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Coenzyme Q. Li. New data on the distribution of coenzyme Q in nature
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS I 0 ~ , 169--172
(1964)
Coenzyme Q. LI. New Data on the Distribution of Coenzyme Q in Nature P. H. GALE, R. E. ERICKSON, A. C. PAGE, Jn., AND I~. FOLKERS From the Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey
Received July 16, 1963 The heart of the rhesus monkey was found to contain coenzyme Q~0. This experimental primate may serve as a model for studies of CoQ~Qas it may relate to human disease. Frog nerve tissue was also found to contain CoQ~0.Such tissue may be a useful system for researches on CoQ in nerve physiology. Only CoQ~0 was identified in human and rodent tumors grown in mice. The tumor strains were HAd-l, HS-1, and S-180. Cells of Ochromonas malhamensis contain CoQ~0. It is of interest that both man and this Ochromonas sp. specifically use both CoQ10 and vitamin BI~. Contrary to an earlier report that a given Polyporous did not contain a CoQ, it has now been found that Polyporous schweinitzii contains both CoQ~ and CoQi0. The "J strain" of a PPLO (Mycoplasma gallisepticum) is of current interest both in respect to its nutrition and its infectious characteristics; this PPLO strain did not contain either a member of either the CoQ or vitamin K groups. INTRODUCTION The isolation and characterization of CoQl0 (ubiquinone 50) (I) (1, 2, 3) opened a field of study which led to the discovery of five homologs (coenzymes Q6-Q10) (4) whose structures differ only in the number of isoprenoid units in the side chain. Two newly discovered analogs of CoQ10 are rhodoquinone ( I I ) , a hydroxy analog, and CoQ10 (H-10) ( I I I ) , which has a terminal saturated isoprenoid unit. Rhodoquinone was isolated from Rhodospirillum rubrum (5) and CoQ10 (H-10) from Gibberella fujckuroi (6). An apparent tetrahydro form of CoQ10 (7) has now been shown to have only one saturated isoprenoid unit and is identical with CoQ10 (H-10) (8). The distribution of these members of the Co Q group has been studied b y several investigators. Lester and Crane (9) examined a wide variety of materials for CoQ. Morton et al. (10) analyzed several tissues from rats during a study of the relationship between CoQ and vitamin A deficiency. Diplock et al. (11) examined fourteen tissues
of rats. An analysis of h u m a n tissues has also been reported (12). The distribution of CoQ in m a n y microbial species has been investigated (9, 13) and the relative amounts of vitamin K and CoQ in a number of microorganisms were reported (14, 15). O CH~O~"~CH3 ~Ha CH30~(CH~CH~C--CH2hoH I
CH~} ~CH3 .CH~[ O
H
(CH~CH~C--CH2)1oH O
II
O
CH,O~CH3 ~H~ ~H~ CH~O(,~ ~#(CH,CH~---C--CH~)~CH2CH2--C--CH,
"go 169
III
170
GALE ET AL.
Mammalian tissues contain CoQ10 (9, 16, 17): However, both CoQ10 and Q9 were found in rat tissue and Q9 may be predominant. The presence of trace amounts of coenzymes Q7 and Qs has also been reported (11). Birds contain CoQ10 (9, 18). Coenzyme Q9 has been identified in walleyed pike (9). Either compound may be present in insects; CoQ9 is found in the housefly and cabbage butterfly (9), and the hawk moth contains CoQ10 (19). Higher plants have been reported to contain CoQ10 (9, 20). Coenzyme Q9 was identified in vegetable oils (21) and algae (9). Microorganisms are a source of all members of the CoQ group. Coenzymes Qs and Q9 are frequently found in bacteria (9), though a few organisms contain CoQ7 or Q10 (13). Yeasts are the usual source of coenzymes Q6 and Q7 (9), and molds contain CoQ~ or Q~0 (9, 13). We now report new data of specific interest to current research on the identity of CoQ in living tissues and cellular material. This biological material includes monkey heart, a P P L O strain, and rodent and human tumor tissue.
