EXPERIhIENTAL
PARASITOLOGY
Entamoeba
47, 180-184
histolytica: HSIN-SHENG
Department 1542
( 19%)
of Biochemistry, Tulane Avenue, (Accepted
Lo
Flavins 1 AND
in Axenic E.
RICHARD
Organisms
REEVES
Louisiana State Unkrsity Medical Center, New Orleans, Louisiana 70112, U.S.A. for publication
16 January
1979)
Lo, H.-S., AND REEVES, R. E. 1979. Entamoeba histolytica: Flavins in axenic organisms. Experimental Parasitology 47, 180-184. The individual flavin species of axenic Entamoeba histolytica were assayed: separated riboflavin was assayed by the lumiflavin method; flavin-adenine dinucleotide ( FAD ) , by an enzymatic method; flavin mononucleotide (FMN) was calculated from the difference, total flavin minus FAD and riboflavin. The amount of flavin in micrograms per grams fresh cells follows: total flavin, 7.6 f 0.9 riboflavin, 1.6 2 0.7; FMN, 6.6 2 0.5; and FAD, 1.2 calculated as riboflavin; k 0.1. Recalculated to nanomoles per milligrams total amebal protein these values were: total flavin, 0.21; riboflavin, 0.04; FMN, 0.15; and FAD, 0.02. The identity of each flavin was confirmed by a paper chromatographic method. Analyses on Panmede, the main source of flavins in the TP-S-1 medium, indicate that it contains all three forms of flavin. Its contribution to growth medium in micrograms per milliliters: riboflavin, 2.1 2 0.3; FMN, 0.6 2 0.1; and FAD, 0.4 f 0.1. The in vivo biosynthesis of FMN and FAD from riboflavin by E. histolytica is demonstrated. A new and convenient method was found to separate riboflavin from flavin nucleotides in tissue extracts. INDEX DESCRIPTORS: Entamoeba histolytica; Protozoa, parasitic; Culture, axenic; Riboflavin; FMN; FAD; Flavin nucleotides; Panmede, flavin content; Flavin, peptide-bound; Riboflavin, separation from flavin nucleotides.
INTRODUCTION
Flavins, being essential components of many biological oxidation-reduction systems, are widely distributed in nature. The role of flavins in the parasitic ameba Entamoeba histolytica had not been considered until recently when Weinbach and Diamond (1974) suggested the presence of flavoproteins in this organism. Weinbach et al. (1976) cite several lines of evidence pointing to the participation of flavins in amebal metabolism. This theme was extended by Weinbach et al. (1977), and in
180 0014-4894/79/020180-05$02.00/O 0 1979 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Growth and harvesting of amebae. tamoeba histolytica, strain 200: NIH, grown axenically in Diamond’s TP-S-1 dium ( Diamond 1968). Trophozoites
1 Present address: Department of Parasitology, National Defense Medical Center, P.O. Box 8244, Taipei, Taiwan, Republic of China. Please address all correspondence to R. E. Reeves in New Orleans.
Copyright All rights
a published conference report (Weinbach et al. 1978). In the last a significant role for flavins in amebal areobic metabolism was envisioned and an assay for total flavin in the ameba was reported. The present work extends Weinbach’s prior findings by reporting assays on the individual flavin species in the ameba and in its growth medium. It also addresses the problem of whether or not amebae synthesize the flavin nucleotides from riboflavin.
