[CANCER RESEARCH 47, 1598-1601, March 15, 1987]
Proteases Occurring in the Cell Membrane: A Possible Cell Receptor for the
Bowman-Birk Type of Protease Inhibitors1
Jonathan Yavelow,2 Michele Caggana, and Kenneth A. Beck3
Rider College, Department of Biology, Lawrenceville, New Jersey 08648
ABSTRACT
The legume-derived Bowman-Birk trypsin and chymotrypsin protease
inhibitors (BBI) are effective anticarcinogens in vivo and in vitro. The
chymotrypsin-inhibitory domain has been shown to be responsible for
this anticarcinogenic action. In this study we identify hydrolytic enzymes
by their ability to hydrolyze the relatively specific chymotrypsin substrate
succinyl-Ala-Ala-Pro-Phe-aminomethyl
coumarin. Results presented
in this study show: (a) there is an approximately 2-fold increase in the
activity of these enzyme(s) between normal and transformed C3H/10T1/?
cells; (b) there are five such enzymes associated with transformed cells
(separated by diethylaminoethyl-cellulose chromatography); (c) only two
of these enzymes are inhibited by BBI; (</)the BBI-inhibitable enzymes
are membrane associated; (e) the BBI-inhibitable enzymes are similar to
each other but different from pancreatic chymotrypsin. BBI has thus
distinguished a subpopulation of enzymes capable of hydrolyzing succinyl-Ala-Ala-Pro-Phe-aminomethyl
coumarin which may mediate the
transformation of C3H/10T1/} cells.
INTRODUCTION
The trypsin and chymotrypsin protease inhibitors, BBI,4 from
soybeans and other legumes are potentially nutritionally rele
vant anticarcinogens—particularly with respect to colon cancer
(1,2). The BBI are small (Mr 8,000 to 10,000), extensively
disulfide-bonded proteins which are heat and acid stable (3).
Metabolic studies suggest BBI is not digested but rather inter
acts with the colon wall as a biochemically active protein (4)
and is ultimately eliminated in the feces (5). Recent studies
have shown that: (a) nanomolar levels of BBI suppress in vitro
malignant transformation (6); (b) BBI is the only molecule
responsible for the anticarcinogenic activity of a crude extract
from acetone-defatted soybeans (6); (c) ingestion of this crude
extract protects animals from dimethylhydrazine-induced colon
tumors (7). It is thus of considerable interest to understand the
mechanism of anticarcinogenic action of BBI.
Protease inhibitors suppress in vitro transformation induced
by a variety of carcinogens (8-10). Thus, presumably there are
cellular functions regulated by proteolysis involved in the mech
anism of carcinogenesis (11). Protease inhibitor-mediated sup
pression of a number of phenomena, including c-myc gene
expression (12), sister chromatid exchange (13), receptor
expression (14), and release of cell-associated plasminogen
activator ( 15) has been reported. Unfortunately, however, little
is known concerning the protease(s) which may be directly or
indirectly regulating these events. The narrow spectrum of
proteases affected by BBI (as compared to other protease inhib
itors) enables it to be used as a tool for the isolation and
Received 8/6/86; revised 11/21/86; accepted 12/10/86.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by a grant from Research Corporation; publication costs were paid
by a grant from the National Cancer Institute (CA43565-01). This paper is part
I of a series.
2To whom requests for reprints should be addressed.
3 Recipient of salary support by NIH Grant CA 22704.
4 The abbreviations used are: BBI. Bowman-Birk inhibitors): TPCK, tosylphenylethyl chloromethyl ketone: ZPCK. ¿V-carbobenzoxyphenylalanine chloromethyl ketone; DFP, diisopropylfluorophosphate; AMC, aminomethyl coumarin;
DMSO. dimethyl sulfoxide.
purification of proteases relevant to the mechanism of
carcinogenesis.
Thus far it is known that the chymotrypsin-inhibitory domain
of BBI is responsible for suppression of in vitro malignant
transformation (6). For this reason we have begun to identify
proteases capable of hydrolyzing a specific chymotrypsin sub
strate that are associated with normal and transformed libro
blasts. In this report we identify a family of membrane-associ
ated enzymes of which only two are inhibited by BBI.
