C. JAMES LOVELACE AND GENE W. MILLER

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Tzz JOURNAL OF HI5TOCHEISTRY AND Cociesneism Copyright 1967 by The Histochemical Society, Inc. Vol. 16, No. 4 Printed in U.S.A. IN VITRO EFFECTS OF FLUORIDE ON TRICARBOXYLIC ACID CYCLE DEHYDROGENASES AND OXIDATIVE PHOSPHORYLATION : PART I C.

1 Tzz JOURNAL OF HI5TOCHEISTRY AND Cociesneism Copyright 1967 by The Histochemical Society, Inc. Vol. 16, No. 4 Printed in U.S.A. IN VITRO EFFECTS OF FLUORIDE ON TRICARBOXYLIC ACID CYCLE DEHYDROGENASES AND OXIDATIVE PHOSPHORYLATION : PART I C. JAES LOVELACE AND GENE W. ILLER Humboldt State College, Arcata, California, and Utah State University, Logan, Utah Studies were conducted on the in vitro effect fluoride on the succinic oxidase system utilizing mitochondria obtained from cauliflower. Preincubation mitochondria with fluoride did not increase inhibition succinic oxidase. Various other tricarboxylic acid cycle substrates were used to determine their sensitivity to fluoride; only succinate oxidation was affected. A series succinate concentrations in the presence and in the absence fluoride showed increased activity succinic dehydrogenase, which indicated competitive inhibition. Various concentrations phosphate in the absence fluoride showed that phosphate had only slight effects on the succinic 2,6-dichlorophenolindophenol reductase component the succinic oxidase system. In the absence phosphate, various concentrations fluoride showed an initial increase in activity followed by a decrease in activity succinic 2,6-dichlorophenolindophenol reductase. In the presence phosphate, fluoride caused marked inhibition succinic 2,6-dichlorophenolindophenol reductase. It is believed that this inhibition results from an enzyme-fluorophosphate complex which has a lower dissociation constant than that the enzyme-substrate complex. An oxidative phosphorylation study indicated that both respiration and phosphorylation were inhibited. The exact mechanism by which fluoride causes injury to plants and animals remains obscure. Nevertheless, it is known that certain physiologic processes are markedly affected by fluoride. Accumulating evidence indicates that the basic metabolism the organism is involved. Battelli and Stern (2) first reported that the electron transport system is inhibited by fluoride. Potter and Schneider (18) suggested that the inhibition succinic dehydrogenase by fluoride was competitive and postulated that the site inhibition was between succinate and succinic dehydrogenase. Slater and Bonner (21), in experiments using the succinic oxidase system isolated from beef heart muscle, concluded that succinic dehydrogenase was the only component the oxidase system susceptible to inhibition by fluoride. Singer et al. (2) showed that purified succinic dehydrogenase from yeast is more easily inactivated by fluoride than is that obtained from beef heart muscle. Bonner and Wildman (4) found that this enzyme system is active in plant tissues by showing its inhibition by malonate. Hiatt (8), in describing properties soluble succinic dehydrogenase from the roots Phaseolus vulgaris L. and from the leaves Nicotiana tabacum L., 1 This research was supported by a special air pollution grant from the Division Air Pollution, Bureau State Services, U.S. Public Health Service. Fellowship nos.: 1FeAP21,91-1, 5F3- AP21,91-2 and AP Journal paper no. 598, Utah Agricultural Experiment Station. found essentially no differences between the two preparations in relation to ph, buffer, substrate concentration, and the action certain inhibitors. Bonner and Thimann (5) noted a reduction in overall plant growth due to fluoride inhibition. A reduction in the photosynthesis rate was reported by Thomas and Hendricks (22). This was further substantiated by cnulty and Newman (13), who showed a marked decrease in the chlorophyll content fluoride-treated plants when compared to control plants. Decreased respiration in plant tissues was noted by Bonner and Wildman (4) and by Laties (9). cnulty and Newman (12), using intact growing plants, demonstrated an increase in respiration. Yu,2 using discs cut from tobacco plant leaves, observed an increase in respiration at lower concentrations fluoride. However, respiration was inhibited with increasing concentrations fluoride. It appears, therefore, that an initial effect fluoride on plants involves either inhibition or enhancement respiration with the specific result depending upon plant species, fluoride concentration and extent injury. This study was undertaken to elucidate further the effects fluoride on oxidative phosphorylation and suecinic dehydrogenase in higher plants. 2ing-Ho Yu, unpublished.s. thesis, Utah State University Library, Logan, Utah. 195