Enwas mewere
Entamoeba
h.istolytica:
harvested by centrifugation for 5 min at 500g after 96 hr growth at 37 C. Cells were washed by centrifugation in a balanced salt buffer containing 10 mit4 potassium phosphate, pH 7.0, 20 mM KCI, 0.5 mM 150 mM NaCI, and 0.1 mM W$&, WNOs)z. Extraction of water-soluble flavins. The extraction of soluble amebal flavins was made with the warm-water method of Yagi (1971). Harvested amebae were suspended in 4 vol of distilled water heated to 80 C, and kept at 80 C for 5 min. Cells were then ruptured at room temperature by 20 passes at a speed of 900 rpm in a Teflon-pestle tissue grinder. These homogenates were kept for an additional 15 min at 80 C. After centrifugation for 20 min at 14,500g aliquots of the supernatant solution were used for the estimation of total and individual flavins. Total flavin assay. Quantitative determination of ffavin was based on its photolytic conversion to lumiflavin and the subsequent extraction of the fluorescence into chloroform as described by Yagi ( 1971). Fluorescence was measured with a Farrand fluorometer, Model A, employing Corning filters as recommended by Cerletti and Giordano (1971) 5543 and 3385. An internal standard of riboflavin was included for each set of determinations. Individual flavin assay. Riboflavin was separated from flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD) on a Bio-Gel P-2 column and determined by Yagi’s lumiflavin method. FAD in the warm-water extract was assayed by an enzymatic method described by Horecker ( 1950). Cuvettes with an optical pathlength of 1.0 cm containing 10 ~01 of pyrophosphate buffer, pH 8.3; 0.13 unit of apoenzyme of n-amino acid oxidase (EC 1.4.3.3) prepared by the method of Massey and Curti ( 1966) ; standard FAD or aliquots of sample; 50 nmol of NADH; 0.9 unit of L-lactate dehydrogenase (EC 1.1.1.27); and water. The reaction mixture
181
FLAVINS
was incubated for 20 min in the thermostated cuvette chamber (30 C). n-Alanine, 100 nmol, was then added to initiate the reaction. The final volume was 0.4 ml. The oxidation of NADH was monitored taking l = 6220 x cm-l x M-l at 340 nm. The amount of FAD in the sample was determined by referring to a standard curve made with known amounts of standard FAD. The relationship between the rate of NADH oxidation and FAD present was linear for quantities up to 40 ng. FMN was calculated by subtracting from total flavin the assayed quantities of FAD and riboflavin. Radioactivity measurement. Radioactivity was measured with a Beckman LS-100 liquid scintillation spectrometer in 10 ml of PCS (Amersham). Quenching was determined by an internal [‘“Cl toluene standard, or for counting segments of chromatograms, by applying a sample of [ 2-14C] riboflavin of known disintegrations per minute to a blank paper strip. All counts were corrected to disintegrations per minute. Estimation of protein. Protein was estimated by the method of Lowry et al. (1951). Reagents used. Riboflavin was obtained from Nutritional Biochemical Corporation; FMN, FAD, n-alanine, and n-amino acid oxidase (hog kidney) from Sigma; [~J~C]riboflavin (31 &i/pol) from Amersham; L-lactate dehydrogenase (pig heart) from Boehringer; and NADH from P-L Biochemicals. Panmede was purchased from Harrisons & Crossfield, P.O. Box 39, Bronxville, New York 10708. All other reagents were of analytical grade. RESULTS For the preparation of 14C-labeled flavin nucleotides trophozoites of Entamoeba histolytica, 1.4 million, were inoculated into each of seven 125-ml screw-cap flasks containing 110 ml of Diamond’s TP-S-1 medium and 0.4 pCi of [2-14C]riboflavin.
182
LO
AND
After 96 hr growth 0.86 g fresh cells was harvested. From these cells was obtained the warm-water amebal flavin extract used below. Bio-Gel P-2 column Fractionation Labeled Amebal Flavins
F&ins
1
of
Preliminary experiments employing standard solutions of each flavin had established conditions for the complete separation of riboflavin from FMN and FAD on a Bio-Gel P-2 column. Two and one-half milliliters of the amebal Aavin extract described above, representing 5.3 pg of total flavin, was applied to a Bio-Gel P-2 column (I3 x 170 mm) and eluted at room temperature with distilled water. Effluent fractions of 4 ml were collected. Each fsaction was assayed for flavin by Yagi’s method. These results are presented as the solid line in Fig. 1. It shows two flavin peaks in the column fractions. The radioactivity of each fraction was also measured, shown by the broken line in Fig. 1. Two labeled peaks appear in the figure. Identification of Amebal Paper Chromatography
REEVES
by
The fractions containing the two flavin peaks in the above experiment were sepa-
FIG. 1. Bio-Gel P-2 column chromatography Z-YLlabeled Entamoeba histolytica flavins. solid line represents the amount of flavin in fraction, calculated as riboflavin. The broken represents the radioactivity in each fraction.