MATERIALS
AND METHODS
Materials. Cells used in this study were kindly provided by Dr. Ann
Kennedy (Harvard School of Public Health, Boston, MA); chymostatin
was kindly provided by Dr. Walter Troll (New York University School
of Medicine, New York); Eagle's basal medium, fetal bovine serum,
and gentamicin were purchased from Grand Island Biological Company
(Grand Island, NY); succinyl-Ala-Ala-Pro-Phe-aminomethylcoumarin was purchased from Enzyme Systems (Livermore, CA); DEAESephacel was from Pharmacia (Piscataway, NJ); protein assay reagent
was purchased from Bio-Rad (Richmond, CA); chymotrypsin was pur
chased from Worthington (Freehold, NJ); other chemicals were of the
highest available grade.
Methods. Cells were grown in 150-cnr flasks (Corning) using Eagle's
basal medium supplemented with 10% fetal calf serum and gentamicin
as previously described (6). Cell homogenates were routinely prepared
from 5 confluent 150-cm2 flasks. Cells were scraped with a sterile
plastic rake, washed twice with Hanks' balanced salt solution (contain
ing Ca2+ and Mg2+), and homogenized in 3 to 5 ml of homogenizing
medium (0.25 M sucrose-10 mM Tris-I mM CaCI2, pH 7.8). The extent
of homogenization was precisely 100 strokes using a Dounce-type
homogenizer. Ninety to 100% of the cells were broken as judged by
phase-contrast microscopy. Crude membranes were isolated, and
marker enzymes were assayed as previously described (16, 17). The
resolubilized membrane pellet was loaded onto a 0.9 x 5-cm column
containing DEAE-Sephacel. (This was accomplished by rehomogenizing the 100,000 x g pellet in column buffer, 250 strokes.) The column
was equilibrated with 25 mM Tris-25 mM glycylglycine-1 mM CaCb,
pH 7.8. After loading the sample, the column was washed thoroughly
and eluted with a linear NaCl gradient to l M over a total volume of
200 ml (5-ml fractions were collected). All operations were performed
at 4'C.
Enzyme activity was assessed using the synthetic fluorescent sub
strate succinyl-Ala-Ala-Pro-Phe-AMC
(18). Assays were performed
in a total volume of 1 ml with a maximum of 5% DMSO (final
concentration) in assay buffer (25 mM Tris-25 mM glycylglycine, pH
7.8, containing 1 mM CaCI2). All samples were tested at three different
concentrations of enzyme to prove turnover of substrate was dose
responsive. Substrate was prepared as a 20 mM stock in DMSO and
diluted to a final concentration of 0.2 or 0.4 mM. The amount of sample
assayed using postnuclear supernatants (1000 x g for 8 min) or cytosol
and crude membrane preparations ranged from 100 to 500 /ig of
protein. Assays performed on fractions after chromatography on
DEAE-Sephacel contained from 2 to 20 ^g of protein. Protein was
determined by the method of Bradford (19). Enzyme activity was
continuously monitored using a Perkin-Elrner spectrofluorometer (ex
citation, 383 nm; emission, 455 nm). The enzyme activities were linear
for up to 60 min. Enzyme was preincubated with inhibitor for 30 min
at 37°Cprior to adding the fluorescent substrate. The BBI was isolated
from defatted soy flour as previously described (20).
1598
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research.
BBI-INHIBITABLE
MEMBRANE-ASSOCIATED
RESULTS
Previous studies using BBI and chemical modifications of
BBI such that the inhibitor contains only chymotrypsin-inhibitory activity have proven the chymotrypsin-inhibitory
site is
capable of suppressing in vitro malignant transformation (6).
Other chymotrypsin inhibitors (e.g., chymotrypsin and TPCK)
also effectively suppress in vitro transformation (21). These
studies have led us to pursue the identification of chymotrypsinlike enzymes associated with mouse fibroblasts. Toward this
end we have utilized the synthetic chymotrypsin substrate succinyl-Ala-Ala-Pro-Phe-AMC.
The synthetic tetrapeptide substrate succinyl-Ala-Ala-ProPhe-AMC is slowly hydrolyzed by crude extracts of both nor
mal and transformed C3H/10T'/2 cells (Table 1). The substrate
is hydrolyzed approximately twice as rapidly in transformed as
compared to normal cell lines. This increase is observed in both
X-ray- and methylcholanthrene-induced
transformants, sug
gesting the increased activity is independent of carcinogen.
Quantitation of enzyme (protease) activity in crude extracts is
difficult, because synthetic substrates are competing with other
cellular proteins present in the samples.
