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Multiple form of L-phenylalanine sensitive alkaline phosphatase in rat fecal

extracts

Article  in  The Japanese journal of experimental medicine · November 1986

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Yoshihiro Shidoji
University of Nagasaki
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勅ultipleFormof L−PhenyJcllcmine、Sens汁iYeAlkclHne
PhosphcfIcISeinRcI FeccrTExIrc．cIs
Soon Hie KIM，Yoshihiro SHIDOJI and Norimasa HOSOYA
pg如rfmg乃f ofⅣ加古γ最0乃J∫‘ん00g Of肋α協∫CZg花紙，ダα√祝坤of〟gdic言花β，ひれ古びgr∫如0f roた叩J
〃0乃gO，劫乃砂0一点祝，rOたγ0 773JJα如花
Mammalianintestinal alkaline phosphatase
isabrush−bordermembraneboundectoenzyme
Whichcatalysesahydrolysisofmonophosphate−
estersinanalkalinecondition・Ahalfcentury
ago，alkaline phosphatase actlVlty has been
foundin mammalian feces［1］，althoughtheir
OrlglnWaSnOtClarified untilthe mid1970’S．
Ithasrecentlybeenreportedthatanimmu−
nologically active alkaline phosphatase derived
fromintestinal mucosais excretedin rat feces
［2］・Fecalexcretion ofthe enzyme has been
SuggeSted to be a final stepinits normal
Catabolism．If so，it seems worthwhile to
Show a molecular relationship betweenintes−
tinaland fecalalkaline phosphatases・How−
ever，nOinformationis now available about
Physicochemical characteristics of the fecal
alkaline phosphatase（S）．In this study，We
investlgated some characteristics of fecalalka−
line phosphatase compared with theintestinal
enzyme，in terms ofinhibition by L−Phenyl−
alanine whichis a specificinhibitor agalnSt
intestinalalkaline phosphatase，and a charge
heterogeneltyOnionexchangechromatography・
Tenmale Wistarrats，Weighing150g，Were
housedinindividualmetaboliccagesfor3days
beforecollectionoffeces・Alaboratorychow
dietand tapplngWaterWerefreeofaccess all
daylong・Fecalpellets were collected with
a minimum contamination of urine and food．
AtlO：00everymornlngthepelletsweresaved
金 順姫，四童子好広，細谷憲政（東京大学医学部保健
栄養学教室）
冒悪等6言・。．款も55書誌］
（Received for Publication，February21，1986）
for9consecutive days and kept At −200C
untiluse・Thecombinedfecalpellets，333・5g
Wet Wt・，Were homogenizedin7vol．of O．9％
NaCI solution by a Polytron homogenizer
（Kinematica，Switzerland）for30sec，tWice．
Thehomogenateswerecentrifugedat8，600×g
for20min and the resultant supernatant was
used to meaSure alkalinephosphatase actlVlty・
Alkaline phosphatase actlVlty WaS meaSured
byamethodofBesseyetat．［3］usingp−nitro−
Phenylphosphateasasubstrate・Theenzyme
activity was expressed as FLmOles of p−nltrO−
Phenolliberated per min・ Organ specific
inhibitors，SuChasL−Phenylalanineforintestinal
alkaline phosphatase and L−homoarglnlne for
hepatic alkaline phosphatase，Were uSed−tO
define theisozyme Characteristics ofIalkaline
Phosphatase activityln the fecalextracts．
The fecalextract was applied to a column
（1×90cm）of Sephacryl S−300 equilibrated
With50mMTris−HClbufferDPH8・Ocontaining
O・2MNaCl・Bluedextran2000（Pharmacia）
WaS uSed to determine a void volume of the
COlumn・Bovine serum albumin（Seikagakn
Kogyo Co・），Catalase（Sigma ChemicalCo．），
and ovalbumin（Schwarz／Mann．，Orange−
burg・）were used as molecular weight
standards．
Protein concentrations were determined by
amethodofLowryetal．［4］withbovineserum
albumin as a standard． Fractions from col＿
umnswereestimatedforproteinbymeasurlng
ultravioletabsorbanceat280nm． Escherichia
COtialkalinephosphatase（typeIII）was pur−
 252 S．H∴KIM gfαJ． ［Vol．56

