Table Of ContentZOOLOGICAL SCIENCE 11: 221-227 (1994) 1994Zoological SocietyofJapan
Rapid and Quantitative Detection of Aspartic Proteinase in Animal
Tissues by Radio-labeled Pepstatin A
Masanori Mukai1 Toshihiko Kondo2 and Katsutoshi Yoshizato1'3
,
1Molecular Cell Science Laboratory, Department ofBiological Science, Faculty of
Science, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima-shi,
Hiroshima 724 and 2Department ofPhysical Biochemistry,
Institute ofEndocrinology, Gunma University,
Maebashi 371, Japan
ABSTRACT—Anewradio-derivativeofpepstatinAwasdevelopedandwasshowntobeusedasaprobeforrapidly and
quantitatively detecting aspartic proteinases in animal tissues. The carboxyl group ofpepstatin A was activated by the
water soluble carbodiimide, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and was then coupled with
N-hydroxysulfosuccinimide (Sulfo NHS). 35S]-Methionyl-pepstatin with a relatively high specific activity was obtained
by coupling the Sulfo NHS-pepstatin with[L-[35S]-methionine. Binding specificity of the pepstatin A derivative was
characterizedusingpepsinasatestenzyme. Bindingexperimentsshowedthattheradio-labeledpepstatincanbeusedas
aprobewhichbindsspecificallytoasparticproteinasesanddetectsthem. Theprobecoulddetectaslowas0.1 nmolesof
pepsin. Toknowwhethertheradio-labeledprobecanbeactuallyusedtodetectasparticproteinasesinanimaltissues, it
was applied to the tail tissue of metamorphosing amphibian tadpole which had been known to show high activity of
cathepsinD. Theassaydemonstratedmarkedincreaseinasparticproteinasesattheclimaxstage. Itwasconcludedthat
theradio-labeledpepstatinderivativedevelopedbythepresentstudyisusefulforquickandquantitativedeterminationof
pepstatin-reactive enzymes in animal tissues.
Chemical modification ofpepstatin hasbeen designed to
INTRODUCTION
utilize the inhibitor as an experimental probe which tightly
The aspartic proteinase is a group of proteinases (EC binds to the active site of aspartic proteinases and carries
3.4. group 23) which has an optimum pH at an acidic region. reporter groups [16]. Bimane-labeled pepstatin [21] and
The family contains pepsin, cathepsin D, cathepsin E, renin, dinitrophenyl-pepstatin [22] have been synthesized and used
chymosin andgastricsin. Amino acid sequencesofmamma- for the determination ofthe subcellular location ofcathepsin
lian aspartic proteinases and the corresponding nucleotide D in cultured human synovial cells. These methods of
sequences of their cDNA clones revealed that they have the modifications require relatively complicated techniques of
active center, which contains two aspartic residues and are organicchemistryandcontainmultiplestepsincludingtechni-
highlyconserved amongasparticproteinases [4, 8, 10, 12, 32, ques of column chromatography. Because both pepstatin
33, 35]. derivatives described above are detected by fluorescence,
Pepstatin A is produced by actinomyces [37] and is a these derivatives cannot be used as a probe in cases where
potent inhibitor of pepsin (Ki=4.5xlO-11 M), cathepsin D samples to be analyzed contain fluorescent materials.
(Ki=10_10M) and other aspartic proteinases [3, 27]. In the present study the authors aimed at developing a
Pepstatin A binds to the active center of the enzyme in a muchsimpleradio-labelingprocedureofpepstatintoobtaina
stoichiometric manner which is surrounded by two aspartic stable derivative. For this purposes, l-ethyl-3-(3-dimethyl-
moieties [3, 19, 37]. aminopropyl) carbodiimide hydrochloride (EDC) [42] and
Radio-labeled pepstatin derivatives have been chemical- N-hydroxy-sulfosuccinimide (Sulfo NHS) [2, 34] were
ly synthesized. Pepstatinwas coupled with 3H]-glycine and selected as activating agents since they are easy to handle
[
used for determining Kd values of a complex ofpepstatinyl- because oftheirhydrophilicity. The activatedpepstatinwas
[3H]-glycine-cathepsin D [15]. A radio-iodinated derivative coupled with L-[35S]-methionine which has longer halflife of
of pepstatin was prepared by introducing a tyrosine residue decay than 125I.