The hydrolytic activity appears to be associated with a crude
membrane fraction subsequent to gentle homogenization and
subcellular fractionation (Table 2). Approximately 75% of the
hydrolytic activity from both normal and transformed cell lines
appears in the fraction enriched for ouabain-sensitive Na+, K+ATPase and cytochrome oxidase. (No attempt has been made
in this study to separate mitochondria from other cellular
membranes.) The percentage of distribution of láclatedehydrogenase suggests minimal contamination of the crude membrane
fraction with cytosolic components. Due to the higher enzyme
activity in methylcholanthrene-transformed
cells we have used
this cell line for initial purification studies.
In order to assess the number of enzymes capable of hydro-
lyzing the synthetic substrate we chromatographed the total
cellular homogenate or the crude membrane fraction on DEAESephacel (Fig. 1, A and B, respectively). Similar results have
been obtained from six separate experiments. Several chymotrypsin-like enzyme activities are routinely observed. The en
zyme activities range from approximately 5 to 800 nmol AMC
released/min/mg protein. The distribution of enzymes is simi
lar when a total cell homogenate is compared to an enriched
membrane fraction (A and B, respectively). Higher specific
activity values from crude membrane preparations reflect the
enrichment of enzyme in the membrane fraction (Table 2). This
suggests that most of the hydrolytic activity is membrane as
sociated or within membrane-bound organdÃ-es (e.g., lysosomes). Differences between the two chromatograms may also
reflect a portion of soluble enzymes absent from the crude
membrane fraction.
In order to assess which enzyme activities are relevant to the
anticarcinogenic mechanism of BBI, assays were performed in
the presence and absence of this protease inhibitor. Since BBI
also is a specific trypsin and chymotrypsin inhibitor these assays
also serve to assess the endopeptidase activity as opposed to
sequential exopeptidase activity which could also lead to the
liberation of AMC. Although multiple chymotrypsin-like en
zyme activities are detected, only two are inhibited by BBI
(Table 3, Panel A, Peaks I and V, and Panel B, Peaks II and
IV). The salt concentration where these BBI-inhibitable en
zymes elute is virtually identical (0.32 and 0.31 M for the "early
peak" and 0.59 and 0.57 M for the "late peak"). BBI-inhibitable
Peak I from Panel A may be artifactually low due to competing
protein present in the assay; however, in Panel B the activity of
the early peak is still considerably lower in activity than the
Table 1 Succinyl-Ala-Ala-Pro-Phe-AMC
Hydrolytic activity in normal and
transformed CÃŒHI10THcells
Cellular homogenates were centrifugea at 1000 x g, and supernatants were
assayed for hydrolytic activity.
pmol/min/mg
ENZYMES
100
us
WXXES/MIN
PERMG
PROTEIN
10
300
PROTEIN
200
protein
100
Experiment 1 Experiment 2
Normal C3H/10T'/!
37.7 ±4.3°
52.0 ±5.0
X-ray-transformed C3H/IOT'/2
76.3 ±6.1
92.0 ±5.0
Methylcholamhrene-lransformed
C3H/IOT1/;
82.6 ±6.3
142.0 ±7.0
" Mean ±SD of samples incubated at three different enzyme concentrations.
0
I 3 5 7 9 II 13 15 17 19 21 23 2527 2931 3335 3739 41 43 4547 19
FRACTION
NUMBER
PANEL B
Table 2 Specific activities and percentage of distribution of total activity of
marker enzymes and succinyl-Ala-Ala-Pro-Phe-AMC
hydrolase in subcellular
fractions
C3H/10T'/2 cells were gently homogenized, and the postnuclear supernatant
was fractionated into a 100,000 X # supernatant and pellet. Assays of marker
enzymes and succinyl-Ala-Ala-Pro-Phe-AMC
hydrolase were performed as
described in "Materials and Methods." The percentage of distribution of total
activity was calculated by multiplying specific activity values by total «ig
of protein
present in each fraction and then calculating percentages. Standard deviations in
all cases were less than 10%.
