Utilization oÃ-Riboflavin Homologues by D-Amino Acid Oxidase and Xanthine Oxidase1
ABSTRACT D-Amino acid oxidase and xanthine oxidase, two enzymes possessing ionically bound flavin coenzymes, have been studied with their flavin coenzymes derived from either 7-ethyl-8-methyl-flavin or 7-methyl-8ethyl-flavin, vitamin-like homologues of riboflavin. 7-Ethyl-8-methyl-flavin caused a significant reduction of both D-amino acid oxidase and xanthine oxidase in the liver, but not in the kidney. 7-Methyl-8-ethyl-flavin caused a significant reduction of D-amino acid oxidase in both the liver and kidney, a significant reduction of xanthine oxidase in the liver, but a large and sig nificant increase of the latter enzyme in the kidney. An improved procedure for the assay of xanthine oxidase has been described. J. Nutr. 107: 645-649, 1977. INDEXING KEY WORDS riboflavin • riboflavin homologues • enzymes •D-amino acid oxidase •xanthine oxidase Of the approximately two dozen ana logues of riboflavin (fig. 1, A) whose bio logical activities have been studied in some detail (1), two are unique; they are 7-ethyl8-methyl-10- ( 1'-o-ribity 1) isoalloxazine ( 7ethyl-8-methyl-flavin; fig. 1, B) and 7methyl-8-ethyl-lO- ( I'-o-ribityl )isoalloxazine (7-methyl-8-ethyl-flavin; fig. 1, C) (2). These two homologues of riboflavin are the only two synthetic analogues of any of the B-complex vitamins which are able to re place the naturally occurring vitamin in the metabolism of mammalian tissue (3). When either is the sole flavin being utilized by the rat, the animals are indistinguish able in terms of growth, food utilization, appearance and survival from those utiliz ing riboflavin. In terms of growth and ef ficiency of food utilization, 7-ethyl-8methyl-flavin and 7-methyl-8-ethyl-flavin have 47% and 36%, respectively, of the activity of riboflavin. It was observed, however, that females fed diets containing either flavin from weaning until adulthood, were unable to 645
rear their normal-appearing, newborn young (4). In the absence of any observ able physical defects in the young, efforts were made to correlate some specific flavin inadequacy with the mortality of the young. It was found that the utilization of 7-ethyl-8-methyl-flavin caused the succinic acid dehydrogenase (EC 1.3.99.1) (SDH) to fall to 56%, 33%, and 10% of normal activity in the kidney, heart, and liver, respectively, in the adult rat (5) and to 8%, 7%, and 6% in the same tissues of the fetuses produced by such females (6). When 7-methyl-8-ethyl-flavin was utilized, the SDH activities of the kidney, heart, and liver fell to 70%, 60%, and 55%, re spectively, of the normal values (7). AnReceived for publication July 16, 1976. 1 This work was supported In part by Grant No. AM 11034 from the National Institute of Arthritis and Metabolic Disease and Grant No. HD 01306 from the National Institute of Child Health and Human Development. "This report constitutes part of the dissertation submitted by J. J. Dombrowskl for the Ph.D. degree, University of Nebraska, 1971. * To whom inquiries concerning this report are to be sent at the University of Maryland.
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JOSEPH J. DOMDROWSKI,2 ROBERT D. FAULKNER, ANDJOHN P. LAMBOOY3 Department of Biochemistry, University of Nebraska College of Medicine, Omaha, Nebraska 68105 and the Department of Biochemistry, University of Maryland School of Dentistry, Baltimore, Maryland 21201
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DOMDROWSKI, FAULKNER AND LAMBOOY
(EC 1.2.3.2) (XO) in the kidneys and livers of rats utilizing either homologue of riboflavin. MATERIALS AND METHODS
Weanling female rats of the Wistar strain 4 weighing between 40 and 45 g were used. The maintenance of the rats and the basal diet fed have been described (8). The riboflavin diet (basal diet) contained 20 mg/kg of riboflavin; each of the homo logue diets contained 20.8 mg/kg of the appropriate homologue. All groups were fed their respective diets ad libitum until they weighed approximately 200 g, at which time the enzyme assays were per formed. Eight groups of from 10 to 12 rats were used. Separate groups of rats were used for each enzyme for each homologue, and all assays were done simultaneously from a riboflavin group and from a homo Fig. 1 Basic flavin structure. logue group. For each rat, the kidney and Flavin RT R* Trivial name liver enzyme preparations were assayed simultaneously. ABCCH,-C,H6-CH,-CH,~CH,-C,H5-(Rb)(7-Et)(8-Et)Riboflavin7-Ethyl-8-methyl-flavin7-Methyl-8-ethyl-flavin Immediately following decapitation and exsanguination, the kidneys and livers were removed from rats and placed in cold buf fer solutions; 0.02 M pyrophosphate buffer 5 was used for the DAAO assays, and 0.1 M phosphate buffer6 was used for