196 LOVELACE AND ILLER ATERIALS AND ETHODS Source enzyme: A modification the method outlined by Wedding and Black (25) was used to obtain mitochondria from cauliflower (Bras8ica oleracea L.) plants.

2 196 LOVELACE AND ILLER ATERIALS AND ETHODS Source enzyme: A modification the method outlined by Wedding and Black (25) was used to obtain mitochondria from cauliflower (Bras8ica oleracea L.) plants. When using a nonparticulate extract for spectrophotometric work, an acetone powder mitochondria prepared from cauliflower was extracted according to the method Hiatt (8). The yield dry powder was about 2 g/kg fresh cauliflower. It was stored at -2#{176}C in an evacuated flask containing anhydrous calcium sulfate. In this laboratory, the enzyme preparation was active after a period more than 7 months. The reaction mixture contained 2.8 X 1 sucrose; 8.3 X 1- glucose; 4.7 X 1- gcl,; 3. KH,POa; 1.6 X 1 adenosine monophosphate (AP); 6.3 X 1 adenosine diphosphate (ADP); 2.9 X 1 adenosine triphosphate (ATP); 5.2 X 1- hexokinase; 5.8 X 1 cytochrome C; 4. X 1- substrate; 5. X 1- Tris; and.2 ml 2% enzyme solution. Determination phosphorylation: The reaction mixture contained radioactive inorganic orthophosphate (4 X 1-1 X 1 cpm/ml). An aliquot.5 ml reaction mixture was withdrawn as soon as possible after adding the mitochondrial suspension and another was withdrawn at the end 3 mm. Esterfied P in each aliquot was analyzed by the reversed phase chromatographic method developed by Hagihara and Lardy (7). The inorganic phosphate was adsorbed while the esterifled phosphate passed through the column and was collected for counting. Counts per minute after zero time incubation were subtracted from counts per minute after incubation reaction mixture in order to determine actual esterifled phosphate. Determination oxygen consumption: Oxygen consumption was measured manometrically using a Gilson edical Electronics refrigerated differential respirometer. All experiments were conducted at 25#{176}C.After temperature equilibration, readings were taken at 1-mn intervals for a period 3 mm for each treatment. icroliters oxygen were corrected to standard conditions. Each flask contained the components listed in Table I. Leloir and Dixon (1) and Tsou (23) found that pyrophosphate was a competitive inhibitor succinic dehydrogenase; thus phosphate was prepared according to the method Slater and Bonner (21). Spectrophotometric methods: A slight modification the procedure used by Ells (6) was used. The rate reduction sodium 2,6-dichiorophenolindophenol was measured at a wavelength 6 me for 3 sec between 15 and 45 sec after initiation the reaction. Enzyme activity was negligible when phenazine methosulfate or succinate was omitted from the reaction mixture. The rate reduction sodium 2,6-dichiorophenolindophenol was proportional to enzyme concentration under the conditions used in these experiments. No activity was obtained using a boiled enzyme preparation. RESULTS AND DISCUSSION Experiments were conducted to determine whether or not cytochrome c was a necessary D + Cytochrome C Rate in 1 O2Iuig protein 4. - Cytochrome c I I Time in minutes FIG. 1. Effect addition cytochrome c to reaction mixture. The flask contained all components the reaction mixture with the exception cytochrome which was the variable. Suocinate at 4 X 1- was present as substrate. The figures represent the results from four replications and the range at 4 mm is shown.

