MOLECULAR REPRODUCTION AND DEVELOPMENT 29:29-39 (1991)

Purification and Preliminary Characterization of Guinea Pig Testicular Proteinase Inhibitors E.A.O. FALASE, B.T. STOREY, AND C. TEUSCHER Division of Reproductive Biology, Department of Obstetrics and Gynecology, University o f Pennsylvania School of Medicine, Philadelphia, Pennsylvania ABSTRACT Three guinea pig testicular, Iowmolecular-weight,acid-stable inhibitors specific for trypsinlike proteinases were isolated, purified, and characterized. The procedure comprised acid extraction of testicular acetone powder, pH precipitation of the extract, gel filtration of the supernatant on Sephadex G-100 and G-50, ion-exchange chromatography on SP-Sephadex, followed by QAE-Sephadex. Final purification was by rechromatography on Sephadex G-50 superfine gel. The three proteinase inhibitors were labeled A, 6, and Cnb, the latter to denote nonbinding of Cnb to the QAE-Sephadex.Components A and Cnb showed competitive, whereas B showed noncompetitive, inhibition against trypsin. All three inhibitors were active against trypsin but were ineffective against chymotrypsin. The inhibition constants, Ki, were obtained using trypsin-catalyzed hydrolysis of the Nbenzyloxycarbonyl-1-arginyl amide of 7-amino-4-trifluoromethylcoumarin (CBZ-Arg-AFC) at pH 8.0. The values were calculated to be, for A, 1.5 x M; for 6, 1.5 x M; and, for Cnb, 2.2 x M. The K, values calculated from inhibition of trypsin-catalyzed hydrolysis of the active site titrant 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) using Easson-Stedman plots were, for A, 7.7 x lo-’ M; for 6, 6.7 x lo-’ M; and, for Cnb, 1.4 x M. The M,s as determined by active site titration with MUG6 were A, 11.2 kDa; 6, 10.5 kDa; Cnb, 17.0 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave M, values for A of 11 kDa, for B of 4 kDa, and for Cnb of 19 kDa. The discrepancy in M, values for B indicates that it may function as a dimer or trimer in the active state.

(Zaneveld et al., 1971). Fink and Fritz (1976) isolated two such inhibitors from the seminal vesicles of guinea pigs, using affinity chromatography on trypsin resin. Most of these proteinase inhibitors inhibit the sperm acrosomal proteinase acrosin (Fritz et al., 19761, as would be expected since this enzyme’s amidohydrolase activity is very similar, though not identical, to that of trypsin with respect to substrate specificity (McRorie and Williams, 1974). Although the functions of these seminal plasma proteinase inhibitors have not been determined, they may protect the epithelia of both the female and the male reproductive tracts, and the intact spermatozoa therein, from proteolytic action of enzymes released from defective and dead spermatozoa (Fritz et al., 1976). They may also play a part in the capacitation of spermatozoa (Zaneveld et al., 1969; Schill et al., 1975)and regulation of acrosomal enzymes involved in zona penetration (Zimmerman and Burch, 1978). Suominen and Setchell (1972) reported that an inhibitor of trypsin-like proteases was present in the rete testis fluid of rams and boars. No further characterization of the inhibitor from this source has been provided. The most extensive examination of this type of trypsin-like proteinase inhibitor has been carried out in the mouse. Poirier and Jackson (1981) isolated two such inhibitors, one of M, 6.2 kDa from seminal vesicles and the other of M, 4.0 kDa from the epididymis. The 6.2 kDa protein has been documented in extensive studies by this group to bind to the anterior portion of ejaculated sperm, to be lost from uterine sperm after a residence time approximating that expected for capacitation, and most importantly to occupy a plasma Key Words: Trypsin-like,Proteinase inhibitors, Testes, membrane binding site recognizing the mouse zona Sperm, Male reproductive tract pellucida (Nicholson et al., 1983; Irwin et al., 1983; Aarons et al., 1984; Poirier et al., 1986; Robinson et al., 1987; Boettger et al., 1989). The proteinase inhibitor INTRODUCTION originating in the seminal vesicles could bind to the Trypsin inhibitors in the sex glands and seminal sperm during ejaculation, protect the sperm during plasma of mammals were first isolated by Haendle et uterine passage, and then dissociate to allow sperm al. (1965). Since then, an assortment of low-molecular- binding to the zona (Robinson et al., 1987). Epididymal weight proteinase inhibitors of this specificity with M, mouse sperm are also fully capable of fertilizing eggs in the range 6-12 kDa have been isolated from the seminal plasma of humans (Fink et al., 1971; Suominen and Niemi, 1972; Fritz et al. 1976), boars (Polakoski Received September 1, 1990; accepted December 14, 1990. and Williams, 1974; Tschesche et al., 1974; Jonakova Address reprint requests to Cory Teuscher, PhD, Department of and Cechova, 1985; Veselsky et al., 1985; Jonakova et Microbiology, 775 WIDB, Brigham Young University, Provo, UT al., 1988), bulls (Cechova and Fritz, 1976), and rabbits 84602.