Treatment with sodium borohydride produced dihydrocoenzyme Q with a maximum at 290 mt~. The ultraviolet spectra of the quinone and hydroquinone forms provided both qualitative evidence for and quantitative measurement of the CoQ (24). Direct comparison of mobilities on reversedphase papergrams (18) with those of reference samples of CoQ's was valuable for detection and identification of the CoQ. The sensitivity of the method (about 10 mcg.) was improved at least threefold by spraying the air-dried papergram with leucomethylene blue (21). Though the spray reagent is not specific, a positive reaction gave additional evidence for CoQ and a negative reaction eliminated the possibility that a particular component, visible as an ultraviolet absorbing spot on the papergram, was a CoQ. Other analytical methods useful both qualitatively and quantitatively, were an assay based upon a modified Craven reaction (25), and thinlayer chromatography (26) on silica gel and polyethylene. The modified Craven assay is specific for these methoxy-benzoquinones. The thin-layer chromatographic technique, like the papergram method, is not specific for members of the CoQ group. However, side-by side comparison with reference samples permitted estimation and virtual identification of as little as 0.1-0.2 mcg. of CoQ.
MATERIALS AND METHODS
The results are summarized in Table I. Coenzyme Q was obtained from the freezedried cells of O. malhamensis by direct solvent extraction; in all other cases, saponification was the initial isolation step. The levels of CoQ previously reported, both between and for different species, cover a range sufficiently broad to include those found in this study. The seemingly high value for 0. malhamensis is well within the range of the amount of CoQ found in microorganisms. The absence of a CoQ in Clostridium sticklandii is consistent with the anaerobic metabolism of that organism. The presence of CoQl0 in both man and O. malhamensis is worthy of note since both utilize vitamin B12. Though many aerobic bacteria contain substantial levels of CoQ10, certain species do not contain detectable amounts, but may instead contain a member of the vitamin K group. The absence of either type of quinone in cells of Mycoplasma gallisepticum need
The crude lipid portion of the source materials was obtained either by direct extraction with a solvent such as hexane or by saponification with sodium hydroxide in ethanol or methanol in the presence of pyrogallol, followed by solvent extraction (22, 23). Further purification was accomplished with chromatography on columns of Davison silica gel (desiccant grade, 100-200mesh), magnesium-alumino silicate (Decalso), or magnesium silicate (Florisil) (22, 23). Steroids, a major component in many of the preparations, could be partially removed by crystallization from isooctane solution at 0~ A combination of these steps was often required in order to obtain concentrates of sufficient purity to permit satisfactory qualitative and quantitative determinations of CoQ. Thin-layer chromatography on silica gel was particularly useful for the purification of microgram amounts of CoQ. Progress in purification was followed by means of ultraviolet spectra in ethanol over the 250310 m~ range. Fractions with a peak in the vicinity of 275 m~ were further purified until concentrates having the typical CoQ absorption were obtained.
RESULTS AND DISCUSSION
COENZYME Q
171
TABLE I DISTRIBUTION OF COENZYME Q Source
Monkey heart Rana pipiens frog (nerve tissue) HAd-1 tumors from mice S-180 tumors from mice HS-1 tumors from mice Shark liver Cecropia moth (thorax) Polyporous schweinitzii Mycoplasma gallisepticum Clostridium sticklandii Ochromonas malhamensis
Wet weight (mg./100 g.)
6.0 1.5 1.3 1.5 1.1 13.0 5.8
60 (dry weight)
CoQ homolog(s)
10 10 10 10 10 9, 10 9 9, 10
Identificationand estimationmethodsa
AUV, C, RI, LMB TLC (S, P) AUV, C, Ry, LMB 5UV, C, Rs, LMB ~UV, C, Rf, LMB TLC(S), UV, Rs, LMB, m.p., NMR AUV, RI, LMB, m.p. Rj, LMB AUV, TLC (S, P) AUV, Rf, LMB AUV, RI, LMB, TLC (S)
10
a Abbreviations: AUV, difference in intensity of absorption spectra of quinone and hydroquinone forms at 275 m~; C, Craven assay; RI, papergram mobility; LMB, leucomethylene blue spray reagent; TLC, thin-layer chromatography; (S), silica gel; (P), polyethylene; m.p., melting point; NMR, nuclear magnetic resonance.