the XO other essential mitochondrial flavoprotein assays. In the cold room,7 the kidney cap enzyme, the electron transport linked disule and medullary portion were removed, phosphopyridine nucleotide dehydrogenase and the blotted kidney weighed. The tissue (EC 1.6.99.3) (DPNHD) (NADHD), in was placed in 9 ml of the appropriate buf which the FAD is not covalently bonded fer for each g of tissue and homogenized as in SDH, but ionically bound, shows the in a Virtis homogenizer at maximum speed same activity in these tissues whether FAD 30 seconds. The homogenate was trans is used or 7-ethyl-8-methyl-flavin-F'AD is for ferred to a Dounce 8 (9) homogenizer and used. Such changes in the activity of SDH 12 passes with the loose-fitting plunger coupled with no change in the activity of were made. The homogenate from the last DPNHD, both mitochondrial enzymes, the step was used directly for the DAAO first enzyme characterized by covalently assays. bonded flavin and the second character ized by ionically bonded flavin, in obvi ' CPN rats, Carworth, Inc. These rats are now from Carworth Division, Charles River ously healthy adult animals, suggested the available Breeding Laboratories, Wilmington, Mass. 01887. 5 Sodium pyrophosphate buffer, 0.2 M. Dissolve possibility that the form of the attachment 22.30 g (0.05 mole) of sodium pyrophosphate decabetween the coenzyme and the apoenzyme hydrate In water and dilute to 950 ml. Concentrated hydrochloric acid (approximately 3.3 ml) was added might be a determining factor. This thought to make pH 8.35 ; dilute to 1 liter. The above stock prompted us to investigate the activities of solution, 200 ml, was diluted to 500 ml for use; was 8.6. two additional enzymes that possessed pH«Phosphate buffer (0.1 M) with 1 mM EDTA. To 500 ml of 0.20 M monobasic potassium phosphate ionically bound flavin coenzymes but en solution was added 452 ml of 0.2 N sodium hydroxide zymes that were present in the cytosol. solution ; adjust to pH 7.8. Add 10 ml of 0.1 M EDTA solution and make to 1 liter. This is a report of our findings concerning ' All operations were performed in the cold room ; the activities of o-amino acid oxidase (EC all 9 Dounce solutions held in ice. homogenlzers are available from Kontes 1.4.3.3) (DAAO) and xanthine oxidase Glass Co., Vlneland, N.J.
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H-C-OH H-Ç-OH H-C-OH H-C-OH H-C-H
RIBOFLAVIN
HOMOLOGUES
647
cuvette, 0.10 ml of enzyme solution was added, the contents mixed and a lag period of 3 minutes permitted to elapse. The spec trophotometer was nulled on the blank and the OD of the reaction cuvette measured at 295 nm at 30 second intervals for a pe riod of approximately 10 minutes. The as say is repeated using 2.80 ml of buffer and xanthine working solution and 0.20 ml of enzyme preparation. The change in OD is plotted against time to obtain the OD change/minute/ml assay solution. This value multiplied by 3 gives the OD change/minute/assay sample which, when divided by the protein content of the en zyme solution used provides the specific activity as OD change/minute/mg protein. The procedure described by Waldman and Burch (13) was used for the assay of DAAO; the activity was expressed as /* moles pyruvate formed/minute/mg pro tein. RESULTS
The assays were done by three different individuals at times separated by 2 years and in different laboratories. Some aspects of the procedures were checked and some of the assays repeated by a fourth indi vidual in a third laboratory. The procedure used for the assay of DAAO was that de scribed by Waldman and Burch (13). The assay of XO made use of a procedure de scribed in the literature ( 10) for the 7ethyl-8-methyl-flavin and its associated riboflavin groups. However, it was con cluded that the procedure could be im proved; the method used for the 7-methyl8-ethyl-flavin and its associated riboflavin groups was the one described here (done by J. J. D.) and which was based on a procedure used by Dr. Ranier Fried of Creighton University for the preparation of XO from milk. For these reasons in the ine International Equipment Company, B-20 Centri fuge. *° It Is necessary that the enzyme solution gives an OD change during the assay of no greater than 0.03 unit/minute to assure a linear reaction rate (11). u It was found that following this Initial period in the frozen state (—25°)the enzyme activity and pro tein concentration were stable for approximately 14 12Xanthine stock solution (0.01 M). Crystalline xanthine, 38.03 mg, are dissolved in a small volume of 0.01 N sodium hydroxide solution, then made to 25 ml with the same solution. The 0.1 mu xanthine \vorklng solution is prepared by diluting 1.00 ml of the above stock solution to 100 ml with 0.1 M phosphate-1 m M EDTA buffer solution.
