Comp. Biochem. Physiol.,
1976,
Vol.
53B,
pp.
141
to
144.
Pergamo,1 Press. Printed in Great Britain
NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE IN ENTAMOEBA HISTOLYTICA, A PROTOZOAN LACKING MITOCHONDRIA DAN R. HARLOW, EUGENEC. WEINBACHAND LOUIS S. DIAMOND Section on Physiology and Biochemistry, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Received 21 October 1974) Abstract--1. A nicotinamide nucleotide transhydrogenase exhibiting both energy-linked and nonenergylinked reactions is present in intact and subcellular fractions of Entamoeba histolytica. 2. Both reactions are localized primarily in the 110,000g sedimented fraction. 3. Even though E. histolytica is eukaryotic, it contains no mitochondria; hence the amoebal enzyme is non-mitochondrial in contrast to mammalian cells where it is located on the inner mitochondrial membrane. 4. Although the enzyme is non-mitochondrial in the amoeba, it responds to palmitoyl-CoA and Ca2÷ in the same way that the mammalian enzyme does. 5. E. histolytica is the first protozoan species in which nicotinamide nucleotide transhydrogenase has been found.
INTRODUCTION NICOTINAMIDE nucleotide transhydrogenase catalyzes hydrogen exchange between NAD +* and NADP + by the reaction: NADH + NADP + ~ NAD + + NADPH (Ernster & Lee, 1964; Colowick et al., 1952). The enzyme has been reported from bacterial (Clark et al., 1955), plant (Hasson & West, 1973) and animal cells (Kaplan et al., 1953). In animal cells, the transhydrogenase is located on the inner mitochondrial membrane (Teixeira da Cruz et aL, 1971) and thus is at least structurally associated with the aerobic metabolism of the cell. Entamoeba histolytica, a tissue invading protozoan of the human intestine, liver and other sites is considered to be an anaerobe (Lwoff, 1951) although recent evidence indicates that it may be a facultative anaerobe. When oxygen is present, the amoeba will utilize it (Montalvo et al., 1971; Weinbach & Diamond, 1974); however, it grows and reproduces under anaerobic conditions (von Brand, 1973). Although the amoeba produces fermentation products such as ethanol and CO2 both in the presence and absence of oxygen, the amounts produced in the presence of oxygen are less than are produced anaerobically (Montalvo et al., 1971). Even though the electron carriers under aerobic conditions have not been characterized, the absence of mitochondria (for review see m
* Abbreviations: NAD +, nicotinamide adenine dinucleotide; NADP ÷, nicotinamide adenine dinucleotide phosphate; ATP, adenosine triphosphate; Tris, tris(hydroxymethyl)aminomethane; Palmitoyl-CoA, palmitoyl coenzyme A; AcPyAD÷, acetylpyridine adenine dinucleotide; AcPyADP ÷ acetylpyridine adenine dinucleotide phosphate; NADH, reduced nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; AcPyADH, reduced acetylpyridine adenine dinucleotide; AcPyADPH, reduced acetylpyridine adenine dinucleotide phosphate; PCMB, parachloromercuribenzoate. , .l~.p.la)53,2B A
von Brand, 1973) and cytochromes (Hilker & White, 1959) as well as the lack of a functional tricarboxylic acid cycle (Weinbach & Diamond, 1974) indicate that typical electron transport mechanisms are not present. We have found a NAD(P)H diaphorase which is more active with NADPH than with NADH (in some preparations, the NADH activity approached zero). In the absence of typical aerobic mechanisms for generating ATP, the amoeba presumably must rely on other mechanisms such as glycolysis for ATP production. The possibility that NADH produced in glycolysis could be oxidized by aerobic mechanisms which differ from those found in typical aerobic cells was considered in investigating the observed oxygen utilization. One such possibility is that the oxidation of NADH is mediated by the transhydrogenase; the oxidation of the NADPH produced by this reaction is catalyzed by the diaphorase resulting finally in the reduction of oxygen or other electron carriers. Such an aerobic supplemental NADH oxidation system is consistent with the observed oxygen utilization and diminished quantities of fermentation products produced aerobically. Ryley (1967) noted that a nicotinamide nucleotide