ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 173, 347-354 (1976)

Estrogen-Dependent Trypsin-Like Activity in the Rat Uterus

Localization of Activity in the 12,000g Pellet and Nucleus’

JOSEPH KATZ, WALTER TROLL, MILTON LEVY, KAREN FILKINS, JOSEPH RUSSO, AND MORTIMER LEVITZ2

The Departments ofobstetrics and Gynecology and Environmental Medicine, New York University School of Medicine, New York, New York 10016

Received August 4, 1975

Direct evidence was obtained for the presence of hormone-stimulated trypsin-like protease activity in the rat uterus. Ovariectomized rats were either untreated (U), treated with estradiol (E), or estradiol plus progesterone (EP). The uteri were excised and subcellular fractions were prepared. Each fraction was assayed for protease activity using protamine as substrate, the cleavage products being quantitated fluorometrically following reaction with 4-phenylspiro[furan-2(3H),l’-phthalanl-3,3’dione (Fluram). Fractions from U rats yielded negative results, whereas the 12,000g pellets and nuclei from the uteri of E and EP rats exhibited appreciable activities. No significant increase in protease activity was observed in thymus and diaphragm following hormone treat- ment, indicating organ specificity. The enzyme (or enzymes) from the 12,000g pellet was solubilized and some characteristics were determined. The apparent K, is about 1.0 x

1Omfi M, the temperature optimum is about 44°C and maximum velocity is achieved in the alkaline range (pH - 8.5). The protease is a plasminogen activator and is inhibited by diisopropyl fluorophosphate, Antipain, and Leupeptin. These properties resemble those of trypsin.

A recent publication from this labora- tory presented indirect evidence for the presence of hormone-stimulated trypsin- like proteolytic activity in rat uterine chromatin. In ovariectomized rats treated with estradiol or estradiol plus progester- one the enzyme activity was reflected in the polyacrylamide gel electrophoresis profiles of uterine histones which showed extensive breakdown of the lysine-rich band provided chromatin was incubated at 37°C prior to extraction of the histones (2). Protamine, added to the incubation mix-

’ This investigation was supported by Grant M74.80 from The Population Council, New York, N.Y., and Grants CA 02071 and ES 00260 from the United States Public Health Service. A preliminary report has appeared (1).

2 To whom all correspondence should be ad- dressed: Dr. Mortimer Levitz, New York University Medical Center, 550 First Avenue, New York, N.Y. 10016.

ture prevented histone breakdown, pre- sumably by acting as a competitive sub- strate (3).

A new sensitive assay for trypsin-like proteases has been developed using prota- mine as the substrate (4). The liberated amino groups are reacted with 4-phenyl- spiro[furan-2(3H),l’-phthalanl-3,3’-dione (Fluram) and the adduct is quantitated fluorometrically (5). The method was ap- plied to the analysis of subcellular frac- tions of rat uterus in different hormonal states. The results presented in this paper indicate that the protease activity ex- pressed is indeed hormonally controlled

3 Common names and abbreviations used: Fluram, 4-phenylspiro [furam-2(3H), l’-phthalanl- 3,3’dione; Antipain, L(s)-1-carboxy-2-phenylethyl] carbamoyl-L-arginyl-L-valyl-L-argininal; Leupeptin, acetyl-L-leucyl-L-leucyl-L-argininal; U, untreated; E, estradiol-treated; EP, estradiol plus progesterone treated; PF, protamine-Fluram assay.

347 Copyright Q 1976 by Academic Press All rights of reproduction in any form reserved.

348 KATZ ET AL.

and is localized in the 12,000g granules as well as in the nucleus. A second aspect of the paper concentrates on the solubiliza- tion and on some of the properties of the granule-associated enzyme.

MATERIALS AND METHODS

Animals. Young adult female rats (CDcR’) weigh- ing 200-250 g were obtained from Charles River Laboratories, Inc. Two weeks following ovariectomy they were divided into three groups, each receiving subcutaneous injections at 10 AM and 4 PM daily for 4 days. The first group (I-7) received only 0.2 ml of sesame oil. The second (E) was treated with 0.3 pg of estradiol in 0.2 ml of oil. The third group (EP) re- ceived the same estradiol regimen but 3 mg of pro- gesterone were added to the injection medium on Days 2,3, and 4. On the fifth day uteri were excised and pooled according to hormone treatment. In one experiment E rats were treated as described above except that rats were sacrificed following 1, 2,3, or 4 days of hormone treatment.