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A portion of the right median lobe (about 2.5 g) of each liver was blotted, weighed and added to 9 ml of the appro priate buffer for each g of tissue weight, in a Dounce homogenizer. Following 12 passes with the loose-fitting plunger, homogenization was complete. This homogenate was used directly for the DAAO assays. The assay of XO for the 7-ethyl-8-methylflavin group and its associated riboflavin group was done as described by Murray and Chaykin (10). The preparation of the enzyme for the assay of XO for the 7-methyl-8-ethyl-flavin group and its associated riboflavin group was done by the following procedure. To the stirred tissue homogenate in 0.1 M phosphate buffer prepared above, was added dropwise, 4 M acetic acid until the pH was 5.0 (at 3°), and the homogenate was then centrifugea at 21,000 X g9 for 30 minutes in 40 ml tubes and the No. 870 head. The pellet was discarded and to the stirred supernatant solution was added 1 N sodium hydroxide solution until the pH was 7.9. To a portion of this stirred solu tion was added dropwise as quickly as possible the predetermined volume of satu rated (for 0°) ammonium sulfate solution (SASS) to make the final concentration 32% saturated; the suspension was permit ted to stand 15 minutes and then centrifuged for 30 minutes in the No. 870 head at 12,000 x g.9 The pellet was discarded and the supernatant solution brought to 52% saturation by the addition of the proper amount of SASS. Again, after stand ing 15 minutes the suspension was centrifuged as immediately above. The pellet was dissolved in sufficient 0.1 M phosphate buffer to provide a clear solution at a concentration of from 0.5 to 1.0 mg10 of protein (12) per 0.1 ml of enzyme solu tion. The enzyme preparations were then frozen and assayed 24 to 48 hours after being frozen.11 The assay procedure for XO was as fol lows. To one quartz cuvette was added 2.90 ml of 0.1 M phosphate buffer and to another 2.90 ml of xanthine working solu tion.12 The cuvettes were placed in the spectrophotometer and allowed to equili brate at 37°for 15 to 20 minutes. To each
AND ENZYMES
648
DOMDROWSKI, FAULKNER AND LAMBOOY TABLE 1
DISCUSSION
D-Amino acid oxidase and xanthine oxidase activities for the liver and kidneys of rats receiving riboflavin or one of its homologues
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Since rats utilizing riboflavin, 7-ethyl-8methyl-flavin or 7-methyl-8-ethyl-flavin are indistinguishable, it might reasonably be Group'Rb-17-EtRb-2 expected that the flavoprotein enzymes would all show essentially normal activity. acid100± This was not found to be the case. 7-Ethyl8%3 8-methyl-flavin caused the SDH activities 67±13%*100±106±2%100±7%< of the kidney, heart, and liver to be greatly 8%5 reduced below what might be considered 8-EtRb-37-EtRb-48-EtRatsno.121211 ±7%100±8%84±7%100±3% 61 1012 5%Xanthine 42± normal (5); while the use of 7-methyl-8oxidase8100± ethyl-flavin, although it caused a less severe drop in the SDH activities in these tissues, 5% 52±12%5100± 12118LiverD-Amino was still responsible for a significant drop (7), yet these reduced activities caused no 4% 150±6%" apparent harm to the rat. The activities of 62±6%sKidneyoxidase'100±6% DPNHD were found to be the same in 1D-Aminoacid oxidase was measured as /imoles of these tissues whether riboflavin or 7-ethylpyruvate formed/minute/mg protein. ! Rb = riboflavin; 7-Et = 7-ethyl-8-methyl-flavin ; 8-Et 8-methyl-flavin was used (6). This sug = ^-methyl-S-ethyl-flayin. ' The value for the gested that inability to form the special activity of the enzyme in the tissue of the riboflavin covalent bond between the flavin and the group expressed as 100% with the SEMexpressed as apoenzyme for SDH, might be responsible percentage of the average value. For example, the value for the designated enzyme activity was for the reduced activities when the homo 0.012±0.001 Mmoles of pyruvate formed; 0.001/ logues were used; however, the nature of 0.012 X 100 = ±8%. 4Significantly different the covalent bond would seem to support from value immediately above [Student's "t" test or suggest that the 7-ethyl-8-methyl-flavin (14) ; P = 0.002 to 0.007]. 5Significantly dif ferent from value immediately above (Student's "t" would have provided more activity for test; P = 0.001 or less). • Xanthine oxidase was SDH because of the more nearly normal measured as AOD/minute/mg protein. configuration of the bond (7). The structural demands for the covalent terests of uniformity and clarity, the re bond are not required for the activity as a sults presented in table 1 have been given coenzyme (as FAD or as homologue-F'AD) in percentage of the value of the corre for DPNHD, which is linked to the apo sponding tissue in the riboflavin control enzyme by ionic bonds. The relative sim group, measured at the same time by the plicity of the ionic bond and the normal activity of DPNHD suggested that en same individual. When the metabolically active flavin in zymes requiring only the ionic bond might the rat was 7-ethyl-8-methyl-flavin, it pro show normal activity when the homologues vided a significantly reduced DAAO activ are used, but this was found not to be the ity in the liver, when compared with ribo case. Both homologues caused a reduction flavin, but it supported the same level of in the activity of DAAO and XO in the activity as the vitamin in the kidney. This liver. The 7-methyl-8-ethyl-flavin caused a homologue also provided a significantly reduction in the activity of DAAO, and a large increase in the activity of XO in the reduced XO activity in the liver, but it pro vided essentially full coenzymic activity kidney. for XO in the kidney. It has been relatively commonplace to When the metabolically active flavin in find a substantial reduction in the enzyme the rat was 7-methyl-8-ethyl-flavin, it pro activities during vitamin deprivation and vided a significantly reduced DAAO activ the resulting deficiency state. We have now ity in the liver and the kidney, and also a shown that a substantial reduction can be significantly reduced XO activity in the produced in the activity in the enzymes de liver. However, it caused a remarkable and scribed above by the use of vitamin-like consistent increase of an average of 50% homologues. That one of these homologues in the XO activity of the kidney. caused an increase in the activity of one of
RIBOFLAVIN
HOMOLOGUES
ACKNOWLEDGMENTS
The authors wish to express sincere ap preciation to Dr. Rainer Fried, Department of Biochemistry, Creighton University, for much valuable discussion and advice con cerning the purification of xanthine oxidase. They also wish to thank Mr. Warren A. Hill for his excellent assistance in confirm ing the utility of the procedure described in this report for the assay of xanthine oxidase.