chasedfromSigmaCo・Homogenatesofrat and the bacterial alkaline phosphatases（Fig・

duodenalmucosawere prepared withlOOvol・
of O．9％NaClasintestinalalkaline phospha−
Anaveragewetweightofdailyfecaloutput
was−5．20±0．23g（mean±S・E・，N＝10）perrat・
Alkalinephosphataseactivitiesweredetermined
to bel14．9FLmOIp−nitrophenolliberated per
minperdayinthefecalextracts（TABL臆1）・
LPhenylalanine，aninhibitor specific for
intestinal alkaline phosphataseDinhibited the
intestinalmucosalalkalinephosphataseactlVlty
inadosedependentmannerasshowninFig・
1A．Incontrast，E・COlialkalinephosphatase
was not foundinhibited at allin the presence
of30mM L−Phenylalanine）at a COnCentration
of which theintestinalenzyme wasinhibited
by86％・ThefecalalkalinephosphataseチC−
tiviti甲Were Partiallyinhibited byincreaslng
cohcentration of the amino acid，but at above
15mMOftheinhibitortheactivityinthefecal
extracts rQmained relatively constant・The
inhibitioncurveofthefecalenzymesapparently
resembledthatofal：1mixtureoftheintestina）
lA）． Then，We teSted whether or not L−
phenylalanine was still active toinhibit the
intestinalenzymebutnotto do with thebac−
terialenzyme evenin the fecalextracts・As
shownin Fig・lB）theintestinal alkaline
phosphataseaddedtothecxtractswasefhcient−
lyinhibited byincreaslng COnCentrations of
L−Phenylalanine，in contrast E・COli alkaline
phosphatase added to the extracts was not
inhibited apparently・
TABLEI Daily excretion of alkaline phospha−
taseactivitylntOratfeces．
Fecal宅慧買忠恕atase A／レPhe（−）
LPhe（一） L−Phe（＋）  』
114．9±18．5 41．0±3．6 73．9±15．4 58．1±5．0
L−Phe（−）or レPhe（＋）：in the absence or
presence of30m別［LNPhenylalanine・A means a
differencein alknline phosphatase activity be−
tween L−Phe（−）andレPhe（＋）・10samples
were collected fromlO rats．Mean±S．E．

Fig．lInhibition of alkaline phosphatase activities by L−Phenylalanine・

一〇Llu8−01uBLむd

0    10     20     30 0     10     20     30

L・Phenylalanine：（mM） L−Phenylalanine：（mM）

Alkalinephosphataseactivitiesweremeasuredin theabsenceorpresence（2・lj
6．4，11，15，21−and30・mM）ofL−Phenylalanine・A，aSaCOntrOl，alkalinephosq
phatase activitywasmeasuredin the absence of L−Phenylalanirle・▲−ローローロー，
E．colialkalinephosphatase（70．7nmol／5min）；−△−△−△，，Intestinalalkaline
phosphatase（74．5）；−○−○−〇一，・Fecalalkalinephosphatase（66・0）；一▲￣▲￣▲￣，
A mixture。f E．C。Ii andintestinal alkaline phosphatase（34・5＋28・Op）・B，
−●−＋−●−，AmiXtureOffecalandintestinalalkalinephosphatase（38・2＋34・6
nmov5min）；，−−→−→ト，AmixtureoffecalandE・COlialkalinephosphatase
（38．2＋41．1）；一〇−Op〇一，Fecalalkalinephosphatase（42・0）・
 1986】 FECAL ALKALINE PHOSPHATASE 253

No fecal alkaline phosphatase actlVlty WaS Phenylalanine，SuggeStlng that most of the

influenced byレhomoarglnlne aS Well as the
intestinaland thebacterialenzyme （result not
Shown）．
The fecal extract was conducted to amm0−
nium sulfate fractionation． 44％
alkaline phosphatase activities were
in30−60％ammonium sulfate．In
tion，alkaline phosphatase activities
enzyme actlVlty WaSintestinal orlgln． The
30−60％ammoniumsulfatefraction，afterdial−
ySIS agalnSt the equilibrating buffer，WaS aP−
Plied to a COlumn of DEAE−Sephadex A−25
0f total （Fig．2）． Several sharp peaks of alkaline
recovered phosphatase actlVltyWereelutedbetweenO．1M
this frac＿ and O．2M NaCl・ Alkaline phosphatasein
Were ln− each peak WaSinhibited by30mM L−Phenyl−

附m地肌皿腑
hibited by77％in the presence of 30mM］レ alaninetothesamedegree（Pl；93．1％inhibi−