whichwas theniodinatedwith 125I. Thiswasutilizedforthe In the amphibian metamorphosis, the larval tail tissue is
determination of Kd of pepsin-pepstatin-[125I]-monoiodo- subjecttothehistolysisbyseveralproteinasessuchascollage-
tyrosine methyl ester complex [41]. However, 3H-labeled nase [11, 25] and cathepsin D [18, 29, 38]. This phe-
compoundischemicallyunstablebecausetritiumisexchange- nomenon was utilized to see validity and usefulness of the
able with surrounding hydrogen. 125I-Labeling has dis- radio-labeledpepstatinderivativefordetectingproteinasesin
advantages because ofits short half life of radioactivity. animal tissues. The present study succeeded in demonstrat-
ingthattheradio-labeledpepstatinisausefulprobeforquick
Accepted March 11, 1994 and quantitative detection of aspartic proteinases in animal
Received January 27, 1994 tissues.
222 M. MUKAI, T. KONDO AND K. YOSHIZATO
MATERIALS AND METHODS buffer, pH5.0, containing 1% bovine serum albumin (BSA) for 10
min on ice for protecting nonspecific binding of pepstatin A to the
membranes. Then, theblockingbufferwasreplacedwith2mlof20
MaterPieaplsstatin A was purchased from Peptide Institute, Inc. (Osa- mM sodium acetate buffer, pH5.0, which contained radio-labeled
ka, Japan). EDC and Sulfo NHS were obtained from Pierce pepstatinA([35S]-methionyl-pepstatinA, 104-106cpm/assay). The
(Rockford, IL). L-[35S]-methionine was from New England Nuc- membranes were incubated for additional 30min on ice. Mem-
lMeAaSrS()B.ostoPne,psiMnA)wa.s aSEprPo-dPucAtKofC1W8awkaosPfurroemChWeamtiecrasls(M(iOlsfaokrad,, bacreatnaetsewbeufrfeert,hepnHw5a.s0,hetdor4etmiomvees orandiioce-lwaibtehle2dmpleposfta2t0inmAMthsaotdihuamd
Japan). All other reagents were of analytical grade. Tadpoles of not bound to pepsin, and were dried and placed in 5ml of a
bullfrog, Rana catesbeiana, were purchased from a local animal scintillation cocktail (Atomlight, New England Nuclear, Boston,
supplier. TheywerestagedasdescribedbyTaylorandKollros(TK MA) to count radioactivities using a liquid scintillation counter
(LSC-3000, Aloka). Nonspecific bindingofradio-labeledpepstatin
stage) [36]. A to the nitrocellulose membrane was estimated through the identi-
Coupling reaction cal procedure described above with an exception that membranes
mM
Pepstatin A (0.7mg, 1//moles) was dissolved in 200//l of were washed with neutral buffer (20 sodium phosphate buffer,
dimethylsulfoxide. SulfoNHS (10//moles) andEDC(100//moles) pH7.4).
were dissolved in 800//lof 16mMsodiumphosphate buffer, pH7.4,
containing20%dimethylsulfoxide. Theseweremixedtogetherand Binding of [35S]-methionyl-pepstatin to the tail tissue of bullfrog
were incubated at room temperature (ca. 20°C) for 1 hr. The tadpole
product ofthis reaction, Sulfo NHS-pepstatin, was diluted with 4ml The tail oftadpole ofbullfrog, Rana catesbeiana, was used as a
of20mM sodium phosphate buffer, pH7.4, and was applied slowly source of aspartic proteinases for the binding assay of [35S]-
to a SEP-PAK C18 column (1 drop/sec) using a syringe. The methionyl-pepstatin A. Tailswere dissectedfrom bullfrogtadpoles
column was washed twice with 5ml of 20mM sodium phosphate at thepremetamorphicstage (TKstage XVII) ortheclimaxstage of
buffer, pH7.4, for removing excess amounts of activating agents metamorphosis(TKstageXXIII)andkeptat —20°Cuntiluse. The
(Sulfo NHS and EDC). Sulfo NHS-pepstatin was eluted from the frozen tail tissues were defrosted on ice and finely minced to small
column twice with2ml ofdimethyl sulfoxide, mixed with4ml of 10 pieces (about 3Mx3x3mm3) atm0M-4°C with scissors in the solution
mMsodiumphosphatebuffer,pH7.4,containingL-[35S]-methionine containing 0.1 NaCl and 20 sodium acetate buffer, pH5.0.