649
LITERATURE CITED 1. Lambooy, J. P. (1975) Biological activities of analogs of riboflavin. In: Riboflavin (Rivlin, R. S., ed.), Chapt. 10, pp. 303-368, Plenum Press, New York. 2. Lambooy, J. P. (1958) The synthesis of 6ethyl-7-methyl-9-( 1'-D-ribityl)isoalloxazine and 6-methyl-7-ethyl-9-(l'-D-ribityl)isoalloxazine. J. Am. Chem. Soc. 80, 110-113. 3. Lambooy, J. P. (1961) Growth promoting properties of 6-ethyl-7-methyl-9-( l'-D-ribityl)isoalloxazine and 6-methyl-7-ethyl-9-( I'-Dribityl)isoalloxazine. J. Nutr. 75, 116-126. 4. Lambooy, J. P. (1958) The biological ac tivity of 6-ethyl-7-methyland 6-methyl-7ethyl-( l'-D-ribityl)isoalloxazine. Biochim. Biophys. Acta 29, 221. 5. Kim, Y. S. & Lambooy, J. P. (1967) Use of a riboflavin homolog in the reduction of succinic dehydrogenase activity in tissues of healthy rats. Arch. Biochem. Biophys. 122, 644-647. 6. Kim, Y. S. & Lambooy, J. P. (1971) In duction of a specific enzyme inadequacy in infant rats by the use of a homologue of ribo flavin. J. Nutr. 101, 819-830. 7. Dombrowski, J. J. & Lambooy, J. P. (1973) The reduction of succinic dehydrogenase in the rat by 7-methyl-8-ethyl-flavin. Arch. Bio chem. Biophys. 159, 378-382. 8. Lambooy, J. P., Smith, C. D. & Kim, Y. S. (1971) Utilization of 7-chloro-8-methylnavin in the rat and its specific antiriboflavin action in the kidney. J. Nutr. 101, 11371146. 9. Dounce, A. L., Witter, R. F., Monty, K. J., Pate, S. & Cottone, M. A. ( 1955) A method for isolating intact mitochondria and nuclei from the same homogenate, and the influence of mitochondrial destruction on the properties of cell nuclei. J. Biophys. Biochem. Cytol. 1, 139-153. 10. Murray, K. N. & Chaykin, S. J. (1966) The reduction of nicotinamide N-oxide by xathine oxidase. J. Biol. Chem. 241, 3468-3473. 11. Avis, P. G., Bergel, F. & Bray, R. C. (1955) Cellular constituents. The chemistry of xathine oxidase. Partoxidase I. The from preparation of aJ. crystal line xathine cow's milk. Chem. Soc. //, 1100-1105. 12. Lowry, O. H., Rosebrough, N. J., Fair, A. L. & Randall, R. J. (1951) Protein measure ment with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. 13. Waldman, R. H. & Burch, H. B. (1963) Rapid method for the study of enzyme distri bution in rat kidney. Am. J. Physiol. 204, 74914. 752. "Student" (1908) The Probable Error of the Mean. Biometrika. 6, 1-25.
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these enzymes in one of the tissues is cer tainly remarkable and might very well be a unique finding. Whether the activities of these several enzymes were reduced, ele vated or maintained within normal limits appeared to be of little consequence to the rats since they were in excellent health. The concept of what constitutes normal en zyme activities would appear to be of lim ited value in situations of this sort; such concepts might be questionable. For ex ample (5), in the case of SDH in the liver of animals utilizing 7-ethyl-8-methyl-flavin, the animal thrives when the activity of a critical enzyme is only 10% of normal, thus implying that the normal level when riboflavin is utilized may be ten times greater than required to sustain good health. Postulating a reason for the atypical in crease in activity of XO in the kidney when 7-methyl-8-ethyl-flavin is utilized is diffi cult. The increase may be due to more ef ficient functioning of the homologue-F'AD coenzyme or to an increase in the XO apoenzyme molecules. Not much is known about the biological function of XO. It has low specificity but its activity is highest for the oxidation of xanthine, however, its most important or natural substrate is un known. The elevated activity might be no more than a fortuitous combination of an abnormal coenzyme and an abnormal sub strate. For all of these reasons, speculation as to the causes for the noteworthy increase described above would seem unpromising at this time.
