APPL1CATI()N 01’ ffl E STEREOSPECIFIC INHIBITOR L-PHENVLALANINE

TO rIII 1 ENZ\ MORPHOLOGY OF INTESTINAL ALKALINE

1HOSPHATASE’

KEIICHI WATANABE AND WILLIAM H. FISHMAN

Department of Pathology (Oncology), Tufts University School of Medicine, and the Cancer Research
Department, .Vew England Center Hospital, Boston, Mass.

Received for publicatiomi July 11, 1963

SUMMARY and terminal web in the presence of n-phen-

yialanine. These reactions were absent in com-
‘l’he imstestimie-sl)ecific inhibitor of alkaline
panion sections incui)ated in L-phenviaianine-
phosphatase, L-phenvlalanine, has h)een emiiployed
in the study of alkaline phosphatase in rat
containing substrate solutions.
intestine. Five substrates were used, -glycero-
The possibility of comparing the morphology

phohate, o-carboxy)henylphosphate, a-naph-

of a tissue in which both test and control solutions
thyl acid phosphate, naphthoi AS-BI phosphate are identical except for the configuratioms of one
and naphthol AS-TR phosphate. The hydrolytic component may contribute to a muore precise
rate and specific L-phenylalanine inhibition was interpretation of the results obtailsed in the test
most nmarked in the case of the first three sub- section.
strates. When formol-Ca fixed, gum-sucrose
INTRODUCTION
treated sections of rat intestine were incubate(l
in these substrates in the presence separately of Partially purified intestinal alkaline phos-
0.05 Al ii- and L-phenvlalanine and Gornori’s phatase of rat (5) and human (6) has been
calcium-cobalt method was employed, a striking reported to undergo specific inhibition by
inhibition by the L-isomer was regularly found. L-phenylalanine but not by m)-phenylalanine.
Azo dye hiiethods were applied under different The enzyme l)rePared from a number of other
conditions of time, temperature and cation tissues was insensitive to 1)0th D- and L-phenyl-
concentration ins somiie instances. The contrast aianine. This behavior contrasts markedly with
was marked but not (iramatic in the case of the previously reported organ-unspecific inhibi-
a-naphthyl acid phosphate, amid it was evident tion of alkaline phosihatase by a-amino acids
but not. marked in the case of naphthol AS (1) and by other reagents (2, 15, 7). Another
phosphate substrates. These findings have been unique property of intestinal alkaline phos-
discussed. phatase is its preference for o-carboxyphenyl-
The alkaline phosphatase of intestinal epithe- phosphate.
hal cells (humuan) grown in tissue culture ex- The method for measuring serum alkaline
hibited great sensitivity to L-phenylalanine. phosphatase of intestinal origin utilizes the
Rat kidney an(l leukocyte alkaline phosphatase release of product from the substrates f3-glycero-
were relatively insensitive under the same phosphate or phenyiphosphate separately in the
conditions. presence of 5 mM L- and 5 mill! m)-phenylalanine.
Morphologically, the L-phenylaianine-sensitive The mi-phenyialanine solution is the best control
alkaline phosphatase in rat intestine is. largely solution since it contains all of the constituents
confine(i to the striate(l border of the epithelial of the test (L-) solution in exactly the same con-
cells. In animuals maintained on a high fat diet, centration. The only difference is the configura-
azo dye methods demomist rated the presence of tion of the amino acid, phenylalanine, as indi-
fine granules in the regiomu of the Golgi apparatus cated in the structural formuulae on page 253.
This investigation was aided in part by the The system described above, if it could be
American Cahucer Society (Mass. Division), Inc., used for enzyme locahizatiomu, would appear to
(1079-C-i), by the Anuericami Cancer Society, Inc., be ideal for the precise interpretation of the
New York (P-106, P-107) and by the National
enzymorphology of intestinal alkaline phos-
Cancer Institute, Natiomual Imistitutes of Health,
Bethesda, Md. (CA-09734). phatase. It would also make it possible to
252

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 L-PHENYLALANINE AND INTESTINAL ALKALI NE PHOSPHATASE 253

Intestine-Specific Inhibitor themi were trmtnsferred to cold Hoit. ‘5 gum sucrose

of Alkaline solution. After 48 hours the tissues were blotted
and embedded in gelatin. Following a period in
the refrigermttor for 3 tI, 4 days, 5 sections were
Phosphatase

p p
cut froni thie tissue block omi thme freezing nnicro-
tome and caught in mt beaker of cold distilled
water. Within 2 hiours, these sections were trans-
CH ferred to ,mse or muore (If thie substrate mmuedia

H-C-NH, listed imi Table I.

