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Formate oxidation by an obligately parasitic fungus Tilletia contraversa
ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Formate
Oxidation Fungus
E. B. VAISEY,2 From
the
VERNON Department Oregon
g&63-65
by Tilletia
(1961)
an Obligately contraversa’
H. CHELDELIN
AR‘D
Parasitic
R. TV. XEWBURGH3
of Chemistry and the Science Research State College, Corualli,s, Oregon Rcceix-ed
April
Institute,
24, 1961
Formate oxidation by enzyme preparations from I[‘illetia contrauersa was optimum at pH 5.1, competitively inhibited by ethanol and nitrite, by either carbon monoxide or pyridine nucleotides. These results formate is oxidized by a catalase-hydrogen peroxide complex. INTRODUCTIOY
Tilletia contraversa is a fungus pathogenic to wheat, other cereals, and grasses. The most prominent symptom of infection in wheat is that the black teliospores of the fungus replace the entire wheat kernel within the seed coat, forming a so-called “bunt ball.” The organism has a diphasic life cycle, and in the dicaryotic stage it is an obligate parasite. Although the monocaryotic stage can be cultured successfully, n-e have been more interested in the met,abolism of the parasitic stage, in the hope that eventually it, might offer clues to the organism’s pathogenicity. Since preliminary chemical analyses of teliospores harrestcd from wheat indicated that they contained up to 62 p.p.m. formic acid on a dry weight basis, we became interested in determining the fate of this acid. 1 Supported by a grant-in-aid from the Frasch Foundation. Published with t,he approval of the Monograph Publication Committee, Research Paper Ko. 400, School of Science, Department of Chemistry. ’ Present address: Food and Drug Directorate, Department of Sat,ional Health and Welfare, Ottawa, Canada. ‘Scholar in Cancer Research from an Ethel M. Craig Memorial Grant, from thr i\meriran Cancer Soc>iety.
63
MATERIALS
AKD
HARVESTING
OF
teliospores and unaffected indicate that
METHODS TELIOSPORES
Bunt balls were removed from ripe wheat plants and winnowed from excess chaff. They were then blended briefly with water in a Waring blendor to release the teliospores from the bunt balls. The teliospores were filtered through eight layers of cheesecloth, collected on filt,er paper, and washed until the wash water was colorless. Teliospores treated in this way were essentially free from plant material as judged by microscopic cxamination.
EXZYME
PREPARATIONS
Teliospores were converted into acetone powders by blending them in a Waring blendor for 10 sec. with 10 vol. acetone at -10°C. Then they were filtered, washed with 2 vol. acet,one, air-dried, and stored at 3°C. The 1hick rcll walls of the teliospores were ruptured at 3-8°C. by shaking an aqueous suspension of the acet,onc powder with 4-mm. glass beads on a Mickle disintegrator. The ratio of acetone powder, water and beads for maximum breakage in a 55 mm. X 25 mm. diameter Mickle cell was 1.5 g.:5 ml.:15 g. Acetone powders contained 22% of ether-soluble materials which werr released into the medium as the teliosporrs were broken. Since this fatty mntrrial interfrrcd with the impact of beads on sports, the suspension was centrifuged t)riefly nftrr 5 min. shaking, and the fat was skimmed from the surface. The suspension was then shaken for anot,her 5 min. -4 sport homogenate was prepared from the
VAISEY,
CHELDELIN
0 ETHANOL-172/m
NO
/
INHIBITOR
I
I
I
I
0.5
1.0
1.5
2.0
“FORMATE
(pc)