AND ENZYMES
ABSTRACT D-Amino acid oxidase and xanthine oxidase, two enzymes possessing ionically bound flavin coenzymes, have been studied with their flavin coenzymes derived from either 7-ethyl-8-methyl-flavin or 7-methyl-8ethyl-flavin, vitamin-like homologues of riboflavin. 7-Ethyl-8-methyl-flavin caused a significant reduction of both D-amino acid oxidase and xanthine oxidase in the liver, but not in the kidney. 7-Methyl-8-ethyl-flavin caused a significant reduction of D-amino acid oxidase in both the liver and kidney, a significant reduction of xanthine oxidase in the liver, but a large and sig nificant increase of the latter enzyme in the kidney. An improved procedure for the assay of xanthine oxidase has been described. J. Nutr. 107: 645-649, 1977. INDEXING KEY WORDS riboflavin • riboflavin homologues • enzymes •D-amino acid oxidase •xanthine oxidase Of the approximately two dozen ana logues of riboflavin (fig. 1, A) whose bio logical activities have been studied in some detail (1), two are unique; they are 7-ethyl8-methyl-10- ( 1'-o-ribity 1) isoalloxazine ( 7ethyl-8-methyl-flavin; fig. 1, B) and 7methyl-8-ethyl-lO- ( I'-o-ribityl )isoalloxazine (7-methyl-8-ethyl-flavin; fig. 1, C) (2). These two homologues of riboflavin are the only two synthetic analogues of any of the B-complex vitamins which are able to re place the naturally occurring vitamin in the metabolism of mammalian tissue (3). When either is the sole flavin being utilized by the rat, the animals are indistinguish able in terms of growth, food utilization, appearance and survival from those utiliz ing riboflavin. In terms of growth and ef ficiency of food utilization, 7-ethyl-8methyl-flavin and 7-methyl-8-ethyl-flavin have 47% and 36%, respectively, of the activity of riboflavin. It was observed, however, that females fed diets containing either flavin from weaning until adulthood, were unable to 645
rear their normal-appearing, newborn young (4). In the absence of any observ able physical defects in the young, efforts were made to correlate some specific flavin inadequacy with the mortality of the young. It was found that the utilization of 7-ethyl-8-methyl-flavin caused the succinic acid dehydrogenase (EC 1.3.99.1) (SDH) to fall to 56%, 33%, and 10% of normal activity in the kidney, heart, and liver, respectively, in the adult rat (5) and to 8%, 7%, and 6% in the same tissues of the fetuses produced by such females (6). When 7-methyl-8-ethyl-flavin was utilized, the SDH activities of the kidney, heart, and liver fell to 70%, 60%, and 55%, re spectively, of the normal values (7). AnReceived for publication July 16, 1976. 1 This work was supported In part by Grant No. AM 11034 from the National Institute of Arthritis and Metabolic Disease and Grant No. HD 01306 from the National Institute of Child Health and Human Development. "This report constitutes part of the dissertation submitted by J. J. Dombrowskl for the Ph.D. degree, University of Nebraska, 1971. * To whom inquiries concerning this report are to be sent at the University of Maryland.
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JOSEPH J. DOMDROWSKI,2 ROBERT D. FAULKNER, ANDJOHN P. LAMBOOY3 Department of Biochemistry, University of Nebraska College of Medicine, Omaha, Nebraska 68105 and the Department of Biochemistry, University of Maryland School of Dentistry, Baltimore, Maryland 21201
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DOMDROWSKI, FAULKNER AND LAMBOOY
(EC 1.2.3.2) (XO) in the kidneys and livers of rats utilizing either homologue of riboflavin. MATERIALS AND METHODS
Weanling female rats of the Wistar strain 4 weighing between 40 and 45 g were used. The maintenance of the rats and the basal diet fed have been described (8). The riboflavin diet (basal diet) contained 20 mg/kg of riboflavin; each of the homo logue diets contained 20.8 mg/kg of the appropriate homologue. All groups were fed their respective diets ad libitum until they weighed approximately 200 g, at which time the enzyme assays were per formed. Eight groups of from 10 to 12 rats were used. Separate groups of rats were used for each enzyme for each homologue, and all assays were done simultaneously from a riboflavin group and from a homo Fig. 1 Basic flavin structure. logue group. For each rat, the kidney and Flavin RT R* Trivial name liver enzyme preparations were assayed simultaneously. ABCCH,-C,H6-CH,-CH,~CH,-C,H5-(Rb)(7-Et)(8-Et)Riboflavin7-Ethyl-8-methyl-flavin7-Methyl-8-ethyl-flavin Immediately following decapitation and exsanguination, the kidneys and livers were removed from rats and placed in cold buf fer solutions; 0.02 M pyrophosphate buffer 5 was used for the DAAO assays, and 0.1 M phosphate buffer6 was used for the XO other essential mitochondrial