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FMN Phosphatase and FAD Pyrophosphatase in RatIntestinal B rush Borders: Role in Intestinal A bsorption ofD ie ta ry R iboflavinTOSHIO AKIYAM A1, JACOB SELHUB ANDIRW IN H. ROSENBERGS ectio n o f G astro en tero lo gy, D epa rtm en t o f M edicin e, Th eU niversity o f C hica go, P ritzker Sc ho ol o f M edicin e, 9 50 E .5 9th S treet, C hica go , IL 60 63 7

ABSTRACT Flavin m ononucleotide (FM N) and flavin adenine dinucleotide (FAD),are tw o m ajor coenzym e form s of dietary riboflavin. Y et little attention has been givento the release of the vitam in from its coenzyme forms during the absorptive process.H om ogenates from rat intestine catalyze the hydrolysis of these flavin coenzym es. Todeterm ine the location of FMN and FA D hydrolases, hom ogenates of intestinal m ucosawere fractioned. FM N and FAD phosphatases were localized in brush border membranes. FAD pyrophosphatase activity was maximal at pH between 6.5 and 8.5 whileFM N phosphatase has a pH optim um of 7.5-8.0. FAD pyrophosphatase is more stableto heat. The two enzym es separate on ion exchange chrom atography of an isobutanole xtra ct o f in testin al b rus h b ord er m em bran e fra ctio n. In hib ition o f 14 C-rib ofla vin u pta keby FMN and FAD in everted rings of rat intestine is directly related to the amount ofconversion of these coenzym es to free riboflavin by intestinal enzym es. W hen FMN andFAD conversion to riboflavin is inhibited by EDTA, com petition w ith 14C -riboflavin fortran sp ort w as c orre sp on din gly d ecrea se d. T he se stu dies a re be st e xp la in ed b y a se que ntialprocess in w hich hydrolysis of FMN and FA D by enzym es of the intestinal brush borderis followed by absorption of free riboflavin. J. Nutr. 112: 263-268, 1982.INDEXING KEY W ORDSdinucleotide riboflavin flav in m on on ucleo tid e fla vin ad enin e

Previous studies in man (1) and more recently in rats (2) have suggested that the intestinal absorption of free riboflavin occursby a saturable mechanism . The mechanismof intestinal absorption of flavin m ononucleotide (FMN) and flavin adenine dinucleotide(FAD), the two major coenzymes which comprise the bulk of dietary riboflavin (1, 3), ispresently unknow n. M ucosal hom ogenatesand intestinal fluid contain enzym es capableof hydrolyzing FMN and FAD to riboflavin(4-6); the subcellular origin of these enzym esis uncertain. T hese enzym atic activities w erenot found in saliva, bile, pancreatic and gastric juices (7).The present study examines the cellulardistribution of these flavin phosphatases inrat intestinal hom ogenates and their possiblefu nctio n in d ietary rib oflav in ab so rp tio n.

MATERIALS AND METHODS[2-14C ]riboflavin (31 / C i/^m ole w as ob

tained (Amersham Searle Corporation, Arlington Heights, IL) as was [3H]inulin (215 tC i/V g,N ew E nglan d N uclear, B oston , MA ).[3H]inulin was purified on Sephadex G-25column and was used to determine adheringwater and water space in the intestine in thetransport studies described below. All radioactive compounds were stored at 30.Un-labeled riboflavin, FM N, FAD, and 2-phen-ylethanol were purchased (Sigm a Chem ical

1 98 2 A m er ic an I ns ti tu te o f N u tr it io n R ec ei ve d f or p ub li ca ti on 1 3 J u l y1981' C ur re nt a dd re a i s 9 1 T iu ts uj ig ao k . 63 0- 53 S hi bu m i- ch o. T su C it y 5 14 .Japan

26 3

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264 AKIY AM A ET AL.Co., St. L ouis, MO ). Other reagents were allof analytical grade.E nzyme studies. U nfa sted Spr ague-D aw-ley rats (200-300 g) were killed by decapitati on. The small i ntesti ne was removed andwashed w ith cold saline and mucosal tissuewas scraped of f at 4.These scrapings werehomogenized for 30 seconds at 4with 15volumes (w/v) of 50 mM mannitol and cen-trifuged at 10,000 X g for 10 minutes. Thesupernatant was decanted and the pellet wasresuspended in 5 volumes of mannitol solution and rehomogenized for 30 seconds, thencombined with the supernatant, filteredthrough cheese cloth and a brush bordermembrane enriched f raction was isol ated bythe procedure of Schmitz, et al. (8).