FIG. 1. Reciprocal plot of the inhibition of formate oxidation by ethanol. Reaction mixtures (5.4 ml.) contained trypsin-treated spore extract, 1 ml.; formate-Cl4 (4.0 &pmole), as indicated; ethanol, as indicated; and 0.15 M citrate-phosphate buffer at pH 5.1. Incubation time, 1 hr. Velocity (V) measured as the radioactivity in respired carbon dioxide. above suspension by removing the glass beads and diluting to 10 ml. with water. Microscopic examination of the homogenate indicated that about 10% of the teliospores were unbroken, about 15% had cracked cell walls, and the remainder were in fragments of various sizes. The trypsin-treated spore extract was prepared by incubating the spore homogenate with 1.5 mg. of crystalline trypsin for about 12 hr. at 0°C. The preparation was then centrifuged at 17,000 X g for 30 min. The supernatant fraction (spore extract) contained about 25% of the formate-oxidizing activity of the spore homogenate. In experiments with pyridine nucleotides which required prolonged dialysis of the extract (over 24 hr.), the trppsin t,reatment was omitted. ASSAY OF FORMATE OXIDATION Since the endogenous respiration of the enzyme preparations was high compared to their specific oxidizing activity, formate oxidation was measured as the radioactivity in carbon dioxide arising from formate-C14. The experiments were performed in 50-ml. Erlenmeyer flasks, each equipped with a center well, a rubber serum bottle stopper, and sometimes a side arm. The reaction
AND NEWBURGH mixtures in the flasks were agitated on a Dubnoff shaker at 3O”C., and respired carbon dioxide was trapped in the center well as sodium carbonate. The reaction was stopped as desired by the addition of 1 ml. of 20% trichloroacetic acid delivered with a hypodermic syringe through the serum bottle stopper. The flasks were then agitated at 70°C. for 15 min. to assure that all the carbon dioxide was trapped. The radioactive sodium carbonate was converted to barium carbonate and counted by standard procedures. The error in assay procedures was generally less than 5%. ASSAY OF CATALASE Spore extracts prepared without the trypsin treatment were assayed for catalase by the iodometric method described by Herbert (1). Such extracts contained 1.5-2.0 mg. protein/ml. CARBON MONOXIDE
INHIBITION
Carbon monoxide-oxygen mixtures were prepared by displacement of water from a bottle. Reaction flasks were filled with the gas mixtures by an evacuation procedure. The reaction flasks with side arms were protected from light with aluminum foil, and the reaction was started by tipping in substrate from the side arm. RESULTS EFFECTS
AND DISCUSSION
OF DPN, TPN AND CO ON FORMATE OXIDATION
Since T. contraversa is a plant pathogen, initially it was anticipated that formate oxidation would be linked to oxygen by a pyridine nucleotide and the cytochrome system as it is in higher plants (2, 3). However, it was found impossible to stimulate formate oxidation in extensively dialyzed spore extracts with either di- or triphosphopyridine nucleotide (DPN, TPN). It was also impossible to inhibit significantly the formate oxidation of spore homogenates with carbon monoxide containing 10% oxygen. These negative results made it necessary to examine other mechanisms for formate oxidation. EVIDENCE
THAT COMPLEX I CATALYZES FORMATE OXIDATION
Another enzyme system that might catalyze format’e oxidation in T. contraversa was the catalase-hydrogen peroxide com-
FORMATE
OXIDATION
plex (complex I) recently shown to be responsible for formate oxidation in higher animals (4). The properties of complex I have been reviewed by Chance (5). It is of special interest to this study that complex I oxidizes ethanol (6) and nitrite (7) as well as formate (8). Thus if formate oxidation by an organism depended on complex I it should be competitively inhibited by ethanol and nitrite. Preliminary experiments with acetone powders showed that formate oxidation was inhibited by ethanol and nitrite. In later experiments employing soluble teliospore extracts, it was shown that these inhibitions were competitive (Figs. 1 and 2). Evidence corroborating the participation of complex I in formate oxidation is the pH optimum of the reaction at 5.1 (Fig. 3). According to Chance (9), the optimum velocit,y for t’his oxidation is to be expected at pH 5.0 through to pH 4.3. The pHactivity curve shown in Fig. 3 is presumably influenced by the unknown enzyme which produces hydrogen peroxide from endogenous substrates to form complex I. The sharp drop in activity below pH 5.1 may be due to inactivation of this hydrogen peroxidc producing system.