of The each line
FIG. 2. Incorporation of [Z1’C]riboflavin into labeled FMN and FAD by Entamoeba histolytica. The chromatogram was irrigated for 96 hr in the descending mode with the organic phase of n-butanol-acetic acid-water (4: 1:5). Arrow indicates the starting line. (A) Reference spots of FMN and FAD were marked under a Mineralight UVS-11 ultraviolet lamp. (B ) Sample was from fraction 5 of Fig. 1 and Z-cm segments were cut from this channel for radioactivity measurement.
rately concentrated by lyophilization. The residues were separately dissolved in 50-J portions of distilled water and spotted onto Whatman No. 1 filter paper. Standard solutions of riboflavin, FMN, and FAD were spotted in reference channels. The first peak from the Bio-Gel P-2 column contained only flavin nucleotides and the second peak contained only riboflavin. The two paper chromatograms were cut into 2-cm segments which were measured for radioactivity. The results of these determinations are shown, diagramatically, in Figs. 2 and 3. Figure 2 demonstrates the in vivo biosyntheses of labeled FMN and FAD from [2-l%] riboflavin. Figure 3 reveals that the recovered riboflavin was in the same form as it was added. The indentities of the three amebal flavins were thus established. Assays for Individual Flavins and in the Growth Medium
in Amebae
The amount of flavin in micrograms per grams fresh amebal cells was as follows
Entamoeba
Mstolytica:
(5 separate extracts): total flavin, 7.6 * 0.9 calculated as riboflavin; riboflavin, 1.6 -+ 0.7; FMN, 6.6 f 0.5; and FAD, 1.2 + 0.1. Recalculated to nanomoles per milligrams protein these values were: total flavin, 0.21; riboflavin, 0.04; FMN, 0.15; and FAD, 0.02. The same assay methods revealed that a 2% solution of Panmede, as prescribed for the TP-S-1 medium, contributes flavin as follows, in micrograms per milliliters of growth medium (three different Panmede batch numbers): riboflavin, 2.1 * 0.3; FMN, 0.6 * 0.1; and FAD, 0.4 + 0.1. Vitamin mixture 107 contributes only 0.18 pg of riboflavin per milliliter to the TP-S-1 medium. Feptide-Bound
Flavins
The residue from the extraction of watersoluble flavins, representing 2.2 g cells, was assayed for peptide-bound flavins employing the method of Cerletti et al. (1963). We found no evidence of peptide-bound flavins in E. histolytica.
FLAVINS
183
FIG. 3. Identification of [2-W]riboflavin in Entamoeba histolytica. This chromatogram was irrigated for 6 hr in the ascending mode with the same solvent as in Fig. 2. Arrow indicates the starting line. (A) Reference spot, marked as described in Fig. 2. (B) The dotted area (ordinate at left) represents a pooled sample from fractions 10 and 11 of Fig. 1. The slanted-line area (ordinate at right) represents a sample from stork [2-“Clriboflavin. Each channel was cut into 2-cm segments for radioactivity measurement.
by either group of workers. The ability of E. histolytica to convert riboflavin into FMN and FAD suggests the presence in DISCUSSION ameba of activities corresponding to the Flavin mononucleotide (FMN) is the enzymes: flavokinase (EC 2.7.1.26) and major flavin in Entamoeba histolytica, FMN adenylyltransferase (EC 2.7.7.2). flavin-adenine dinucleotide (FAD) and Previously reported enzymes of E. hisriboflavin are present in lesser amounts. tolytica which are able to reduce flavins Although the growth medium provides all include pyruvate synthase, EC 1.2.7.1 three forms of flavin, amebae make flavin (Reeves et al. 1977), pyruvate oxidase, nucleotides from riboflavin during growth EC 1.2.3.6 (Takeuchi et al. 1975), NADPH: in TP-S-1 medium. The relatively high flavin oxidoreductase (Lo and Reeves concentration of riboflavin in E. histolytica 1978), and an enzyme called DT-diaphomay be a result of the pinocytotic activity rase, EC 1.6.99.2 (Weinbach et al 1977). of this organism (Serrano and Reeves None of these enzymes has been shown 1975; Lo and Reeves 1979). Weinbach et to be specific for one of the flavin nucleoal. (1978) reported that the total flavin tide forms therefore it would be premature content of E. histolytica is 0.1 nmol/mg to regard any of them as a flavoprotein enprotein. Our results are twofold greater. zyme. Rather, the flavins, riboflavin as The difference may be due to different well as the nucleotides, may simply be methodology or to strain differences (strain electron-accepting substrates of the vari200: NIH was used in this work while ous enzymes. The wide range of nonphysioWeinbach et al. employed strain HK-9). logical electron acceptors which can reNo evidence for the existence of peptide- place flavin in the reactions catalyzed by bound flavins in E. histolytica was found some of these enzymes lends credence to
184
LO AND REEVES
this view. The vital physiological role of flavins in amebal aerobic metabolism, as visualized by Weinbach et al. (1978), may be correct in principle. However, since reduced flavins avidly combine with oxygen to form hydrogen peroxide, a known product of amebal aerobic metabolism, it is not necessary to postulate any other intermediate steps between reduced free flavins and oxygen. ACKNOWLEDGMENTS A preliminary report of this work was presented at the 69th Annual Meeting of the American Society of Biological Chemists in Atlanta, 5-8 June 1978 (Abstract 418). Some of the data are from the doctoral dissertation by H.-S. Lo submitted to Louisiana State University in 1978. The work was supported, in part, by Grant AI02591 from the U.S. National Institutes of Health. The senior author was recipient of a fellowship from the National Science Council, Republic of China on Taiwan. Mrs. Betty West cultured the Entamoeba histolytica.