Supernatant
Pellet
Specific % of distri- Specific % of distriactivity
button
activity
bution
II 13 IS 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49
FRACTION
NUM6ER
Ouabain-sensitive
Na*,K*-ATPaseLáclate
Fig. 1. DEAE-Sephacel elution profile of a methylcholanthrene-transformed
dehydrogenaseCytochrome
cell lysate (A) and a crude membrane preparation (B). The total cell lysate was
oxidaseSuccinyl-Ala-Ala-Pro-Phe-AMChydrolase0.60°5.38*0.05C104.0^11.592.38.521.94.80°0.85*1.07f329.6''88.57.791.578.1
prepared by extensive homogenization (400 strokes), and a 20,000 x g supernatant
was applied to the column (.1). The crude membrane was prepared by limited
homogenization (100 strokes), and a resolubilized 100,000 x g pellet was applied
" Phosphate (fimol/min/^g protein).
to the column (/?). A NaCI gradient from 0 to l M was started at Fraction 10.
* AAj4o/min/ng protein.
Succinyl-Ala-Ala-Pro-Phe-AMC
hydrolytic activity is depicted as the solid bars.
r AAsM/min/jig protein.
Black dots reflect either micrograms protein (A) or A .»„
nm (B). Seven-hundred
d AMC (pmol/min/mg protein).
fifty ti] of each fraction were assayed in B as compared to 250 //I for A.
1599
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research.
BBI-INHIBITABLE
MEMBRANE-ASSOCIATED
ENZYMES
Table 3 Sensitivity of various succinyl-Ala-Ala-Pro-Phe-AMC
hydrolases from
Fig. 1 to BBI
Peaks I to V (from Fig. IA) denote Fractions 13, 14, 19, 25, 32, and 33; Peaks
1 to V (from Fig. IB) denote Fractions 14, 20, 24, 37, and 40. BBI was
preincubated with enzyme samples for 30 min prior to the addition of substrate.
for A and B the BBI concentrations used were 10 and 40 /<\t. respectively.
in this study prove: (a) there is an approximate 2-fold increase
in the activity of enzyme(s) capable of hydrolyzing the relatively
specific chymotrypsin substrate succinyl-Ala-Ala-Pro-PheAMC between normal and transformed C3H/10T'/2 cells; (b)
there are five such enzymes associated with transformed cells;
proteinPeakPanel
nmol/min/mg
(c) only two of these enzymes are inhibited by BBI; (d) the BBI(M)0.320.590.310.57Control3.903.8067.40362.70718.90151.1040.10530.10712.50260.70BBI2.85(73.1)°5.80(152.6)60.40
inhibitable enzymes are membrane associated; and (e) the BBIAIIIIIIIVVPanel
inhibitable enzymes are similar to each other but different and
mediate the transformation of C3H/10T'/2 cells. The large
(89.6)479.30(132.1)479.30 number of cells used for enzyme isolation most likely results in
the micromolar level of inhibitor needed in these studies as
(66.7)150.0(99.3)7.7(18.9)530.0(100.0)178.1
compared to nanomolar levels shown to be effective in trans
formation studies where fewer cells are present (6).
BIIIHIIVV[NaCI]
A chymostatin-sensitive enzyme activity has also been iden
tified in a crude membrane fraction from virally transformed
chick embryo fibroblasts (17). Interestingly, this enzyme me
(25.0)263.1
(100.9)
diates the release of cell-associated to extracellular plasminogen
°Numbers in parentheses, percentage of control.
activator. Although the BBI-sensitive enzymes described in this
study are also inhibited by chymostatin, other protease inhibi
Table 4 Sensitivity of each of two BBI-inhibitable hydrolases to various protease
tors differentially affect the two enzymes. More specifically,
inhibitors
BBI-inhibitable
Peaks 1 and 2 were separated using DEAE-Sephacel.
All
dithiothreitol, EDTA, and iodoacetamide do not inhibit the
enzyme assays were performed using the substrate succinyl-AIa-Ala-Pro-Phechick enzyme (17) but are effective inhibitors of the enzymes
AMC. Enzymes were preincubated with various inhibitors for 30 min prior to the
described in this study. However, because inhibitors were tested
addition of substrate. Numbers represent the average percentage of control values
using a crude extract from the chick cells as opposed to the
of samples incubated in triplicate.
partially purified samples in our study, further analysis com
controlAdditionDithiothreitolChymostatinZPCKDFPIodoacetamideEDTABBIConcentration5
% of
paring these two enzymes is needed. The similarities and dif
ferences between mouse- and chick-derived enzymes will be
166.744.433.361.444.472.580.052.034.7BBIPeak
254.345.736.236.254.354.385.357.422.7Chymotrypsin54.30.040.90.091.352.20.0ND"ND
established
only after the enzymes are purified from both
mM20
ng/ml200
sources and characterized.