not be considered unreasonable, particularly in view of its rather unusual and, as yet, unknown characteristics. Coenzyme Q was not detected in an earlier examination of a species of Polyporous (9). Present evidence for coenzymes Q9 and Qi0 in the cells of Polyporous schweinitzii is given by the papergram data. I t is of interest t h a t only CoQi0 was identified in h u m a n and rodent tumors grown in mice. Assays on tissues from normal mice have shown the presence of both coenzymes Q9 and Q10. Walleyed pike are known to contain CoQ~, and CoQ10 has been reported, though not completely identified in several organs from cod. The presence in tiger shark liver of a small amount of CoQtQ, in addition to CoQg, was rather unexpected. The shark's diet includes, however, a number of creatures, such as crabs, snails, and rays, which have not been analyzed for CoQ. I t has been reported (27) t h a t the anemia and muscular dystrophy t h a t develop in rhesus monkeys maintained on a purified diet low in vitamin E are rapidly progressive states that lead to death unless a-tocopherol or the 6-chromanol of hexahydrocoenzyme Q4 is given. I t was desirable to determine the particular CoQ in the tissue of the rhesus monkey because of the curative activity of the CoQ-chroinanol in the anemic and
dystrophic states. Only CoQ10 was identified in the heart of the rhesus monkey. This primate m a y serve as an experimental model for a study of CoQ in h u m a n disease. Nerve tissue of the frog is commonly used in fundamental studies of physiology. I t was of interest to establish the presence and identity of the CoQ in frog nerve tissue. Coenzyme QI0 was found. Frog nerve tissue might be a model for research on CoQi0 and nerve physiology. ACKNOWLEDGMENTS We are indebted to Dr. T. C. Stadtman, National Institute, National Institutes of Health, for the cells of Clostridium sticklandii; to Dr. W. J. Robbius, New York Botanical Garden, for the cells of Polyporous schweinitzii; and to Dr. J. H. Heller, New England Institute for Medical Research, for the tiger shark liver. The PPLO strain was isolated by P. Smith at the University of Pennsylvania and designated "J strain." REFERENCES 1. CRANE, F. L., HATEFI, Y., LESTER, R. L., AND WIDMER, C., Biochim. Biophys. Acta 25, 220
(1957). 2. MORTON, R. A., GLOOR, U., SCHINDLER, O., WILSON, G. M., CHOPARD-DIT-JEAN,L. H., HEMMING, F. W., ISLER, O., LEAT, W. M. F., PISNNOCK, J. F., Ri.~EGG, R., SCHWIETER,U., AND W I S S ,
~195S).
O.,
Helv. Chim. Acta 41, 2343
172
GALE ET AL.
3. MORTON, R. A., Nature 182, 1764 (1958). 4. LESTER, R. L., CRANE, F. L., AND HATEFI, Y., J. Am. Chem. Soc. 80, 4751 (1958). 5. CLOVER, J., AND THRELFALL, D. R., Biochem. J. 85, 14P (1962). 6. GALE, P. H., ARISON, B. H., TRENNER, N. R., PAGE, A. C., JR., AND FOLKERS, K., Biochemistry 2, 196 (1963). 7. LAVATE,W. V., DYER, J. R., SPRINGER,C. M., AND BENTLEY, R., J. Biol. Chem. 237, PC 2715 (1962). 8. GALE, P. I-I., TRENNER, N. R., ARISON, B. H., PAGE, A. C., JR., AND FOLKERS, K., Biochem. Biophys. Res. Commun. 12, 414 (1963). 9. LESTEI% R. L., AND CRANE, F. L., J. Biol. Chem. 234, 2169 (1959). 10. MORTON, R. A., WILSON, G. M., LOWE, J. S., AND LEAT, W. M. F., Chem. Ind. (London) 1957, 1649. 11. DIPLOCK, A. T., EDWIN, E. E., GREI~]N, J., BUNYAN~J., ANDMARCINKIEWICZ,S., Nature 186, 554 (1960). 12. GALE, P. H., KONIUSZY, F. R., PAGE, A. C., JR., AND FOLKERS, K., Arch. Biochern. BiGphys. 93,211 (1961). 13. PAGE, A. C., JR., GALE, P. H., WALLICK, H., WALTON, 1:~. B., McDANIEL, L. E., WOODRUFF, H. B., AND FOLKERS, K., Arch. Biochem. Biophys. 89,318 (1960). 14. PANDYA,K. P., BISHOP, D. H. L., AND KING, H. K., Biochem. J. 78, 35P (1961). 15. BISHOP, D. H. L., PANDYA, K. P., AND KING, H. K., Bioehem. J. 83,606 (1962).