REFERENCES CERLETTI, P., AND GIORDANO, M. G. 1971. Determination of flavin compounds in tissues. In “Methods in Enzymology” (D. McCormick and L. D. Wright, eds.), Vol. 18B, pp. 285-290. Academic Press, New York. CERLETTI, P., STROM, R., AND GIORDANO, M. G. 1963. Flavin peptides in tissues: The prosthetic group of succinic dehydrogenase. Archives of Biochemistry and Biophysics 101, 423428. DIAMOND, L. S. 1968. Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae. Journal of Parasitology 54, 1047-1056. HORECKER, B. L. 19508. Triphosphopyridine nucleotide-cytochrome c reductase in liver. Journal of Biological Chemistry 183, 593-605. Lo, H.-S., AND REEVES, R. E. 1978. Flavin metabolism in Entamoeba histolytica. Federation Proceedings 37, 1345. Lo, H.-S., AND REEVES, R. E. 1979. Riboflavin requirement for the cultivation of axenic Entamoeba histolytica. American Journal of Tropical Medicine and Hygiene, in press.
LOWRY, 0. H., ROSEBHOUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin-phenol reagent. Journal of Biological Chemistry 193, 265-275. MASSEY, V., AND CURTI, B. 1966. A new method of preparation of n-amino acid oxidase apoprotein and a conformational change after its combination with flavin adenine dinucleotide. Journal of Biological Chemistry 241, 3417-3423. MONTALVO, F. E., REEVES, R. E., AND WARREN, L. G. 1971. Aerobic and anaerobic metabolism in Entamoeba histolytica. Experimental Parasitology 30, 249-256. REEVES, R. E., WARREN, L. G., SUSSKIND, B., AND Lo, H.-S. 1977. An energy-conserving pyruvateto-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase. Journal of Biological Chemistry 252, 726-731. SERRANO, R., AND REEVES, R. E. 1975. Physiological significance of glucose transport in Entamoeba histolytica. Experimental Parasitology 37, 411-416. TAKEUCHI, T., WEINBACH, E. C., AND DIAMOND, L. S. 1975. Pyruvate oxidase (CoA acetylating) in Entamoeba histolytica. Biochemical and Biophysical Research Communications 65, 591596. WEISBACH, E. C., AND DIAMOND, L. S. 1974. Entamoeba histolytica. I. Aerobic metabolism. Experimental Parasitology 35, 232-243. WEINBACH, E. C., HARLOW, D. R., TAKEUCHI, T., DIAMOKD, L. S., CLAGGETT, C. E., AND KON, H. 1976. Aerobic metabolism of Entamoeba histoZytica: Facts and fallacies. In “Proceedings of the International Conference on Amebiasis, Mexico City, 1975” (B. Sepulveda and L. S. Diamond, eds. ), pp. 190-203. Instituto Mexicane de1 Seguro Social, Mexico City. WEIXBACH, E. C., HARLOW, D. R., CLAGGETT, C. E., AND DIAMOND, L. S. 1977. Entamoeba histolytica: Diaphorase activities. Experimental Parasitology 41, 186-197. WEIXLIACH, E. C., CLAGGETT, C. E., TAKEUCHI, T., AND DIAMOND, L. S. 1978. Biological oxidations and flavoprotein catalysis in Entamoeba histolytica. Archives de Inoestigaclon Medica 9 ( Suppl 1 ), 89-98. YAGI, K. 1971. Simultaneous microdetermination of riboflavin, FMN, and FAD in animal tissues. In “Methods in Enzymology” (D. McCormick and L. D. Wright, eds.), Vol. 18B, pp. 290296. Academic Press, New York.
