Properties of an estrogen-induced hydrolytic enzyme from mouse uterus

0022-4731/83 $3.00+0.00

J. steroid Biochrctl.Vol. 19, No. I. pp. 743-749. 1983 Printed in Great Britain. All rights reserved

Copyright %, 1983Pergamon Press Ltd

25. Sex Steroid Induced Proteins

PROPERTIES OF AN ESTROGEN-INDUCED HYDROLYTIC ENZYME FROM MOUSE UTERUS* TAOMASH. FINLAY, JOSEPH KATZ, SUSAN KADNER and MORTIMERLEVITZ Department of Obstetrics and Gynecology, New York University Medical Center. New York, NY 10016, U.S.A. SUMMARY The purification and properties of an estradiol-sensitive hydrolytic activity from mouse uterus which fits several criteria for being an induced protein are described. The activity in the uteri of immature animals can be stimulated 2-4-fold by estradiol to that approaching the adult level. Stimulation is blocked by puromycin. The enzyme which we have designated hydrolase II, was purified approx. 400-fold to apparent homogeneity by chromatography on Affigei Blue, DEAE-~eiiuiose and octyi-Sepharos~. Hydrolase II is a single chain polypeptide with an estimated mol. wt = 65,000 daitons and has an N-terminal serine residue. A variety of N-blocked L-amino acid nitrophenyl esters are cleaved by the enzyme. K,‘s at pH 7.2 were all approx. 40 pm. Of substrates tested, phenylaianine nitrophenyl ester had the highest I’,,,,,. Cbz-fi-alanine nitrophenyl ester, which is not a normal protease substrate was cleaved with a K, of 145 PM. The enzyme had no detectable activity against peptide nitroanilide substrates for trypsin-, chymotrypsin- or elastase-like enzymes. It is inhibited by ZPCK and DIFP but not by TLCK and Ala-Ala-Pro-Ala chloromethyi ketone, a potent inhibitor of eiastase-like enzymes. Mouse plasma protein protease inhibitors were without effect as was SBTI. Our results rule out hydrolase II being a carnosinase, non-serine esterase, plasminogen activator, collagenase or coiiagenase activator and suggest that it is a chymotrypsin-like protease.

During the course of the above studies we found that the 28,OOOg supernatant from mouse uterine homogenates also contained an activity which could cleave /I-aianine nitrophenyl ester (/I-Ala-NPE). This activity (hydrolase II) was also stimulated by estradiol in immature animals. In this communication we describe the isolation, purification and properties of hydrolase II and show that although hydrolase I and II have many features in common they are distinctly different enzymes.

Administration of estrogens to the hormone-deficient female rodent results in the synthesis of specific estrogen-induced proteins (IPs). In the rat these are detectable 3@45 min after estrogen administration and 3 h prior to the general stimulation of uterine protein synthesis [I, 23. It was initially proposed that a unique “key” IP was responsible for triggering a subsequent cascade of uterine transcriptional events [3]. However, demonstration of the existence of at least three different IPs in the rat uterus has cast doubts on the “key” IP hypothesis [4]. Recently, two of the rat uterine induced proteins, IP-1 and IP-2 have been identified as creatine kinase and as a nonspecific enolase respectively [S], Several years ago we reported the isolation and partial purification of a hydrolytic activity from mouse uterine homogenates which fit the criteria for being an IP [S]. In immature animals an increase in activity was detectable 30 min after estrogen administration and activity doubled after 2 h. Although the natural substrate for this enzyme was not identified, the hydrolase (hydrolase I) cleaved peptide aminomethyl coumarins and was inhibited by standard serine protease inhibitors. Interestingly, the physical properties of hydrolase I appeared to be similar to those reported for IP-3 [4], the third of the rat uterine IPs, and a protein for which no function has been reported.