Mg/ml1
Recently another BBI-inhibitable enzyme was identified in
mM10
C3H/10TVÌ
cells (22). This enzyme is a Val-Pro-Arg-AMC
mM10
mM10
hydrolase which is inhibited by BBI, chymostatin, and TPCK.
MM20
Clearly the chymotrypsin-like enzyme based upon inhibitor
pM40
studies departs considerably from pancreatic chymotrypsin in
MMBBIPeak
* ND, not determined.
this case. The cytosolic location of this enzyme, as well as the
different substrate hydrolyzed, suggests it is different from the
later peak, and protein levels are equivalent. The enzymes which two membrane-associated enzymes identified in this study.
are not inhibited by BBI are stimulated by thiols, and not Thus, there may be at least three fibroblast enzymes affected
inhibited by typical serine protease inhibitors, ZPCK and DFP by BBI.
The fact that BBI-inhibitable enzymes reside in a crude
(data not shown). These enzymes are therefore assumed to be
membrane fraction is consistent with the model that BBI exerts
nonserine proteases.
The two BBI-inhibitable enzymes have been compared to its action initially on a protease acting as a receptor in the
each other and to pancreatic chymotrypsin using a series of plasma membrane (6). Parallel studies have been performed
protease inhibitors (Table 4). A similar effect is observed for tracking the fate of fluorescently labeled BBI in transformed
the two fibroblast-derived enzymes regardless of the protease
fibroblasts (see accompanying paper). These studies reveal BBI
is taken up by cells in a time- and temperature-dependent
inhibitor used. BBI also inhibits the enzymes in a dose-respon
manner. It is tempting to speculate that BBI acts as a trigger
sive manner. Results of this experiment fall into three general
for internalization of the membrane-associated protease.
categories: (a) similar inhibition of the fibroblast chymotrypsin
and pancreatic chymotrypsin; (¿>)
lesser inhibition of the fibro
blast chymotrypsin than pancreatic chymotrypsin; and (c) ACKNOWLEDGMENTS
greater inhibition of the fibroblast chymotrypsin than pan
The authors appreciate helpful discussions with Dr. Ann Kennedy,
creatic chymotrypsin. More specifically, dithiothreitol, ZPCK,
Dr. Walter Troll, and Dr. Paul Billings.
and EDTA affect the hydrolytic activity of these enzymes
toward the synthetic substrate approximately in the same range.
Next, chymostatin, DFP, and BBI are much more potent inhib
REFERENCES
itors of pancreatic chymotrypsin than they are inhibitors of the
1. Troll, W. and Yavelow, J. Protease inhibitors in the diet as anticarcinogens.
fibroblast chymotrypsin enzyme. Finally, iodoacetamide inhib
In: D. A. Roe (ed.). Diet, Nutrition, and Cancer: From Basic Research to
its the fibroblast enzymes and not pancreatic chymotrypsin.
Policy Implications, pp. 167-176. New York: Alan R. liss. Inc., 1983.
2. Yavelow, J. Diet and cancer: role of legume-derived anticarcinogens. NJ
The significance of these differences will have to await purifi
Med., 83: 233-234, 1986.
cation of the BBI-sensitive enzymes from fibroblasts.
3. Birk, Y. The Bowman-Birk inhibitor. Int. J. Peptide Protein Res., 25: 113131, 1985.
4. Yavelow, J., Beck, K. A., Levitz, M., and Troll, W. In vitro effects of soybean
protease inhibitors. In: ). W. Finley and D. E. Schwass (eds.), Xenobiotic
Metabolism: Nutritional Effects, pp. 283-292. Washington, DC: American
Chemical Society, 1985.
DISCUSSION
The chymotrypsin-inhibitory domain of BBI effectively sup
presses in vitro malignant transformation (6). Results presented
1600
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research.
BBI-INHIBITABLE MEMBRANE-ASSOCIATED
5. Yavelow. J., Finlay, T. H.. Kennedy, A. R.. and Troll, W. Bowman-Birk
soybean protease inhibitor as an anticarcinogen. Cancer Res.. -Iy 2454s2459s. 1983.