transhydrogenase would be of importance in protozoan cells for maintaining the redox balance between the nicotinamide nucleotides, although he reported that the enzyme had not been found in any protozoan. MATERIALS AND METHODS Entamoeba histolytica trophozoites were grown in axenized culture according to the method of Diamond (1968). The amoebae were harvested at 72 hr and washed twice by centrifugation in buffered sucrose (50 mM Tris, pH 7.4; 250 mM sucrose). Suspensions of washed intact cells or cells homogenized in buffered sucrose were assayed for nicotinamide nucleotide transhydrogenase according to the method of Kaplan (1967). The analogs of NAD ÷, 141
142
D. R. HARLOW, E. C. WEINBACH AND L. S. DIAMONI)
Table 1. Transhydrogenase of intact E. histolytica Conditions
nmoles reduced, min ~- mg proteinNADPH ~ AcPyAD ~ NADH ~ AcPyADP +
Fresh After 2 min After 10 min. After 2 mM ADP After 2 mM ATP Overnight at 4°C After 2 mM ATP Overnight at - 10°C After 2 mM ATP 30 Days at -10c'C After 2 mM ATP
7.4 6.8 3.l 4.2 11.0 12.0 10.8 16.3
2.4 1.6 30 4-3 2.2 3.2 2.5 3.4 0.9 0-7
The assay medium contained 100/~moles potassium phosphate buffer, pH 6-5; 1/~mole nicotinamide nucleotides and analogs; and 0.1 ml of the appropriate sample (1.8 to 2.7 mg protein) in a final vol of 1.0 ml. The suspensions were assayed at room temp as described by Kaplan (1967).
NADP ÷ and the palmitoyl--CoA were obtained from P-L Biochemicals, Milwaukee, U.S.A. Native coenzymes were purchased from Sigma Chemical Company, St. Louis, U.S.A. RESULTS
The acetylpyridine analogs of N A D + a n d N A D P + (AcPyAD + a n d A c P y A D P +) were used since the activities with these analogs were higher t h a n with the thionicotinamide, acetylpyridine d e a m i n o a n d pyridinealdehyde analogs. Intact E. histolytica exhibited a transhydrogenase activity which decreased on storage in the "forward" direction ( N A D H + AcP y A D + ~ N A D + + A c P y A D P H ) a n d was e n h a n c e d by the addition of 2 m M A T P (Table 1). In the "reverse" reaction ( N A D P H + Z c P y A D + ~ N A D P + + AcPyADH), the activity increased with storage but was not enhanced by 2 m M ATP. The different responses to storage may be related to the energy requirement of the forward reaction (Danielson & Ernster, 1963). Increasing storage results in progressive lysis of the a m o e b a (Weinbach & Diamond, 1974)
a n d thus may cause disruption of the energy supply to the transhydrogenase. The same disruption may enhance the reverse reaction since there is no energy requirement for it. Initial experiments determined that the transhydrogenase activity was located in the particulate rather than the s u p e r n a t a n t fraction of the disrupted trophozoites (Table 2). Several particulate fractions were obtained by differential centrifugation: 600, 15,000 and 110,000 g sediments (Fig. 1). The enzyme activity was located primarily in the 110,000 ,q or "small particulate" sediment. A finding as yet unexplained is the high level of activity of the forward reaction in the s u p e r n a t a n t fraction which is inhibited by 2 m M ATP. This finding appears to be inconsistent with the localization of the enzyme activity in the 110,000 g sediment fraction; however, if the forward reaction rate without A T P is subtracted from the rate with added A T P (Fig. 1, upper graph), the highest positive value (denoting stimulation by ATP) is located in the 110,000 g sediment coinciding with the highest activities of the other enzyme parameters measured (Fig. 1, lower graph).
Table 2. Localization of transhydrogenase after fractionation of homogenized E. histolytica Fraction Supernatant Fresh After 2 mM ATP Overnight at 4°C After 2 mM ATP Overnight at - 10"C After 2 mM ATP Sediment Fresh After 2 mM ATP Overnight at 4°C After 2 mM ATP Overnight at - 10°C After 2 mM ATP
nmoles reduced, min ~• mg proteinNADPH--* AcPyAD + NADH --~ AcPyADP 36 4.0 1.6 1'6 2.8 2.0
4.2 2.9 3.2 1"6 3.2 2.4
18.4
2-4 5-3 1-8 2.7 0.9 2.3
8.0 8.0 15.3 11.4
Amoebae were homogenized for 1 rain at 4"C with a Teflon pestle homogenizer and centrifuged at 110,000 g for 2 hr. Assay conditions as described in Table 1.