H,N-C-H
Preparations of substrate media and stains-
COOH COOH mug procedures: Substrate nnedia were always
D- made freshly before use in 2-aminso-2-muethyl-1 ,3-
Phenylalanine propitmiediol buffer, 0.05Al at pH 9.75. A sunumary
of the conditions ehluployed withs each substrate
identify tise cellular structures or organdies appears in Table I.
which are (lie source of thw L-phenylaianimie- Examination of histochemical substrates
sensitive alkaline l)1i051)hatase. with regard to L-phenylalanine inhibition:
Five substrates were eachi prepared ins propanediol
MATERIALS ANI) METhODS buffer in time concentrations employed ihi the
I’reparatiomu of tissues: Freshi tissues were respective tissue staining procedure. The conucen-
taken frllmis time upper portion of jejununi and trations of D- and L-phienylalaniine were each 0.05
kidmiey of nllrnsal adult male albino rats killed by .1!. Purified rntt mt estinal :tl kal ihie phiosphiatase
exsanguinat illIu under lighmt ethier anesthesia. w:ts added. At the end of time incubatiomu time
Tissues were fixed iii c(Iid 10 fornuol-calciunsu for imidicated ims Table II, ansalysis for inuorganic P
24 hours, carefully I)l(It ted on filter paper, and were performed according to Sisihillwara et a!.

TABLE I
(‘ondilions of Stain my Reactions

Calcium-Cobalt Azo Dye Coupling

Substrate Mixture Method (after . Method, Fast Blue
(;omori 5)) RR as couh)lert

Cation
Times Temp. Time Temp.
Substrate (Supplied i Concentration
(mm) #{176}c (mm) ‘C
Chloride)

i-;lcerlllIim(Isihtt’ (8) 0.0190 A! Mg 0.006 A! 30-45 37#{176}

Ca 0.055 A!
Na-a-miapht imyl acid phos- 0.0044 .11 Mg4 0.006 A! 20-30 25#{176}
phiate Ca 0.055 A!
Na-a-napiit liyl t(i(l phi(Is- 0.0044 .1! Noise 2 25#{176}
phate (11)
o-Carboxyphmemmyi-phiosphiate 0.0020 A! Mg 0.006 Al 20-30 37#{176}
(8) Ca 0.055 Al
Naphtiuol AS-B! phosphate 0.001 .1! Nonue 3 . 25#{176}
(Na) (3(
Naphuthol AS-TR phiosphitute 0.0013 3! Noise 3 25#{176}
(3)
Naphuthsol AS-lB phosphate 0.0013 A! Mg 0.006 .1! 60 37#{176}
(8) Ca 0.011 Al

* Ihe timime of ihidubatiohi was decided after deterniinimig the imictih)atioms period required to produce
a good positive react ioni in a sectioni imscubated ins substrate solution lacking phienyialanimue.
tIn order to insure the pllsitive reaction in eosinophilic leukocytes, sectiomus were placed ihi a sub-
st.rate, anmimso acid aimd propamsediol mixture at. 4#{176}C
for 1 huour. The digests were thems placed ins a Wa-
(er incubator, fast i)ltle HR salt added, and the reactioms terhuiihiate(I 2 nninutes later.

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254 WATANABE AND FISHMAN

TABLE II
A Study of L-Phenylalanine Inhibition Using Histochemical Substrates

Alkaline Phosphatase Activity

(pg of P liberatedt 15 minutes)

Molar Incubation
Substrates Concen- Time . Phenylalanmne
(ration (mm.)