flavoprotein assays. In the cold room,7 the kidney cap enzyme, the electron transport linked disule and medullary portion were removed, phosphopyridine nucleotide dehydrogenase and the blotted kidney weighed. The tissue (EC 1.6.99.3) (DPNHD) (NADHD), in was placed in 9 ml of the appropriate buf which the FAD is not covalently bonded fer for each g of tissue and homogenized as in SDH, but ionically bound, shows the in a Virtis homogenizer at maximum speed same activity in these tissues whether FAD 30 seconds. The homogenate was trans is used or 7-ethyl-8-methyl-flavin-F'AD is for ferred to a Dounce 8 (9) homogenizer and used. Such changes in the activity of SDH 12 passes with the loose-fitting plunger coupled with no change in the activity of were made. The homogenate from the last DPNHD, both mitochondrial enzymes, the step was used directly for the DAAO first enzyme characterized by covalently assays. bonded flavin and the second character ized by ionically bonded flavin, in obvi ' CPN rats, Carworth, Inc. These rats are now from Carworth Division, Charles River ously healthy adult animals, suggested the available Breeding Laboratories, Wilmington, Mass. 01887. 5 Sodium pyrophosphate buffer, 0.2 M. Dissolve possibility that the form of the attachment 22.30 g (0.05 mole) of sodium pyrophosphate decabetween the coenzyme and the apoenzyme hydrate In water and dilute to 950 ml. Concentrated hydrochloric acid (approximately 3.3 ml) was added might be a determining factor. This thought to make pH 8.35 ; dilute to 1 liter. The above stock prompted us to investigate the activities of solution, 200 ml, was diluted to 500 ml for use; was 8.6. two additional enzymes that possessed pH«Phosphate buffer (0.1 M) with 1 mM EDTA. To 500 ml of 0.20 M monobasic potassium phosphate ionically bound flavin coenzymes but en solution was added 452 ml of 0.2 N sodium hydroxide zymes that were present in the cytosol. solution ; adjust to pH 7.8. Add 10 ml of 0.1 M EDTA solution and make to 1 liter. This is a report of our findings concerning ' All operations were performed in the cold room ; the activities of o-amino acid oxidase (EC all 9 Dounce solutions held in ice. homogenlzers are available from Kontes 1.4.3.3) (DAAO) and xanthine oxidase Glass Co., Vlneland, N.J.
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H-C-OH H-Ç-OH H-C-OH H-C-OH H-C-H
RIBOFLAVIN
HOMOLOGUES
647
cuvette, 0.10 ml of enzyme solution was added, the contents mixed and a lag period of 3 minutes permitted to elapse. The spec trophotometer was nulled on the blank and the OD of the reaction cuvette measured at 295 nm at 30 second intervals for a pe riod of approximately 10 minutes. The as say is repeated using 2.80 ml of buffer and xanthine working solution and 0.20 ml of enzyme preparation. The change in OD is plotted against time to obtain the OD change/minute/ml assay solution. This value multiplied by 3 gives the OD change/minute/assay sample which, when divided by the protein content of the en zyme solution used provides the specific activity as OD change/minute/mg protein. The procedure described by Waldman and Burch (13) was used for the assay of DAAO; the activity was expressed as /* moles pyruvate formed/minute/mg pro tein. RESULTS
The assays were done by three different individuals at times separated by 2 years and in different laboratories. Some aspects of the procedures were checked and some of the assays repeated by a fourth indi vidual in a third laboratory. The procedure used for the assay of DAAO was that de scribed by Waldman and Burch (13). The assay of XO made use of a procedure de scribed in the literature ( 10) for the 7ethyl-8-methyl-flavin and its associated riboflavin groups. However, it was con cluded that the procedure could be im proved; the method used for the 7-methyl8-ethyl-flavin and its associated riboflavin groups was the one described here (done by J. J. D.) and which was based on a procedure used by Dr. Ranier Fried of Creighton University for the preparation of XO from milk. For these reasons in the ine International Equipment Company, B-20 Centri fuge. *° It Is necessary that the enzyme solution gives an OD change during the assay of no greater than 0.03 unit/minute to assure a linear reaction rate (11). u It was found that following this Initial period in the frozen state (—25°)the enzyme activity and pro tein concentration were stable for approximately 14 12Xanthine stock solution (0.01 M). Crystalline xanthine, 38.03 mg, are dissolved in a small volume of 0.01 N sodium hydroxide solution, then made to 25 ml with the same solution. The 0.1 mu xanthine \vorklng solution is prepared by diluting 1.00 ml of the above stock solution to 100 ml with 0.1 M phosphate-1 m M EDTA buffer solution.