A protozoan lacking mitochondria
• NADH ~ Ac PyADP++ ATF' minus NAOH-- AcPyADP +
143
It30
80 A ~60 c 4O • NAOPH~AcPyAD +
3k
D N A D H ~ AcPyADP +
20
50
I00
150
2O0
250
3~O
3~
[Polrnitoyl-CoAl (~M)
'~ 25
Fig. 2. Inhibition of transhydrogenase by palmitoyl--CoA. The small particulate fraction (110,000O) prepared as described in Fig. 1 was used. Same assay conditions as in Table 1; the amount of protein per assay was 2.1 mg.
/" /
~'2o. ~ 5E z ~ ~ to
5
/V\ L Intoct
I 600 xg
I 15000 xg
I I10000 xg
I SPNT
I
Fig. 1. Transhydrogenase localization after differential centrifugation of homogenized E. histolytica. Amoebae were homogenized for 1 min at 4°C with a Teflon pestle homogenizer and centrifuged as follows: 600 0 for 10min; 15,0000 for 5min; 110,000o for 2hr. Sediments from each centrifugation, and the supernatant fraction (SPNT) from 110,000 O centrifugation were assayed according to conditions described in Table 1. E. histolytica transhydrogenase in the 110,000o sediment was not inhibited by 1 mM KCN or 1 mM Ca 2+ but was inhibited (72'~;) by 1 mM PCMB. The enzyme was sensitive to palmitoyl~'oA at low concentrations (Fig. 2). The palmitoyl CoA concentration necessary for inhibition was 25-200-fold higher than that required for inhibition of the mammalian enzyme (Rydstr6m, 1972); however, the amount of protein used per assay for the amoebal enzyme was 25-100fold higher. Nonspecific binding of the palmitoyl-CoA to the amoebal non-enzyme protein may account for the greater concentration required for inhibition,
DISCUSSION
The response of the nicotinamide nucleotide transhydrogenase of E. histolytica to palmitoly-CoA and Ca 2+ is similar to the response of the analogous enzyme of beef heart sub-mitochondrial particles (Rydstr6m, 1972; Hoek et al., 1974). However, the amoeba has no mitochondria, and the 110,000 g sediment fraction contains no structures resembling mitochondria or mitochondrial fragments when examined by electron microscopy. The similarity of the amoebal enzyme to the mammalian analog even though the amoeba lacks mitochondria and is either anaerobic or facultatively anaerobic raises interesting questions concerning the
function of the transhydrogenase in the amoeba. In mammalian mitochondria, the enzyme is located on the inner membrane and is associated with the classical components of aerobic metabolism located there. However, the amoebal enzyme may instead be associated with a NADPH diaphorase thus providing an aerobic means for NADH oxidation which is fundamentally different from the cytochrome-containing NADH oxidation system of mammaliam mitochondria. Although the habitat of the amoeba is deficient in oxygen (von Brand, 1973), the precise concentration of available oxygen is not known. Thus the possibility of this aerobic NADH oxidation system must be considered. If, on the other hand, the habitat is anaerobic; the presence of this system is enigmatic although it has been suggested that such an oxygen utilizing system may operate as a "scavenger" to remove small amounts of oxygen which are toxic to the anaerobic organism. Finally, Van de Stadt et al. (1971) have reported that mammalian mitochondrial transhydrogenase may be involved in energy conservation (ATP production). In the forward reaction, mammalian transhydrogenase requires energy (e.g. ATP) while in the reverse reaction energy is released which at least theoretically could be coupled to ATP production. The amoebal analog also responds to ATP in the forward reaction but it is not yet known if energy capable of being coupled to ATP production is released in the reverse reaction. The possibility that transhydrogenase participates in energy conservation in E. histolytica, an anaerobe or facultative anaerobe, presents an intriguing question for future bioenergetic research.
REFERENCES
CLARK W. M., KAPLAN N. O. & KAMENM. D. (1955) Symposium on electron transport in the metabolism of microorganisms. Bact. Rev. 19. 234-262. COLOWICK S. P., KAPLAN N. O., NEUFELD E. F. t~ CIOTTI
M. M. (1952) Pyridine nucleotide transhydrogenase I. Indirect evidence for the reaction and purification of the enzyme. J. biol. Chem. 195, 95-105.