Materials. Protamine chloride was obtained from Sigma and succinylated (6). Fluram was bought from Fisher and diisopropyl fluorophosphate from Aldrich. Human plasminogen, plasmin, and uroki- nase were generously donated by Dr. Alan Johnson. Antipain and Leupeptin were kindly donated by Dr. T. Sugimura, Tokyo, Japan.

Preparation of subcellular fractions. Extranuclear fractions. With all operations at 0-4”C, rat uterine samples (either pooled or separate) were homoge- nized by hand (25 strokes in a Dounce homogenizer) in 0.25 M sucrose solution (3 ml/uterus). The result- ing homogenates were centrifuged for 20 min at SOOg and the resulting pellets were resuspended in 0.25 M

sucrose (0.4 ml/uterus) and saved for use in the protamine-Fluram (PF) assay. In some studies the washes were tested for enzyme activities. In other studies the supernatant fractions from the 12,000g centrifugation were centrifuged at 100,OOOg. The re- sulting microsomal and soluble fractions were sub- mitted to the PF assay.

Nuclei. Three types of nuclear preparations, A, B, and C, were tested for protease activity in the PF assay. A was a crude nuclear fraction prepared by washing the 600g pellet described above twice and resuspending it in 0.25 M sucrose. B and C were purified nuclear fractions prepared as described by Sepherd et al. (7) and Szego et al. (8), respectively. Both nuclear preparations were purified by high speed centrifugation in 2.4 M sucrose. The major difference was in the mode of homogenization. In C the uteri were homogenized by hand in the Dounce apparatus (9), whereas in B homogenization was achieved in a Polytron PT 10 (Brinkmann, Great

Neck, N.Y.). The nuclei were suspended in 0.25 M

sucrose (2 uteri/ml). Small aliquots of nuclei were assayed for DNA (10).

The protamine-Fluram (PF) assay. In a typical assay 0.36 ml of 0.1 M phosphate buffer, pH 8.5, to which 100 pg protamine and 20 ~1 of the test solu- tion or suspended pellet were added, was incubated at 37°C for 0, 2 or 4 h. Then 0.6 ml of 0.2 M borate buffer, pH 8.0, was added. While vigorously shaking this mixture on a vortex shaker, 0.3 ml of a 1% solution of Fluram in dry acetone was added. The sample was centrifuged and the fluorescence deter- mined in a Farrand Ratio Fluorometer equipped with Corning filter 7-51 in the exciting beam and Corning tilters 3-72 and 4-70 in the secondary light path. Assays were performed in duplicate. In paral- lel incubations the test substance or protamine was withheld from the medium. The data are usually reported as fluorescence units/4 h/100 pg DNA, per- mitting normalization of protease values in extra- nuclear fractions to the approximate cell number.

Assay for plasminogen activation. The PF assay was adapted to the measurement of plasminogen activation as outlined by Kessner et al. (11). These authors kindly provided the details of the assay prior to publication. The assay was carried out in two stages. First, a mixture of 0.07 CTA units of plasminogen, the test substance (usually 25% of that used for the direct PF assay) and 0.3 ml of 0.1 M

phosphate buffer, pH 8.5, was incubated for 1 h at 37°C. In the second stage, designed to measure the released plasmin, 100 pg of succinylated protamine in 0.06 ml of the same buffer were added and the incubation was continued for 0,2, or 4 h. The sample was then submitted to the standard PF assay as described in the preceding section. Several controls were run in which the test substance was either omitted or replaced by 0.05 units of urokinase or 0.01 casein units of plasmin.