6. Yavelow, J., Collins, M., Birk, Y., Troll. W.. and Kennedy, A. R. Nanomolar
concentrations of Bowman-Birk soybean protease inhibitor suppress X-rayinduced transformation in vitro. Proc. Nati. Acad. Sci. USA, 82:5395-5399,
1985.
7. Weed. H. G.. McGandy, R. B., and Kennedy. A. R. Protection against
dimethylhydrazine-induced adenomatous tumors of the mouse colon by
dietary addition of an extract of soybeans containing the Bowman-Birk
protease inhibitor. Carcinogenesis (Lond.), 6: 1239-1241. 1985.
8. Kennedy, A. R., and Little, J. B. Effects of protease inhibitors on radiation
transformation in vitro. Cancer Res.. 41: 2103-2108. 1981.
9. Geard, C. R., Rutledge-Freeman. M., Miller, R. C, and Borek, C. Antipain
and radiation effects on oncogenic transformation and sister chromatid
exchanges in Syrian hamster embryo and mouse C3H/10T'/¡ cells. Carcino
genesis (Lond.), 2: 1229-1233, 1981.
10. Anderson. K. B. Leupeptin inhibits retrovirus infection in mouse fibroblasts.
J. Virol., 48: 765-769. 1983.
11. Quigley. J. P. Proteolytic enzymes of normal and malignant cells. In: R. O.
Hynes (ed.). Surfaces of Normal and Malignant Cells, pp. 247-285. Sussex.
England: John Wiley and Sons. 1979.
12. Chang, J. D., Billings. P. C., and Kennedy, A. R. c-myc expression is reduced
in antipain-treated proliferating C3H/10T'A cells. Biochem. Biophys. Res.
Commun., 133: 830-835, 1985.
13. Kennedy. A. R.. Radner, B. S.. and Nagasawa. H. Protease inhibitors reduce
the frequency of spontaneous chromosome abnormalities in cells from pa
tients with Bloom's syndrome. Proc. Nati. Acad. Sci. USA, 81: 1827-1830.
ENZYMES
14. Baldassare, J. J., Bakshian, S.. Knipp. M. A., and Fisher, G. J. Inhibition of
fibrinogen receptor expression and serotonin release by leupcptin and antipain. J. Biol. Chem., 260: 10531-10535. 1985.
15. O'Donnell-Tormey, J., and Quigley, J. P. Inhibition of plasminogen activa-
16.
17.
18.
19.
20.
21.
22.
1984.
tory release from transformed chicken fibroblasts by a protease inhibitor.
Cell, 27:85-95, 1981.
Quigley. J. P. Association of a protease (plasminogen activator) with a specific
membrane fraction isolated from transformed cells. J. Cell Biol., 71: 472486, 1976.
O'Donnell-Tormey, J., and Quigley, J. P. Detection and partial characteri
zation of a chymostatin-sensitive endopeptidase in transformed fibroblasts.
Proc. Nati. Acad. Sci. USA, SO: 344-348, 1983.
Powers, J. C., Tanaka, T., Wade Harper, J. Minematsu. Y., Barker, L.,
Lincoln, D., Crumley, K. V., Fraki, J. E., Schechter, N. M., Lazarus, G. G.,
Nakajima, K., Nakashino, I... Neurath, H., and Woodbury, R. G. Mammalian
chymotrypsin-like enzymes. Comparative reactivities of rat mast cell pro
teases, human and dog skin chymases, and human cathepsin G with peptide
4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl
fluoride inhibitors. Biochemistry. 24: 2048-2058, 1985.
Bradford, M. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal.
Biochem., 72: 248-254, 1976.
Birk. Y., Gertler, A., and Khalef, S. A pure trypsin inhibitor from soya beans.
Biochem. J., 87: 281-284. 1963.
Kennedy, A. R. The conditions for the modification of radiation transfor
mation in vitro by a tumor promoter and protease inhibitors. Carcinogenesis
(Lond.), 6: 1441-1445, 1985.
Billings, P. C, and Kennedy, A. R. A protease activity in C3H/10T'/2 cells
which may be a target of the anticarcinogenic Bowman-Birk inhibitor. Proc.
Am. Assoc. Cancer Res., 27:485. 1986.
1601
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research.
Proteases Occurring in the Cell Membrane: A Possible Cell
Receptor for the Bowman-Birk Type of Protease Inhibitors
Jonathan Yavelow, Michele Caggana and Kenneth A. Beck
Cancer Res 1987;47:1598-1601.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/47/6/1598
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research.