65 ~0
m 0-
20
X I k
15,
I 0" u 2 a z cn 2
I
I 0
IO 5. -
0i
0
0’ \ 5.0
O-0 6.0
7.0
FH
3. Effect of pH on formate oxidation. Reaction mixtures (5.0 ml.) contained spore homogenate, 2 ml.; formate-Cl’, 1.0 PC. (0.25 pmole); 0.15 di citrate-phosphate buffer at the pH indicated. Incubation time, 2 hr. FIG.
The specific catalase activity (I) of the spore extracts was in the order of 0.24 1. X g.-I X sec.-I The foregoing results t’hus indicate that teliospores of T. contmversa oxidize formate by means of a catalase-hydrogen peroxide complex. *
I 0
REFERENCES 1.
D., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.)> Vol. II, p. 784. Academic Press, New York, 1955. 2. M.~THEwS, M. B., AND T’ESI-ESLAXD, B., J. Viol. Chem. 186,667 (1950).
X > \
I
I
I
I
I
0.5
1.0
1.5
2.0
‘/FORMATE
(/Lc)
Reciprocal plot of the inhibition mate oxidation by nitrite. Experimental tions similar to those shown in Fig. 1. FIG.
2.
I
of forcondi-
HERBERT,
3. DAVISOA-, D. C., Biochem. J. 49, 520 (1951). 4. ORO, J., AND RMPOPORT, D. A., J. Biol. Chcm. 234, 1661 (1959). 5. CHANCE, B., Advnrxcas itl Ettzpnol. 12, 153 (1951). 6. KEILIS, D., AND HARTREE, E. F.. PTOC. Roy. Sot. (London) B119, 141 (1936). 7. HEWEL, I,. A., AA-D PORTERFIIZD, V. T., J. Biol. Chern. 178,549 (1949). 8. CHANGE. B., J. niol. Chcnz. 179, 1341 (1949). 9. cH-\VCE, B., J. Viol. chcwk. 194, 4i1 (1952).
OF BIOCHEMISTRY
AND BIOPHYSICS
Formate
Oxidation Fungus
E. B. VAISEY,2 From
the
VERNON Department Oregon
g&63-65
by Tilletia
(1961)
an Obligately contraversa’
H. CHELDELIN
AR‘D
Parasitic
R. TV. XEWBURGH3
of Chemistry and the Science Research State College, Corualli,s, Oregon Rcceix-ed
April
Institute,
24, 1961
Formate oxidation by enzyme preparations from I[‘illetia contrauersa was optimum at pH 5.1, competitively inhibited by ethanol and nitrite, by either carbon monoxide or pyridine nucleotides. These results formate is oxidized by a catalase-hydrogen peroxide complex. INTRODUCTIOY
Tilletia contraversa is a fungus pathogenic to wheat, other cereals, and grasses. The most prominent symptom of infection in wheat is that the black teliospores of the fungus replace the entire wheat kernel within the seed coat, forming a so-called “bunt ball.” The organism has a diphasic life cycle, and in the dicaryotic stage it is an obligate parasite. Although the monocaryotic stage can be cultured successfully, n-e have been more interested in the met,abolism of the parasitic stage, in the hope that eventually it, might offer clues to the organism’s pathogenicity. Since preliminary chemical analyses of teliospores harrestcd from wheat indicated that they contained up to 62 p.p.m. formic acid on a dry weight basis, we became interested in determining the fate of this acid. 1 Supported by a grant-in-aid from the Frasch Foundation. Published with t,he approval of the Monograph Publication Committee, Research Paper Ko. 400, School of Science, Department of Chemistry. ’ Present address: Food and Drug Directorate, Department of Sat,ional Health and Welfare, Ottawa, Canada. ‘Scholar in Cancer Research from an Ethel M. Craig Memorial Grant, from thr i\meriran Cancer Soc>iety.