I n this procedure the homogenate (H ) wastreated with CaCl2 (final concentration 10mM ) and centrifuged twice, once at lowspeed (2000 X g, 10 minutes) for the removalof a debris pellet (Pi) followed by high speed(20,000 X g, 15 minutes) to separate a supernatant (SO from a second pel let (P2) . Fraction P2 was further f ractionated by resuspen-sion in the mannitol solution (5 ml/originalgm of tissue), rehomogenization with a motor-driven teflon pestle at 1200 rpm (3 upand down strokes), treatment with M gSO4(10 mM f inal concentration) and two centri f-ugations, one at 4000 X g for 10 minutes (P3)and one at 30,000 X g for 15 minutes whichyielded supernatant (S2) and pellet (P4). Byelectron microscopy P2 is enriched, whereasP4 is composed of nearly pure brush bordermembrane vesicles. The various pel let f ractions were resuspended in mannitol and analyzed for protein (9), and for enzyme activi ties as described below.FMN phosphatase was determined by incubation of the protein fraction (4-10 ngprotein) for 10 minutes at 37with 1 /MFM N in 0.1 M Tris-HCl buffer, pH 9.0, ina final volume of 1 ml. T he incubation mixture was then extracted with 3 .5 ml 2-phenyl-ethanol and the fluorimetrie intensity at520nm (excitation at 380 nm) in the alcoholf raction was determined. Ribof lav in concentration was calculated using the distributioncoefficient of 3.06 between 2-phenylethanoland water compared to 0.014 for FMN (10).FAD pyrophosphatase was estimated onthe basis of increase in the fluorimetrie in

tensity at 520 nm (excitation at 380 nm),because at equivalent concentrations FADyields only 15% of the f luorimetrie intensityproduced by FMN or riboflavin, the hydrolysis products (10). The protein fraction (4-10 fig) was incubated for 10 minutes at 37 in 0.1 M Tris-HCl buffer pH 9.0 with 1 /MFAD. Hydrolytic products were determinedfrom the fol lowing expression:Ip = 0 .85 - 1.176I 0

Ip represents the f luorimetrie intensi ty dueto product formation, I t the fluorometric intensity after incubation and I 0 the initial intensi ty before incubation. For the separatedetermination of riboflavin and FMN in suchincubations, the fluorimetrie intensity wasdetermined before and af ter extracti on w ith2-phenylethanol . Activi ty uni ts for both f lav inphosphatases are expressed in nmoles ofproducts formed per minute. Maltase activi tywas determined by the procedure of D ahl-qvist [11].Solubiliza tion and DE-52 chroma togr a -phy of phospha ta ses fr om brush bordermembrane prepa ra tions. A 5 ml a liquot (6.2mg protein) of P2 fraction was mixed with3.75 ml isobutanol and stirred for 30 minutesat room temperature, then 6 minutes at 40.The mixture was centrifuged (2000 X g, 20minutes) and the water layer removed anddialyzed overnight against 2 liters 0.01 MT ris-HCl containing 0.005 M MgCl2 (pH 8.8).The dialyzed solution, which contained virtually all the flavin phosphatases from themembranes, was applied on DE-52 column(125 ml bed volume) which was prequili-brated with the same T ris buffer. Enzymeswere eluted from the column by a lineargradient consisting of 140 ml of the Trisbuffer in the mixing chamber and 140 ml ofthe same buffer containing 0.1 M sodium acetate. F ractions collected were analyzed forthe flavin phosphatases activities.

Tra nspor t studies. To eva lua te the function of intestinal f lav in phosphatases, experiments were designed to study the effects ofFMN and FAD on the intestinal transport ofriboflavin. These experiments uti l ized evertedjej unal rings f rom 200-300 g Sprague-Daw-ley unfasted male rats studied by the method