EXPERIMENTAL Materials Amino acid nitrophenyl esters, diisopropyl flurophosphate (DIFP), tosyl lysyl chloromethyl ketone (TLCK) and soybean trypsin inhibitor were from Sigma. N-Cbz phenylalanyl chloromethy1 ketone, tetrapeptide nitroanilide protease substrates and t-BooAla-Ala-Pro-Phe-aminomethyl coumarin (AlaAla-Pro-Phe-AMC) were obtained from Vega Biochemicals. t-Boc-Ala-Ala-Pro-Ala-AMC and acetylAla-Ala-Pro-Val-chloromethyl ketone were gifts from Dr M. Zimmerman (Merck, Sharp & Dohme Research Labs) and Dr J. C. Powers (Georgia Institute of Technology) respectively. Radiochemicals were obtained from New England Nuclear. Na”‘I (carrierfree) was from Amersham-Searle. [ 1-3H]-DIFP was from New England Nuclear. Octyl-Sepharose was prepared as previously described [7]. All other chemicals were of the highest purity commercially available.

*This work was supported in part by NIH Grant CA-02071 from the National Cancer Institute. 743

744

‘rtiOM.AS

H. FI\I.*\

Female Swiss mice were obtained from Blue Spruce (Alta Mont, NY) either as weanlings (3-4 weeks old) or as young adults (9-12 weeks old). Animals were caged with controlled light-dark cycles and had free access to food and water. In experiments where the effects of estradiol on the activity of uterine enzymes were tested. animals received a single injection of estradiol in 0.2 ml 0.15 M saline containing I”,, ethanol at various intervals before sacrifice. Control animals received saline alone. After sacrifice uteri and or other organs were dissected and homogenized in 2 ml 0.25 M sucrose/‘uterus. centrifuged for 30 min at 2X,000 0 and the supernatant collected. All operations were conducted at 0-4’C Protein concentration of the supernatant fractions was determined with Fluram using bovine serum albumin as standard [X].

CI rf/

0

T

12

6

8

4

4

x

2 40

60

80

%

b

80 60 40 i

Peptide-AMC hydrolase activity was determined from the change in fluorescence at 460nm due to release of 7-amino-4-methyl coumarin (AMC) as previously described [63. Amino acid nitrophenyl ester hydrolase activity was determined from the change in absorbance at 405 nm as a function of time. Incubations were carried out at room temperature and contained 0.3 mM substrate, generally P-Ala-NPE, in 0.02 M sodium phsophate buffer, pH 7.2 with 5% DMSO. Nitrophenyl esterase activity was calculated from the initial slopes of the recorder tracings. Spontaneous hydrolysis of the nitrophenyl esters. usually insigni~~~~nt under the assay conditions, was corrected for by subtracting the absorbance changes determined in the absence of enzyme.

Fig. I. Chromatography of hydrolase II on octyl-Sepharose. (A) Combined fraction from DEAE-Cellulose containing protein-bound [“HI-DIP and P-Ala-NPE &erase activity (5.0 ml. 3.42 x IO“ c.p.m.:ml. 27.5 units/ml) were loaded on to a 0.9 x 30cm column of octyl-Sepharose equilibrated with 0.1 M Tris--Cl, pH 7.4. After a wash with 40ml of equilibrating buffer the column was eluted with a O-30% gradient of dioxane. (B) Fractions containing protein-bound C3H)-DIP and &Ala-NPE esterase from the above column (A) were combined and concentrated to 5 ml by ultra~itration. Half was treated with fresh [“HI-DIP and then chromatogr~~phed on Sephadex G-25. Treated and untreated halves were combined and 5 ml (2.62 x IO” c.p.m./ml, 3.4 it/ml) was chromatographed on octyl Sepharose as described in (A).

llteri from 30 to 40. young adult mice were excised and homogenized in 0.25 M sucrose (2 ml/uteri). The homogenate was centrifuged at 105.000 8 for 90 min and the pellet discarded. A portion of the supernatant (Z&250,;) was treated with lO-3 M [“HI-DIFP to inactivate and covalently label the enzyme with diisopropyl phosphate (DIP). After chromatography on Sephadex G-25. the labeled hydrolase was recombined with the remaining unlabeled material. Albumin was removed by passage through a 1.5 x 25 cm column of Affigel Blue equilibrated with 0.02 M potassium phosphate, pH 7.4. Hydrolase containing fractions were loaded onto a 0.9 x 30cm column of DEAE-cellulose equilibrated with 0.05 M Tris-Cl. 0.05 M NaCl, pH 7.4. After a wash with equilibrating buffer, the column was eluted with a linear gradient of NaCl. Hydrolase II (as D-Ala-NPE esterase activity) and protein bound C3H]-DIP eiuted as a single sharp peak with 0.2 M NaCI. The final step in the purification procedure was hydrophobic chromatography. Hydrolase II containing fractions from the DEAE-ceIlulose column were loaded onto 0.9 x 30cm column of octyt-Sepharose