(lOOpmoles, 4.1 MBq, 1.6xl08cpm) and was incubated at room Tissue pieces were collected in microfuge tubemsM(Ca. 100mg wet
temperature (ca. 20°C) for 2hr. The reaction mixture in which weight tissue/tube) aMnd suspended in 1 mlof20 sodium acetate
35S]-methionyl-pepstatin was produced was diluted with deionized buffercontaining0.1 NaClat0-4°C. Theradio-labeledpepstatin
w[ater and loaded on a SEP-PAK C18 column in the same way as A (2.0xl04cpm/assay) was added to the tissue suspension and
described above. The column was washed twice with 10ml of allowed to stand for binding at 0-4°C for 30min. For removal of
deionized water. This step separated the labeled pepstatin from unbound pepstatin probe, tubes were then centrifuged at l,700xg
uncoupled L-[35S]-methionine and other by-products which did not for5min at4°Candthe supernatants were discarded. The precipi-
mM
mcoevtahlieonntylly-pebpisntdatitno(0p.e7p2stMaBtiqn,A2..8XT1h0e7cpelmu/a4temlcodnitmaeitnhiynlgsu[l3f5oSx]-- ctaotnetsaiwneirnegw0.a1shMedNa3Ctli,mepsHwi5t.0h.1 mTlheonf,20forrecsoovdeiriunmgarcaedtiaot-elabbueflfeedr
ide) was kept at —20°C before use. pepstatin A bound totissues, precipitateswere lysed in 600//lof0.1
NNaOHat37°Cfor2hrandwerecentrifugedat7,000Xgfor 10min
Separation of i5S]-methionylpepstatin Afromfreepepstatin A at room temperature. Radioactivity in the supernatant was
taine[d35Se]x-ceMsesth[aiomnoyulntpsepsotfatpienpsptraetpianreAd.asFdreesecrpiebpesdtaatbionveAstwilalscorne-- tchoeunptreodc.eduTrheedepsrcortiebiendcboyncLeonwtrryateitoanl.oufsilnygsaBteSsAwaassadsettaenrdmairnded[20b].y
moved mainly by HPLC as follows. The preparation was 4-fold
diluted with deionized water and loadedon a SEP-PAK C18column RESULTS
which was then eluted with acetonitrile. The eluate containing
pepstatin A and its 35S]-methionyl derivative was evaporated to Thecarboxylgroupofpepstatin Awasactivatedby EDC
[
drynessat40°Cin vacuoandwasdissolvedinanappropriateamount to form o-acylurea which was then coupled with Sulfo NHS.
ofdimethyl sulfoxide. The concentrated radio-labeled pepstatin A Sulfo NHS-pepstatin thus obtained was more resistant to
was subjected to reversed phase high-performance liquid chroma- hydrolysis than o-acylurea and was hydrophobic. Excess
tography (HPLC, Inertsil 300-C8, 4.6x100mm, Gasukuro Kogyo amounts of hydrophilic activating reagents were easily re-
Inc. Tokyo, Japan) and eluted at 50°C with a linear gradient of moved from the reaction mixture using a reversed phase
acetonitrile (from 25 to 55%) containing 0.1% trifluoroacetic acid column ofSEP-PAK C Coupling ofthe activated pepsta-
(byTF2A2)0.nmThfeorfpleopwstraatteinwaAsa0.n4dmbly/mtihne.radTihoeacteilvuiattyefwoarstmheonliatboerleedd tin A with the amino g]r8o.up of L-[35S]-methionine produced
one. Radioactive fractions were collected and concentrated by [3?S]-methionyl-pepstatin A with specific activity of 0.72
evaporation. MBq///mole (2.8X107cpm///mole).
Specificity of the radio-labeled pepstatin A was charac-
Binding assays terized by analyzing its binding properties to pepsin that had
Appropriate amounts of pepsin (10-440ftg/20/A HiO/mem- been immobilizedon a nitrocellulose membrane. The radio-
brane) were spotted on nitrocellulose membranes (1.5x1.5cm2). labeled pepstatin was bound to the enzyme at pH 5.0, but not
Membranes were dried and incubated in 20mM sodium acetate at pH 7.4, same as pepstatin A (Fig. 1). The binding of
Radio-labeled Pepstatin A 223
[35S]-methionyl-pepstatin A to pepsin is proportional to pep-
6- sin concentrations in the range of 1-12nmoles. Radio-
labeledpepstatinAboundincreasinglytothefixedamountof
/\
/ pepsin as the amount of labeled pepstatin increased up to
5 pH5.0 around the dose of 2X104cpm and then the binding was
A
^x leveled off (Fig. 2), suggesting that the labeled pepstatin
binds to limited and specific sites of pepsin.