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INTESTINAL FLAVIN PHOSPHATASES 265of Crane and Mandlestam (12) as modifiedby Rosenberg et al. (13). T he jejunum fromthree rats were everted, cut into rings 4-6mm wide which were placed in cold oxygenated saline soluti on unti l thei r transferinto the incubation f lasks.T he incubation flasks consisted of 25 mlErlenmeyer with 2.5 ml K rebs-R inger phosphate solution (pH 7.0) containing 0.3 iM[14C] ribof lav in, 0 .05 f id [3H] inul in and otheradditions specified in the text. T he flaskswere gassed in the cold with 100% oxygenfor a minimum of 15 minutes. T hereaftertwo rings selected at random, were placedin each flask proceeded by incubation for 15minutes in a 37Dubnof f metabol ic shakingincubator oscillating at 90 min"1. The reaction was terminated by transferring the f lasksinto ice cold water, removing the rings rapidly and rinsing in cold saline. T he tissueswere then blotted, weighed, and autoclavedfor 30 minutes in 2.5 ml distilled water torelease radioactive compounds. After mixing,0.5 ml of this solution was solubilized in 10ml Scintisol- compete ( Isolab Inc., Akron,OH ) and analyzed for [3H ] and [14C ]in a scintillation counter. I n experiments with FMNand FAD, the incubation medium was analyzed for ribof lav in released f rom these coen-zymes. A 1 ml aliquot was treated with 1.4ml 10% trichloroacetic acid and centri fugedfor 10 minutes at 2000 X g. R iboflavin (total)was then determined fluorimetrically afterextraction with 2-phenylethanol (see above)from which total the concentration of riboflavin deriving from carbon-14 was subtracted.The condi tions of these experiments wereset af ter preliminary studies which showedthat riboflavin uptake by rat jejunum is independent of the distance from the duodenum and that at 0.3 tMhe uptake proceeds primarily by a saturable mechanismacross the brush border surface in agreementwith the studies by M einen et al. (2).

RESULTSEnzyme studies. FMN phospha ta se a ndFAD pyrophosphatase activi ti es are presentin the rat mucosal homogenates as shown intable 1. These activities reside mostly in the

particulate f ractions w ith highest activity inf raction P2 which represents enriched brushborder membranes (8) . Subsequent fraction-ati on of P2 resul ted in one f raction (P4) whichhas 16- fold the specif ic acti vi ties of the original homogenates. The enrichment and distribution of these flavin phosphatase activitiesin the vari ous f racti ons fol low closely thoseof the brush border marker, maltase.The two brush border fl avi n phosphataseactivities have both common and distinctproperties as shown in table 2 and figure 1.Both activities are strongly inhibited byEDTA , whereas L -phenylalanine, a knowninhibi tor of intestinal alkal ine phosphatase(14), had no ef fect on FAD pyrophosphataseactivity and a small effect on FM N phosphatase activity. FAD pyrophosphatase activity has a broad pH optimum extendingbetween pH 6.5-8.5 whereas FMN phosphatase has a peak activity at pH 7.5-8.0. TheFAD hydrolyzing enzyme is more stable toheat than the FMN enzyme.The two activities are partially resolvedaf ter isobutanol extraction and chromatog-raphy of the water phase on DE-52 column( fig. 1) . FAD pyrophosphatase activity is represented in two peaks one eluting in f racti on27, the other in fraction 36. FMN phosphatase ltess a major peak in fractions 31-32 with some shoulder between fractions34-37.

Tr anspor t studies. To eva lua te the possiblerole of the brush border f lavin phosphatasesin the absorption of dietary riboflavin, experiments were designed to examine the fateof riboflavin derived from FM N and FADand whether such riboflavin competes w ithfree [14C] riboflavin for uptake by evertedjejunal rings. I ncubation of FMN with theserings resul ted in the inhibi tion [14C]ribof lavinuptake in a dose dependent fashion (fig. 2).ED TA , which inhibits FMN hydrolysis, antagonized this effect of FMN on [14C]ribo-flavin uptake.

Studies with FAD yielded similar results.Uptake of [14C]riboflavin by the intestinalring preparations is inhibited by FAD andthis inhibition is antagonized by ED TA( fig. 3 ).EDTA had no effect on the uptake of[14C]riboflavin directly. [14C]riboflavin uptakeat zero concentration of FMN and FAD (figs.

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266 AKI YAMA ET A L.TA BL E 1

F MN ph osp ha ta se a nd F AD pyr op hos pha ta se in r a t intes tin al h omo gen ates b efo re a nd a fte r fr a ctio na tio nH P4 S,

Protein, total mg 416 209 186 16.1 5.34 9.2 2.0FMN phosphataseTotal units 556 233 37 277 79 203 10Specific activity 1.4 1.10 0.2 17.2 14.8 22 5Fold purification 1.0 8.78 0.14 12.3 10.5 15.7 3.5FAD pyrophosphataseTotal activity 288 140 34 136 38 103 6Specific activity 0.69 0.67 0.18 8.4 7.1 11.3 4Fold purification 1.0 0.94 0.26 12.1 10.25 16.3 4.3M altaseTotalactivitySpecificactivityFoldpurification2200.531.087.50.420.79260.140.261026.3312285.249.977.58.4215.93.21.63.0

1Preparation of the homogenate and subsequent fractionation were performed as described in Methods. Thefractions PI, Si and P2were obtained from the homogenate (H) after CaCl2treatment and subsequent centrifugations.Fractions P3, P4and 82 were derived from P2after treatment with M gSO4and subsequent centrifugations.