equilibrated with 0.1 M Tris-Cl. pH 7.4. After a preliminary wash, the column was eluted with a linear gradient of from 0 to 30”/, dioxane (Fig. IA). The majority of the protein (as AZsO) passed through the column with the wash. Protein-bound t3H]-DIP and p-Ala-NPE esterase activity eluted with 20-25% dioxane. It was routinely observed that bound C3H]-DIP eluted at a slightly higher dioxane concentration than did esterase activity. The difference in elution was attributed to an increase in hydrophobicity resulting from conversion of the active-site serine hydroxyl to the less polar diisopropyl phosphate ester. To show that this was the case the following experiment was conducted. All fractions from the octyl-Sepharose column containing either esterase or radioactivity were combined. Half was inactivated with [“HI-DIFP as described above. After chromatography on Sephadex G-25, the inactive and active halves were combined and rechromatographed on the octylSepharose column under essentially identical conditions as in Fig. 1A. No change in the elution pattern was observed (Fig. IS) except for the increase in the radioactivity peak and decrease in the esterase activity peak.

40

8’0

FRACTION NUMBER

745

Estrogen-induced uterine hydrolase

A summary of the purification procedure appears in Table 1. Purification was approx. 30&400-fold over the 105,000 g supernatant with a yield of 15-400/, whether enzymatic activity or bound [‘HI-DIP was used as a basis for comparison. SDS-gel electrophoresis in the presence of mercaptoethanol (data not shown) showed a single protein staining and 3H-containing band with an estimated mol. wt of 65,000. A single protein and radioactive peak with an estimated mol. wt of 65,000 was also seen for the native protein on HPLC gel-exclusion chromatography using a TSK-3000 column. N-Terminal analysis with dansyl chloride using reverse phase HPLC for identification of the DNS-amino acid [9] showed serine ( > 9504) to be the only N-terminal amino acid. The above experiments provided additional evidence for homogeneity and indicated that the hydrolase has only a single peptide chain. RESULTS

Hydrolase I and hpdrolase II are djferetrt enzymes

In earlier studies we had demonstrated the presence of an estrogen-induced activity in mouse uterine homogenates capable of hydrolyzing Ala-Ala-Pro-Alaand Ala-Ala-Pro-Phe-AMC [6]. During the course of the experiments reported on here, we found that the uterine homogenate also contained a /I-Ala-NPE hydrolase activity. PreIim~nary ex~riments suggested that the peptide-AMC and &Ala-NPE hydrolase activities did not copurify. Indeed, highly purified preparations of the B-Ala-NPE hydrolase were entirely without detectable peptide-AMC hydrolase activity, Two explanations for this observation were considered: either the uterus contained two distinct enzymes which did not copurify or else it contained a single enzyme with two activities one of which was more sensitive to denaturation than the other. To distinguish between these two possibilities, we examined the effects of a series of protease and esterase inhibitors on the peptide-AMC and the /I-Ala-NPE hydrolase activities in the uterine homogenate supernatant (Table 2). The results suggested the presence of two different enzymes. Both hydrolases in the homogenate were inhibited by DIFP and ZPCK. Neither was inhibited by standard non-serine esterase inhibitors such as sodium fluoride or bis nitrophenylphosphate or by ethyl acetate. These data were consistent with both enzymes being chymotrypsin-like serine esterases. However, while hydrolase I (peptide-AMC hydrolase) was inhibited by leupeptin and antipain, hydrolase II (P-Ala-NPE esterase) was not. Further confirmation for the existence of two enzymes comes from a study of the heat stability of the hydrolase activities in uterine homogenates (Table 3). p-Ala-NPE esterase activity (hydrolase II) was found to be stable to heating at 50°C for 10 min while Ala-Ala-Pro-Ala-AMC hydrolase activity (hydrolase I) lost 50% of its initial activity within 2min. Moreover, when the hydrolase II preparation at various