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A portion of the right median lobe (about 2.5 g) of each liver was blotted, weighed and added to 9 ml of the appro priate buffer for each g of tissue weight, in a Dounce homogenizer. Following 12 passes with the loose-fitting plunger, homogenization was complete. This homogenate was used directly for the DAAO assays. The assay of XO for the 7-ethyl-8-methylflavin group and its associated riboflavin group was done as described by Murray and Chaykin (10). The preparation of the enzyme for the assay of XO for the 7-methyl-8-ethyl-flavin group and its associated riboflavin group was done by the following procedure. To the stirred tissue homogenate in 0.1 M phosphate buffer prepared above, was added dropwise, 4 M acetic acid until the pH was 5.0 (at 3°), and the homogenate was then centrifugea at 21,000 X g9 for 30 minutes in 40 ml tubes and the No. 870 head. The pellet was discarded and to the stirred supernatant solution was added 1 N sodium hydroxide solution until the pH was 7.9. To a portion of this stirred solu tion was added dropwise as quickly as possible the predetermined volume of satu rated (for 0°) ammonium sulfate solution (SASS) to make the final concentration 32% saturated; the suspension was permit ted to stand 15 minutes and then centrifuged for 30 minutes in the No. 870 head at 12,000 x g.9 The pellet was discarded and the supernatant solution brought to 52% saturation by the addition of the proper amount of SASS. Again, after stand ing 15 minutes the suspension was centrifuged as immediately above. The pellet was dissolved in sufficient 0.1 M phosphate buffer to provide a clear solution at a concentration of from 0.5 to 1.0 mg10 of protein (12) per 0.1 ml of enzyme solu tion. The enzyme preparations were then frozen and assayed 24 to 48 hours after being frozen.11 The assay procedure for XO was as fol lows. To one quartz cuvette was added 2.90 ml of 0.1 M phosphate buffer and to another 2.90 ml of xanthine working solu tion.12 The cuvettes were placed in the spectrophotometer and allowed to equili brate at 37°for 15 to 20 minutes. To each
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DOMDROWSKI, FAULKNER AND LAMBOOY TABLE 1
DISCUSSION
D-Amino acid oxidase and xanthine oxidase activities for the liver and kidneys of rats receiving riboflavin or one of its homologues
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Since rats utilizing riboflavin, 7-ethyl-8methyl-flavin or 7-methyl-8-ethyl-flavin are indistinguishable, it might reasonably be Group'Rb-17-EtRb-2 expected that the flavoprotein enzymes would all show essentially normal activity. acid100± This was not found to be the case. 7-Ethyl8%3 8-methyl-flavin caused the SDH activities 67±13%*100±106±2%100±7%< of the kidney, heart, and liver to be greatly 8%5 reduced below what might be considered 8-EtRb-37-EtRb-48-EtRatsno.121211 ±7%100±8%84±7%100±3% 61 1012 5%Xanthine 42± normal (5); while the use of 7-methyl-8oxidase8100± ethyl-flavin, although it caused a less severe drop in the SDH activities in these tissues, 5% 52±12%5100± 12118LiverD-Amino was still responsible for a significant drop (7), yet these reduced activities caused no 4% 150±6%" apparent harm to the rat. The activities of 62±6%sKidneyoxidase'100±6% DPNHD were found to be the same in 1D-Aminoacid oxidase was measured as /imoles of these tissues whether riboflavin or 7-ethylpyruvate formed/minute/mg protein. ! Rb = riboflavin; 7-Et = 7-ethyl-8-methyl-flavin ; 8-Et 8-methyl-flavin was used (6). This sug = ^-methyl-S-ethyl-flayin. ' The value for the gested that inability to form the special activity of the enzyme in the tissue of the riboflavin covalent bond between the flavin and the group expressed as 100% with the SEMexpressed as apoenzyme for SDH, might be responsible percentage of the average value. For example, the value for the designated enzyme activity was for the reduced activities when the homo 0.012±0.001 Mmoles of pyruvate formed; 0.001/ logues were used; however, the nature of 0.012 X 100 = ±8%. 4Significantly different the covalent bond would seem to support from value immediately above [Student's "t" test or suggest that the 7-ethyl-8-methyl-flavin (14) ; P = 0.002 to 0.007]. 5Significantly dif ferent from value immediately above (Student's "t" would have provided more activity for test; P = 0.001 or less). • Xanthine oxidase was SDH because of the more nearly normal measured as AOD/minute/mg protein. configuration of the bond (7). The structural demands for the covalent terests of uniformity and clarity, the re bond are not required for the activity as a sults presented in table 1 have been given coenzyme (as FAD or as homologue-F'AD) in percentage of the value of the corre for DPNHD, which is linked to the apo sponding tissue in the riboflavin control enzyme by ionic bonds. The relative sim group, measured at the same time by the plicity of the ionic bond and the normal activity of DPNHD suggested that en same individual. When the metabolically active flavin in zymes requiring only the ionic bond might the rat was 7-ethyl-8-methyl-flavin, it pro show normal activity when the homologues vided a significantly reduced DAAO activ are used, but this was found not to be the ity in the liver, when compared with ribo case. Both homologues caused a reduction flavin, but it supported the same level of in the activity of DAAO and XO in the activity as the vitamin in the kidney. This liver. The 7-methyl-8-ethyl-flavin caused a homologue also provided a significantly reduction in the activity of DAAO, and a large increase in the activity of XO in the reduced XO activity in the liver, but it pro vided essentially full coenzymic activity kidney. for XO in the kidney. It has been relatively commonplace to When the metabolically active flavin in find a substantial reduction in the enzyme the rat was 7-methyl-8-ethyl-flavin, it pro activities during vitamin deprivation and vided a significantly reduced DAAO activ the resulting deficiency state. We have now ity in the liver and the kidney, and also a shown that a substantial reduction can be significantly reduced XO activity in the produced in the activity in the enzymes de liver. However, it caused a remarkable and scribed above by the use of vitamin-like consistent increase of an average of 50% homologues. That one of these homologues in the XO activity of the kidney. caused an increase in the activity of one of
RIBOFLAVIN
HOMOLOGUES
ACKNOWLEDGMENTS
The authors wish to express sincere ap preciation to Dr. Rainer Fried, Department of Biochemistry, Creighton University, for much valuable discussion and advice con cerning the purification of xanthine oxidase. They also wish to thank Mr. Warren A. Hill for his excellent assistance in confirm ing the utility of the procedure described in this report for the assay of xanthine oxidase.