PARASITOLOGY
Entamoeba
47, 180-184
histolytica: HSIN-SHENG
Department 1542
( 19%)
of Biochemistry, Tulane Avenue, (Accepted
Lo
Flavins 1 AND
in Axenic E.
RICHARD
Organisms
REEVES
Louisiana State Unkrsity Medical Center, New Orleans, Louisiana 70112, U.S.A. for publication
16 January
1979)
Lo, H.-S., AND REEVES, R. E. 1979. Entamoeba histolytica: Flavins in axenic organisms. Experimental Parasitology 47, 180-184. The individual flavin species of axenic Entamoeba histolytica were assayed: separated riboflavin was assayed by the lumiflavin method; flavin-adenine dinucleotide ( FAD ) , by an enzymatic method; flavin mononucleotide (FMN) was calculated from the difference, total flavin minus FAD and riboflavin. The amount of flavin in micrograms per grams fresh cells follows: total flavin, 7.6 f 0.9 riboflavin, 1.6 2 0.7; FMN, 6.6 2 0.5; and FAD, 1.2 calculated as riboflavin; k 0.1. Recalculated to nanomoles per milligrams total amebal protein these values were: total flavin, 0.21; riboflavin, 0.04; FMN, 0.15; and FAD, 0.02. The identity of each flavin was confirmed by a paper chromatographic method. Analyses on Panmede, the main source of flavins in the TP-S-1 medium, indicate that it contains all three forms of flavin. Its contribution to growth medium in micrograms per milliliters: riboflavin, 2.1 2 0.3; FMN, 0.6 2 0.1; and FAD, 0.4 f 0.1. The in vivo biosynthesis of FMN and FAD from riboflavin by E. histolytica is demonstrated. A new and convenient method was found to separate riboflavin from flavin nucleotides in tissue extracts. INDEX DESCRIPTORS: Entamoeba histolytica; Protozoa, parasitic; Culture, axenic; Riboflavin; FMN; FAD; Flavin nucleotides; Panmede, flavin content; Flavin, peptide-bound; Riboflavin, separation from flavin nucleotides.
INTRODUCTION
Flavins, being essential components of many biological oxidation-reduction systems, are widely distributed in nature. The role of flavins in the parasitic ameba Entamoeba histolytica had not been considered until recently when Weinbach and Diamond (1974) suggested the presence of flavoproteins in this organism. Weinbach et al. (1976) cite several lines of evidence pointing to the participation of flavins in amebal metabolism. This theme was extended by Weinbach et al. (1977), and in
180 0014-4894/79/020180-05$02.00/O 0 1979 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Growth and harvesting of amebae. tamoeba histolytica, strain 200: NIH, grown axenically in Diamond’s TP-S-1 dium ( Diamond 1968). Trophozoites
1 Present address: Department of Parasitology, National Defense Medical Center, P.O. Box 8244, Taipei, Taiwan, Republic of China. Please address all correspondence to R. E. Reeves in New Orleans.
Copyright All rights
a published conference report (Weinbach et al. 1978). In the last a significant role for flavins in amebal areobic metabolism was envisioned and an assay for total flavin in the ameba was reported. The present work extends Weinbach’s prior findings by reporting assays on the individual flavin species in the ameba and in its growth medium. It also addresses the problem of whether or not amebae synthesize the flavin nucleotides from riboflavin.
