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Ubiquinone in four genera of protozoa
Comp. Biochem. Physiol., 1963, Vol. 8, pp. 331 to 334. PergamonPress Ltd., London. Printed in Great Britain
UBIQUINONE IN FOUR GENERA OF PROTOZOA* C A T H E R I N E V A K I R T Z I - L E M O N I A S , t G. W. K I D D E R and V I R G I N I A C. DEWEY Biological Laboratories, Amherst College, Amherst, Massachusetts (Received 10 September 1962)
Abstract--1. UQ was isolated from four genera of Protozoa and identified by chromatography. 2. The ciliate, Tetrahymena pyriformis, contains UQs. 3. The flagellates, Crithidia fasciculata, Strigomonas oncopelti and Astasia Mebsii, contain UQg. 4. The nature of the UQ synthesized by an organism would appear to have no taxonomic significance. INTRODUCTION WHILE coenzyme Q or ubiquinone (UQ) (Thompson, 1962) from various plants, animals and micro-organisms has been isolated and characterized as to the number of isoprenoid units contained in the side chain (Lester & Crane, 1959; Page et al., 1960), there have been few reports regarding its occurrence in the Protozoa (Fuller et al., 1961 ; Crane, 1962). We have carried out the isolation and identification of UQ from four species of Protozoa selected because of their diversity of type and the availability of axenic cultures. MATERIAL AND METHODS Tetrahyraena pyriformis W., a ciliate with animal-like nutritional pattern, was grown in a medium consisting of 3% proteose-peptone with 0.5% glucose. Crithidia fasciculata, a trypanosomid parasite of mosquitoes, has similar nutritional requirements (Kidder & Dutta, 1958) and was cultured in a like medium to which had been added 0.025% hemin. Strigoraonas oncopelti, a flagellate parasitic in certain insects, has relatively simple nutritional requirements (Newton, 1957) but was cultured for this study in the medium used for T. pyriformis. The fourth organism used, Astasia Mebsii, is a free-living, colorless "plant flagellate" and was cultured in the medium given in Table 1. The cultures were grown with aeration at room temperature in 2 1. of medium contained in 12 1. pyrex bottles, placed on their sides in a roller apparatus. When maximum growth was reached (4-7 days) the cells were harvested by low-speed centrifugation, saponified and extracted according to Crane et al. (1959). A second * Supported by research grants (A-1005) and (CY-2924) from the National Institutes of Health, U.S. Public Health Service, and a grant (CH-27) from the American Cancer Society. t Present address: Greek Atomic Energy Commission, Athens, Greece. 331
332
CATHERINE VAKIRTZI-LEMONIAS,G. W. KIDDERAND VIRGINIAC. DEWEY
series of experiments was carried out in which the saponification was performed in methanolic KOH, because there was evidence that there might have been exchange of the methoxy groups of UQ for ethoxy groups when ethanolic K 0 H was used. TABLEl--CULTUREMEDIUMFOR.,4STAS1A
Butyric acid Na glutamate Na citrate (NH4)2SO4 KH2PO4
/zg/ml
/~g/ml
1600 1000 124 460 905
MgSO~.7H20 200 CaClv2H~O 100 Thiamine 0.05 Cobalamine 0'005 Salt mixture* 1 : 100 pH to 6"5 with NaOH
* As used by Hutner et al. (1956). The crude extracts were dried in a flash-evaporator, redissolved either in ethanol or isooctane and then placed at - 2 0 ° to freeze out sterols. Crittu'dia in particular contains large amounts of sterol. Further purification of the UQ was accomplished by chromatography on columns of Decalso F, using first isooctane and then increasing amounts of ether in isooctane. Silicic acid gave less satisfactory separation of the UQ from the other lipids than did Decalso F. It was found necessary to use an all-glass apparatus for the chromatography; if connections were made with Tygon tubing the solvents used removed materials (apparently plasticizer) from the tubing which absorbed in the ultraviolet with a maximum at 282 m/x and thus interfered with UQ determinations. Except in the case of Astasia, 5% ether in isooctane was used for the elution of fractions containing UQ. The UQ from Astasia was eluted with 2.5% ether in isooctane to avoid contamination with a pink-purple pigment, possibly a carotenoid, which was removed from the columns by 5% ether-isooctane. Fractions from the columns which contained UQ were identified by their u.v. absorption spectra in a Beckman DK-2 recording spectrophotometer before and after reduction with NaBH4. At least two passages over a column were required before the UQ could be crystallized. Recrystallization at - 2 0 ° from absolute ethanol was carried out until constant melting point was achieved. UQ was also isolated from beef heart and from baker's yeast in order to have known compounds for comparison. Samples of UQs and UQ 7 were obtained through the kindness of Dr. Otto Isler of Hoffmann-La Roche Co., Basel.