16. CUNNINGHAM,N. F., ANDMORTON, R. A., BiGchem. J. 72, 92 (1959). 17. LowE, J. S., MORTON, R. A., AND VERNON, J., Biochem. J. 67,228 (1957). 18. LINN, B. O., PAGE, A. C., JR., WONG, E. L., GALE, P. H., SHUNK, C. H., AND FOLKERS, K., J. Am. Chem. Soc. 81, 4007 (1959). 19. HELLER, J., SZARKOWSKA,L., AND MICHALEK, H., Nature 188, 491 (1960). 20. JAYARAMAN,J., AND RAMASARMA,T., J. Sci. Ind. Res. (India) 20C, 69 (1961). 21. PAGE, A. C., JR., GALE, P. H., KONIUSZY, F. R., AND FOLKERS, K., Arch. Biochem. BiGphys. 85, 474 (1959). 22. CRANE, F. L., LESTER, R. L., WIDMER, C., AND HATEFI, Y., Biochim. Biophys. Acta 32, 73 (1959). 23. LESTER, R. L., AND CRANE, F. L., Biochim. Biophys. Acta 32,492 (1959). 24. LESTER, R. L., HATEFI, Y., WIDMER, C., AND CRANE, F. L., Biochim. Biophys. Acta 33, 169 (1959). 25. KoNIUSZY, F. R., GALE, P. H., PAGE, A. C., JR., AND FOLKERS, K., Arch. Biochem. BiGphys. 87, 298 (1960). 26. Technical Bulletin No. 22, November 1962, BrinkInann Instruments, Inc., and literature references cited. 27. DINNING, J. S., FITCH, C. D., SHUNK, C. H., AND FOLKERS, K., J. Am. Chem. Soc. 84, 2007 (1962).
(1964)
Coenzyme Q. LI. New Data on the Distribution of Coenzyme Q in Nature P. H. GALE, R. E. ERICKSON, A. C. PAGE, Jn., AND I~. FOLKERS From the Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey
Received July 16, 1963 The heart of the rhesus monkey was found to contain coenzyme Q~0. This experimental primate may serve as a model for studies of CoQ~Qas it may relate to human disease. Frog nerve tissue was also found to contain CoQ~0.Such tissue may be a useful system for researches on CoQ in nerve physiology. Only CoQ~0 was identified in human and rodent tumors grown in mice. The tumor strains were HAd-l, HS-1, and S-180. Cells of Ochromonas malhamensis contain CoQ~0. It is of interest that both man and this Ochromonas sp. specifically use both CoQ10 and vitamin BI~. Contrary to an earlier report that a given Polyporous did not contain a CoQ, it has now been found that Polyporous schweinitzii contains both CoQ~ and CoQi0. The "J strain" of a PPLO (Mycoplasma gallisepticum) is of current interest both in respect to its nutrition and its infectious characteristics; this PPLO strain did not contain either a member of either the CoQ or vitamin K groups. INTRODUCTION The isolation and characterization of CoQl0 (ubiquinone 50) (I) (1, 2, 3) opened a field of study which led to the discovery of five homologs (coenzymes Q6-Q10) (4) whose structures differ only in the number of isoprenoid units in the side chain. Two newly discovered analogs of CoQ10 are rhodoquinone ( I I ) , a hydroxy analog, and CoQ10 (H-10) ( I I I ) , which has a terminal saturated isoprenoid unit. Rhodoquinone was isolated from Rhodospirillum rubrum (5) and CoQ10 (H-10) from Gibberella fujckuroi (6). An apparent tetrahydro form of CoQ10 (7) has now been shown to have only one saturated isoprenoid unit and is identical with CoQ10 (H-10) (8). The distribution of these members of the Co Q group has been studied b y several investigators. Lester and Crane (9) examined a wide variety of materials for CoQ. Morton et al. (10) analyzed several tissues from rats during a study of the relationship between CoQ and vitamin A deficiency. Diplock et al. (11) examined fourteen tissues
of rats. An analysis of h u m a n tissues has also been reported (12). The distribution of CoQ in m a n y microbial species has been investigated (9, 13) and the relative amounts of vitamin K and CoQ in a number of microorganisms were reported (14, 15). O CH~O~"~CH3 ~Ha CH30~(CH~CH~C--CH2hoH I
CH~} ~CH3 .CH~[ O
H
(CH~CH~C--CH2)1oH O
II
O
CH,O~CH3 ~H~ ~H~ CH~O(,~ ~#(CH,CH~---C--CH~)~CH2CH2--C--CH,
"go 169
III
170
GALE ET AL.