Solubilization of the protease in the 12,000g gran- ule. The PF assay showed the protease activity to be concentrated in the 12,000g extranuclear granules and the nuclei. In attempts to solubilize the granule- localized enzyme, aliquots of the pellet were sus- pended in 1 ml of 0.25 M sucrose and subjected to the following conditions: (1) freeze-thaw in dry ice (three times) in the presence of 0.1% Triton X-100; (2) soni- cation for 1 min at 0°C using the sonifier cell disrup- ter Model W 140 (Branson Sonic Power Co.) at con- trol setting 3; (3) shearing in a Polytron, PT 10 on setting 3 for intervals totaling 1 min at 0°C; (4) incubation at 37°C for 0.5, 1, or 2 h. After each procedure the mixture was centrifuged at 12,000g for 20 min at 0°C and the resulting supernate and pellet were assayed by the PF method. In one experiment the 12,OOOg pellet from E rats was lysed for 2 h at 37°C and the mixture was centrifuged at 50,OOOg.

ESTROGEN-DEPENDENT RAT UTERINE PROTEASE 349

The resulting supernate and pellet were assayed for protease activity. The relative efficiencies of solubi- lization were assessed.

Enzyme properties. Several properties of the en- zyme from E and EP rats were studied. The enzyme was solubilized by autolysis at 37°C for 1 h. In each experiment the assay was run in the usual way except that one of the conditions was varied. To study substrate dependence, the protamine concen- tration was varied from 5 to 100 pg/incubation. In other studies the temperature was varied from 25 to 60°C. In a third series of experiments the pH was varied between 4 and 10.

Inhibition studies. The influence of Antipain and Leupeptin on protease derived from estradiol- treated rata was examined. The assay was run in the customary way except that the final volume was 0.38 ml and the enzyme was exposed to 5-160 pg of inhibitor prior to the addition of the protamine sub- strate. The incubations were run for 4 h. The data are given as percent inhibition. In another inhibi- tion study the influence of diisopropyl fluorophos- phate on the protease activity was determined in the following way. The solubilized enzyme was preincu- bated in the absence or presence of 1 x lo-* M diisopropyl fluorophosphate at pH 8.0 for 1 hat 37°C. Then the sample was submitted to the PF assay and the results with and without inhibitor were com- pared. Suspensions of nuclei were also submitted to the diisopropyl fluorophosphate inhibition test.

RESULTS

Protease Activity in Rat Uteri. Comparison with Other Tissues

Subcellular fractions prepared from uterine homogenates of hormone-treated ovariectomized rats were assayed for pro- tease activity by the protamine-Fluram (PF) method. Hormone-dependent activity was demonstrable in the 12,000g pellet. As shown in Table I, there is virtually no activity in the U rats. There is considera- ble activity in the E animals and slightly less in the EP rats. In two studies a fourth group of rats were treated with progester- one without estradiol. The results were similar to those obtained with the U rats. No activities were detectable in the 100,OOOg pellets or supernatant fractions in any rats.

In two experiments crude and purified nuclei were submitted to the PF assay. The results in Table II indicate apprecia- ble activities in specimens derived from the hormone-treated rats but virtually

none was detected in uterine nuclei ob- tained from the untreated animals.

To determine the course of onset of pro- tease activity, rats were treated with es- tradiol for shorter times than usual and uterine extracts were assayed. The data in Table III show that the response is not acute. More than 1 day of treatment is required for the expression of enzyme ac- tivity in the 12,000g pellet and an addi- tional day is required for appearance of the activity in the nucleus.

In one experiment the enzyme activity in the uterus was compared to those in diaphragm and thymus. The results in Ta- ble IV indicate that only the uterus ex- hibited a significant response to hormone treatment. The hormone induction thus, is organ specific. Curiously, this is the only experiment in which the value for the uterine extract from EP rat exceeded that of the E rat. Whereas the difference in the protease concentration in the extranuclear fraction between the E and EP rat uterus appears to be statistically significant (Ta- ble I), the physiological importance of this observation is obscure.

Solubilization of Protease Activity

The 12,000g uterine pellets from U, E, and EP rats were submitted to several con- ditions designed to solubilize the enzyme

TABLE I

PROTEA~E ACTIVITY IN THE 12,000~ PELLETS OF HOMOGENATES OF UTERI FROM HORMONE-TREATED

OVARIECTOMIZED RATS”

Experi- Hormone treatment ment

Untreated Estradiol Estradiol + progesterone

1 3.00 33 16 2 2.5 19 13 3 5.0 30 20 4 2.5 30 19 5 0.3 18 10 6 1.6 22 9

a In each experiment the 12,000g pellets from three to five uteri were pooled and suspended in 0.25 M sucrose (0.4 ml/uterus).

b Values are in fluorescence units per 4 h normal- ized to 100 pg of tissue DNA. Each assay was done in duplicate and the average deviations were invaria- bly less than 10% for the hormone-treated rata.