(D-L)/
None (0.05 .if) (0.05 if) D X100(

/3-(lvcerophiosphsate 0.0190 15 9.7 8.00 0.90 89

o-Carboxyphsenyl-phsllsphate 0.0020 15 14.6 8.95 1.75 80
Nit a-Naphithivl acid phosphate 0.0044 15 10.05 8.40 1.05 87
Naphsthol AS HI phsllsphiate (slldiumsu salt)* . .. 0.0010 60 1.89 1.23 0.45 63
Naphthsol AS-ill phllsphntte* 0.0013 60 1.36 1.00 0.40 60

Values are calculated (IH the basis of 15 muiinutes incubation.

t
Rat intestinal alkaline phosphatase with a specific activity of 48,980 (mug phensol liberated from
0.016 Al phenyiphsosphate/15 minutes/mg protein).
The percentage of inhuibition by L-phehiylalalsifle versus D-phenylaialuinue.

TABLE III
Activity of the Rat Intestine Mucosa Alkaline Phosphatase after Incubation with D- and
L -Phenylalanine

Calcium-Cobalt Method Azo Dye Coupling Method

Phenylalanine (0.05 .11) Phenylalanmne (((.05 .11)

Substrates D- L-

Epithelial Leuko- Epithelial Leuko- Epithelial Leuko- Epithelial Leuko-

cells cytes cells cytes cells cytes cells cytes

-Glycerophosphat.e 3+
o-Carboxypheisyl-phosphate 3+
Na a-Naphthsyl phosphuate 3+ C? 4+G 3+ 2-F-U 3+
C C
Naphthsol AS-HI phosphate 3+U? 2+ 2+ 2+
Naphthilll AS-TR phsosphate 2+ 4+U 2+ 3+G 2+
C C

C; Alkaline phsosphsatase activity in Golgi zone.

C; Alkalimue l)lilIsPhatase activity in other area of cytoplasm.

(16). Values for imiorganic pluospisorus were RESULTS

obt.aimsed ins tile absemsce of phemsylalamsine, in tue A summsiary results of of tue
time biochemical
presence (If D- anud in (lie presemice of L-phen- experiments appears in Table II and of the stain-
ylalamsine. Frons these, the degree of inhibition has imig remtctions in Table III. Pimotomicrographs
been consuputed imi each case. (Figures 1 to 10) illustrmtte thue findings.
Studies omu cultured Iuuman intestinal
It nutty be noted froni Table II timat time hydrol-
cells: ‘Fise L-plmenylalanine imsisibitor of alkaline ysis of -giycerophiosphuate, o-carboxypiuen-
phosphsatase was also emisployed Ofl cultured isumsian ylphsosphate and a-napisthiyl acid phosphate
intestinal epitlielial cells originitlly obtained from proceeds nsore rapidly than tise rates noted for
Microbiological (‘onspany amid propagated in naphthol AS-BI and AS-TR pimosphmat.es. The
Leighton tuh)es. concentration of phsenylalanuimse, 0.05 Al, WaS tlue