Mammalian tissues contain CoQ10 (9, 16, 17): However, both CoQ10 and Q9 were found in rat tissue and Q9 may be predominant. The presence of trace amounts of coenzymes Q7 and Qs has also been reported (11). Birds contain CoQ10 (9, 18). Coenzyme Q9 has been identified in walleyed pike (9). Either compound may be present in insects; CoQ9 is found in the housefly and cabbage butterfly (9), and the hawk moth contains CoQ10 (19). Higher plants have been reported to contain CoQ10 (9, 20). Coenzyme Q9 was identified in vegetable oils (21) and algae (9). Microorganisms are a source of all members of the CoQ group. Coenzymes Qs and Q9 are frequently found in bacteria (9), though a few organisms contain CoQ7 or Q10 (13). Yeasts are the usual source of coenzymes Q6 and Q7 (9), and molds contain CoQ~ or Q~0 (9, 13). We now report new data of specific interest to current research on the identity of CoQ in living tissues and cellular material. This biological material includes monkey heart, a P P L O strain, and rodent and human tumor tissue.
Treatment with sodium borohydride produced dihydrocoenzyme Q with a maximum at 290 mt~. The ultraviolet spectra of the quinone and hydroquinone forms provided both qualitative evidence for and quantitative measurement of the CoQ (24). Direct comparison of mobilities on reversedphase papergrams (18) with those of reference samples of CoQ's was valuable for detection and identification of the CoQ. The sensitivity of the method (about 10 mcg.) was improved at least threefold by spraying the air-dried papergram with leucomethylene blue (21). Though the spray reagent is not specific, a positive reaction gave additional evidence for CoQ and a negative reaction eliminated the possibility that a particular component, visible as an ultraviolet absorbing spot on the papergram, was a CoQ. Other analytical methods useful both qualitatively and quantitatively, were an assay based upon a modified Craven reaction (25), and thinlayer chromatography (26) on silica gel and polyethylene. The modified Craven assay is specific for these methoxy-benzoquinones. The thin-layer chromatographic technique, like the papergram method, is not specific for members of the CoQ group. However, side-by side comparison with reference samples permitted estimation and virtual identification of as little as 0.1-0.2 mcg. of CoQ.
MATERIALS AND METHODS
The results are summarized in Table I. Coenzyme Q was obtained from the freezedried cells of O. malhamensis by direct solvent extraction; in all other cases, saponification was the initial isolation step. The levels of CoQ previously reported, both between and for different species, cover a range sufficiently broad to include those found in this study. The seemingly high value for 0. malhamensis is well within the range of the amount of CoQ found in microorganisms. The absence of a CoQ in Clostridium sticklandii is consistent with the anaerobic metabolism of that organism. The presence of CoQl0 in both man and O. malhamensis is worthy of note since both utilize vitamin B12. Though many aerobic bacteria contain substantial levels of CoQ10, certain species do not contain detectable amounts, but may instead contain a member of the vitamin K group. The absence of either type of quinone in cells of Mycoplasma gallisepticum need
The crude lipid portion of the source materials was obtained either by direct extraction with a solvent such as hexane or by saponification with sodium hydroxide in ethanol or methanol in the presence of pyrogallol, followed by solvent extraction (22, 23). Further purification was accomplished with chromatography on columns of Davison silica gel (desiccant grade, 100-200mesh), magnesium-alumino silicate (Decalso), or magnesium silicate (Florisil) (22, 23). Steroids, a major component in many of the preparations, could be partially removed by crystallization from isooctane solution at 0~ A combination of these steps was often required in order to obtain concentrates of sufficient purity to permit satisfactory qualitative and quantitative determinations of CoQ. Thin-layer chromatography on silica gel was particularly useful for the purification of microgram amounts of CoQ. Progress in purification was followed by means of ultraviolet spectra in ethanol over the 250310 m~ range. Fractions with a peak in the vicinity of 275 m~ were further purified until concentrates having the typical CoQ absorption were obtained.