63
MATERIALS
AKD
HARVESTING
OF
teliospores and unaffected indicate that
METHODS TELIOSPORES
Bunt balls were removed from ripe wheat plants and winnowed from excess chaff. They were then blended briefly with water in a Waring blendor to release the teliospores from the bunt balls. The teliospores were filtered through eight layers of cheesecloth, collected on filt,er paper, and washed until the wash water was colorless. Teliospores treated in this way were essentially free from plant material as judged by microscopic cxamination.
EXZYME
PREPARATIONS
Teliospores were converted into acetone powders by blending them in a Waring blendor for 10 sec. with 10 vol. acetone at -10°C. Then they were filtered, washed with 2 vol. acet,one, air-dried, and stored at 3°C. The 1hick rcll walls of the teliospores were ruptured at 3-8°C. by shaking an aqueous suspension of the acet,onc powder with 4-mm. glass beads on a Mickle disintegrator. The ratio of acetone powder, water and beads for maximum breakage in a 55 mm. X 25 mm. diameter Mickle cell was 1.5 g.:5 ml.:15 g. Acetone powders contained 22% of ether-soluble materials which werr released into the medium as the teliosporrs were broken. Since this fatty mntrrial interfrrcd with the impact of beads on sports, the suspension was centrifuged t)riefly nftrr 5 min. shaking, and the fat was skimmed from the surface. The suspension was then shaken for anot,her 5 min. -4 sport homogenate was prepared from the
VAISEY,
CHELDELIN
0 ETHANOL-172/m
NO
/
INHIBITOR
I
I
I
I
0.5
1.0
1.5
2.0
“FORMATE
(pc)
FIG. 1. Reciprocal plot of the inhibition of formate oxidation by ethanol. Reaction mixtures (5.4 ml.) contained trypsin-treated spore extract, 1 ml.; formate-Cl4 (4.0 &pmole), as indicated; ethanol, as indicated; and 0.15 M citrate-phosphate buffer at pH 5.1. Incubation time, 1 hr. Velocity (V) measured as the radioactivity in respired carbon dioxide. above suspension by removing the glass beads and diluting to 10 ml. with water. Microscopic examination of the homogenate indicated that about 10% of the teliospores were unbroken, about 15% had cracked cell walls, and the remainder were in fragments of various sizes. The trypsin-treated spore extract was prepared by incubating the spore homogenate with 1.5 mg. of crystalline trypsin for about 12 hr. at 0°C. The preparation was then centrifuged at 17,000 X g for 30 min. The supernatant fraction (spore extract) contained about 25% of the formate-oxidizing activity of the spore homogenate. In experiments with pyridine nucleotides which required prolonged dialysis of the extract (over 24 hr.), the trppsin t,reatment was omitted. ASSAY OF FORMATE OXIDATION Since the endogenous respiration of the enzyme preparations was high compared to their specific oxidizing activity, formate oxidation was measured as the radioactivity in carbon dioxide arising from formate-C14. The experiments were performed in 50-ml. Erlenmeyer flasks, each equipped with a center well, a rubber serum bottle stopper, and sometimes a side arm. The reaction
AND NEWBURGH mixtures in the flasks were agitated on a Dubnoff shaker at 3O”C., and respired carbon dioxide was trapped in the center well as sodium carbonate. The reaction was stopped as desired by the addition of 1 ml. of 20% trichloroacetic acid delivered with a hypodermic syringe through the serum bottle stopper. The flasks were then agitated at 70°C. for 15 min. to assure that all the carbon dioxide was trapped. The radioactive sodium carbonate was converted to barium carbonate and counted by standard procedures. The error in assay procedures was generally less than 5%. ASSAY OF CATALASE Spore extracts prepared without the trypsin treatment were assayed for catalase by the iodometric method described by Herbert (1). Such extracts contained 1.5-2.0 mg. protein/ml. CARBON MONOXIDE