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 L-PHENYLALANINE AND INTESTINAL ALKALINE PHOSPHATASE 255

(Ihid fouisd to give a reproducih)le inhibition of time Tlse enzyme activity of the rat kidney:
st a using react II 115 wit is sodium $-glyceropiuos- Alkaline phiosphsat.ase activity wtts confimied to thue
Pilate. At this required higim concemstratiois of brush border of thue proximal coisvoluted tubules
I)-pimeisylalahsine, 0.05 A!, an appreciable ihshibition and tise adventitia of vessels ims every methsod
(l7-39) occurs, which, however, is much less in employed. Kidney alkaline phiosphsatase activity
extent tlm:uii time (IflC il(Ited witlu L-piueniylalanine. was relatively insensitive to m-phsenylahtnimie.
Thse figures (last columssn) indicate the relative Effects of the inluibitor on staining re-
iii hmibi t iois produced h- I. - versus D-pisenylalanine actions : Intestinal alkaline phsosphsatase was
amid demsa Inist rate t lust. ( lie phens(Imuenon is exhibited shiarply depressed by 0.05 31 L-phemiyiaianine
by all 5 substrates. It appears thmat the eiszyme in hut not at all by D-phemsylalanine imi t.hse case of (hue
time presence lIf naphthiol AS-HI phosphsate and four substrates where the liberated phuosphate
miaphm(.hsol AS-TB. PhosPhate is the least sensitive radical was visualized by t.hue Comori calcium-
(II L-phsemiylal:unine under these experiniental cobalt methiod.2 However, (he imiluibition of thse
(( lImdit.iOmiS. enzyme by L-phsenylalanine was less complete in
Localization of alkaline phospisatase in rat all azo dye c(Iuphing methods, especially in (hue
imitestine : Time posit ive reaction obtainsed usinug case of naphthol AS-HI and naph(hol AS-TIt
t hue caiciumsi -col)alt msuet hod wit ii ends subst rate phosphsates. ‘Flue incompleteness of the imuactiva-
i#{149}as
largely coisfined (lie region of the striated (ion was also more noticeah)le whsen (lie imshiibi(ion
border (for ex:tniple, Fig. 1). ‘Flue intercellular studies were applied to (hue int.estine of a rat
mumembraise slioved weak activity in many sections. previously fed on a high fat diet. The enzyme
Tue Colgi regioms and other cytoplasmuic regions activity of thue eosinophiilic leukocy(es ins thme
were almost always negative for activity if (lie lamina propria
of the intestinal vihli, which was
(issues were hIlt. (Iverihscuhated or if the tissues demonstrated only in azo dye coupling methiods,
were not t:mken froni time animals onu (lie huigh fat remained unaffected its judged h)y (hue sanse
diet. intensity of stain in (lie presence of eithier D- or
Following time applications (If (lie azo coupling L-phenylalanine in substrate mueditt (Figs. 3, 4,
method, alkalimse phiospisatase was visualized as 7, 8). ‘Flue alkaline phosphuatase activity of tue
very flue black granules whichm were nsost intense cultured human intestinual epithiehial cells was
iii (lie striated border, in thse regions of thue Colgi most dramatically inactivated eves by :t conuceni-
apparatus, amid, iii thse terminttl web (cytoplasmic (ration of L-phienyialanine (0.01 A!) lower than
region right below the striated border). Medium thiat whiichu was employed on (hue rat inutestine
immtenssity :mmsd faimit activity in o(hser regions of (Figs. 9-10). The enzynie activity of (hue kidniey
supramiuclear cyt oplasnu (If t hie epithselial cells was less greatly affected by (lie imshuibi(or, espe-
were oI)served when sodium a-napisthsyl acid cially in (hue distal portions of (hue pr(Ixinsttl cons-
phosphate was (lie substrate (Fig. 3). However, in voluted tubules. Little difference imi time ini(ensity
(lie case (If naplm(iscll AS-HI pisosphuate, enzynue of tue stain for kidney alkahinse pisosphuatase was
activity was largely confined to tise striated produced by the imihihitor when thie muaphuthsol AS
border and visualized as a purple dye deposit (Fig. phosphates were (hue substrates -

7). Weak Colgi regions activity was on1y shown

wisen tissues were taken from (lie rats omu (hue high I)ISCUSSION
fat diet. Ims (lie case of msapisthsol AS-TR phosphate,
Stereospecific, intestine -specific L -phen -
tile wisllle picture was alns(Ist thie same as tisat of
ylalanine inhibition: This phenomenon has
AS-BI pimosphsate excel)t that (lie deposits were
been demonstrated in the rat and human and
fimsely granular iii ai)pearance. L-Phsenylalanine
imsisibited (hue emszymne activity in (hue organehles
has been found to possess lrecise structural

described above. Withs regard to time eosinophsilic requirements. For exanul)le, D-phenylalanine,

leukocvtes and eisdothiehial cells of lacteals in thue formyl-DL-phenylalanine, DL-a-phenyl-a-alanine,