•E£• 4 if Binding of 35S]-methionyl-pepstatin A to pepsin was
tQO. //£$ competitively sup[pressed by pepstatin A (Fig. 3), indicating
^
X^^ 3 /// that the radio-labeled derivative shows the same binding
"CD / specificity as pepstatin A.
3 y^ i
n o */
•
/
1 o
C^O pH7.4 '
n i i 1 1 112 15 EQO.
Pepsin (nmoles/assay)
Fig p1.epsipnH.-DeTpheendreandtio-blianbdeilnegd odfer[i35vaSt]i-vmeesthi(o3n.y6lX-lpe0p4stcaptmi/naAssayt)o co3
m
were incubated with various amounts of pepsin (0.7-14.6
nmoles) that had been dotted on nitrocellulose membranes.
For removing unbound pepstatin A probes, membranes were
washed with 20mM sodium acetate buffer, pH5.0, (circle) or
with20mM sodium phosphate buffer, pH7.4, (square). Each
point represents the mean oftriplicate assays and bars indicate
standard errors ofthe mean.
Concentration of pepstatin A /xM)
(
Fig. 3. Suppression ofthe bindingofthe radio-labeledpepstatin A
topepsinbypepstatinA. Pepsin(5.7nmoles/assay)spottedon
membraneswasincubatedwiththefixedamountofthepepstatin
derivatives (8.8X103cpm/assay) andvaried amountsofpepsta-
tin A (0-48juM) as a competitor. The radioactivity bound to
pepsin was counted. Each point represents the mean oftripli-
cate assays with its standard error indicated by a bar.
Figure 1 shows that the 35S]-methionyl-pepstatin A pre-
[
pared as above can detect pepsin when its amount is more
than 1.0nmoles. To get the probe for aspartic proteinases
showing a higher specific radioactivity, the labeled pepstatin
A was further purified by subjecting it to reversed phase
HPLC (Fig. 4). Pepstatin A was eluted at 38% acetonitrile
as a single peak. The radio-labeled pepstatin A was eluted
at 44% acetonitrile also as a single peak. The pepstatin
5 10 derivative thus purified had a specific activity of 3.6MBq/
Concentration of labeled pepst4atin A //mole and could detect as low as 0.1 nmoles of pepsin. Its
(X10 cpm)
bindingtopepsinwasproportionaltotheconcentrationofthe
Fig. 2. Binding of 35S]-methionyl-pepstatin Ato the fixed amount enzyme in the range of0.1-0.3nmoles (Fig. 5).
of pepsin. Pep[sin (5.7nmoles) was immobilized on a nitro- Theradio-labeledpepstatinAwastriedtouseasaprobe
caemloluulnotssemofemlabbrealendep(e1p.s5tXati1n.5Acm(24).a4xnd10i3n-c8u.ba8tXed10w4ictphmi/nadsiscaayt)e.d to detect aspartic proteinases in the animal tissue. The
Radioactivity bound to membranes was counted as amounts of metamorphosing tadpole tail was quantitated for the pepsta-
labeled pepstatin A bound to pepsin. The radioactivity of 103 tin-reactive enzymes (Fig. 6). The specific binding of the
cpmcorrespondsto35pmolesofthepepstatin Aprobe. Each probe to the tail of a metamorphosing tadpole was 18-fold
point represents the mean of triplicate assays. Bars indicate higher than the binding to the tail of a premetamorphic
standard errors ofthe mean. animal. Ifwe postulate that all the bound probe recovered
224 M. MUKAI, T. KONDO AND K. YOSHIZATO
••- --
"
DMSO I 75
I
0.7 labeled pepstatlnA
~
0.6 pepstatinA ,--,-x'103cpm 50
,-'v ^ 4
0.5 \ _,-**" I
oEc 0„.4 ivoity 25 £co
CM 2.2
CN o
0.3 <
+(*0 ._', ^} i|\\ tr -
g 0.2
c H,
nCO J j °\
S 0.1
w
n
I
10 20 30 40 50
Retention time (min)
Fig. 4. Separationof[35S]-methionyl-pepstatinAandpepstatinAbyreversedphaseHPLC . Theradio-labeledpepstatinA
fraction obtained at the second reaction was applied to a Inertsil 300-C8 (4.6x100mm) and eluted at 50°Cwith alinear
gradient of25-55% of acetonitrile (dotted line) containing 0.1% TFA. The flow rate was 0.4ml/min. The solid line
indicates the absorbance at 220nm. The radioactivityofeach fraction (0.4ml) wascounted (circle). The first peak of
220nm containsdimethyl sulfoxide whichwasused asthe solventofpepstatinA. Theclosedarrow at38% acetonitrile
showsthepeakofpepstatinAandtheopenarrowat44% acetonitrilethepeakof 35S]-methionyl-pepstatin. Theshaded
[
fractions were collected as 35S]-methionyl pepstatin and used as aprobe for aspartic proteinases (3.6 MBq/^mole).