INHIBITION
Carbon monoxide-oxygen mixtures were prepared by displacement of water from a bottle. Reaction flasks were filled with the gas mixtures by an evacuation procedure. The reaction flasks with side arms were protected from light with aluminum foil, and the reaction was started by tipping in substrate from the side arm. RESULTS EFFECTS
AND DISCUSSION
OF DPN, TPN AND CO ON FORMATE OXIDATION
Since T. contraversa is a plant pathogen, initially it was anticipated that formate oxidation would be linked to oxygen by a pyridine nucleotide and the cytochrome system as it is in higher plants (2, 3). However, it was found impossible to stimulate formate oxidation in extensively dialyzed spore extracts with either di- or triphosphopyridine nucleotide (DPN, TPN). It was also impossible to inhibit significantly the formate oxidation of spore homogenates with carbon monoxide containing 10% oxygen. These negative results made it necessary to examine other mechanisms for formate oxidation. EVIDENCE
THAT COMPLEX I CATALYZES FORMATE OXIDATION
Another enzyme system that might catalyze format’e oxidation in T. contraversa was the catalase-hydrogen peroxide com-
FORMATE
OXIDATION
plex (complex I) recently shown to be responsible for formate oxidation in higher animals (4). The properties of complex I have been reviewed by Chance (5). It is of special interest to this study that complex I oxidizes ethanol (6) and nitrite (7) as well as formate (8). Thus if formate oxidation by an organism depended on complex I it should be competitively inhibited by ethanol and nitrite. Preliminary experiments with acetone powders showed that formate oxidation was inhibited by ethanol and nitrite. In later experiments employing soluble teliospore extracts, it was shown that these inhibitions were competitive (Figs. 1 and 2). Evidence corroborating the participation of complex I in formate oxidation is the pH optimum of the reaction at 5.1 (Fig. 3). According to Chance (9), the optimum velocit,y for t’his oxidation is to be expected at pH 5.0 through to pH 4.3. The pHactivity curve shown in Fig. 3 is presumably influenced by the unknown enzyme which produces hydrogen peroxide from endogenous substrates to form complex I. The sharp drop in activity below pH 5.1 may be due to inactivation of this hydrogen peroxidc producing system.
65 ~0
m 0-
20
X I k
15,
I 0" u 2 a z cn 2
I
I 0
IO 5. -
0i
0
0’ \ 5.0
O-0 6.0
7.0
FH
3. Effect of pH on formate oxidation. Reaction mixtures (5.0 ml.) contained spore homogenate, 2 ml.; formate-Cl’, 1.0 PC. (0.25 pmole); 0.15 di citrate-phosphate buffer at the pH indicated. Incubation time, 2 hr. FIG.
The specific catalase activity (I) of the spore extracts was in the order of 0.24 1. X g.-I X sec.-I The foregoing results t’hus indicate that teliospores of T. contmversa oxidize formate by means of a catalase-hydrogen peroxide complex. *
I 0
REFERENCES 1.
D., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.)> Vol. II, p. 784. Academic Press, New York, 1955. 2. M.~THEwS, M. B., AND T’ESI-ESLAXD, B., J. Viol. Chem. 186,667 (1950).
X > \
I
I
I
I
I
0.5
1.0
1.5
2.0
‘/FORMATE
(/Lc)
Reciprocal plot of the inhibition mate oxidation by nitrite. Experimental tions similar to those shown in Fig. 1. FIG.
2.
I
of forcondi-
HERBERT,
3. DAVISOA-, D. C., Biochem. J. 49, 520 (1951). 4. ORO, J., AND RMPOPORT, D. A., J. Biol. Chcm. 234, 1661 (1959). 5. CHANCE, B., Advnrxcas itl Ettzpnol. 12, 153 (1951). 6. KEILIS, D., AND HARTREE, E. F.. PTOC. Roy. Sot. (London) B119, 141 (1936). 7. HEWEL, I,. A., AA-D PORTERFIIZD, V. T., J. Biol. Chern. 178,549 (1949). 8. CHANGE. B., J. niol. Chcnz. 179, 1341 (1949). 9. cH-\VCE, B., J. Viol. chcwk. 194, 4i1 (1952).