lansiina proprits (If villi, naplmthsol AS phosphate I)L--phenyl--alanine, L-tyrosine amid n.L-aianine
met hsods gave a positive reactions within 3 minutes. are not inhibitors under the same conditions in
Ots the other hand, activity in these cells could be which L-phenylalamne is (5).
demonstrated tmsimsg sodium a-nsaphmtlsol acid phsos- The organ-specific inhibition is a funuction of
phmate as stmbs(ra(e and fast blue HR as coupler
concentration of phenyialamiine amid substrate.
only after preincubatiouu its described in Table I;
the calciumis-cobalt techmmiiqtie gave no reaction. 2 The possibility of a positive reaction, invisible
Its the case of time sections slsowimsg positive in the light microscope but visible mi the electron
microscope, remains in view of thue findings of
react ioiss for leukocytes ntn(I endo( hselial cells, (lie
Clark (4), who studied (he alkaline I)hiosPhatase in
ireseisce (If L-phsenylal anine mi (lie imicubat ion muouse intestimittl cells wit Is the caiciumis-cobalt
muixture had no iusimil)i( ing act fllhi nisethod using both miuicrosd( Ipes.

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256 WATANABE AND FISIJMAN

Fi ;s. I S. Al k:mlmime i)hm( spima.tase activity If formmuol-calciunu fixed , frozemu sect il Ihued rat j ej unumsi.
Coumm(erst aihi(d h)\ I’ mmiet imvl greemm in vemommal l)tmfler p!1 4.0.

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 L-PHENYLALANINE AND INTESTINAL ALKALINE PHOSPHATASE 257

Thus, with rat intestinal alkaline phosphatase A number of 1)ossibilities suggest themselves for
and f3-glycerophospha(e, a concentration of 0.01 consideration.
Al inhibited intestinal enzyme 60% and non- Phosphoryl transfer to an acceptor alcohol
intestinal enzyme 0-10% (5). At a concentration (propanediol buffer) will reduce the amount of
of 0.05 Al of )henylalanine, the 1)-isomer is inorganic phosphate which would otherwise
inhibitory (20-40%) with regard to alkaline accumulate in an alkaline phosphatase digest (9,
phosphatase from intestinal and non-intestinal 13, 14). We have verified the fact that at concen-
sources; inhibition by the L-isomer is, however, (rations of propanediol of 1.0 M or higher the
more marked, approaching 90%. Preliminary staining reaction by the calcium-cobalt method
experimnents in this laboratory indicate that (he was decreased whereas t.hat produced by azo dye
inhibition by L-phenvlalanine follows a kinetic technique was unchanged (substrate: naphthyl
course which fits neither classical competitive nor acid phosphate, veronal buffer). This finding is
non-competitive inhibition. explained on the basis of Ph05Ph0mYi transfer.
Tue (lata imi this l)l)d extend (he phenomenon However, since the l)resence of propanediol or
to a variety of histochemical substrates3 for other alcohol acceptors is unnecessary in order
alkaline phosphatase which show between 60 to to demonstrate L-phenylaianine inhibition, there
90%. inhibition with respect to L- over D-phen- are no grounds to implicate this type of phos-
ylalanine effects. }ohiowing the application of phate group transfer in the explanation.
histochemnical methods under much the same The presence of divalent cations in the digests
condmtions, the over-all phenomenon is clearly prepared for the calcium-cobalt. method, amid t.heir
evidemu(, but there are sonne challenging findings absence in the mixtures destined for the azo-dye
which mnerit further discussion. procedure, could be a pertinent circumstance.
Comparison of the results obtained with Fromn biochemical data, it has been proven that
calcium-cobalt method and azo dye method: stereospecific inhibi(iomi by L-phenvlalanine
For 1)0th a-naphthyl acid phosphate and naphthol requires no magnesium ions (5). We have added
AS-TB phiosphate, L-phenylaianine produces 1 if magnesium chloride to naphthyl acid
almost cotusplete inhibition when the inorganic phosphate plus fast blue RR amid found the same
Pi505PI5it(d is tral)l)ed by calciumn ions and degree of inhibition by L-phenyialahuine of the
visualized by (hue Comori calcium-cobalt. method. staining reaction as in the absence of huagnesium

However, when the organic radical is captured by chloride.