RESULTS Comparisons of the UQ isolated from the protozoans with those of known composition have permitted conclusions as to the number of isoprene units synthesized by each type. Chromatographyon silicone-treatedpaper (Lester & Ramasarma, 1959), developedin propanol-water(4 : I, v/v), gave results whichare
333
UBIQUINONE IN FOUR GENERAOF PROTOZOA
recorded in Table 2. T h e R/values of the unknowns were compared with those of known compounds and ratios of the R I values to those for UQ10 and U Q 6 w e r e calculated, to compensate for differences in temperature, time of development and other factors. TABLE 2--CFIBOMATOGRAPHY OF U Q
R1
O r g a n i s m or
Rt U Q × / U Q t 0
Rt U Q s / U Q x
compound
Found
Reported
Found
Reported
Found
Reported
Beef heart (UQ10) UQ9 UQs UQ7 Yeast (UQ6) Tetrahymena Crithidia Strigomonas Astasia
0"30 (11)* -0"45 (9) 0"53 (8) 0.58 (18) 0.44 (23) 0.35 (11) 0.37 (2) 0"37 (2)
0"27 0"36 0"42 0"49 0'54 -----
1-00 -1-50 1"77 1.93 1 "46 1"17 1.23 1"23
1"00 1"33 1"56 1"81 2'00 -----
1"93 -1"29 1"09 1-00 1"32 1"66 1 "57 1 '57
2"00 1"50 1-28 1"10 1.00 -----
* N u m b e r in p a r e n t h e s e s is the n u m b e r of d e t e r m i n a t i o n s m a d e .
T h i n layer chromatography on silicic acid using the isooctane-benzene-acetone solvent of Shunk et al. (1961) was very successful and confirmed the conclusion that the ciliate U Q contains 8 isoprene units (Crane, 1962) while the flagellates all possess the compound with 9 units. It should be stated that the zinc-amine paper described by Diplock et al. (1960) gave us poor results while the petroleum jelly paper (Page et al., 1960) was unsatisfactory due to its high background fluorescence. Nor were melting point comparisons with those of known compounds (Lester et al., 1959) satisfactory, for even after repeated crystallizations matching was equivocal. TABLE 3--UBIQUINONE CONTENT OF FOUR GENERA OF PROTOZOA
Organism Tetrahymena pyriformis Crithidia fasciculata Strigomonas oncopelti Astasia klebsii
/zmole/g dry weight 0"65 0"23 0.40 0'14
Inasmuch as these protozoans are all highly aerobic organisms it was of interest to determine the U Q content of the cells (Sugimura & Rudney, 1960). As may be seen by a comparison with the data of Lester & Crane (1959) the protozoans are, as might be expected, relatively rich in the coenzyme (Table 3). T h e values for the ~'-'1% A / p l c m (ox-red 275 m/z) of Lester et al. (1959) were used for these determinations.
334
CATHERINEVAKIRTZI-LEMONIAS, G. W. KIDDER AND VIRGINIA C. DEWEY
DISCUSSION Fuller et al. (1961) have characterized the U Q of Euglena gracilis as U Q s. Since Astasia is considered to have been derived from the green euglenoid stock by loss of chloroplasts and stigma, it would have seemed likely that it would form the same isoprene analog of U Q as the parent stock. T h e fact that it does not serves to emphasize again the lack of relationship between phyletic position and the nature of the U Q synthesized by a given organism.
