formation accounted for approximately 40% of the oxygen consumed. These findings, coupled with evaluations of the oxidases, indicated that both systems use in common 2 mechanisms for oxygen reduction, 1 of which is peroxide-forming. Neither system was sensitive to cyanide, azide, or antimycin A. Rotenone inhibition of NADH oxidation resulted in equivalent decreases in oxygen consumption by the peroxide-forming and nonperoxide-forming mechanisms. In contrast, malonate inhibition occurred via disruption of the peroxide-forming mechanism. Fumarate stimulated membrane-catalyzed NADH oxidation, despite aerobic conditions, and this fumarate reductase was rotenone-sensitive. NADH- or succinate-dependent peroxide formation virtually was abolished and oxygen consumption was minimal in the presence of fumarate. Malonate also inhibited fumarate-dependent NADH oxidation and succinate-dependent peroxide formation/oxygen consumption. Collectively, these findings clearly indicate that NADH- or succinate-dependent hydrogen peroxide accumulation involves the malonate-sensitive fumarate reductase, in the absence of fumarate. A model of the H. diminuta electron transport system is presented.

PHOSPHOLIPID DEPENDENCE OF THE HYMENOLEPIS DIMINUTA MITOCHONDRIAL NADPH:NAD TRANSHYDROGENASE

The mitochondrial, membrane-associated, nonenergy -linked NADPH:NAD transhydrogenase of adult Hymenolepis diminuta exhibited a phospholipid dependence. This lipid dependence was suggested when mitochondrial membranes were subjected to organic solvent or phospholipase treatments. Although hexane extraction of lyophilized membranes enhanced transhydrogenase activity, subsequent aqueous acetone extraction significantly inhibited the transhydrogenase. An acetone/water-dependent extraction of phospholipids was reflected in the phosphorus content of released material. Incubation of mitochondrial membranes with phospholipase A2 or C markedly reduced transhydrogenase activity, and phospholipase A2 treatment resulted in the greater reduction in activity. The mitochondrial, membrane-associated, NADH-utilizing oxidase and fumarate reductase activities were diminished significantly by hexane as well as phospholipase treatments. Phospholipase A2 caused the greater inhibition of the NADH-utilizing systems. Thus, in contrast to the transhydrogenase, neutral lipids and phospholipids apparently were required by the electron transport-coupled activities. The transhydrogenase activity of organic solvent- or phospholipase-treated membranes was not stimulated effectively by phospholipid addition. However, phospholipid-dependent stimulation of transhydrogenase was accomplished employing a partially lipid-depleted preparation of the enzyme obtained by detergent treatment and ammonium sulfate precipitation. Of the phospholipids tested, only phosphatidylcholine significantly stimulated transhydrogenase activity. The stimulation noted with phosphatidylcholine was not duplicated by cholate or deoxycholate.

The in vitro effects of farnesol and derivatives on Hymenolepis diminuta.

Employing an in vitro maintenance system, in which 8-day-old Hymenolepis diminuta survives for 24 hr (Fioravanti and MacInnis, 1976), it was found that farnesol or farnesal supplementation of the medium had no beneficial effects on maintenance and these substances induced necrosis at higher concentrations. Similar experiments utilizing Schillers (1965) culture system demonstrated that neither farnesol, farnesal, nor farnesyl methyl ether exhibited growth promoting effects and were toxic to the worms at higher concentrations. In addition, neither the 2-cis, 6-trans nor the 2-trans, 6-trans-isomers of farnesol promoted growth in the Schiller system and at higher concentrations resulted in severe necrosis within 24 hr.

Mitochondrial NADH-->NAD transhydrogenation in adult Hymenolepis diminuta.

Adult Hymenolepis diminuta mitochondria catalyze a transhydrogenation reaction between NADPH and NAD and between NADH and NAD. The NADPH-->NAD reaction is catalyzed by an inner membrane-associated pyridine nucleotide transhydrogenase, whereas the NADH-->NAD reaction is ostensibly catalyzed by another system(s). The source(s) of NADH-->NAD activity was evaluated by assessments of its intramitochondrial distribution and thermal lability and by comparisons with the distribution/thermal lability of NADH dehydrogenase, lipoamide dehydrogenase, and NADPH-->NAD transhydrogenase. The occurrence of NADH and lipoamide dehydrogenase components was readily demonstrable. Like NADPH-->NAD transhydrogenase, NADH dehydrogenase was essentially membrane bound. Lipoamide dehydrogenase and NADH-->NAD activities were, at different levels, in the membrane and soluble fractions. Based on thermal profiles, NADH and lipoamide dehydrogenase differed from each other and from NADPH-->NAD transhydrogenase. Although the NADH-->NAD profile closely paralleled that for lipoamide dehydrogenase, it also was similar to the NADH dehydrogenase profile. Collectively, these data are consistent with the supposition 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.