Enwas mewere
Entamoeba
h.istolytica:
harvested by centrifugation for 5 min at 500g after 96 hr growth at 37 C. Cells were washed by centrifugation in a balanced salt buffer containing 10 mit4 potassium phosphate, pH 7.0, 20 mM KCI, 0.5 mM 150 mM NaCI, and 0.1 mM W$&, WNOs)z. Extraction of water-soluble flavins. The extraction of soluble amebal flavins was made with the warm-water method of Yagi (1971). Harvested amebae were suspended in 4 vol of distilled water heated to 80 C, and kept at 80 C for 5 min. Cells were then ruptured at room temperature by 20 passes at a speed of 900 rpm in a Teflon-pestle tissue grinder. These homogenates were kept for an additional 15 min at 80 C. After centrifugation for 20 min at 14,500g aliquots of the supernatant solution were used for the estimation of total and individual flavins. Total flavin assay. Quantitative determination of ffavin was based on its photolytic conversion to lumiflavin and the subsequent extraction of the fluorescence into chloroform as described by Yagi ( 1971). Fluorescence was measured with a Farrand fluorometer, Model A, employing Corning filters as recommended by Cerletti and Giordano (1971) 5543 and 3385. An internal standard of riboflavin was included for each set of determinations. Individual flavin assay. Riboflavin was separated from flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD) on a Bio-Gel P-2 column and determined by Yagi’s lumiflavin method. FAD in the warm-water extract was assayed by an enzymatic method described by Horecker ( 1950). Cuvettes with an optical pathlength of 1.0 cm containing 10 ~01 of pyrophosphate buffer, pH 8.3; 0.13 unit of apoenzyme of n-amino acid oxidase (EC 1.4.3.3) prepared by the method of Massey and Curti ( 1966) ; standard FAD or aliquots of sample; 50 nmol of NADH; 0.9 unit of L-lactate dehydrogenase (EC 1.1.1.27); and water. The reaction mixture
181
FLAVINS
was incubated for 20 min in the thermostated cuvette chamber (30 C). n-Alanine, 100 nmol, was then added to initiate the reaction. The final volume was 0.4 ml. The oxidation of NADH was monitored taking l = 6220 x cm-l x M-l at 340 nm. The amount of FAD in the sample was determined by referring to a standard curve made with known amounts of standard FAD. The relationship between the rate of NADH oxidation and FAD present was linear for quantities up to 40 ng. FMN was calculated by subtracting from total flavin the assayed quantities of FAD and riboflavin. Radioactivity measurement. Radioactivity was measured with a Beckman LS-100 liquid scintillation spectrometer in 10 ml of PCS (Amersham). Quenching was determined by an internal [‘“Cl toluene standard, or for counting segments of chromatograms, by applying a sample of [ 2-14C] riboflavin of known disintegrations per minute to a blank paper strip. All counts were corrected to disintegrations per minute. Estimation of protein. Protein was estimated by the method of Lowry et al. (1951). Reagents used. Riboflavin was obtained from Nutritional Biochemical Corporation; FMN, FAD, n-alanine, and n-amino acid oxidase (hog kidney) from Sigma; [~J~C]riboflavin (31 &i/pol) from Amersham; L-lactate dehydrogenase (pig heart) from Boehringer; and NADH from P-L Biochemicals. Panmede was purchased from Harrisons & Crossfield, P.O. Box 39, Bronxville, New York 10708. All other reagents were of analytical grade. RESULTS For the preparation of 14C-labeled flavin nucleotides trophozoites of Entamoeba histolytica, 1.4 million, were inoculated into each of seven 125-ml screw-cap flasks containing 110 ml of Diamond’s TP-S-1 medium and 0.4 pCi of [2-14C]riboflavin.
182
LO
AND
After 96 hr growth 0.86 g fresh cells was harvested. From these cells was obtained the warm-water amebal flavin extract used below. Bio-Gel P-2 column Fractionation Labeled Amebal Flavins
F&ins
1
of
Preliminary experiments employing standard solutions of each flavin had established conditions for the complete separation of riboflavin from FMN and FAD on a Bio-Gel P-2 column. Two and one-half milliliters of the amebal Aavin extract described above, representing 5.3 pg of total flavin, was applied to a Bio-Gel P-2 column (I3 x 170 mm) and eluted at room temperature with distilled water. Effluent fractions of 4 ml were collected. Each fsaction was assayed for flavin by Yagi’s method. These results are presented as the solid line in Fig. 1. It shows two flavin peaks in the column fractions. The radioactivity of each fraction was also measured, shown by the broken line in Fig. 1. Two labeled peaks appear in the figure. Identification of Amebal Paper Chromatography
REEVES
by
The fractions containing the two flavin peaks in the above experiment were sepa-
FIG. 1. Bio-Gel P-2 column chromatography Z-YLlabeled Entamoeba histolytica flavins. solid line represents the amount of flavin in fraction, calculated as riboflavin. The broken represents the radioactivity in each fraction.