IN VITRO EFFECTS OF FLUORIDE ON TCA CYCLE DEHYDROGENASES 197 Effect Preincuoation with Sodium on the Succinic Ozidase S1/8tem Sodium 1 x 1-1 x 1- No Preincubation I 1d,/hr/sd 86. ± 14.9b 77.2 ± 6.

3 IN VITRO EFFECTS OF FLUORIDE ON TCA CYCLE DEHYDROGENASES 197 Effect Preincuoation with Sodium on the Succinic Ozidase S1/8tem Sodium 1 x 1-1 x 1- No Preincubation I 1d,/hr/sd 86. ± 14.9b 77.2 ± ± ± ± ± ±.2 Preincubation in Sodium for 3 mm extract 8.2 ± ± ± ± ± ±4.5 a Each flask contained all components the reaction mixture with 4. X 1 disodium succinate as substrate. b These figures represent the average three cactor in the oxidation reaction. Cytochrome c can be washed from the mitochondria without disrupting the phospholipid membranes or the integrity the mitochondria. Thus, it can be lost during preparation procedures and in this manner become a limiting factor. The results these experiments are shown in Figure 1. It is evident (Fig. 1) that oxygen uptake was linear with respect to time in both the absence and presence cytochrome c. However, cytochrome c enhanced the oxygen uptake approximately 2%. Consequently, cytochrome c was added to all reaction mixtures in experiments involving mitochondria and oxidation or phosphorylation. To determine conditions maximum inhibition oxidation succinate to fumarate, an experiment was conducted to see whether penetration fluoride into the mitochondria was a factor. The mitochondrial fraction was preincubated for 3 mm in the same concentration sodium fluoride that would be added to the reaction mixture. Comparative data for preincubation as opposed to no preincubation are shown in Table I. The results indicate that the differences are slight and are probably attributable to experimental variation. In subsequent experiments mitochondrial extracts were not preincubated with fluoride. A series experiments was conducted to determine the sensitivity various oxidases to fluoride. In all cases, fluoride was added as sodium fluoride to the reaction mixture in the Warburg flasks (Table II). alate and reduced nicotinamide adenine dinucleotide (NADH) + H oxidase systems were inhibited 2% at 5 x 1- fluoride. This inhibition probably represented a general osmotic effect rather than specific fluoride inhibition. Succinic oxidase was markedly affected at 1- fluoride, and at 5 x 1- fluoride was almost totally inhibited. At this concentration, all systems were affected to some extent. Since other tricarboxylic acid (TCA) cycle oxidase systems follow the pathway involving NADH + 11+, they were not studied. Oxidative phosphorylation studies: The important role which oxidative phosphorylation plays in growth, maintenance and development organisms is well recognized (Beevers (3)). Phosphorylation inhibition results in a general disruption cellular metabolism and finally in the death the cell. Results from in vitro oxidative phosphorylation studied indicate that both oxidation and phosphorylation increase in the presence 5 x 1 fluoride. In the presence The Effect Sodium on TCA Oxidase Systems II Sodium. Substrate NADH + H1 alate Succinate,J,/kr/sd extract 9.1 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.5 Each flask contained all components the reaction mixture with 4 X 1- substrate. These figures represent the average three replications in each case.

198 LOVELACE AND ILLER Sodium 5 X l III The Effect on Oxidative Phosphorylation Oxygen g atoms/hr/mg Uptake protein 1.5 ±.41#{176} 11.5 ±.59 1.2 ±.37 2.1 ±.