[
4
£ 3 -
*0o*)
oc) a.
ScoE _
2
o
£L
II
O
(A) TX- 1
I II I
XVII XXIII
TK stage of tadpole
Fig. 6. Detection ofaspartic proteinase in the tailoftadpole bythe
35S]-methionyl-pepstatin A. The specific bindingofthe probe
[
Pepsin (nmoles/assay) wascalculated bysubtractingthevalueofradioactivityobtained
Fig. 5. Binding of the 35S]-methionyl-pepstatin A to pepsin. in the presence ofexcess amounts ofpepstatin A (30^M) from
HPLC-purified radio-l[abeled pepstatin A (104cpm/assay) was that without it. Tail tissues of bullfrog tadpole (about 100mg
incubated for 30min at 0-4°C with nitrocellulose membranes wet weight/assay) at premetamorphosis (TK stage XVII) orthe
containingvariedamountsofpepsin. Membraneswerewashed climaxofmetamorphosis (TKstage XXIII)were incubatedwith
with acidic buffer and counted for radioactivity (circle). Non- the radio-labeled pepstatminMA (HPLC-purified, 2.0xl04cpmM/
specificbindingtonitrocellulosemembraneswasobtainedasthe assay) in solution of 20 sodium acetate buffer and 0.1
radioactivity remaining on membranes when they were washed NaCl, pH5.0, with or without a competitor, pepstatin A (30
with neutral pH (square). The radioactivity of 103cpm is ,«M). The radioactivity of 103cpm is equivalent to 7.1 pmoles
equivalent to 7.1 pmolcsofthe pepstatin A probe. Each point of the pepstatin probe as in Fig. 5. Each value represents the
represents the mean of triplicate assays and bars indicate stan- mean of triplicate assays with its standard error indicated by a
dard errors of the mean. bar.
Radio-labeled Pepstatin A 225
from tissues, the tail contained bindingsitesof0.7nmoles/10 wastherelativelylowcapacityofnitrocellulosemembranesto
mgproteinat theclimaxstage and0.04nmoles/10mgprotein hold tissue proteins. We first tried to assay enzymes in the
at the premetamorphic stage. These values ofbinding sites tissue samples using nitrocellulose membranes as we did for
are expressed as those for "pepsin equivalent". the test enzyme. However, reliable and reproducible values
A
of binding of the radio-labeled pepstatin could not be
obtained in the membrane assay.
DISCUSSION
This probe is shown to detect pepstatin-sensitive pro-
PepstatinAisaproteinaseinhibitorthat isnotspecificto teinase^) in the tail of bullfrog tadpole. The amount of
one species of enzyme but shows the activity toward several specific bindingto the tail tissue muchincreases at the climax
species ofenzymes grouped as the aspartic proteinase family stageofmetamorphosis (TKstage XXIII) ascomparedtothe
including pepsin, cathepsin D, cathepsin E, renin, chymosin premetamorphic one. Several reports demonstrated that
and gastricsin. Therefore, 35S-]-methionyl-pepstatin A de- cathepsin D like proteinase activityin the tail increasesat the
[
veloped in the present study can be applied to animal tissue climax ofmetamorphosis [9, 18, 29, 38, 39, 43] andpepstatin
forthefirstscreeningofproteinaseswhichisgroupedintothis sensitive proteinase plays important roles in the regression of
A
family. tail during metamorphosis [31]. Pepstatin bindingsitesat
The radio-labeled pepstatin A keepsthe same activity as the climax stage of metamorphosis increases 18-fold as com-
pepstatin A. We used the carboxyl group ofpepstatin A as pared to those at the premetamorphic stage. The ratio of
D
the site of modification, as pepstatin-conjugated resins have cathepsin activity of the tail at the climax stage to that at
been usually prepared by modifying this residue and utilized the premetamorphic stage is 18 in Rana catesbeiana [29] and
for purification of aspartic proteinases such as cathepsin D 16inXenopuslaevis[39]. Thesevaluesareveryclosetothat
[1]. Thepresentstudyalso confirmsthatthe carboxylgroup obtained by our method.