Acknowledgements--The authors gratefully acknowledge the skilful technical assistance of Carol L. Meehan. The thin layer chromatography was carried out by Dr. Richard Braun. REFERENCES CRANE F. L. (1962) Quinones in lipoprotein transport systems. Biochem. 1, 510-517. CRANEF. L., LESTERR. L., WIDMERC. • HATEFIY. (1959) Studies on the electron transport system. XVIII. Isolation of coenzyme Q (Q275) from beef heart and beef heart mitochondria. Biochim. Biophys. Acta 32, 73-79. DIPLOCKA. T., GREENJ., EDWIN E. E, & BUNYANJ. (1960) Studies on vitamin E. 4. The simultaneous determination of tocopherols, ubiquinones and ubichromenols (substance SC) in animal tissues: a reconsideration of the Keilin-Hartree heart preparation. Biochem. J. 76, 563-571. FULLERR. C., SMILLIER. M., RICOPOULOSN. & YOUNTV. (1961) Comparative study of some quinones in photosynthetic systems. Arch. Biochem. Biophys. 65, 197-202. HUTNER S. H., BACHM. K. & Ross G. I. M. (1956) A sugar-containing basal medium for vitamin B1,-assay with Euglena; application to body fluids. J. Protozool. 3, 101-112, KIDDER G. W. & DUTTA B. N. (1958) The growth and nutrition of Crithidia fasciculata. y. Gen. MicrobioL 18, 621-638. LESTER R. L. & CRANE F. L. (1959) The natural occurrence of coenzyrne Q and related compounds. J. Biol. Chem. 234, 2169-2175. L~TER R. L., HATEFIY., WXDMERC. & CRANEF. L. (1959) Studies on the electron transport system. XX. Chemical and physical properties of the coenzyme Q family of compounds. Biochem. Biophys. Acta 33, 169-185. LESTER R. L. & RaMASARMAT. (1959) Chromatography of the coenzyme Q family of compounds on silicone-impregnated paper. J. Biol. Chem. 234, 672-676. NEWTONB. A. (1957) Nutritional requirements and biosynthetic capabilities of the parasitic flagellate Strigomonas oncopelti. J. Gen. Microbiol. 17, 708-717. PAGEA. C., Jr., GALEP., WALLCIKR. B., WALTONL. E., MCDANIELL. E., WOODRUFFH. B. and FOLKERSK. (1960) Isolation of coenzyme Qx0 from bacterial fermentation. Arch. Biochem. Biophys. 89, 318-321. SHUNKC. H., MCPHERSONJ. F. & FOLKERSK. (1961) Coenzyme Q. XXIX. Monophosphate of dihydrocoenzyme Q10. Biochem. Biophys. Res. Comm. 6, 124--128. SUGIMURAT. & RUDNEYH. (1960) The adaptive formation of ubiquinone 30 (coenzyme Q6) in yeast. Biochem. Biophys. Acta 37, 560-561. THOMPSON R. H. S. (1962) Classification and nomenclature of enzymes and coenzymes. Nature, Lond. 193, 1227-1231.
UBIQUINONE IN FOUR GENERA OF PROTOZOA* C A T H E R I N E V A K I R T Z I - L E M O N I A S , t G. W. K I D D E R and V I R G I N I A C. DEWEY Biological Laboratories, Amherst College, Amherst, Massachusetts (Received 10 September 1962)
Abstract--1. UQ was isolated from four genera of Protozoa and identified by chromatography. 2. The ciliate, Tetrahymena pyriformis, contains UQs. 3. The flagellates, Crithidia fasciculata, Strigomonas oncopelti and Astasia Mebsii, contain UQg. 4. The nature of the UQ synthesized by an organism would appear to have no taxonomic significance. INTRODUCTION WHILE coenzyme Q or ubiquinone (UQ) (Thompson, 1962) from various plants, animals and micro-organisms has been isolated and characterized as to the number of isoprenoid units contained in the side chain (Lester & Crane, 1959; Page et al., 1960), there have been few reports regarding its occurrence in the Protozoa (Fuller et al., 1961 ; Crane, 1962). We have carried out the isolation and identification of UQ from four species of Protozoa selected because of their diversity of type and the availability of axenic cultures. MATERIAL AND METHODS Tetrahyraena pyriformis W., a ciliate with animal-like nutritional pattern, was grown in a medium consisting of 3% proteose-peptone with 0.5% glucose. Crithidia fasciculata, a trypanosomid parasite of mosquitoes, has similar nutritional requirements (Kidder & Dutta, 1958) and was cultured in a like medium to which had been added 0.025% hemin. Strigoraonas oncopelti, a flagellate parasitic in certain insects, has relatively simple nutritional requirements (Newton, 1957) but was cultured for this study in the medium used for T. pyriformis. The fourth organism