4 198 LOVELACE AND ILLER Sodium 5 X l III The Effect on Oxidative Phosphorylation Oxygen g atoms/hr/mg Uptake protein 1.5 ±.41#{176} 11.5 ± ± ±.74 Phosphorus Esterified &moles/mg protein 28. ± ± ± ± 1.4 Ratio Phosphorus to Oxygen #{176}Each reaction vessel contained all the reaction mixture and utilized 4 X 1- disodium succinate as substrate. These figures represent the results from three replications. 5 x 1- fluoride, a decrease in phosphorylation and a corresponding decrease in oxidation were noted (Table III). Both phosphorylation and oxidation decreased markedly at 5 x 1-2 fluoride. The phosphorus to oxygen (P :) ratios remained essentially the same from -5 x 1 fluoride. These ratios account for all radioactive phosphorus used to phosphorylate any organic compound, including those other than ADP. They do not, however, account for any 32 cleaved during the course these experiments. With 5 x 1 fluoride there was a marked increase in the P: ratio. If, as suggested by some workers (Reiner (19), Pierpoint (17), Beevers (3)), high concentrations fluoride inhibit ATPase, then the above results could be accounted for by ATPase inhibition. The results seem to indicate an increased phosphate uptake over the amount oxygen used at 5 x 1 fluoride. However, quantities esterifled phosphate could have been magnified in the presence fluoride resulting from inhibition ATPase. This seems to be the case since all P : values obtained were somewhat above the theoretical value 2. These data are similar to those Slater and Bonner (21), who found an increased P: ratio when using 4 x 1 fluoride as an inhibitor the succinic oxidase system obtained from pig heart. The sharp decrease in oxygen uptake and phosphorus esterification shown at the 5 x 1-2 fluoride level probably reflected a concentration fluoride that was highly effective as an inhibitor succinic dehydrogenase. In oxidation experiments, an increase in the amount oxygen taken up was noted consistently at the lowest concentration fluoride. This substantiated the results Reiner (19), Pierpoint (17), Akazawa and Beevers (1) and Yu.2 However, with the increased fluoride concentrations, a sharp reduction was noted in oxygen uptake. Inhibition enolase (Warburg and Christian (24), iller (15)) and inhibition phosphoglucomutase (Najjar (16), Yang and iller (26)) would probably not be involved since these enzymes are found in the soluble rather than the particulate fraction. Therefore, the succinic oxidase system and particularly succinic dehydrogenase (Slater and Bonner (21)) must account for the major part this reduced oxygen uptake. Possibly adverse effects on several enzymes, including inhibition added as well as endogenous hexokinase (elchior and elchior (14)), are intensified with concentrations fluoride at 5 x 1- and higher. Spectrophotometric studies for dehydrogenase determination: Experiments were designed to determine the effects phosphate per se on succinic dehydrogenase (succinic 2,6- dichlorophenolindophenol reductase). Only the The Effect Varying s Phosphate on Succinic,6-Dichlorophenolindophenol Reduckzse in the Absence Phosphate IV Change in Ontical Density per inute per illigram Protein at 6 mi 2.7 ±.3b 2.69 ± ± ± ±.2 #{176}The reaction cuvette contained.5 Tris buffer, ph 7.4;.1 potassium cyanide; 3 X 1- sodium 2,6-dichlorophenolindophenol;.3 mg/ml phenazine methosulfate;.4 disodium succinate; enzyme to give an optical density change.2-.4/3 sec. All experiments were b These figures represent the average three

IN VITRO EFFECTS OF FLUORIDE ON TCA CYCLE DEHYDROGENASES 199 The Effect Varying s in the Absence Phosphate on Succinic 2,6-Dichlorophenolindophenol Reductase 1 X 1-i 5X1-2 1 x 1- V Change in Outical