0 1991 WILEY-LISS, INC.

30

E.A.O. FALASE ET AL.

and require a period of in vitro capacitation in order to do so (Miyamoto and Chang, 1973),yet these sperm are never in contact with the 6.2 kDa seminal protein (Irwin et al., 1983). The protein was not found in the testis or the epididymis by immunofluorescence (Irwin et al., 1983),but the 4.0 kDa inhibitor derived from the epididymis was found to be associated with epithelial cells in the apical portion of the caput epididymidis and, in the testis, also by immunofluorescence (Poirier and Nicholson, 1984). The epididymal inhibitor appears to be lost during epididymal transit (Poirier and Nicholson, 1984). This report of a low-molecular-weight trypsin inhibitor present in the mouse testis raised the interesting question of the number and nature of this type of low-molecular-weight proteinase inhibitor that might be present in the mammalian testis. This question had not heretofore been examined. In this paper, we report the presence of three such low-molecularweight proteins in the guinea pig testis, one of which may be analogous to the previously reported 4.0 kDa inhibitor in the mouse testis (Poirier and Jackson, 1981; Poirier and Nicholson, 1984), whereas the two others are clearly different.

The lyophilized material containing inhibitor activity was redissolved in 1mM HC1 and chromatography carried out on Sephadex G-50, utilizing a 2.5 x 90 cm column. Fractions were collected while absorbance was monitored at 280 nm. The fractions exhibiting inhibitory activities were again pooled and lyophilized. The lyophilized material from the preceeding steps was dissolved in and exhaustively dialyzed against 0.05 M sodium acetate buffer containing 0.2 M NaC1, pH 4.2. This was applied to a 1.5 x 25 cm SP-Sephadex column equilibrated with 0.05 M sodium acetate buffer, 0.2 M NaC1, pH 4.2. The column was washed with the same buffer until the absorbance at 280 nm returned to baseline, at which point the column was eluted with a linear gradient of 0.2-0.8 M NaCl in 0.05 M sodium acetate, pH 4.2. Three peaks of inhibitory activity were obtained and were pooled separately. The three peaks of inhibitory activity were labeled A, B, and C. Each of the peaks was dialyzed against distilled water, lyophilized, redissolved in and dialyzed against 0.05 M bicarbonate-carbonate buffer at pH 10, and applied to a 1.5 x 25 cm QAE-Sephadex column equilibrated with 0.05 M bicarbonate-carbonate buffer, pH 10. No inhibitory activity was detected in the nonbinding effluent when peaks A and B were applied to the column. Elution of the column with a linear gradient of NaCl up to 1.0 M in 0.05 M bicarbonatecarbonate buffer, pH 10, yielded fractions with inhibitory activity which were again pooled. Peak C did not bind to the column, and so was designated Cnb. The pooled fractions from A, B, and Cnb were exhaustively dialyzed against distilled water and lyophilized. Further chromatographic purification was carried out on Sephadex G-50 superfine in a 2.5 x 90 cm column for all pooled peaks exhibiting proteinase inhibitory activity after redissolving the lyophilized powder in 1 mM HC1 containing 0.02% sodium azide. Fractions with inhibitory activity were pooled separately and concentrated. The samples were stored at -70°C.