used, Astasia Mebsii, is a free-living, colorless "plant flagellate" and was cultured in the medium given in Table 1. The cultures were grown with aeration at room temperature in 2 1. of medium contained in 12 1. pyrex bottles, placed on their sides in a roller apparatus. When maximum growth was reached (4-7 days) the cells were harvested by low-speed centrifugation, saponified and extracted according to Crane et al. (1959). A second * Supported by research grants (A-1005) and (CY-2924) from the National Institutes of Health, U.S. Public Health Service, and a grant (CH-27) from the American Cancer Society. t Present address: Greek Atomic Energy Commission, Athens, Greece. 331
332
CATHERINE VAKIRTZI-LEMONIAS,G. W. KIDDERAND VIRGINIAC. DEWEY
series of experiments was carried out in which the saponification was performed in methanolic KOH, because there was evidence that there might have been exchange of the methoxy groups of UQ for ethoxy groups when ethanolic K 0 H was used. TABLEl--CULTUREMEDIUMFOR.,4STAS1A
Butyric acid Na glutamate Na citrate (NH4)2SO4 KH2PO4
/zg/ml
/~g/ml
1600 1000 124 460 905
MgSO~.7H20 200 CaClv2H~O 100 Thiamine 0.05 Cobalamine 0'005 Salt mixture* 1 : 100 pH to 6"5 with NaOH
* As used by Hutner et al. (1956). The crude extracts were dried in a flash-evaporator, redissolved either in ethanol or isooctane and then placed at - 2 0 ° to freeze out sterols. Crittu'dia in particular contains large amounts of sterol. Further purification of the UQ was accomplished by chromatography on columns of Decalso F, using first isooctane and then increasing amounts of ether in isooctane. Silicic acid gave less satisfactory separation of the UQ from the other lipids than did Decalso F. It was found necessary to use an all-glass apparatus for the chromatography; if connections were made with Tygon tubing the solvents used removed materials (apparently plasticizer) from the tubing which absorbed in the ultraviolet with a maximum at 282 m/x and thus interfered with UQ determinations. Except in the case of Astasia, 5% ether in isooctane was used for the elution of fractions containing UQ. The UQ from Astasia was eluted with 2.5% ether in isooctane to avoid contamination with a pink-purple pigment, possibly a carotenoid, which was removed from the columns by 5% ether-isooctane. Fractions from the columns which contained UQ were identified by their u.v. absorption spectra in a Beckman DK-2 recording spectrophotometer before and after reduction with NaBH4. At least two passages over a column were required before the UQ could be crystallized. Recrystallization at - 2 0 ° from absolute ethanol was carried out until constant melting point was achieved. UQ was also isolated from beef heart and from baker's yeast in order to have known compounds for comparison. Samples of UQs and UQ 7 were obtained through the kindness of Dr. Otto Isler of Hoffmann-La Roche Co., Basel.
RESULTS Comparisons of the UQ isolated from the protozoans with those of known composition have permitted conclusions as to the number of isoprene units synthesized by each type. Chromatographyon silicone-treatedpaper (Lester & Ramasarma, 1959), developedin propanol-water(4 : I, v/v), gave results whichare
333
UBIQUINONE IN FOUR GENERAOF PROTOZOA
recorded in Table 2. T h e R/values of the unknowns were compared with those of known compounds and ratios of the R I values to those for UQ10 and U Q 6 w e r e calculated, to compensate for differences in temperature, time of development and other factors. TABLE 2--CFIBOMATOGRAPHY OF U Q
R1
O r g a n i s m or
Rt U Q × / U Q t 0
Rt U Q s / U Q x
compound
Found
Reported
Found
Reported
Found
Reported
Beef heart (UQ10) UQ9 UQs UQ7 Yeast (UQ6) Tetrahymena Crithidia Strigomonas Astasia
0"30 (11)* -0"45 (9) 0"53 (8) 0.58 (18) 0.44 (23) 0.35 (11) 0.37 (2) 0"37 (2)
0"27 0"36 0"42 0"49 0'54 -----
1-00 -1-50 1"77 1.93 1 "46 1"17 1.23 1"23
1"00 1"33 1"56 1"81 2'00 -----
1"93 -1"29 1"09 1-00 1"32 1"66 1 "57 1 '57
2"00 1"50 1-28 1"10 1.00 -----
* N u m b e r in p a r e n t h e s e s is the n u m b e r of d e t e r m i n a t i o n s m a d e .