5 IN VITRO EFFECTS OF FLUORIDE ON TCA CYCLE DEHYDROGENASES 199 The Effect Varying s in the Absence Phosphate on Succinic 2,6-Dichlorophenolindophenol Reductase 1 X 1-i 5X1-2 1 x 1- V Change in Outical Density per inute per illigram Protein at 6 m 2.7 ±.lob 2.75 ± ± ± ± ±.4 #{176}The reaction cuvette contained.5 Tris buffer, ph 7.4;.1 potassium cyanide; 3 x 1-s sodium 2,6-dichlorophenolindophenol;.3 mg/mi phenazine methosulf ate;.4 disodium succinate; enzyme to give an optical density change.2-.4/3 sec. All experiments were b These figures represent the average three 1 x 1- and higher concentrations phosphate influenced the enzyme activity (Table IV). Definite inhibitory effects on the enzymes were evident under these conditions. The increased inhibitory effect at the higher concentrations was probably nonspecific, but could have been caused by pyrophosphate contamination in the reaction mixture, although precautions were taken to eliminate it. Slater and Bonner (21), however, found that the phosphate ion itself competitively inhibited an enzyme preparation obtained from beef heart. In the absence phosphate, fluoride at 1 x 1- and 5 x 1- enhanced succinic 2,6- dichlorophenolindophenol reductase activity (Table V). At higher concentrations inhibition occurred. The decreases were similar to those found with phosphate and could indicate that the inhibitory effects fluoride or phosphate are nonspecific. Both factors, however, have been shown to be competitive inhibitors succinic dehydrogenase (Slater and Bonner (21)). Inhibition induced by either fluoride or phosphate was slight until high concentrations were used. The effects various concentrations fluoride in the presence 5 x 1- phosphate on succinic 2, 6-dichlorophenolindophenol reductase are tabulated (Table VI). In these experiments, fluoride had a marked inhibitory effect at concen- The Effect Varying s in the Presence 5 X 1 Phosphate on Succinic #{163},6-Dichlorophenolindophenol reductase 1 X 1-2 VI Change in Optical Density per inute oar illigram Protein 2.57 ± ± ± ± ± ±.6 #{176}The reaction cuvette contained.5 Tris buffer, ph 7.4;.1 potassium cyanide; 3 X 1- sodium 2,6-dichlorophenolindophenol;.3 mg/mi phenazine methosulfate;.4 disodium succinate; enzyme to give an optical density change.2-.4/3 sec. All experiments were These figures represent the average three trations as low as 1 x 1- in the presence phosphate. Slater and Bonner (21) postulated that such inhibition was caused by 1 molecule phosphate and 1 molecule fluoride reacting with the enzyme molecule. With 5 x 1 fluoride, succinic 2, 6-dichlorophenolindophenol reductase activity was inhibited 57%. By contrast, 5 x 1- fluoride, in the absence phosphate, inhibited the activity only 18%. These results (Fig. 2) thus support those Slater and Bonner (21) in implicating a fluorophosphate complex as the major causative agent in succinic 2, 6-dichlorophenolindophenol reductase inhibition. Slater and Bonner (21) postulated that the fluorophosphate complex acts as an inhibitor succinic dehydrogenase in the same manner as malonate; i.e., the complex competes with succinate for the active site on the enzyme molecule. A series experiments was conducted to determine whether the inhibition noted in these studies was competitive or noncompetitive. The results were plotted by the method Lineweaver and Burk (11) (Fig. 3). The fluorophosphate complex competes with the substrate for the active site on the enzyme molecule. The dissociation constant for the enzymephosphate complex is slightly less than the dis-

6 2 LOVELACE AND ILLER 3. fluoride phosphate = fluoride 5 x 1 phosphate OD/min/nig protein in x 1O4 Fia. 2. The effects fluoride, phosphate and fluorophosphate on succinic 2,6-dichiorophenolindophenol reductase activity. The reaction cuvette contained.5 Tm buffer, ph 7.4;.1 potassium cyanide; 3 X 1 sodium 2,6-dichlorophenolindophenol;.3 mg/nil phenazine methosu.lfate;.4 sodium succinate enzyme to give an optical density change.2-.4/3 sec. All experiments were x1O2NaF 1/v 4. 3 ci a ONaF Fio. 3. Lineweaver-Burk plot the inhibition succinic 2,6-dichlorophenolindolphenol reductase activity mitochondria from cauliflower by sodium fluoride. Assay mixture in a final volume 3 ml contained.5 Tris buffer ph 7.4 phosphate;.1 potassium cyanide; 3 X 1- sodium 2,6-dichlorophenolindoiphenol;.3 mg/mi phenazine methosulfate;.4 disodium succinate; enzyme to give an optical density change.2-.4/3 sec. All experiments were conducted at room temperature. l s sociation constant for the enzyme-substrate complex and about the same as the dissociation constant for the enzyme-fluoride complex (Tables IV and V). The dissociation constant for the enzyme-fluorophosphate complex, however, is much less than the dissociation constant for the enzymesubstrate complex. Thus it competes effectively with the substrate for the active site on the enzyme molecule (Table VI; Figs. 2 and 3). REFERENCES 1. Akazawa, T. and Beevers, H.: itochondria in the endosperm castor beans: a developmental study. Biochem. J. 65: 115, Battelli, F. and Stern, L.: Biochem. Z. 3: 172, 191. Quoted by Slater and Bonner, Beevers, H.: Respiratory etabolism in Plants. Row, Peterson and Co., New York, p Bonner, J. and Wildman, S. G.: Enzymatic mechanisms in the respiration spinach leaves. Arch. Biochem. 1: 497, Bonner, W. D. and Thimann, K. V.: The