350 KATZ ET AL.

TABLE II

PROTEASE ACTIVITY IN UTERINE NUCLEIC FROM HORMONE-TREATED OVARIECTOMIZED RATS

Experi- Hormone treatment ment

Untreated Estradiol Estradiol + progesterone

Nuclear preparations

A B C A B C A B C

1 l.Ob 1.1 0.8 7.9 9.0 7.2 14.6 18.4 12.3 2 1.0 1.0 1.3 13.9 10.8 9.1 11.8 13.4 9.8

’ Nuclei preparation A (crude) and B (purified according to Shepherd et al., 7) were from the same animal, one horn being used for each preparation. Nuclei C (purified, according to Szego et al., 8) were obtained from different animals. In each experiment three to four uteri or horns were pooled.

’ Values are in fluorescence units per 4 h (not normalized as in Table I). The suspended nuclei were assayed for DNA and each incubation tube contained about 15 pg DNA. Assays were done in duplicate, the average deviations being about 10%. About 1-2 ng of trypsin afford the values shown for the hormone- treated animals.

TABLE III

TIME COURSE OF ONSET OF PROTEASE ACTIVITY IN ESTRADIOL-TREATED OVARIECTOMIZED RAT@

E treat- Uterine ment weight (days) (mg)

Protease activity

Nuclei 12,000g pellet

Ob 80 0.0’ 1.1 1 172 0.0 0.5 2 195 1.9 10.5 3 260 9.8 13.0 4 238 19.2 21.2

a Following treatment with estradiol (El for 1, 2, 3, or 4 days, uteri were excised and homogenized. The 12,OOOg pellets and nuclei were analyzed for protease activities.

b Treated with sesame oil only. c Values are in fluorescence units per 4 h normal-

ized to 100 pg DNA and are averages of three uteri. The standard errors were less than 10%. Values less than 4-5 are indistinguishable from zero.

(or enzymes). These included mechanical destruction, the combined application of thermal shock and detergent action, and autolysis. The results are shown in Table V. Shearing released only 5% of the pro- tease from the granules. Greater solubili- zation was achieved by sonication, but the results were variable. Freeze-thaw cycles in the presence of Triton X-100 caused the solubilization of about 40 and 25%, respec- tively, of the protease activity contained in the pellets from E and EP rats. Incubation of the 12,OOOg pellet at 37°C for 1 h consist- ently solubilized about 50% of the activity.

This procedure was adopted for the routine solubilization of the enzyme except that in some instances the incubation time was extended to 2 h, resulting in slightly higher yield. All the procedures described in Table V were also applied to uterine extracts from U rats. In no case was pro- tease detected in either the soluble frac- tion or the pellet.

Properties of the Protease

Some of the properties of the crude solu- bilized enzyme are shown in Fig. 1. It is apparent that under the conditions of the assay nearly maximum reaction velocity is achieved at a concentration of 50 pg of protamine/incubation, one-half the con- centration routinely used in the assay. The

TABLE IV

PROTEOLYTIC ACTIVITY IN 12,000G PELLETS OF TISSUE HOMOGENATES FROM HORMONE-TREATED

OVARIECTOMIZED RATS

Tissue Hormone treatment

Untreated Estradiol Estradiol +

progester- one

Uterus 0.0” 33.6 41.5 Thymus 0.4 0.7 1.0 Diaphragm 3.3 8.0 4.4

D Values are in fluorescence units (per 100 pg of tissue DNA per 4 h) as determined by the prota- mine-Fluram method.