2 and 3) was the same in the presence orabsence of EDTA.In Fig. 4a the [ I4C]ribof lavin uptake datafrom the studies in figures 2 and 3 are re-plotted against the accumulated unlabeledriboflavin derived from FMN and FAD.These plots produce an inhibition curvewhich is independent of the coenzyme concentration or EDTA and is similar to thatobtained when uptake of [14C]riboflavin bythe rings was determined in presence of increasing concentrations of added unlabeledriboflavin (fig. 4b).DISCUSSION

The data presented here show that enzymes that release ribof lavin from dietarycoenzyme forms, FAD and FMN , are concentrated in the brush border membranefraction of rat intestinal homogenates. Therelative distribution and enrichment in thevarious fractions of these flavin phosphatasesresemble the behavior of the brush bordermembrane enzyme, maltase.The two activi ties appear to be catalyzedby different protein entities. Though they areboth inhibited by EDTA , FAD pyrophosphatase significantly differs from FMN phosphatase with respect to pH optima, stability

to heat and Chromatographie behavior onDE-52 column.The discrepancy between the present findings and those of Okuda (15), who suggesteda location for these phosphatases in the cy-tosol f raction, can be best explained on the

0 .3r

02i

26 28 30 32 34 36F ra ctio n n um b er (6 m l e ac h)

38 40Fig. 1 DE-52 cel lulose fractionation of solubi lizedflavin phosphatases from P2 fraction. The membranepreparation wassolubilized with isobutanol, then appliedon DE-52 column and eluted with sodium acetate gradient asdescribed in M ethods. Fractions collected wereanalyzed for FMN phosphatase O O, and FAD pyrophosphatase .Activity units were as definedin the M ethods section.

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INTESTINAL FLAV IN PHOSPHATASES 267TABLE 2

P r ope rtie s of th e two fla vin ph osp ha ta se s in th ee nr ic he d b ru sh b or de r m embr a ne fr a ct io n

0.4r

pHptimumResidualactivityfter30mi n at6Presenceof I O"1EDTAPresenceof.5mMphenylalanineFMN

phosphatase17.5-8.0SO100%

inhibition10%inhibitionFADpyrophosphatase16.5-S.598%100%

inhibitionnoinhibition

1 Incubation conditions were asdescribed in the text.

basis of incomplete separation between soluble and membrane fractions. Okuda's cy-tosol fraction was obtained after subjectingthe mucosal homogenates to two centrifu-gations at low speed, one (520 X g, 10 minutes) to remove "nuclei" and one (11,000 X g,20 minutes) to remove "mitochondria" fractions. The remaining cytosol fraction probably contained brush border membrane particles.

0 .4 r

M ed iu m FM N (/ M )Fig. 2 The ef fect of increasing concentrations ofFMN on jejunal uptake of [14C]riboflavin.Incubation

conditions were as described in the text. EDTA was (pH7.0) when added at 10 mM final concentration. Curvesat the top part of the figure represent [14C]riboflavinuptake in absence ---, and in presence O O of EDTA. Linesat the bottom part of the figure representunlabeled riboflavin accumulation in the incubationmedia, as determined after incubation, in the absence(-* -* -* -) and, in the presence (0 0 )ofEDTA.

- ,1 . 0 -

E0.8 B

025

IOM ed iu m FA D (/ M)

Fig. 3 The effect of increasing concentration of FADon jejuna! uptake of [14C]riboflavin.Conditions of incubations, including EDTA addition were as describedin figure 2 except for FAD which replaced FMN .Curves in the upper part of the f igure represent [14C]-ribof lavin uptake in absence ( --- ) and in presence ( - O -- O - -) of EDTA . The strai ght l ines at thelower part of the figure represent unlabeled riboflavinaccumulati on i n absence ( -$- $-) and in presence(.. 0-0 --) of EDTA.