7 IN VITRO EFFES OF FLUORIDE ON TCA CYCLE DEHYDROGENASES 21 action some inhibitors concerned with pyruvate metabolism. Amer. 1. Bot. 37: 66, Ella, H. A.: A colorimeteric method for the assay soluble succinic dehydrogenase and pyridine-nucleotide-linked dehydrogenases. Arch. Biochem. 85: 561, Hagihara, B. and Lardy, H. A.: A method for the separation orthophosphate from other phosphate compounds. J. Biol. Chem. 235: 889, Hiatt, A. J.: Preparation and some properties soluble succinic dehydrogenase from higher plants. Plant Physwl. 36: 552, Laties, G..: The role pyruvate in the aerobic respiration barley roots. Arch. Biochem. 2: 284, Leloir, L. F. and Dixon,.: Enzymologia 2: 81, Quoted byslater and Bonner, Lineweaver, H. and Burk, D.: The determination enzyme dissociation constants. J. Amer. Chem. Soc. 56: 658, cnulty, I. B. and Newman, D. W.: Effects atmospheric fluoride on the respiration rate bush bean and gladiolus leaves. Plant Physiol. 32: 121, cnulty, I. B. and Newman, D. W.: echanism fluoride induced chlorosis. Plant Physiol. 36: 385, elchior, N. C. and eichior, J. B.: Inhibition yeast hexokinase by fluoride ion. Science 124: 42, iller, G. W.: Properties enolase in extracts from pea seeds. Plant Physiol. 33: 199, Najjar, V. A.: The isolation and properties phosphoglucomutase..1. Biol. Chem. 175: 281, Pierpoint, W. S.: Phosphatase and meta- hosphatase activities pea extracts. Biochem : 67, Potter, V. R. and Schneider, W. C.: Studies on the mechanism hydrogen transport in animal tissues..1. Biol. Chem. 142: 543, Reiner, J..: The kinetics multiple enzyme inhibition. J. Gen. Phys. $: 367, Singer, T. P., Thimot, N. Z., assey, V. and Kearney, E. B.: Purification and properties succinic dehydrogenase from yeast. Arch. Biochem. 62: Slater, E. C. and Bonner, W. D., Jr.: The effect fluoride on the succinic oxidase system. Biochem : 185, Thomas,. D. and Hendricks, R. H.: Effect air pollution onplants. In Handbook air Pollution, edited by P. Z. agill. cgraw Hill Book Co., Inc., New York, 1956, Sect Tsou, C. L.: On the cyanide inactivation succinic dehydrogenase and the relation succinic dehydrogenase to cytochrome b. Biochem. J. 49: 512, Warburg. and Christian, W.: Isolation and crystallization the fermentation enzyme enolase. Biochem. Z. $1: 384, Wedding, R. T. and Black,. K.: ethod for isolating the mitochondrial from cauliflower. Plant Physiol. $7: 364, Yang, S. F. and iller,. W.: Biochemical studies on the effect fluoride on higher plants. Biochem : 55, 1963.

Higher Biology. Unit 2: Metabolism and Survival Topic 2: Respiration. Page 1 of 25

Higher Biology. Unit 2: Metabolism and Survival Topic 2: Respiration. Page 1 of 25 Higher Biology Unit 2: Metabolism and Survival Topic 2: Respiration Page 1 of 25 Sub Topic: Respiration I can state that: All living cells carry out respiration. ATP is the energy currency of the cell