MATERIALS AND METHODS Reagents Guinea pig testicular acetone powder was obtained from Rockland Inc. (Gilbertsville, PA). Sephadex G100, Sephadex G-50, SP-Sephadex, QAE-Sephadex, and the electrophoresis calibration kits for molecular weight determination of low- and intermediate-molecular-weight proteins were obtained from LKB-Pharmacia Fine Chemicals Inc. (Piscataway, NJ). The N-benzyloxycarbonyl-arginyl amide of 7-amino-4-trifluoromethylcoumarin (CBZ-Arg-AFC),7-amino-4-triflouromethylcoumarin (AFC), and the AFC amide of the dipeptide Glu-Tyr (Glu-Tyr-AFC) were obtained from Enzyme System Products (Livermore, CAI. N,Ndimethylformamide (DMF), phototrex spectrometric grade, was from J.T. Baker Chemical CO. (Phillipsburg, NJ). Trypsin (from bovine pancreas), chyFluorimetric Assays motrypsin, and 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) were obtained from Sigma Chemical The activity of the inhibitors on bovine pancrease Co. (St. Louis, MO). All other reagents were obtained trypsin was assayed fluorimetrically, using the Nfrom standard commercial sources. benzyloxycarbonyl-arginyl amide of 7-amino-4-trifluoromethyl coumarin (CBZ-Arg-AFC)as the fluoroPurification of Inhibitors genic substrate. The appearance of 4-trifluoromethyl All purification steps were carried out at 4°C unless coumarin (AFC) was determined with an Eppendorf stated otherwise. Acid extraction of guinea pig testic- fluorimeter B 120E (Brinkmann Instruments., Westular acetone powder, precipitation of the extract at pH bury, NY), using the 405-436 nm primary filter for 6.0, dialysis of the supernatant against 1 mM HCl, excitation and a 500 nm low-wavelength cut-off filter lyophilization, and subsequent chromatography on (Wratten No. 12; Eastman Kodak, Rochester, NY) as a 5 X 90 cm Sephadex G-100 columns were carried out as secondary filter for emission. The assays were perpreviously described (Adekunle et al., 1987). To ensure formed at pH 8.0 in a 1.0 ml reaction volume containing retention of low-molecular-weight proteins during the 100 mM tris(hydroxymethy1) aminomethane (Tris) dialysis steps, Spectro/Por 6 dialysis membrane with a HC1,50 mM CaCl,, 1 kg trypsin, and between 10 and molecular weight cut-off of 1,000 was used throughout 100 ~1 of protein inhibitor solution, either undiluted or this study. Fractions exhibiting maximal inhibition of diluted, to provide a range of inhibitor concentrations trypsin were pooled and lyophilized. in the reaction volume. The assay solution was allowed

TESTICULAR PROTEINASE INHIBITORS

0

10

20

30

40 50 FRACTION NUMBER

Fig. 1. Sephadex G-100 column (5 x 90 cm) chromatography of the soluble extract of guinea pig testicular acetone powder. After acid extraction, the lyophilized extract was redissolved in 1 mM HCl containing %lo% sucrose. This was applied to a Sephadex G-100 column and eluted with 1 mM HCl; 25 ml fractions were collected; 1

60

70

31

ao

p1 of sample from each fraction was assayed for its inhibitory activity in 1.0 ml reaction volume against the proteinase action of 1mgiml of trypsin with 100 mM CBZ-Arg-AFC as substrate at 23”C, pH 8.0. Tubes containing maximal inhibitory activity were pooled.

to stand for 5 min a t room temperature, at which point p-guanidinobenzoate (MUGB).The fluorescent product 1 ml of CBZ-Arg-AFCdissolved in dimethylformamide of the hydrolysis, 4-methylumbelliferone (MU), was (DMF) was added. The concentrations of the CBZ- determined fluorimetrically with an excitation waveArg-AFC stock solutions in DMF were adjusted to give length of 365 nm and emission wavelength of 450 nm. the desired substrate concentration while maintaining The data were incorporated into an Easson-Stedman the added DMF a t 0.1%, a concentration shown in plot (Easson and Stedman, 1936-1937), according to control experiments to have no effect on the assay. For the equation I,/i = Ki/(l - i) + E,, where E, is the total the assay of the inhibitor fractions obtained by chro- concentration of trypsin, Ki is the inhibitor constant of matography, the concentration of CBZ-Arg-AFC was the inhibitor under test, and i is the fractional inhibiset at 100 pM. The assays were carried out at room tion of enzymatic activity at a given total inhibitor temperature, 23°C 2 1°C. The fluorescence increase concentration, I,. The experimental values of I, for the due to the release of free AFC from amidohydrolase protein inhibitors were in micrograms per milliliter. At activity of trypsin was linear for at least 5 min. The rate constant trypsin concentration, E,, and varying total was obtained from the linear increase after calibration concentrations of inhibitor, I,, the Easson-Stedman plot of the system with additions of a stock solution of AFC intersects the y axis at the value (I,) = E,. The molecin DMF. Inhibitor constants, Ki, were determined from ular weight of each inhibitor was caiculated using the the plot of the average of reciprocals of velocity and equation M, = (Io)y/Eo.The fluorimeter was calibrated substrate concentration at varying concentrations of by additions of 1 pl aliquots of 0.1 mM MUB. The purified inhibitor (Segel, 1976). Samples were assayed sensitivity of the assay is ready detection of 1 nM MU. in triplicate. The concentration range of the substrate, Assays were performed at pH 8.0 in a 1.0 ml reaction CBZ-Arg-AFC, was 20-100 pM, whereas that for the volume containing 100 mM Tris HC1, 50 mM CaC12,2 inhibitors was 0.07-1.05 pgiml. The effect of the inhib- pg trypsin and between 0 and 4.2 kg of the inhibitor itors on chymotrypsin activity was assayed in the same being tested. The inhibitor was added first and incumanner, substituting 1 pg chymotrypsin in place of bated for 5 min at the room temperature, after which trypsin and using Glu-Tyr-AFC as substrate in the the MUGB was added to a final concentration of 4 pM. The “burst” of fluorescence corresponding to the single concentration range 20-100 pM. turnover hydrolysis of MUGB was recorded. Blanks Active Site Titration to Estimate Inhibitor containing no trypsin were recorded so that the small Molecular Weight fluorescence intensity of MUGB itself, less than 10%of The apparent molecular weight of each inhibitor was the total, could be subtracted. estimated by the decrease in trypsin active sites titratElectrophoretic Procedure able by the method of Jameson et al. (19731, with Aliquots of the active fractions of peaks A, B, and increasing inhibitor concentration. The active site titration utilizes the single turnover hydrolysis of the Cnb from the final chromatography on Sephadex G-50 fluorogenic active site titrant 4-methylumbelliferyl- superfine column were prepared by redissolving 10 and