IBs II

0 2 0 4 0 6 0 8A c c u m u la t e d R i bo f la v i

02 04 06 08A d de d R ib o llo v m ( i

Fig. 4 The ef fect of unlabeled ribof lavin on jejunaluptake of [14C]riboflavin.a) The uptake of [HC]riboflavinasdetermined in the studiesof figures 2 and 3 wasplottedagainst the concentration of unlabeled riboflavin releasedfrom the coenzymes. (),uptake in presence of FMN;(4) uptake in presence of FMN and EDTA; (O) , uptakein presence of FAD; (0) uptake in presence of FAD andEDTA. b) uptake of [14C]riboflavinin presence of addedunlabeled riboflavin.

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268 AKIY AM A ET AL.Location of both f lavin phosphatases on thebrush border membrane would be consistentwith a two stage process of digestion andabsorption in which uptake of riboflavin occurred after its release from dietary coen-zymes at the brush border. Supportive evidence for this latter possibil ity derives fromthe transport studies. Our studies show a saturable intestinal uptake of [HC]riboflavin

consistent with that described by Meinen etal. (2). Depression of the uptake of[14C ]riboflavin by rat intestinal rings in thepresence of increasing concentration of f lavincoenzymes was largely due to competitionfor transport by unlabeled ribof lav in derivedfrom FM N and FAD. EDTA caused a signi fi cant inhibi tion of the release of unlabeledriboflavin from the two coenzymes andthereby blunted the effect of these coenzymes on the transport of free [14C]ribof lavin.

LITERATURE CITED1. Christensen, S. (1973) The biological fate of riboflavin in mammals. Acta Pharmacol. Toxicol. 32(Supplement 2), 1-79.2 . Me inen, M . , Aeppl i, R . & Rehner, G . (1977) Studies on the absorption of thiamine, ribof lavin andpyridoxine in vitro. Nutr. M etabol. 21 (Supplment1), 266-268.3. Bessey,O . A ., L owry, O. H . & Love, R. H . (1949)The fluorometric measurement of the nucleotide ofriboflavin and their concentration in tissues. J. Biol.Chem. 180, 755-762.4. Okuda, J. (1958) Metabolism of flavin nucleotides.

2. Decomposition of flavin nucleotides in the smallintestine. Chem. Pharm. Bull. 6, 665-669.5 . Christensen, S. (1969) Studies on the ribof lav inmetabolism in the rat. 2. M etabolic flavin elimination after oral for intraperitoneal administration ofriboflavin-5'-phosphate. Acta Pharmacol. Toxicol.27, 34-40.6. Turner, J. B. & Hughes, D. E. (1962) The absorption of bound forms of B-group vitamins by ratintestine. Quart. J. Expt. Physiol. 47, 124-132.7. Okuda, J. (1958) Metabolism of flavin nucleotides1. Decomposition of flavin nucleotides in digestivejuice. Chem. Pharm. Bull. 6, 662-665.8 . Schmi lz, J., Preiser, H ., Maestracci , D ., Ghosh,B. K ., Cerda, J. J. & Grane, R. K . (1973) Purification of the human intestinal brush border membrane. Biochem. Biophys. Acta 323, 98-112.9 . Lowry , O . H ., Rosebrough, N . J., Farr, A . L . & Randal l, R . J. (1951) Protein measurement with fol inphenol reagent. J. Biol. Chem. 193, 265-275.10. Stripp, B. (1975) Intestinal absorption of riboflavinin man. Acta Pharmacol. Toxicol. 22, 353-362.11. Dahlqvist, A. (1964) Methods of assay of intestinaldisaccharidases. Anal. Biochem. 7, 18-25.12. Crane, R .K . & Mandelstam, P. (1960) The activetransport of sugars by various preparations of hamster intestine. Biochim. Biophys. Acta 45, 460-476.13. Rosenberg, I . H ., Col eman, A . L . & Rosenberg,L . E . (1965) The role of sodium ions in the transport of amino acids by the intestine. Biochim. Biophys. Acta 102, 161-171.14. Fishman, W . H ., Green, S. & I nglis, N . I . (1963)L-Phenylalanine: an organ specific sterospecific inhibitor of human intestinal alkaline phosphatase.Nature 19,85-686.15. Okuda, J. (1959) Metabolism of flavinnucleotides.3. Distribution of enzymes in the cellsof the mucosaof the small intestine causing dephosphorylation offlavin mononucleotides. Chem. Pharm. Bull. 7, 295-296.