T h i n layer chromatography on silicic acid using the isooctane-benzene-acetone solvent of Shunk et al. (1961) was very successful and confirmed the conclusion that the ciliate U Q contains 8 isoprene units (Crane, 1962) while the flagellates all possess the compound with 9 units. It should be stated that the zinc-amine paper described by Diplock et al. (1960) gave us poor results while the petroleum jelly paper (Page et al., 1960) was unsatisfactory due to its high background fluorescence. Nor were melting point comparisons with those of known compounds (Lester et al., 1959) satisfactory, for even after repeated crystallizations matching was equivocal. TABLE 3--UBIQUINONE CONTENT OF FOUR GENERA OF PROTOZOA
Organism Tetrahymena pyriformis Crithidia fasciculata Strigomonas oncopelti Astasia klebsii
/zmole/g dry weight 0"65 0"23 0.40 0'14
Inasmuch as these protozoans are all highly aerobic organisms it was of interest to determine the U Q content of the cells (Sugimura & Rudney, 1960). As may be seen by a comparison with the data of Lester & Crane (1959) the protozoans are, as might be expected, relatively rich in the coenzyme (Table 3). T h e values for the ~'-'1% A / p l c m (ox-red 275 m/z) of Lester et al. (1959) were used for these determinations.
334
CATHERINEVAKIRTZI-LEMONIAS, G. W. KIDDER AND VIRGINIA C. DEWEY
DISCUSSION Fuller et al. (1961) have characterized the U Q of Euglena gracilis as U Q s. Since Astasia is considered to have been derived from the green euglenoid stock by loss of chloroplasts and stigma, it would have seemed likely that it would form the same isoprene analog of U Q as the parent stock. T h e fact that it does not serves to emphasize again the lack of relationship between phyletic position and the nature of the U Q synthesized by a given organism.
Acknowledgements--The authors gratefully acknowledge the skilful technical assistance of Carol L. Meehan. The thin layer chromatography was carried out by Dr. Richard Braun. REFERENCES CRANE F. L. (1962) Quinones in lipoprotein transport systems. Biochem. 1, 510-517. CRANEF. L., LESTERR. L., WIDMERC. • HATEFIY. (1959) Studies on the electron transport system. XVIII. Isolation of coenzyme Q (Q275) from beef heart and beef heart mitochondria. Biochim. Biophys. Acta 32, 73-79. DIPLOCKA. T., GREENJ., EDWIN E. E, & BUNYANJ. (1960) Studies on vitamin E. 4. The simultaneous determination of tocopherols, ubiquinones and ubichromenols (substance SC) in animal tissues: a reconsideration of the Keilin-Hartree heart preparation. Biochem. J. 76, 563-571. FULLERR. C., SMILLIER. M., RICOPOULOSN. & YOUNTV. (1961) Comparative study of some quinones in photosynthetic systems. Arch. Biochem. Biophys. 65, 197-202. HUTNER S. H., BACHM. K. & Ross G. I. M. (1956) A sugar-containing basal medium for vitamin B1,-assay with Euglena; application to body fluids. J. Protozool. 3, 101-112, KIDDER G. W. & DUTTA B. N. (1958) The growth and nutrition of Crithidia fasciculata. y. Gen. MicrobioL 18, 621-638. LESTER R. L. & CRANE F. L. (1959) The natural occurrence of coenzyrne Q and related compounds. J. Biol. Chem. 234, 2169-2175. L~TER R. L., HATEFIY., WXDMERC. & CRANEF. L. (1959) Studies on the electron transport system. XX. Chemical and physical properties of the coenzyme Q family of compounds. Biochem. Biophys. Acta 33, 169-185. LESTER R. L. & RaMASARMAT. (1959) Chromatography of the coenzyme Q family of compounds on silicone-impregnated paper. J. Biol. Chem. 234, 672-676. NEWTONB. A. (1957) Nutritional requirements and biosynthetic capabilities of the parasitic flagellate Strigomonas oncopelti. J. Gen. Microbiol. 17, 708-717. PAGEA. C., Jr., GALEP., WALLCIKR. B., WALTONL. E., MCDANIELL. E., WOODRUFFH. B. and FOLKERSK. (1960) Isolation of coenzyme Qx0 from bacterial fermentation. Arch. Biochem. Biophys. 89, 318-321. SHUNKC. H., MCPHERSONJ. F. & FOLKERSK. (1961) Coenzyme Q. XXIX. Monophosphate of dihydrocoenzyme Q10. Biochem. Biophys. Res. Comm. 6, 124--128. SUGIMURAT. & RUDNEYH. (1960) The adaptive formation of ubiquinone 30 (coenzyme Q6) in yeast. Biochem. Biophys. Acta 37, 560-561. THOMPSON R. H. S. (1962) Classification and nomenclature of enzymes and coenzymes. Nature, Lond. 193, 1227-1231.