simsiuml(amucous coupling ith fast blue RH under It is possible that the j. activity in (he
somsiewhuat different incubat ions conditions, the presence of L-phenylalanine may not succeed
imshibi(ion is less evident (see Table III). under the conditions of (he staining reaction in
accumulating sufficient. calcium phosphate at the
The point for discussiolu, therefore, in view of
enzyme site to exceed the soluhihitv product
the results in Table III, is, why has the inhibition
constant for calcium phuispha(e. Consequemitly,
1)cenu niore marked (calcium-cobalt method) for
the negative reaction seen may not reflect a low
visualizing the inorgamc Phosphate and less
alkaline phosphatase activity.
n(I(iceal)le whemi the organic radical is localized?
Another circummstance is the factor of time of
incubation. The azo dye coumplimig mnethods
Another substrate, glucose-1-phuosplsa(e, for
intestimsal alkaline phsosl)huataSe was examined with require 2 to 3 mimites; the calcium-cobalt
sectiamss ihicUh)ated its tue presence of 0.05 Al D- method, 20-60 minutes. It. was possil)le to
amid m-phsemuvlalanine usinig thie calciumsi-cobalt
prolong the incubation timue for the azo-dye
nmethod. Promuotmnsced inhibitions by the L-aniino
acid was no(ed. procedure to 45 mninutcs by carrying ou( the

FIG. 1. (‘omudiions; Na -glycerophiosphate nsediuhu with 0.05 Al D-phenylalanimue (calcium-cobalt

muse(hod). Tue intense activity is shown mainly in the striated border of the epithelial cells with black
anuorphuous deposits (400X
FIt;. 2. Cousditions; the sanise substrate medium with 0.05 Al r-phenylalanine. The activity is alnnost
conupleteiv eiimsuinated. (400X)
FIG. 3. Cohiditions; Na a-miaphthvl acid phosphate muedium with 0.05 Al D-phenylalanine (:tzo’ dye
coupling muuet.huod). Imitense activity is in the striated border, Golgi region and other regions of cytoplasm
with boths vei-y finely granular and coarsely particulated deposits. Leukocytes show thue activity. (400X)
FIG. 4. Conditions; the same substrate with 0.05 A! L-phenylalanine. Sharp depressiomi of thie epithe-
hal cell :mctivit.v occurs. Leukocvte activity remains in(ttct. (400X)

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258 WATAXABE ANI) FISI-IMAN

.-,....“.

. . .1

.) -I

1.

#{149}1 4 pm)

j-
I..

JH
-. :
.

*‘;‘
- .

. - .t.
. - .
.ti
-

Ho. 5. Tue jejummummu frommm a r:tt whiirhu was givemi highs fat diet for 10 days. Incubation in o-carboxy-
phuemsyl pllos1lhlat c mmmcdi 1 hi wit hI 0.05 .1! I) -phiemivlal:tnimue (calciummi-cobalt method) - Intense activity is
showmm hI I he st riate(l hI(lh(lPh mmmdthme ( olgi regioms. (41X)X

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 L-PIIENYLALANIXE AN!) 1N’rESrl NAL ALKALINE P1IOSPIIATASE 259

11(15. -10. Alkahitie phospisatttse activity of cimitured humsuami intestimue epithieiial cells. Formusol cal-
ciimmuu fixatioms for 15 nuinsutes, and then treatnienut with Holt. ‘s gumuu sucrose overnight.
Fio. U. Imucubatiomm its Na a-tiaphthsyl acid phiosphiate hnediumim with 0.05 .11 D-phemiylalahmihle. TIme itmtemsse
activity is shown as bothu very fine dark greemi gramiules which are accummiutmiated along cell hlorders atud
large hlack particles. (400X)
Fmo. 10. 1 uscub:ttion its thie samsue substrate mssedium with 0.05 .1! L-phuemsylalamiimue. Tue activity is CWhi-
pletely elimmsimsated. (4(X)X)