144
D.R. HARLOW, E. C. WEINBACH AND L. S. DIAMOND
DANIELSON L. & ERNSTER L. (1963) Energy-dependent reduction of triphosphopyridine nucleotide by reduced diphosphopyridine nucleotide, coupled to the energytransfer system of the respiratory chain. Biochem. Z. 338. 188-205. DIAMOND L. S. (1968) Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae. J. Parasit. 54, 1047 1056. ERNSTER L. & LEE C.-P. (1964) Biological oxidoreductions. A. Rev. Biochem. 33, 729-788. HASSON E. P. 8z WEST C. A. (1973) A microsomal ATPactivated pyridine nucleotide transhydrogenase. Archs Biochem. Biophys. 155, 258 269. HILKER D. M. t~ WHITE A. G. C. (1959) Some aspects of the carbohydrate metabolism of Entamoeba histolytica. J. Parasit. 8, 539-548. HOEK J. B., RYDSTROMJ. & HOJEBERG B. (1974) Comparative studies on nicotinamide nucleotide transhydrogenase from different sources. Biochim. hiophys. Acta 333, 237-245. KAPLAN N. O. (1967) Beef heart TPNH-DPN pyridine nucleotide transhydrogenases. In Methods in Enzymology (Edited by COLOWlCK S. P. & KAPLAN N. O.), Vol. X, pp. 317-322. Academic Press, New York. KAPLAN N. O., COLOWICK S. P, 8~ NEUFELD E. F. (1953)
Pyridine nucleotide transhydrogenase--lll. Animal tissue transhydrogenases. J. biol. Chem. 205, 1-15. LWOEF M. (1951) Nutrition of parasitic amebae. In Biochemistry and Physiology of Protozoa (Edited by LWOFF A.), Vol. I, pp. 235 250. Academic Press, New York. MONTALVO F. E., REEVES R. E. • WARREN L. G. (1971) Aerobic and anaerobic metabolism in Entamoeba histolytica. Exp. Parasit. 30, 249 256. RYDSTRt3M J. (1972) Site-specific inhibitors of mitochondrial nicotinamide-nucleotide transhydrogenase. Eur. J. Biochem. 31, 496-504. RYLEY J. F. (1967) Carbohydrates and respiration. In Chemical Zoology (Edited by KIDDER G. W.), Vol. I, pp. 55-92. Academic Press, New York. TEIXEIRA DA CRUZ A., RYDSTRi3M J. & ERNSTER L. (1971) Steady-state kinetics of mitochondrial nicotinamide nucleotide transhydrogenase--l. The nonenergy-linked reaction. Eur. d. Biochem. 23, 203 211. VAN DE STADT R. J., NIEUWENHU1S E. J. R. M. & VAN DAM K. (197t) On the reversibility of the energy-linked transhydrogenase. Biochim. biophys. Acta 234, t73-176. YON BRAND T. (1973) Biochemistry o[ Parasites, 2nd Edn., pp. 368-425. Academic Press, New York. WEINBACH E. C. & DIAMOND L. S. (1974) Entamoeba histolytica: 1. Aerobic metabolism. Exp. Parasit. 35, 232-243.
1976,
Vol.
53B,
pp.
141
to
144.
Pergamo,1 Press. Printed in Great Britain
NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE IN ENTAMOEBA HISTOLYTICA, A PROTOZOAN LACKING MITOCHONDRIA DAN R. HARLOW, EUGENEC. WEINBACHAND LOUIS S. DIAMOND Section on Physiology and Biochemistry, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Received 21 October 1974) Abstract--1. A nicotinamide nucleotide transhydrogenase exhibiting both energy-linked and nonenergylinked reactions is present in intact and subcellular fractions of Entamoeba histolytica. 2. Both reactions are localized primarily in the 110,000g sedimented fraction. 3. Even though E. histolytica is eukaryotic, it contains no mitochondria; hence the amoebal enzyme is non-mitochondrial in contrast to mammalian cells where it is located on the inner mitochondrial membrane. 4. Although the enzyme is non-mitochondrial in the amoeba, it responds to palmitoyl-CoA and Ca2÷ in the same way that the mammalian enzyme does. 5. E. histolytica is the first protozoan species in which nicotinamide nucleotide transhydrogenase has been found.