Table 2. ERect of various Inhibitors on the hydrolysis of alanine nltrophcn~ I cater\ and Ala-Alit-Pro-Ala-AMC by mouse uterine homogenate ~j’*,Acti+ Inllibitor /jAla-NPE Ala-Ala-Pro-Ala-AlAC No additions DIFP (IO-‘M) NaF (1Om3M) Ethylacetate (10 3 M) Ris-(p-nitrophenyl) phosphate (IO- 3 M) ZPCK (IO-” M) TLCK (10m3 M) Acetyl-Ala-Ala-Pro-Val-CH,Cl (lo-” M) Antipain (19. ’ M) Leupeptin (IO ‘M)

100

100

5.9 141.2

7.3 I00

I 00 I 11.x

IO0 100 0

15.6

IO0

loif

3x I00

21.5 I.:!

I00

21

--

Uterine homogenate supernatant, prepared as described in the text (50 /II) was incubated for 5 min with the inhibitors at the concentration indicated in 0.1 M sodium phosphate. pH i.4. With the chloromethyl ketones. the buffer also contained Xl”, DMSO which was required for solubility. This concentration of DMSO had no apparent effect on esterase or Ala-Ala-Pro-Ala-AMC hydroiase activity. Aliquots from the reaction then were used to determine hvdrolase activity as described in the rxperimentill section.

stages OF purification was examined for its ability to cleave /?-Ala-NPE, Ala-Ala-Pro-Ala-AMC or AlaAla-Pro-Phe-AMC (Table 3), activity against the peptide-AMC substrates was lost while nitrophenyl esterase activity increased. The most compelling argument for the existence of two distinct uterine hydrolases comes from an ex~~mjnation of the time-course of their stimulation by estradioi (Fig. 2). As we reported earlier, hydrolasc I shows two peaks of activity at 2 and 6 h [6]. Hydrolase II, on the other hand, shows a single peak of activity 4 h after estrogen administration. These results clearly show that the two hydrolases differ in their chemical and biological properties and also in their response to stimulation by estradiol, Stimulation

of hydrolasr

tamoxifen (alone and in combin~ltion with estradioi) on the stimulation of hydrolase 11 activity in weanling animals was examined (Fig. 3). Of the three estrogens tested, estradiol was most effective in stimulating hydrolase II activity. Progesterone (3 ,ug/aminal) and testosterone (3 @g/animal) had little stimulatory effect. Tamoxifen when administered with estradiol. reduced estradiol-mediated stimulation by approx. 400;,.

Since we had already demonstrated that hydrolase 1 was an IP. i.e. its stimulation by estradiol could be btocked by puromy~in, it was of some interest to learn whether the same was true for hydrolase II. Im-

II actioity

In preliminary experiments we found that administration of estradiol to weanling mice resulted in a 2-4-fold increase in uterine hydrolase II activity. To compare the stimulation of hydrolase II activity with other estrogen-mediated phenomena, the effects of a series of steroid hormones and the antiestrogen

Table 3. Heat stabilities of hydrolase activities in mouse uterine homogen~tte Time at SYC (min) 0

I 5 I0

“0 Maximal activity Ala-Ala-Pro-Ala-AM&se p-Ala-NPEase (Hydrolase II) (Hydrolase I) 100 74 13 5

100 100 104 109

Uterine homogenate supernatant was diluted I:4 with 0.2 M sodium phosphate, pH 7.0 and heated at 50°C. At the indicated intervals samples were removed and immediately chilled to 0°C. Aliquots were tested for hydrolase activity as described in the experiment~~l section.

0123456

18 Time (hrs)

Fig. 2. Time-course for the stimulation of uterine hydrolase I and hydrolase II activity by estradiol. Weanling mice (3-5 per group) received 0. I5 log estradiol or vehicle only. At the times indicated animals were killed and hydrolase I (Ala-Ala-Pro-Ala-AMCase) and hydrolase II (/?-AlaNPEase) activity were determined in the uterine homogenate supernatants as described in the experimental section.