itucubat illhu at 2#{176}(’(h-c-chills). Ihe results were huvdrol-zed musore slowly I hams I hue (It her stub-
nO (Iiffercms( thaus the omues obtained its 2 to 3 st t-ates. Ihiis c-ircuhssstausce mssav IXhll’Lims the
mitsimtes imscubatiomi. relative i mucomiuj Ileteusess (If the imuhi ii lit 11115 hI
It is 111lssih)le, also, t lwtt t hue reactiomi bet weemi I_-I liiehuylntlahuihli.
ims(Ih-gamsic I I1iO5iIhiit( ami(I cal(ilmtui ion or tue azo- Other correlationus h)etwecmi h. -anuihso acids
(lye rolljlhihug reactiomu may be imsfluenced by amid intestinal alkaline phosphatase: It is

iihiemilaltmusimse. It is millt l.Iossibie, however, ((I kmuown tiua( L-formsus of mus(Is( amssimio a(-i(ls are
exlliaiuu vhu- (hue L-isohiuer should be utuiquelv actively absorbed t hroughs I lie ihut est imia 1 ej lit lie-
(liffemehut fromsi the mI-isomsuer its (heir effects omi (lie hum of cemtaims animusals (rat, guiuuea pig (log). In
COh5Sl Iotueti(s (If the digest other than (hue sites of a(l(Ii(i(Imi, imutest inal abs(IrJIt ion (If L-amsuihmo aci(Is is
euszvmiie 1(11 ivity. a I Ih(Icess imi which alkaline Iliiosl Ihuatase is believe(l
Ii mmliv, at (emil iots slu(luld be givets to t hue fact to llarti(-illa(e. This belief is based (Ihi I hue sigmiifi-
I hat t lie Iwo tiaj iluthuol .S J)h01 (hates are (fl5 t imucreases its intest imual alkali tue I (hI ISI usa I ase

FIll. 6. ‘Fhse sammse tissue its tue

substrate samue
nmuediunu withi 0.05 ii m.-pluemuyl:mlatuiuie shmowing almiuost
comislulete ClihiuitittiOhi (If activity.
) (400X
Fmo. 7. Inucubat.ioms its tiaphithol
AS-HI phuosl)hsate hmue(hillhmi wit.hu 0.03 .1! t)-phuemiylalamsimie (.&zll (lye
nuietii(I(I ) . I ustehuse activity is showms strictly ims (lie striated border of (hue epitiselial cells as l)ilrple,
ahts(IrhlhilIUs deposits. Thue leukocytes amid (hue endothieli:ml cells of the lacteals show thue :ictivit. (G(X)X
Fmo. 8. Itscuhatiotu in (lie satsue substrate mssediumiu withi 0.05 .1! L-plsenylalaniuse. (‘omusiderahlle activity
still remmsaimis in the st ri:(te(i h)(Irdeu-. Leukocvte activity remmuaimss immtact. (GOOX

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260 WATANABE AND FISHMAN

activity accompaniedi by a diminution in serum 4. CLARK, S. L., JR. The localization of alkaline
phosphatase in tissue of nnice, using the
inorganic phosphate which Tuba (18) observed electron microscope. Amer. J. Anat. 109:
after force-feeding certain L-amino acids. 57-83, 1961.
5. FISHMAN, W. H., GREEN, S. AND INGLIS, N. I.
It is also recognized that L-forms of amino
Organ specific behavior exhibited by rat
acids undergo carboxyi activation by phosphate intestine and liver alkaline phosphatase.
through (he agen(y of adenosine triphosphate or Biochim. Biophys. Acta
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6. FISHMAN, W. H., GREEN, S. AND INOLIS, N. I.
its generating systemn in liver microsomes (10,
L-Phenylalanine : an organ specific , stereo-
12). Of interest, is the fact also that the same specific inhibitor of human intestinal a!-
systems are present in intestinal homogenate kaline phosphatase. Nature 198: 685-686,
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The formnation of carboxvl-activat.ed L-amino agent in histochemical study of alkaline
acids leaves free itn amino group which can be
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9. GREEN, H. AND MEYERHOF, 0. Synthetic ac-
It is also muecessarv to recall that the transfer of tion of phosphatase. III. Transphosphoryia-
(ion with intestinal and senuen phosphatase.
lIhsosl)ha(e groups catalyzed by alkaline phos-
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1956.
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AdKNowLu:h)GMF;NTS
13. MEYERHOF 0. H. Synthetic
AND GREEN,ac-
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