Metabolic indices for evaluating the in vitro maintenance of Hymenolepis diminuta in the presence and absence of various additives.

An in vitro maintenance system for H. diminuta was devised by modifying the cultivation procedure of Schiller (1965). In this diphasic maintenance system, tissue culture medium (Triple Eagles or NCTC-135) was used, in lieu of whole blood, as the 30% supplement to the agar phase. This provides a more defined system suitable for studying the effects of various additives. Morphological criteria were established which aided in assessing the efficacy of media used for the maintenance of 6- and 8-day-old H. diminuta. When successfully maintained, worms exhibited an intact scolex and neck region, undulatory movements along the strobila as well as integumentary and strobilar integrity. A more sensitive method for evaluating the maintenance of 8-day-old worms employed metabolic indices. Wet weight, protein and glycogen levels for 8-day-old H. diminuta served as base-line data allowing estimation of protein and glycogen contents of each worm prior to maintenance. Following maintenance, ratio of final to initial protein and final to initial glycogen levels (metabolic indices). A metabolic index approaching or exceeding unity suggested a reasonably intact metabolism. The addition of sodium taurocholate to the maintenance media appeared beneficial to the worms by prolonging the retention of normal signs. A combination of additives, taurocholate-nucleosides-lipids, improved the maintenance of H. diminuta for periods exceeding 24 hr as determined by observational criteria and metabolic indices. However, addition of a lipid mixture, or a lipid mixture prepared with a low concentration of taurocholate was not beneficial over a 24 hr period. The maintenance system, observational criteria, base-line data and metabolic indices should be useful for future in vitro studies requiring long-term incubation.

The identification and characterization of a prenoid constituent (farnesol) of Hymenolepis diminuta (Cestoda).

Abstract 1. 1. Some non-saponifiable materials from H. diminuta were characterized with particular emphasis given to the prenoid alcohol, farnesol. 2. 2. The cestodes non-saponifiables contained farnesol which was identified and quantified using column chromatography, gas-liquid chromatography, mass spectrometry, and isotope dilution. 3. 3. Only the 2- trans , 6- trans isomer of farnesol was found, being present at a mean concentration of 0.65 μg/g wet weight tissue. No 2- cis , 6- trans isomer was detected. 4. 4. Two non-prenoid materials with apparent molecular weights of 224, possibly representing isomeric variants, were found. One of these materials co-chromatographed with 2- cis , 6- trans farnesol on a 15% FFAP gas-liquid chromatographic column. 5. 5. The cestodes non-saponifiables contained copious quantities of cholesterol.

Hydroxymethylglutaryl coenzyme A reductase activity of adult Hymenolepis diminuta.

The occurrence of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase in adult Hymenolepis diminuta was demonstrated. This activity was negligible in the cestodes cytosolic fraction but was noted when the mitochondrial or microsomal fraction served as the enzyme source. The predominant localization of HMG-CoA reductase activity was with the microsomal fraction. This fraction did not contain appreciable mitochondrial contamination based on the distribution of marker enzymes. The enzymatic nature of HMG-CoA conversion to mevalonic acid by either fraction was apparent because the reaction was heat labile and responded linearly to time of assay and protein content. The enzymatic reduction of HMG-CoA absolutely required NADPH when either fraction was assayed. The lesser activity of the mitochondrial fraction was membrane-associated. The predominant localization of HMG-CoA reductase activity with microsomal membranes and its separation with the membranous component of the mitochondrial fraction suggest that mitochondrial activity reflects the presence of microsomal membranes. In its predominant localization and pyridine nucleotide requirement, the cestodes HMG-CoA reductase activity resembles that of mammalian systems. The finding of HMG-CoA reductase provides an enzymatic mechanism for the intermediate conversion of HMG-CoA to mevalonic acid that would be needed for acetate-dependent isoprenoid lipid synthesis by adult H. diminuta.