ESTROGEN-DEPENDENT RAT UTERINE PRGTEASE 351

TABLE V

COMPARISON OF METHODS FOR SOLUBILIZING PROTEASE IN THE 12,000~ EXTRANUCLEAR PELLETS FROM RAT UTERI IN DIFFERENT HORMONAL STATES’

Method Hormonal state

Estradiol Estradiol + progesterone

Pellet Soluble Pellet Soluble

None 100 100 Shearing 45 k lib 525 39 + 13 52-5 Sonication 26 f 7 34 + 14 37 + 20 15 + 13 Freeze-thaw in Triton X-100 36 * 13 38 + 8 69 c 30 21 t 9 Incubate 1 h at 37°C 37 t I 51 ? 3 60 2 20 47 k 19

n Details of the procedures for solubilization are in the text. b Values are in percent of activity in the nontreated pellet which is taken as 100. All assays are by the

protamine-Fluram assay. Averages are of two to four determinations.

pH Optimum

1

7 .-‘“, : 1 : /’ _,’ 4 6 8 10 PH

FIG. 1. Properties of the rat uterine protease. Ovariectomized rats were treated with estradiol or estradiol plus progesterone. The uteri were excised and subcellular fractions prepared. The 12,000g pel- let was lysed for 2 h at 37°C and the soluble superna- tant submitted to the protamine-Fluram (PF) assay as described in the text except that in each study one condition was varied. The influences of substrate concentration, temperature, and pH on the fluores- cence yields are shown. The arrows in the third panel indicate the pH at the start and end of the incubation. The PF assay is constrained to buffers containing no primary amines or borate in the incu- bation medium. Rather than change buffers, phos- phate, a poor buffer in the high pH ranges was used throughout.

apparent K, calculated from this and simi- lar studies is 1.0 x lop6 M and 1.1 x lo-” M for the proteases, respectively, from E and EP rat uteri. Figure 1 also shows that maximum velocity is achieved by carrying out the incubation at about 44°C. Finally, the third panel in Fig. 1 indicates that optimum rates are obtained at alkaline PI-I.

The data in Table VI indicate that the protease is an effective plasminogen acti- vator. Preincubation of the protease in the presence of plasminogen resulted in ap- proximately fivefold amplification of en- zyme activity. Plasminogen activation is also hormone-dependent as indicated by lack of activity in the uterine extracts from untreated rats.

TABLE VI

PLASMINOGEN ACTIVATION BY PROTEASE FROM 12,000~ PELLETS OF RAT UTERINE HOMOGENATES~

Experi- Hormone Protease activity .- mentb treatment

- Plas- + Plas- minogen minogen

1 None 0.0’ 0.3 E 5.0 13.8 EP 2.2 10.2

2 None 0.0 1.6 E 5.3 15.5 EP 1.6 12.6

3 None 0.1 0.0 E 2.7 8.8 EP 0.3 9.2

0 Ovariectomized rats were either untreated or treated with estradiol (E) or with estradiol + proges- terone (EP) as described in the text. The extranu- clear 12,000g pellets were lysed at 37°C for 1 h and the soluble supernates were submitted to the PF as- say with or without prior incubation with plasmino- gen as described in the text.

b In each experiment analyses were done on ex- tracts of two to three rat uteri, pooled according to hormonal treatment.

c The values are in fluorescence units/4 h.

352 KATZ ET AL.

Inhibition Studies

Antipain and Leupeptin were tested for their ability to inhibit the enzyme ex- tracted from E rat uterus. The data shown in Table VII indicate that they are both effective inhibitors. Diisopropyl fluoro- phosphate inhibited the hormonally in- duced rat uterine protease of the 12,OOOg pellet (Table VIII) and nucleus (results not shown) with equal effectiveness.

DISCUSSION

The sensitive method of Brown et al. (4) was adapted for the assay of trypsin-like protease activity in subcellular fractions of uterine homogenates. The method de- pends on the hydrolysis of protamine and the assay of the newly formed amino groups with Fluram. Under the conditions described in this paper, 1 ng of trypsin is readily detected, the precision is good, the blanks are negligible, and the fluorescence yield increases in reproducibly regular fashion for at least 4 h (Fig. 2). The inter- experiment variance is quite small for a biological study of this kind in which pre- cise control was exercised only over the injection regimen and the time, postooph- erectomy, of utilization of animals. Fur- thermore, some 50 assays can be per- formed on extracts of a single rat uterus and they can be handled routinely by one technician. Exploiting this novel method, a dramatic increase in estradiol-mediated