RESULTS AND DISCUSSION
COENZYME Q
171
TABLE I DISTRIBUTION OF COENZYME Q Source
Monkey heart Rana pipiens frog (nerve tissue) HAd-1 tumors from mice S-180 tumors from mice HS-1 tumors from mice Shark liver Cecropia moth (thorax) Polyporous schweinitzii Mycoplasma gallisepticum Clostridium sticklandii Ochromonas malhamensis
Wet weight (mg./100 g.)
6.0 1.5 1.3 1.5 1.1 13.0 5.8
60 (dry weight)
CoQ homolog(s)
10 10 10 10 10 9, 10 9 9, 10
Identificationand estimationmethodsa
AUV, C, RI, LMB TLC (S, P) AUV, C, Ry, LMB 5UV, C, Rs, LMB ~UV, C, Rf, LMB TLC(S), UV, Rs, LMB, m.p., NMR AUV, RI, LMB, m.p. Rj, LMB AUV, TLC (S, P) AUV, Rf, LMB AUV, RI, LMB, TLC (S)
10
a Abbreviations: AUV, difference in intensity of absorption spectra of quinone and hydroquinone forms at 275 m~; C, Craven assay; RI, papergram mobility; LMB, leucomethylene blue spray reagent; TLC, thin-layer chromatography; (S), silica gel; (P), polyethylene; m.p., melting point; NMR, nuclear magnetic resonance.
not be considered unreasonable, particularly in view of its rather unusual and, as yet, unknown characteristics. Coenzyme Q was not detected in an earlier examination of a species of Polyporous (9). Present evidence for coenzymes Q9 and Qi0 in the cells of Polyporous schweinitzii is given by the papergram data. I t is of interest t h a t only CoQi0 was identified in h u m a n and rodent tumors grown in mice. Assays on tissues from normal mice have shown the presence of both coenzymes Q9 and Q10. Walleyed pike are known to contain CoQ~, and CoQ10 has been reported, though not completely identified in several organs from cod. The presence in tiger shark liver of a small amount of CoQtQ, in addition to CoQg, was rather unexpected. The shark's diet includes, however, a number of creatures, such as crabs, snails, and rays, which have not been analyzed for CoQ. I t has been reported (27) t h a t the anemia and muscular dystrophy t h a t develop in rhesus monkeys maintained on a purified diet low in vitamin E are rapidly progressive states that lead to death unless a-tocopherol or the 6-chromanol of hexahydrocoenzyme Q4 is given. I t was desirable to determine the particular CoQ in the tissue of the rhesus monkey because of the curative activity of the CoQ-chroinanol in the anemic and
dystrophic states. Only CoQ10 was identified in the heart of the rhesus monkey. This primate m a y serve as an experimental model for a study of CoQ in h u m a n disease. Nerve tissue of the frog is commonly used in fundamental studies of physiology. I t was of interest to establish the presence and identity of the CoQ in frog nerve tissue. Coenzyme QI0 was found. Frog nerve tissue might be a model for research on CoQi0 and nerve physiology. ACKNOWLEDGMENTS We are indebted to Dr. T. C. Stadtman, National Institute, National Institutes of Health, for the cells of Clostridium sticklandii; to Dr. W. J. Robbius, New York Botanical Garden, for the cells of Polyporous schweinitzii; and to Dr. J. H. Heller, New England Institute for Medical Research, for the tiger shark liver. The PPLO strain was isolated by P. Smith at the University of Pennsylvania and designated "J strain." REFERENCES 1. CRANE, F. L., HATEFI, Y., LESTER, R. L., AND WIDMER, C., Biochim. Biophys. Acta 25, 220
(1957). 2. MORTON, R. A., GLOOR, U., SCHINDLER, O., WILSON, G. M., CHOPARD-DIT-JEAN,L. H., HEMMING, F. W., ISLER, O., LEAT, W. M. F., PISNNOCK, J. F., Ri.~EGG, R., SCHWIETER,U., AND W I S S ,
~195S).