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LITERATURE CITED 1. Lambooy, J. P. (1975) Biological activities of analogs of riboflavin. In: Riboflavin (Rivlin, R. S., ed.), Chapt. 10, pp. 303-368, Plenum Press, New York. 2. Lambooy, J. P. (1958) The synthesis of 6ethyl-7-methyl-9-( 1'-D-ribityl)isoalloxazine and 6-methyl-7-ethyl-9-(l'-D-ribityl)isoalloxazine. J. Am. Chem. Soc. 80, 110-113. 3. Lambooy, J. P. (1961) Growth promoting properties of 6-ethyl-7-methyl-9-( l'-D-ribityl)isoalloxazine and 6-methyl-7-ethyl-9-( I'-Dribityl)isoalloxazine. J. Nutr. 75, 116-126. 4. Lambooy, J. P. (1958) The biological ac tivity of 6-ethyl-7-methyland 6-methyl-7ethyl-( l'-D-ribityl)isoalloxazine. Biochim. Biophys. Acta 29, 221. 5. Kim, Y. S. & Lambooy, J. P. (1967) Use of a riboflavin homolog in the reduction of succinic dehydrogenase activity in tissues of healthy rats. Arch. Biochem. Biophys. 122, 644-647. 6. Kim, Y. S. & Lambooy, J. P. (1971) In duction of a specific enzyme inadequacy in infant rats by the use of a homologue of ribo flavin. J. Nutr. 101, 819-830. 7. Dombrowski, J. J. & Lambooy, J. P. (1973) The reduction of succinic dehydrogenase in the rat by 7-methyl-8-ethyl-flavin. Arch. Bio chem. Biophys. 159, 378-382. 8. Lambooy, J. P., Smith, C. D. & Kim, Y. S. (1971) Utilization of 7-chloro-8-methylnavin in the rat and its specific antiriboflavin action in the kidney. J. Nutr. 101, 11371146. 9. Dounce, A. L., Witter, R. F., Monty, K. J., Pate, S. & Cottone, M. A. ( 1955) A method for isolating intact mitochondria and nuclei from the same homogenate, and the influence of mitochondrial destruction on the properties of cell nuclei. J. Biophys. Biochem. Cytol. 1, 139-153. 10. Murray, K. N. & Chaykin, S. J. (1966) The reduction of nicotinamide N-oxide by xathine oxidase. J. Biol. Chem. 241, 3468-3473. 11. Avis, P. G., Bergel, F. & Bray, R. C. (1955) Cellular constituents. The chemistry of xathine oxidase. Partoxidase I. The from preparation of aJ. crystal line xathine cow's milk. Chem. Soc. //, 1100-1105. 12. Lowry, O. H., Rosebrough, N. J., Fair, A. L. & Randall, R. J. (1951) Protein measure ment with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. 13. Waldman, R. H. & Burch, H. B. (1963) Rapid method for the study of enzyme distri bution in rat kidney. Am. J. Physiol. 204, 74914. 752. "Student" (1908) The Probable Error of the Mean. Biometrika. 6, 1-25.
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these enzymes in one of the tissues is cer tainly remarkable and might very well be a unique finding. Whether the activities of these several enzymes were reduced, ele vated or maintained within normal limits appeared to be of little consequence to the rats since they were in excellent health. The concept of what constitutes normal en zyme activities would appear to be of lim ited value in situations of this sort; such concepts might be questionable. For ex ample (5), in the case of SDH in the liver of animals utilizing 7-ethyl-8-methyl-flavin, the animal thrives when the activity of a critical enzyme is only 10% of normal, thus implying that the normal level when riboflavin is utilized may be ten times greater than required to sustain good health. Postulating a reason for the atypical in crease in activity of XO in the kidney when 7-methyl-8-ethyl-flavin is utilized is diffi cult. The increase may be due to more ef ficient functioning of the homologue-F'AD coenzyme or to an increase in the XO apoenzyme molecules. Not much is known about the biological function of XO. It has low specificity but its activity is highest for the oxidation of xanthine, however, its most important or natural substrate is un known. The elevated activity might be no more than a fortuitous combination of an abnormal coenzyme and an abnormal sub strate. For all of these reasons, speculation as to the causes for the noteworthy increase described above would seem unpromising at this time.
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