INTRODUCTION NICOTINAMIDE nucleotide transhydrogenase catalyzes hydrogen exchange between NAD +* and NADP + by the reaction: NADH + NADP + ~ NAD + + NADPH (Ernster & Lee, 1964; Colowick et al., 1952). The enzyme has been reported from bacterial (Clark et al., 1955), plant (Hasson & West, 1973) and animal cells (Kaplan et al., 1953). In animal cells, the transhydrogenase is located on the inner mitochondrial membrane (Teixeira da Cruz et aL, 1971) and thus is at least structurally associated with the aerobic metabolism of the cell. Entamoeba histolytica, a tissue invading protozoan of the human intestine, liver and other sites is considered to be an anaerobe (Lwoff, 1951) although recent evidence indicates that it may be a facultative anaerobe. When oxygen is present, the amoeba will utilize it (Montalvo et al., 1971; Weinbach & Diamond, 1974); however, it grows and reproduces under anaerobic conditions (von Brand, 1973). Although the amoeba produces fermentation products such as ethanol and CO2 both in the presence and absence of oxygen, the amounts produced in the presence of oxygen are less than are produced anaerobically (Montalvo et al., 1971). Even though the electron carriers under aerobic conditions have not been characterized, the absence of mitochondria (for review see m
* Abbreviations: NAD +, nicotinamide adenine dinucleotide; NADP ÷, nicotinamide adenine dinucleotide phosphate; ATP, adenosine triphosphate; Tris, tris(hydroxymethyl)aminomethane; Palmitoyl-CoA, palmitoyl coenzyme A; AcPyAD÷, acetylpyridine adenine dinucleotide; AcPyADP ÷ acetylpyridine adenine dinucleotide phosphate; NADH, reduced nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; AcPyADH, reduced acetylpyridine adenine dinucleotide; AcPyADPH, reduced acetylpyridine adenine dinucleotide phosphate; PCMB, parachloromercuribenzoate. , .l~.p.la)53,2B A
von Brand, 1973) and cytochromes (Hilker & White, 1959) as well as the lack of a functional tricarboxylic acid cycle (Weinbach & Diamond, 1974) indicate that typical electron transport mechanisms are not present. We have found a NAD(P)H diaphorase which is more active with NADPH than with NADH (in some preparations, the NADH activity approached zero). In the absence of typical aerobic mechanisms for generating ATP, the amoeba presumably must rely on other mechanisms such as glycolysis for ATP production. The possibility that NADH produced in glycolysis could be oxidized by aerobic mechanisms which differ from those found in typical aerobic cells was considered in investigating the observed oxygen utilization. One such possibility is that the oxidation of NADH is mediated by the transhydrogenase; the oxidation of the NADPH produced by this reaction is catalyzed by the diaphorase resulting finally in the reduction of oxygen or other electron carriers. Such an aerobic supplemental NADH oxidation system is consistent with the observed oxygen utilization and diminished quantities of fermentation products produced aerobically. Ryley (1967) noted that a nicotinamide nucleotide transhydrogenase would be of importance in protozoan cells for maintaining the redox balance between the nicotinamide nucleotides, although he reported that the enzyme had not been found in any protozoan. MATERIALS AND METHODS Entamoeba histolytica trophozoites were grown in axenized culture according to the method of Diamond (1968). The amoebae were harvested at 72 hr and washed twice by centrifugation in buffered sucrose (50 mM Tris, pH 7.4; 250 mM sucrose). Suspensions of washed intact cells or cells homogenized in buffered sucrose were assayed for nicotinamide nucleotide transhydrogenase according to the method of Kaplan (1967). The analogs of NAD ÷, 141
142
D. R. HARLOW, E. C. WEINBACH AND L. S. DIAMONI)
Table 1. Transhydrogenase of intact E. histolytica Conditions
nmoles reduced, min ~- mg proteinNADPH ~ AcPyAD ~ NADH ~ AcPyADP +
Fresh After 2 min After 10 min. After 2 mM ADP After 2 mM ATP Overnight at 4°C After 2 mM ATP Overnight at - 10°C After 2 mM ATP 30 Days at -10c'C After 2 mM ATP
7.4 6.8 3.l 4.2 11.0 12.0 10.8 16.3
2.4 1.6 30 4-3 2.2 3.2 2.5 3.4 0.9 0-7
The assay medium contained 100/~moles potassium phosphate buffer, pH 6-5; 1/~mole nicotinamide nucleotides and analogs; and 0.1 ml of the appropriate sample (1.8 to 2.7 mg protein) in a final vol of 1.0 ml. The suspensions were assayed at room temp as described by Kaplan (1967).