O.,
Helv. Chim. Acta 41, 2343
172
GALE ET AL.
3. MORTON, R. A., Nature 182, 1764 (1958). 4. LESTER, R. L., CRANE, F. L., AND HATEFI, Y., J. Am. Chem. Soc. 80, 4751 (1958). 5. CLOVER, J., AND THRELFALL, D. R., Biochem. J. 85, 14P (1962). 6. GALE, P. H., ARISON, B. H., TRENNER, N. R., PAGE, A. C., JR., AND FOLKERS, K., Biochemistry 2, 196 (1963). 7. LAVATE,W. V., DYER, J. R., SPRINGER,C. M., AND BENTLEY, R., J. Biol. Chem. 237, PC 2715 (1962). 8. GALE, P. I-I., TRENNER, N. R., ARISON, B. H., PAGE, A. C., JR., AND FOLKERS, K., Biochem. Biophys. Res. Commun. 12, 414 (1963). 9. LESTEI% R. L., AND CRANE, F. L., J. Biol. Chem. 234, 2169 (1959). 10. MORTON, R. A., WILSON, G. M., LOWE, J. S., AND LEAT, W. M. F., Chem. Ind. (London) 1957, 1649. 11. DIPLOCK, A. T., EDWIN, E. E., GREI~]N, J., BUNYAN~J., ANDMARCINKIEWICZ,S., Nature 186, 554 (1960). 12. GALE, P. H., KONIUSZY, F. R., PAGE, A. C., JR., AND FOLKERS, K., Arch. Biochern. BiGphys. 93,211 (1961). 13. PAGE, A. C., JR., GALE, P. H., WALLICK, H., WALTON, 1:~. B., McDANIEL, L. E., WOODRUFF, H. B., AND FOLKERS, K., Arch. Biochem. Biophys. 89,318 (1960). 14. PANDYA,K. P., BISHOP, D. H. L., AND KING, H. K., Biochem. J. 78, 35P (1961). 15. BISHOP, D. H. L., PANDYA, K. P., AND KING, H. K., Bioehem. J. 83,606 (1962).
16. CUNNINGHAM,N. F., ANDMORTON, R. A., BiGchem. J. 72, 92 (1959). 17. LowE, J. S., MORTON, R. A., AND VERNON, J., Biochem. J. 67,228 (1957). 18. LINN, B. O., PAGE, A. C., JR., WONG, E. L., GALE, P. H., SHUNK, C. H., AND FOLKERS, K., J. Am. Chem. Soc. 81, 4007 (1959). 19. HELLER, J., SZARKOWSKA,L., AND MICHALEK, H., Nature 188, 491 (1960). 20. JAYARAMAN,J., AND RAMASARMA,T., J. Sci. Ind. Res. (India) 20C, 69 (1961). 21. PAGE, A. C., JR., GALE, P. H., KONIUSZY, F. R., AND FOLKERS, K., Arch. Biochem. BiGphys. 85, 474 (1959). 22. CRANE, F. L., LESTER, R. L., WIDMER, C., AND HATEFI, Y., Biochim. Biophys. Acta 32, 73 (1959). 23. LESTER, R. L., AND CRANE, F. L., Biochim. Biophys. Acta 32,492 (1959). 24. LESTER, R. L., HATEFI, Y., WIDMER, C., AND CRANE, F. L., Biochim. Biophys. Acta 33, 169 (1959). 25. KoNIUSZY, F. R., GALE, P. H., PAGE, A. C., JR., AND FOLKERS, K., Arch. Biochem. BiGphys. 87, 298 (1960). 26. Technical Bulletin No. 22, November 1962, BrinkInann Instruments, Inc., and literature references cited. 27. DINNING, J. S., FITCH, C. D., SHUNK, C. H., AND FOLKERS, K., J. Am. Chem. Soc. 84, 2007 (1962).