Estrogen-induced

147

uterine hydrolase

Table 4. Effect of puromycin on estradiol-stimulated activity

uterine hydrolase

% Stimulation* Ala-Ala-Pro-Ala-AMCase o-Ala-NPEase (Hydrolase I) (Hydrolase II)

Addition Estradioi Estradiol + puromycin

149 0

262 53

* Over control animals not treated with estradioi. Five week old mice were treated with 400 yg puromycin 30 min prior to administration of 0.3 gg estradiol. After 6 hr animals were sacrificed and levels of hydrolase enzymes determined in uterine homogenates.

mice were treated with estradiol in various as shown in Table 4. After 6 h hydrolase I and II activities in the uterine homogenate supernatanl were determined. This time was chosen because both activities were closest to their maximum. Uterine levels of both enzymes were stimulated by estradiol to levels approaching those found in adults. In each case stimulation by estradiol was blocked by prior administration of puromycin. mature

combinations

Substrate and inhibitor specificity of hydrolase II

To better characterize the enzyme, its cleavage of a series of N-blocked amino acid nitrophenyl esters (Table 5) and its inhibition by a series of standard protease inhibitors (Table 6) was examined. The purified enzyme was found to cleave a number of L-W amino acid nitrophenyl esters with &‘s all approx. 40pM. Of the substrates tested, the phenylalanine ester had the highest V,,,. The enzyme was also found to cleave @Ala-NPE although with a K, 4-fold higher than those obtained with the a-amino acid esters. In control experiments fi-Ala-NPE, at 1 mM, was not found to be a substrate or inhibitor of porcine pancreatic trypsin, chymotrypsin or elastase or of human leukocyte elastase. Like the enzymatic activity Hormone specificity

300 t

I

2

3

Hormonal I-Estrone

( 3upJ

4-Tamarifm(.3ugl 6-Ptqestemm(3ugug)

4

5

6

IY-I@+--c

Substrate Cbz-glycine-NPE Cbz-L-tyrosine-NPE t-Boc-L-a-alanine-NPE t-Boc-fi-alanine-NPE Cbz-L-phenylalanine-NPE Cbz-L-tryptophan-NPE Cbz-L-methionine-NPE r-N-r-Boc-L-lysine-NPE Cbz-L-leucine-NPE Cbz-L-proline-NPE

I

in the uterine supernatant, the purified hydrolase was inhibited by ZPCK, DIFP and not by Ala-Ala-ProVal chloromethyl ketone (Table 6). These results are consistent with the hydrolase being a serine hydrolase with chymotrypsin-like specificity. However, we were not able to demonstrate activity against two sensitive peptide substrates for chymotrypsin-like enzymes, succinyl-Aia-Ala-Pro-Phe- and succinyl-Gly-Gly-Phe~-nitroanilide. Purified mouse plasma protease inhibitors, ~~-macroglobulin, a,-protease inhibitor and antithrombin III at 0.1 mgiml and SBTI at 1 mg/ml

Inhibitor bTancxiAn (.3ug) 7-Tzstos~(3ug)

57.7 93.8 18.7 100.0 124.1 101.4 68.3 5 5

* Not determined. Assays were carried out in 0.02 M Na phosphate buffer, pH 7.0. Correction was made for spontaneous hydrolysis (essentially 0 for these substrates under the above conditions). K, and V,,. were determined from reciprocal plots of I/vi against l/(s).

treatment

P-EstmdiolL3ug)

31 33 41 145 35 32 41 ND ND ND

Table 6. Inhibition of purified hydrolase II by standard protease inhibitors

7

Activity (as 7” of control)

3- Eetriol (.3u(l) +

Estmdioll.Bug

1

Fig. 3. Hormonal stimuiation of hydrolase II activity. Wean~in~ mice (3 per group) received the hormones in the amounts indicated. After 3 h the animals were killed and hydrolase II activity (~-Ala-NPEase) was determined in the uterine supernatant homogenate as described in the experimental section. S.B.

Table 5. Hydrolysis of N-blocked amino acid nitrophenyl esters by hydrolase II

Control TLCK ZPCK DIFP Acetyl-Ala-Ala-Pro-Val-CH*Ci

100 89.3 24.9 0 100

Assays were performed with &Ala-NPE as substrate as described in Table 1. All inhibitors were tested at 10m3M.