Fig・2 DEAE−Sephadex A−25ionqexchange column chroIpatOgraphy of fecal

alkaline phosphatase．
︵⊥∈UO記﹂0︵◆l上EUO至芸Uu眉OSqく

∈亡〇一三和むUu月LOSq︵

0 50       100 150       0

FractionNo．

FractionNo．

A，A column（2．3×53・5cm）of DEAE−Sephadex A−25was equilibrated with

lOmM＝Tris儀HClbufferIpH7・4in thecold room・The fecalalkaline phospha−

tase（95m1，186pmol／min）fractionated with ammonium sulfate of 30−60％

Saturation was applied to the column and washed with675ml oflO・mM Tris−
HCl bu仔er，PH 7．4，then followed by anl．11iterlinear NaCI concentration
gradient from O to O・5Min the buffer・Afterward，remalnlngalkaline phos−
Phatase was elutedwithlM NaClin the buffer．Fractions of15ml each were
COllected at a且ow rate of30ml／hr・A portion（50pl）of each fraction was
used for alkaline phosphatase measurementin the absence（−●−●−●−）Or
PreSenCe（▲▲▲▲▲）of30mM＝L−Phenylalanine．5％hPmOgenate Of duodenal
mucosa，Whichwas solubilized with O・1％Triton X−100at4OC forlhr，WaS
Centrifuged atlOJ500×g for90min・The supernatant was applied to the
COlumncontainingO．1％TritonX・10。inthebuffer．Theboldarrow（↓）shows
thepositionwheretheduodenalalkalinephosphataseeluted・B，Peaklfraction
（＃70−75）；C，Peak3fraction（＃81−88）；D，Mixture of Peakl and Peak3
WeredialysedandrechromatographedonDEAE−SephadexA−25column O．3×45
cm）withalineargradient ofO・tO O・5M NaCIconcentration，reSpeCtively．
 254 S．H∴KIMβfαg．
tion，P2；86・7％，P3；82．6％，P4；80．4％，P5；
90・7％inFig．2）・Peakl（Pl），Peak3（P3）
and their mixture were rechromatographed as
Shownin Fig・2B，C，D． Peakl alkaline
Phosphatasewasrecoveredatthesameposition
aspeakl onthefirst chromatogram，and did
not glVe any Other peaks on the rechromat0−
gram・ Peak3 alkaline phosphatase eluted
mostly at the same position as peak3．The
elution position of peakl was certainly dif−
ferentfromthatofpeak3，because amixture
Ofpeaklandpeak3gave two distinctpeaks
Ontherechromatogram（Fig．2D）．Duodenal
alkaline phosphatase，Which was solubilized
With O．1％ Triton X−100，WaS eluted as a
SinglebroadpeakataroundO．25MNaClfrom
the same DEAE−Sephadex column．
When the fecal extract was applied to a
COlumn of Sephacryl S−300，0ne maJOr Peak
Ofalkalinephosphatasewasdetectedataround
160K dalton・Peakl and peak3from the
DEAE−Sephadex column were eluted at the
Same POSitionI SuggeStlng that both enzyme
had the戸ame mOlecular size of about160K
dalton（results not shown）．
SeveralinvestlgatOrShavereportedthatalka−
line phosphatase was excreted as an active
formin rat fecalpelletsl5］and human stool
l1］・Daily output of fecalalkaline phospha−
taseishighlyvariable，forinstance，Duckworth
and Goddenl5］claimed that dietary fiber
increaseddailyfecalexcretionoftotalalkaline
phosphatase activity by7−fold．Even though
there are many differencesin preparations of
SaTPle，Strain（Sprague−Dawley vs Wistar），
aglng，andfoods，aCertainsimilaritybetweena
previous workl6］and this、rePOrt WaS found
indaily昌ⅩCretionlevel（80−1700rl14．9FLmOl／
min／day）oftotalalkalinephosphataseactivity
into feces．
Althoughfecalalkalinephosphatasehasbeen
Shown to be partiallyinhibited by L−Phenyl−
alanine［6］，nO quantitative analysis has so far
been donein detail． Here，in this report，
itis clearly shown that the fecal alkaline
Phosphatase wasinhibited by L−Phenylalanine
inits concentration dependent manner up to
30mM．InFig．1B，Wehaveconfirmlyshown
［Vol．56
that theintestinal alkaline phosphatase even
inthefecalextractswasquantitativelyinhibited
byincreaslngCOnCentrationsofLqPhenylalanine
and that E・COli alkaline phosphatase which
WaS eXOgenOuSly added to the extracts did not
make any effect oninhibition of the fecal
alkalinephosphatasebytheaminoacid．The
inhibitor sensitive alkaline phosphatase was
Calculated to be 58．1％ of total alkaline
Phophatase．inthefecalextracts（TABLEl）・
This valuelSin comparable range with those
（45−60％）reported by Thomas and Henton
l6］．SincetheyusedL−Phenylalanineat5mM，
atwhichconcentrationitsinhibitoryeffectwas
about a half of that at30mMin this report）
the percentage of LPhenylalanine sensitive
alkaline phosphatase might becbme rather