NADP ÷ and the palmitoyl--CoA were obtained from P-L Biochemicals, Milwaukee, U.S.A. Native coenzymes were purchased from Sigma Chemical Company, St. Louis, U.S.A. RESULTS
The acetylpyridine analogs of N A D + a n d N A D P + (AcPyAD + a n d A c P y A D P +) were used since the activities with these analogs were higher t h a n with the thionicotinamide, acetylpyridine d e a m i n o a n d pyridinealdehyde analogs. Intact E. histolytica exhibited a transhydrogenase activity which decreased on storage in the "forward" direction ( N A D H + AcP y A D + ~ N A D + + A c P y A D P H ) a n d was e n h a n c e d by the addition of 2 m M A T P (Table 1). In the "reverse" reaction ( N A D P H + Z c P y A D + ~ N A D P + + AcPyADH), the activity increased with storage but was not enhanced by 2 m M ATP. The different responses to storage may be related to the energy requirement of the forward reaction (Danielson & Ernster, 1963). Increasing storage results in progressive lysis of the a m o e b a (Weinbach & Diamond, 1974)
a n d thus may cause disruption of the energy supply to the transhydrogenase. The same disruption may enhance the reverse reaction since there is no energy requirement for it. Initial experiments determined that the transhydrogenase activity was located in the particulate rather than the s u p e r n a t a n t fraction of the disrupted trophozoites (Table 2). Several particulate fractions were obtained by differential centrifugation: 600, 15,000 and 110,000 g sediments (Fig. 1). The enzyme activity was located primarily in the 110,000 ,q or "small particulate" sediment. A finding as yet unexplained is the high level of activity of the forward reaction in the s u p e r n a t a n t fraction which is inhibited by 2 m M ATP. This finding appears to be inconsistent with the localization of the enzyme activity in the 110,000 g sediment fraction; however, if the forward reaction rate without A T P is subtracted from the rate with added A T P (Fig. 1, upper graph), the highest positive value (denoting stimulation by ATP) is located in the 110,000 g sediment coinciding with the highest activities of the other enzyme parameters measured (Fig. 1, lower graph).
Table 2. Localization of transhydrogenase after fractionation of homogenized E. histolytica Fraction Supernatant Fresh After 2 mM ATP Overnight at 4°C After 2 mM ATP Overnight at - 10"C After 2 mM ATP Sediment Fresh After 2 mM ATP Overnight at 4°C After 2 mM ATP Overnight at - 10°C After 2 mM ATP
nmoles reduced, min ~• mg proteinNADPH--* AcPyAD + NADH --~ AcPyADP 36 4.0 1.6 1'6 2.8 2.0
4.2 2.9 3.2 1"6 3.2 2.4
18.4
2-4 5-3 1-8 2.7 0.9 2.3
8.0 8.0 15.3 11.4
Amoebae were homogenized for 1 rain at 4"C with a Teflon pestle homogenizer and centrifuged at 110,000 g for 2 hr. Assay conditions as described in Table 1.
A protozoan lacking mitochondria
• NADH ~ Ac PyADP++ ATF' minus NAOH-- AcPyADP +
143
It30
80 A ~60 c 4O • NAOPH~AcPyAD +
3k
D N A D H ~ AcPyADP +
20
50
I00
150
2O0
250
3~O
3~
[Polrnitoyl-CoAl (~M)
'~ 25
Fig. 2. Inhibition of transhydrogenase by palmitoyl--CoA. The small particulate fraction (110,000O) prepared as described in Fig. 1 was used. Same assay conditions as in Table 1; the amount of protein per assay was 2.1 mg.