of The each line
FIG. 2. Incorporation of [Z1’C]riboflavin into labeled FMN and FAD by Entamoeba histolytica. The chromatogram was irrigated for 96 hr in the descending mode with the organic phase of n-butanol-acetic acid-water (4: 1:5). Arrow indicates the starting line. (A) Reference spots of FMN and FAD were marked under a Mineralight UVS-11 ultraviolet lamp. (B ) Sample was from fraction 5 of Fig. 1 and Z-cm segments were cut from this channel for radioactivity measurement.
rately concentrated by lyophilization. The residues were separately dissolved in 50-J portions of distilled water and spotted onto Whatman No. 1 filter paper. Standard solutions of riboflavin, FMN, and FAD were spotted in reference channels. The first peak from the Bio-Gel P-2 column contained only flavin nucleotides and the second peak contained only riboflavin. The two paper chromatograms were cut into 2-cm segments which were measured for radioactivity. The results of these determinations are shown, diagramatically, in Figs. 2 and 3. Figure 2 demonstrates the in vivo biosyntheses of labeled FMN and FAD from [2-l%] riboflavin. Figure 3 reveals that the recovered riboflavin was in the same form as it was added. The indentities of the three amebal flavins were thus established. Assays for Individual Flavins and in the Growth Medium
in Amebae
The amount of flavin in micrograms per grams fresh amebal cells was as follows
Entamoeba
Mstolytica:
(5 separate extracts): total flavin, 7.6 * 0.9 calculated as riboflavin; riboflavin, 1.6 -+ 0.7; FMN, 6.6 f 0.5; and FAD, 1.2 + 0.1. Recalculated to nanomoles per milligrams protein these values were: total flavin, 0.21; riboflavin, 0.04; FMN, 0.15; and FAD, 0.02. The same assay methods revealed that a 2% solution of Panmede, as prescribed for the TP-S-1 medium, contributes flavin as follows, in micrograms per milliliters of growth medium (three different Panmede batch numbers): riboflavin, 2.1 * 0.3; FMN, 0.6 * 0.1; and FAD, 0.4 + 0.1. Vitamin mixture 107 contributes only 0.18 pg of riboflavin per milliliter to the TP-S-1 medium. Feptide-Bound
Flavins
The residue from the extraction of watersoluble flavins, representing 2.2 g cells, was assayed for peptide-bound flavins employing the method of Cerletti et al. (1963). We found no evidence of peptide-bound flavins in E. histolytica.
FLAVINS
183
FIG. 3. Identification of [2-W]riboflavin in Entamoeba histolytica. This chromatogram was irrigated for 6 hr in the ascending mode with the same solvent as in Fig. 2. Arrow indicates the starting line. (A) Reference spot, marked as described in Fig. 2. (B) The dotted area (ordinate at left) represents a pooled sample from fractions 10 and 11 of Fig. 1. The slanted-line area (ordinate at right) represents a sample from stork [2-“Clriboflavin. Each channel was cut into 2-cm segments for radioactivity measurement.