higherin the same condition as ours．In
human Samples，a PerCentage Of theinhibitor
SenSitive alkaline phosphatasein total fecal
alkaline phosphatase varied from30tolOO％
at5mML−Phenylalaninel7］．Anyhow，aSSum−
lng that theinhibitor sensitive alkaline phos−
Phatasein the fecal extractsis allintestinal
Orlgln，ltStOtalactlVltylnOnedayfecalextracts
iscalculatedabout13％ofthe smallintestinal
enzyme aCtivity（594・3lLmOl／minin total）in
L−Phenylalanineinhibition study strongly
SuggeStS that the fecalalkaline phosphataseis
a mixture ofintestinal and bacterial alkaline
Phosphatases・Further exploration should be
done to elucidate what L−Phenylalanine resist−
antalkalinephosphataseisinthefecalextracts．
A maJOrfindingin this studyis that the
fecalalkaline phosphatase，Whichis L−Phenyl−
alaninesensitive，Showedachargeheterogenelty
On a DEAE−Sephadex A−25ion exchange
COlumn． The multiple sharp peaks of the
enzyme actlVlty CannOt be artificial during a
COlumnchromatography，becausearechromato一
graphy study tells that atleast thefirst peak
（peakl）and the third peak（peak3）Were
notinterconvertible duringchromatography as
Shownin Fig・2・A percentage of LqPhenyl−
alanineinhibition was almost the samein each
Peak（80−90％），SuggeSting that all of these
Peaks were generated from a single source
 1986］ FECAL ALKALINE PHOSPHATASE
that might be anintestinalalkaline phospha−
tase・Thesolubilizedduodenalalkalinephos−
Phatase seemed more acidic than the fecal
enzymes as shownin Fig・2・ Hence）itis
likely to assume that the released duodenal
alkaline phosphatase mightlooseits negative
Charges during catabolisminintestinallumen
or feces．
Nakataetal・［8］haverecentlyrevealed that
a charge heterogenelty Of E．Coli alkaline
Phosphataseis produced by the bacterialpr0−
teolytic enzyme・ Hence，ltis not strange to
SPeCulate thatintestinalalkaline phosphatase）
which has been released from microvillus
誓embranes，iscatabolizedbysomeenzyme（S）
lnluminalcontents to glVe a Charge heter0−
geneltyOnion−eXChangechromatography・One
Can Still argue thatits charge heterogenelty
mightbe produced during storage of feces or
theywould be derived fromdifferentparts of
intestine（duodenTm，jejunum，etC・）・Struc−
tural differencesln microheterogeneousalka−
line phosphatasesin the fecalextract are now
underinvestlgation，in terms of carbohydrate
COmpOSitionandN−terminalaminoacidresidue．
Summary：Alkaline phosphatase activity
was detectedin fecal extracts of male rats．
58％ofthetotalenzyrpeactivitywasinhibited
by30mMレPhenylalanlne，Whichisaninhibitor
SpeCific forintestinal alkaline phosphatase・
L−Phenylalanine sensitive alkaline phospha−
tasein the fecal extracts revealed multiple
View publication stats
255
peaks onDEAE−SephadexA−25ion exchange
COlumn with alinear gradient of NaCl・
Itisdiscussedthatapartofthefecalalkaline
Phosphatase might be a set of catabolites of
theintestinalenzyme・
REFERENCES
l1】Lawrie，N・R・：The excretion of phosphatase
in the faeces． Biochem．）．，37，31ト312
（1943）．
［2】Lehmann，F．−G．，Hufnagel，H．and Lorenか
MeyerI H・：Fecalintestinal alkaline phos−
Phatase：Aparameterfor toxic damage of the
Smallintestizlalmucosa． Digestion，21，156→
162（1981）．
［3】B・eSSey，0・A・，Lowry，0．H・，Brock，M．J．：
A method ofalkaline phosphatase with 丘ve
Cubic millimeters of serum．）．Biol．Chem．，
164，32ト329（1946）．
［4］Lowry，0・H・，Rosebrough，N．J．，Farr，A．L
andRandall，R．J．：Proteinmeasurement with
thefolinphenolreagent．）．Biol．Chem．，193，
265−275（1951）．
［5］Duckworth，J．and Godden，W．J．：Thein一
触ence of dietaryfibre on secretory activities
Ofthe alimentary tract：Observations of faecal
Phosphataseexcretionandcalciumandnitrogen
balances of rats． Biochem．）．，35，16−23
（1941）．
［61Thomas，D W．and Henton，D．H．：The use
Of fecal alkaline phosphatase as anindicator
Ofintestinal damage． Digcstion，31，82−88
（1985）．
［7】Ho・rrigan，F．D．and Danovitch，S．H．：The
Orlgln Of human fecal alkaline phosphatase．
dm・J・かな・かむリ19，60−3−608（1974）．
［8］Nakata，A・，Shinagaw？，H・and Shima，H・：
Alkaline phosphataselSOzyme COnVerSion by
Cell−free extract of EScherichia coli． FEBS
エg払，175，343−348（1984）．