TABLE VII

EFFECT OF ANTIPAIN AND LEIJPEPTIN ON THE PROTEA~E EXTRACTED FROM UTERI OF ESTRADIOL-

TREATED OVARIECTOMIZED RATS

Inhibitor added” (CLB)

Percent of initial activity

Antipain Leupeptin

0 100 100 5 32* 63

10 71 58 20 56 35 40 48 30

160 23 16

p The protamine-Fluram assay was run as de- scribed in the text with the addition of the inhibitors in amounts indicated. The molecular weights are: Antipain, 696; Leupeptin (acetyl form), 481.

b The estimatedKi is about 1 x 10e5 M.

TABLE VIII

INHIBITION OF HORMONALLY-INDUCED RAT UTERINE PROTEASE ACTIVITY BY DIISSOPROPYL

FLIJOROPHOSPHATE (DFPY

Experi- Hormone treatment!’ Protease activity’ ment

-DFP +DFP

Estradiol 12.0 0.4 E&radio1 + progester- 9.5 0.1

one E&radio1 20.0 0.2 E&radio1 + progester- 8.5 0.3

one

a In each experiment the 12,OOOg pellets from three pooled uteri were lysed for 1 h at 37”C, centri- fuged, and an aliquot of the supernate was analyzed for protease activity. The test sample was preincu- bated for 1 h at pH 8.5 in the absence or presence of DFP (1 x 1O-2 M) at 37°C. Then the solutions were submitted to the protamine-Fluram assay as de- scribed in the text.

b Ovariectomized rats were treated with estradiol or estradiol plus progesterone as described in the text.

r The values are in fluorescence units/4 h.

14

0 L 1 05 10 1.5 2.0 25 30 35 40

Time (hr)

FIG. 2. Typical assay of rat uterine protease by the protamine-Fluram method. The protease was obtained by lysing the 12,000g extranuclear pellet obtained from estradiol-treated rats. The curve shows the averages of assays done in duplicate.

proteolytic activity was demonstrated. Moreover, the activity is specific to the uterus.

Although the application of gentle tech- niques does not preclude the translocation of macromolecules during homogeniza- tion; it is a reasonable supposition that in the hormone-treated rat the uterine pro-

ESTROGEN-DEPENDENT RAT UTERINE PROTEASE 353

tease is concentrated in the nucleus. This view is reinforced by the time-course study (Table III) in which after 2 days of estra- diol treatment, protease was found in the 12 ,OOOg extranuclear granule, but not in the nucleus. After another day of hormone administration, protease activity was de- tected in both subcellular fractions. The protease activity in the nuclei was inde- pendent of the purity of the preparations (Table II), suggesting that the enzyme is intimately associated with the nuclear matrix rather than being loosely bound to the membrane.

The extranuclear granule harboring the protease activity merits elucidation. Mito- chondria, lysosomes, plasma membrane fragments, and less-defined particles sedi- ment at 12,000g. Ordinarily, mitochondria are not associated with trypsin-like en- zymes , whereas lysosomes concentrate acid proteases (12) and to a lesser extent neutral proteases (13). A resolution of the problem may result from the fractionation of the 12,000g sediment followed by com- parisons of concentrations of well-estab- lished marker enzymes for each of the sub- cellular fractions with those of the pro- tease.

Studies are underway to purify and characterize the enzyme more completely. Nevertheless, the properties exhibited by the crude preparations are stongly sugges- tive of the enzyme type. The high pH opti- mum (8.591, the preference for basic amino acid peptides (arginine comprises more than 50% of the amino acid residues in protamine), and the effective inhibition by diisopropyl fluorophosphate indicate the presence of a serine trypsin-like en- zyme (14). Plasminogen activation is also associated with cleavage of arginine pep- tides by serine protease (15).

The protease is present during the es- trus cycle of the rat and mouse (unpub- lished results) and in the normal human uterus (16), but its biological role has not been clarified. The enzyme possesses the capability of degrading histones (2) and of enhancing RNA synthesis (9). These phe- nomena may be related but our studies do not permit quantifications relative to pro- tease activity. Uterine nuclei from the EP

rat exhibited far greater histone break- down and enhancement of RNA synthesis than those seen in the E rat, yet the pro- tease activity exhibited by the EP rat nu- clei was only slightly greater than in E rats (Table II). Perhaps progesterone ad- ministration to the estradiol-primed rat produces changes in the physical structure of chromatin, rendering it susceptible to derepression by proteases or other agents. Investigations along these lines are being pursued.