by either group of workers. The ability of E. histolytica to convert riboflavin into FMN and FAD suggests the presence in DISCUSSION ameba of activities corresponding to the Flavin mononucleotide (FMN) is the enzymes: flavokinase (EC 2.7.1.26) and major flavin in Entamoeba histolytica, FMN adenylyltransferase (EC 2.7.7.2). flavin-adenine dinucleotide (FAD) and Previously reported enzymes of E. hisriboflavin are present in lesser amounts. tolytica which are able to reduce flavins Although the growth medium provides all include pyruvate synthase, EC 1.2.7.1 three forms of flavin, amebae make flavin (Reeves et al. 1977), pyruvate oxidase, nucleotides from riboflavin during growth EC 1.2.3.6 (Takeuchi et al. 1975), NADPH: in TP-S-1 medium. The relatively high flavin oxidoreductase (Lo and Reeves concentration of riboflavin in E. histolytica 1978), and an enzyme called DT-diaphomay be a result of the pinocytotic activity rase, EC 1.6.99.2 (Weinbach et al 1977). of this organism (Serrano and Reeves None of these enzymes has been shown 1975; Lo and Reeves 1979). Weinbach et to be specific for one of the flavin nucleoal. (1978) reported that the total flavin tide forms therefore it would be premature content of E. histolytica is 0.1 nmol/mg to regard any of them as a flavoprotein enprotein. Our results are twofold greater. zyme. Rather, the flavins, riboflavin as The difference may be due to different well as the nucleotides, may simply be methodology or to strain differences (strain electron-accepting substrates of the vari200: NIH was used in this work while ous enzymes. The wide range of nonphysioWeinbach et al. employed strain HK-9). logical electron acceptors which can reNo evidence for the existence of peptide- place flavin in the reactions catalyzed by bound flavins in E. histolytica was found some of these enzymes lends credence to
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this view. The vital physiological role of flavins in amebal aerobic metabolism, as visualized by Weinbach et al. (1978), may be correct in principle. However, since reduced flavins avidly combine with oxygen to form hydrogen peroxide, a known product of amebal aerobic metabolism, it is not necessary to postulate any other intermediate steps between reduced free flavins and oxygen. ACKNOWLEDGMENTS A preliminary report of this work was presented at the 69th Annual Meeting of the American Society of Biological Chemists in Atlanta, 5-8 June 1978 (Abstract 418). Some of the data are from the doctoral dissertation by H.-S. Lo submitted to Louisiana State University in 1978. The work was supported, in part, by Grant AI02591 from the U.S. National Institutes of Health. The senior author was recipient of a fellowship from the National Science Council, Republic of China on Taiwan. Mrs. Betty West cultured the Entamoeba histolytica.
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LOWRY, 0. H., ROSEBHOUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin-phenol reagent. Journal of Biological Chemistry 193, 265-275. MASSEY, V., AND CURTI, B. 1966. A new method of preparation of n-amino acid oxidase apoprotein and a conformational change after its combination with flavin adenine dinucleotide. Journal of Biological Chemistry 241, 3417-3423. MONTALVO, F. E., REEVES, R. E., AND WARREN, L. G. 1971. Aerobic and anaerobic metabolism in Entamoeba histolytica. Experimental Parasitology 30, 249-256. REEVES, R. E., WARREN, L. G., SUSSKIND, B., AND Lo, H.-S. 1977. An energy-conserving pyruvateto-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase. Journal of Biological Chemistry 252, 726-731. SERRANO, R., AND REEVES, R. E. 1975. Physiological significance of glucose transport in Entamoeba histolytica. Experimental Parasitology 37, 411-416. TAKEUCHI, T., WEINBACH, E. C., AND DIAMOND, L. S. 1975. Pyruvate oxidase (CoA acetylating) in Entamoeba histolytica. Biochemical and Biophysical Research Communications 65, 591596. WEISBACH, E. C., AND DIAMOND, L. S. 1974. Entamoeba histolytica. I. Aerobic metabolism. Experimental Parasitology 35, 232-243. WEINBACH, E. C., HARLOW, D. R., TAKEUCHI, T., DIAMOKD, L. S., CLAGGETT, C. E., AND KON, H. 1976. Aerobic metabolism of Entamoeba histoZytica: Facts and fallacies. In “Proceedings of the International Conference on Amebiasis, Mexico City, 1975” (B. Sepulveda and L. S. Diamond, eds. ), pp. 190-203. Instituto Mexicane de1 Seguro Social, Mexico City. WEIXBACH, E. C., HARLOW, D. R., CLAGGETT, C. E., AND DIAMOND, L. S. 1977. Entamoeba histolytica: Diaphorase activities. Experimental Parasitology 41, 186-197. WEIXLIACH, E. C., CLAGGETT, C. E., TAKEUCHI, T., AND DIAMOND, L. S. 1978. Biological oxidations and flavoprotein catalysis in Entamoeba histolytica. Archives de Inoestigaclon Medica 9 ( Suppl 1 ), 89-98. YAGI, K. 1971. Simultaneous microdetermination of riboflavin, FMN, and FAD in animal tissues. In “Methods in Enzymology” (D. McCormick and L. D. Wright, eds.), Vol. 18B, pp. 290296. Academic Press, New York.