ofpepstatin can be modified as a connectingsite forreporter Pepstatin-sensitive proteinases are found also in a wide
groups without destroying properties which are required for varietyoflife includingvertebratessuch ashuman [5], bovine
the inhibitor to bind aspartic proteinases. Radioactive [30],porcine [14],rabbit[6],rat [10],chicken [5,26],frog[23]
methionine can be successfully introduced by this modifica- andfish [7], and invertebratessuch asmarine mussel [24] and
tion into pepstatin A. hemipteran insect [13]. Plant [28], bacteria [17] and retro-
Since this additional chain of methionine is neutral in virus [40] have also been reported to contain pepstatin-
charge, it does not disturb the ionic condition ofthe binding sensitiveproteinases. Therefore, itis consideredthat aspar-
site of aspartic proteinases. The 35S]-methionyl-pepstatin tic proteinases might play fundamental roles in metabolic
[
Awe prepared is more hydrophobic than pepstatin A, which processes ofvarieties of life. However, the information on
may help the derivative to approach more easily for the the enzyme other than mammalian origin has been poor.
A
bindingsite ofthe enzymes thanpepstatin because the site There is a possibility that the binding site recognized by
is thought as a hydrophobic pocket in pepsin [27]. pepstatin A is highly conservedin the molecular evolution of
Pepstatin A binds to aspartic proteinases in a asparticproteinases. Thepepstatinderivative reportedhere
stoichiometric manner. The present study shows that the is expected to be useful in detecting unknown aspartic
binding of the radio-labeled pepstatin A to pepsin is pro- proteinasesinthewiderangeoflifeandstudyingthemfroma
portionaltoconcentrationsofpepsin, indicatingthatitcanbe comparative point ofview.
used as a sensitive probe for the quantitative analysis of It has been shown that aspartic proteinase of HIV virus
aspartic proteinases. Utilizing this probe, we could detect can be converted by the gene technology into a mutant
0.1 nmoles of pepsin. It seems that other methods such as enzyme that has no enzymatic activity but can bind to
radio-immuno assay are more sensitive than the method pepstatin A [40]. This indicates that the catalytic site ofthe
described in the present study. However, the radio-labeled enzyme is different from the pepstatin binding site, although
pepstatin A is much useful as compared to the antibody both sites are in the active center. The radio-active pepsta-
againstasparticproteinases. Wehavetopreparethespecific tin developed in the present study is expected to be useful in
antibody in each case of the study against the enzyme of detecting enzymes of the aspartic proteinase family that lose
A
target. In contrast, the radio-labeled pepstatin can be their catalytic activity.
used for detecting the enzyme belonging to the family of We could not develop pepstatin A derivative which
aspartic proteinases. shows higher sensivity of detection than the method to
We made some alterations in the assay ofbinding ofthe directly measure the enzyme activity or conventional im-
pepstatin A derivative when we tried to detect aspartic munological detection method using specific antibody. It
proteinases in tadpole tissues. Binding was assayed on remains as a future study to develop a method to prepare
nitrocellulose membranes for thetestenzyme (pepsin), while 35S]-methionyl-pepstatin A with much more higher specific
[
the assay for the tissue sample was done in tissue pieces activity.
suspended in acid solution. The pepstatin A derivative
bound to enzymes in the tissue was then removed by centri-
fugationinalkalinecondition. Thereasonforthisalteration
226 M. MUKAI, T. KONDO AND K. YOSHIZATO
19 Kunimoto S, Aoyagi T, Nisizawa R, Komai T, Takeuchi T,
ACKNOWLEDGMENTS Umezawa H (1974) Mechanism of inhibition of pepsin by
pepstatin II. J Antibiot 27: 413-418
T. OhTohekaauatnhdorSs.wTooumlidnloikefotrotehxepirresksintdhediirstchusasnikosnstooDnrst.hiSs.sYtausdyu.gi, 20 PLroowtreyinOmHe,asuRroesmeebnrtouwgihthNJth,eFfaorlrinApLh,enoRlanrdeaalglentR.J J(19B5i1o)l
Chem 193: 265-275
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