/" /
~'2o. ~ 5E z ~ ~ to
5
/V\ L Intoct
I 600 xg
I 15000 xg
I I10000 xg
I SPNT
I
Fig. 1. Transhydrogenase localization after differential centrifugation of homogenized E. histolytica. Amoebae were homogenized for 1 min at 4°C with a Teflon pestle homogenizer and centrifuged as follows: 600 0 for 10min; 15,0000 for 5min; 110,000o for 2hr. Sediments from each centrifugation, and the supernatant fraction (SPNT) from 110,000 O centrifugation were assayed according to conditions described in Table 1. E. histolytica transhydrogenase in the 110,000o sediment was not inhibited by 1 mM KCN or 1 mM Ca 2+ but was inhibited (72'~;) by 1 mM PCMB. The enzyme was sensitive to palmitoyl~'oA at low concentrations (Fig. 2). The palmitoyl CoA concentration necessary for inhibition was 25-200-fold higher than that required for inhibition of the mammalian enzyme (Rydstr6m, 1972); however, the amount of protein used per assay for the amoebal enzyme was 25-100fold higher. Nonspecific binding of the palmitoyl-CoA to the amoebal non-enzyme protein may account for the greater concentration required for inhibition,
DISCUSSION
The response of the nicotinamide nucleotide transhydrogenase of E. histolytica to palmitoly-CoA and Ca 2+ is similar to the response of the analogous enzyme of beef heart sub-mitochondrial particles (Rydstr6m, 1972; Hoek et al., 1974). However, the amoeba has no mitochondria, and the 110,000 g sediment fraction contains no structures resembling mitochondria or mitochondrial fragments when examined by electron microscopy. The similarity of the amoebal enzyme to the mammalian analog even though the amoeba lacks mitochondria and is either anaerobic or facultatively anaerobic raises interesting questions concerning the
function of the transhydrogenase in the amoeba. In mammalian mitochondria, the enzyme is located on the inner membrane and is associated with the classical components of aerobic metabolism located there. However, the amoebal enzyme may instead be associated with a NADPH diaphorase thus providing an aerobic means for NADH oxidation which is fundamentally different from the cytochrome-containing NADH oxidation system of mammaliam mitochondria. Although the habitat of the amoeba is deficient in oxygen (von Brand, 1973), the precise concentration of available oxygen is not known. Thus the possibility of this aerobic NADH oxidation system must be considered. If, on the other hand, the habitat is anaerobic; the presence of this system is enigmatic although it has been suggested that such an oxygen utilizing system may operate as a "scavenger" to remove small amounts of oxygen which are toxic to the anaerobic organism. Finally, Van de Stadt et al. (1971) have reported that mammalian mitochondrial transhydrogenase may be involved in energy conservation (ATP production). In the forward reaction, mammalian transhydrogenase requires energy (e.g. ATP) while in the reverse reaction energy is released which at least theoretically could be coupled to ATP production. The amoebal analog also responds to ATP in the forward reaction but it is not yet known if energy capable of being coupled to ATP production is released in the reverse reaction. The possibility that transhydrogenase participates in energy conservation in E. histolytica, an anaerobe or facultative anaerobe, presents an intriguing question for future bioenergetic research.
REFERENCES
CLARK W. M., KAPLAN N. O. & KAMENM. D. (1955) Symposium on electron transport in the metabolism of microorganisms. Bact. Rev. 19. 234-262. COLOWICK S. P., KAPLAN N. O., NEUFELD E. F. t~ CIOTTI
M. M. (1952) Pyridine nucleotide transhydrogenase I. Indirect evidence for the reaction and purification of the enzyme. J. biol. Chem. 195, 95-105.
144
D.R. HARLOW, E. C. WEINBACH AND L. S. DIAMOND
DANIELSON L. & ERNSTER L. (1963) Energy-dependent reduction of triphosphopyridine nucleotide by reduced diphosphopyridine nucleotide, coupled to the energytransfer system of the respiratory chain. Biochem. Z. 338. 188-205. DIAMOND L. S. (1968) Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae. J. Parasit. 54, 1047 1056. ERNSTER L. & LEE C.-P. (1964) Biological oxidoreductions. A. Rev. Biochem. 33, 729-788. HASSON E. P. 8z WEST C. A. (1973) A microsomal ATPactivated pyridine nucleotide transhydrogenase. Archs Biochem. Biophys. 155, 258 269. HILKER D. M. t~ WHITE A. G. C. (1959) Some aspects of the carbohydrate metabolism of Entamoeba histolytica. J. Parasit. 8, 539-548. HOEK J. B., RYDSTROMJ. & HOJEBERG B. (1974) Comparative studies on nicotinamide nucleotide transhydrogenase from different sources. Biochim. hiophys. Acta 333, 237-245. KAPLAN N. O. (1967) Beef heart TPNH-DPN pyridine nucleotide transhydrogenases. In Methods in Enzymology (Edited by COLOWlCK S. P. & KAPLAN N. O.), Vol. X, pp. 317-322. Academic Press, New York. KAPLAN N. O., COLOWICK S. P, 8~ NEUFELD E. F. (1953)
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