The ability of the protease to act as a plasminogen activator affords a mecha- nism for the amplification of activity. High concentrations of plasminogen activators have been found in endocrine organs (17, 18) and are associated with experimental tumors (19). Like trypsin (20), the protease described in this paper is inhibited by An- tipain and Leupeptin. These low molecu- lar weight inhibitors are excreted, in part, unmetabolized after ingestion by experi- mental animals, and have been shown to delay the onset of chemically-induced tu- mors (21, 22). If the trypsin-like protease activity described in this paper is essential for the expression of hormone action in the uterus, Antipain and Leupeptin may be useful agents to alter the normal or abnor- mal physiological functions of that impor- tant target organ.

REFERENCES

1. KATZ, J., TROLL, W., LEVY, M., Russo, J., FILK- INS, K., AND LEVITZ, M. (1974) Fed. Proc. 33, 1511.

2. LEVITZ, M., KATZ, J., KRONE, P., PROCHOROFF, N. N., ANDTROLL, W. (1974)EndocrinoZogy 94, 633-640.

3. KATZ, J., TROLL, W., Russo, J., FILKINS, K., AND LEVITZ, M. (1974) Endocrine Res. Commun. 1, 331-337.

4. BROWN, F., FREEDMAN, M. L., AND TROLL, W. (1973) Biochem. Biophys. Res. Commun. 53, 75-81.

5. UDENFRIEND, S., STEIN, S., BOHLEN, P., AND DAIRMAN, W. (1972) Science 178, 871-872.

6. KLOTZ, I. M. (1967) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. XI (Hirs, H. W., ed.), pp. 576-580, Aca- demic Press, New York.

7. SHEPHERD, R. E., HUFF, K., AND MCGUIRE, W. L. (1974) Endocrine Res. Commun. 1, 73-85.

8. SZEGO, C. M., RAKICH, D. R., SEELER, B. J., AND

354 KATZ ET AL.

GROSS, R. S. (1974) Endocrinology 95, 863-874. 9. KATZ, J., LEVITZ, M., GOR~TEIN, F., AND TROLL,

W. (1970) Endocrinology 87, 294-301. 10. BURTON, K. (1956) Biochem. J. 62, 315-323. 11. KESSNER, A., ONG, E. B., AND TROLL, W. (1975)

Fed. Proc. 34, 532. 12. DE DWE, C., PRESSMAN, B. C., GIANETTO, R.,

WATTIAX, R., AND APPELMANS, F. (1955) Bio- &em. J. 60, 604-617.

13. DAVIES, P., RITA, G. A., KRAKAUER, K., AND WEISSMANN, G. (1971) Biochem. J. 123, 559- 569.

14. FAHRNEY, D. E., AND GOLD, A. M. (1963) J. Amer. Chem. Sot. 85, 997-1000.

15. SUMMARIA, L., HSIEH, B., AND ROBBINS, K. C. (1967) J. Biol. Chem. 242, 4279-4283.

16. NOTIDES, A. C., HAMILTON, D. E., AND RUDOLPH, J. H. (1973) Endocrinology 93, 210-216.

17. ALBRECHTSEN, 0. K. (1956) Actu Endocrinol. 23, 207-218.

18. ASTRUP, T. (1966) Fed. Proc. 25, 42-51. 19. UNKELESS, J., DAN@, K., KELLERMAN, G. M.,

AND REICH, E. (1974) J. Biol. Chem. 249,4295- 4305.

20. SUDA, H., AOYAGI, T., HAMADA, M., TAKEUCHI, T., AND UMEZAWA, H. (1972) J. Antibiotics 25, 263-266.

21. TROLL, W., KLASSEN, K. A., AND JANOFF, A. (1970) Science 169, 1211-1213.

22. UMEZAWA, H. (1972) Enzyme Inhibitors of Mi- crobial Origin, pp. 15-52, University Park Press, Baltimore.