THOMAS H

73x

did not inhibit /&Ala-NPE hydrolase activity. Ca'+ mM), EDTA (0.1 mM) and dithiothrcitol (I mM) were all without stimulatory or inhibitory effect.

(I

DISCUSSION

In this communication we describe the properties of an unusual hydrolytic enzyme purified from mouse uterine homogenates. Our results suggest that it is different from an activity in this tissue, which we have called hydrolase I, originally identified by its ability to cleave Ala-Ala-Pro.Alaand Ala-Ala-Pro-Phe-AMC 161. Both enzymes are apparently chymotrypsin-like. however, the enzyme reported on here, hydrolase II. does not cleave either of the two peptide-AMC substrates mentioned above although it does cleave a number of amino acid nitrophenyl esters. Hydrolase I and II also differ in their stability on heating and in the time course of their increase in activity following administration of estradiol. Both activities are present at higher levels in uterine homogenates from mature or estrogen-stimulated immature mice than in homogenates from unstimulated. immature animals. The estrogen-stimulated increase in the activity of both hydrolase I and II can be blocked by puromycin suggesting that both enzymes are induced proteins. Of the steroids tested, estradiol was the most effective in stimulating hydrolase II activity in immature mice although estrone and estriol also had activity. Although a single dose of estriol is less potent than estradiol in promoting true uterine growth. certain early events including the induction of IP synthesis [IO] are expressed by both estrogens. Neither progesterone nor testosterone appeared to have hydrolase II stimulatory activity. Hydrolase II was purified approx. 400-fold with a recovery of ISo/,,,from mouse uterine homogenates by affinity chromatography on Affigel Blue. ion exchange chromatography on DEAE-cellulose and hydrophobic chromatography on octyl-Sepharose. SDSpolyacrylamide gel electrophoresis. HPLC-gel-permeation chromatography and W-terminal analysis indicated that the enzyme was a single chain polypeptide of mol. wt 65,000. Its behavior on ion-exchange and hydrophobic chromatography suggest that the hydrolase is an acidic protein with some degree of hydrophobicity. The physical properties of hydrolase 11 (like those of hydrolase I). particularly molecular weight and isoelectric point, resemble those of the estrogen-induced proteins from the rat uterus (IP-1.2,3) reported by Skipper c’t tr/.[4]. A variety of N-blocked L-cc-amino acid nitrophenyl esters were cleaved by the purified enzyme at pH 7.2 with similar K,‘s. The phenylalanine nitrophenyl ester was found to be the best substrate. The purified enzyme had no detectable activity against peptide nitroanilide substrates for trypsin-, chymotrypsinor elastase-like enzymes. However Cbz-P-Ala-NPE, which in control experiments was not hydrolyzed by trypsin, chymotrypsin or elastase was cleaved by hydrolase II with a

K,, of I45 /tM. Inhibition studies shoL\cd that hqdrolysis of fl-Ala-NPE was blocked by ZPCK (an inhibitor of chymotrypsin-like enzymes) and DIFP (a general inhibitor of serine proteases) but not by TLCK (an inhibitor of trypsin-like enrymes) or acetklAla-Ala-Pro-Val-chloromethyl ketone (an inhibitor of elastase-like enzymes [t t]. The nature of its substrates and its inhibition pattern are consistent with hydrolase II being a serine esterase with chymotrypin-like specificity. Its molecular weight, substrate specificity and inhibition pattern suggest that it is not a collagenase [ 121or collagenase activator [ 131. plasminogen activator [14]. carnosinase [ 151or a knowIn cathepsin [ 161.If it proves to be a protease it might function as a post-transcriptional modifier of other proteins synthesized and secreted by the uterus. Over the past few years it has become evident that most secretory proteins undergo one or more proteolytic processing steps during biosynthesis [ 171. Available evidence indicates this processing occurs after translation and following transport to the Golgi complex. However, the precise nature of the processing enzymes are unknown as Golgi proteases have not been purified or characterized.

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uterine hydrolase

749

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Archs bioehem. ~~~p~~s. 202 (1980) 3 14-3

17.