32

E.A.O. FALASE ET AL. n

x

n

-

loo

z

v

z

v

0

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-

50

k Z

+ 2

I

W

0

a W

0

20

40

60

80

100

- 0 140

120

e

FRACTION NUMBER Fig. 2. SP-Sephadex ion-exchange chromatography of the pooled active fractions from the Sephadex G-50 chromatography. The lyophilized material was dissolved in 0.05 M sodium acetate buffer containing 0.2 M NaCl, pH 4.2, and applied to a SP-Sephadex column (1.5 x 25 cm), then eluted with a linear gradient of 0.2-0.8 M NaCl,

as shown. Assays of the fractions for inhibitory activity against trypsin resulted in three peaks of inhibitory activity labeled peaks A, B, and C, respectively. Tubes containing maximal inhibitory activity across each peak were pooled.

1.o

0.5

2 0.0

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m

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1.0

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0 0

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FRACTION NUMBER Fig. 3. QAE anion-exchange chromatography of the separate peaks of inhibitory activity from SP-Sephadex chromatography. The peaks were pooled separately, lyophilized after desalting, and redissolved in 0.05 M bicarbonate-carbonate buffer a t pH 10 and applied to a QAE Sephadex column (1.5 x 25 em). The column was eluted with a linear gradient of NaCl up to 1.0 M in 0.05 M bicarbonate-carbonate buffer.

Fractions were assayed for inhibitory activity as described for Figure 1. Fractions A and B represent the peaks A and B from the SPSephadex, respectively. Peak C did not bind to the QAE column and so was designated fraction Cnb. Tubes containing maximal inhibitory activity across each peak were pooled.

TESTICULAR PROTEINASE INHIBITORS

33

1.o

0.5

0.0 0

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0.30

100

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0.00 0

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FRACTION NUMBER Fig. 4. Sephadex G-50 superfine chromatography of the active fractions from the QAE chromatography. The separate fractions A, B, and Cnb were lyophilized, resuspended in 1 mM HC1 plus 0.02% sodium azide, applied to a Sephadex G-50 superfine chromatography

column (2.5 x 90 cm), and eluted with the same 1 mM HCl solution; 20 ml fractions were collected. Tubes containing maximal inhibitory activity across each peak were pooled. The close congruence of inhibitory activity and protein content is evident in A-C.

TABLE 1. Purification of Guinea Pig Testicular Proteinase Inhibitors

Procedure Sephadex G-50 P e a k Ab

QAE Sephadex G-50 Superfine P e a k Bb QAE Sephadex G-50 Superfine P e a k Cb Q A E (nonbinding) Sephadex G-50 Superfine

Protein content Total protein (mg) Yield (%)

5,226a 438 40 16 350 37 29 135 15 2

100.0 8.4 0.8 0.3 6.7 0.7 0.6 2.8 0.3 0.04

Proteinase inhibitorv activity Specific Total activity activity (units) (unitdmg)

78,390 26,303 21,408 17,566 35,584 31,218 27,825 9,629 5,682 2.626

15 60 535 1.097 102 837 959 71 392 1,250

aYield of protein determined spectrophotometrically, as described by L a y n e (1957). bAfter SP Sephadex chromatography.

Recovery (%)

Purification (mean)

33.6 27.3 22.4 45.4 39.8 35.0 12.3 7.2 3.3

4 35 73 7 56 64 5 26 83

34

E.A.O. FALASE ET AL.

Fig. 5. Analysis of purified fractions A and B by SDS-PAGE. Samples containing 10 kg protein were separated on a 15% polyacrylamide gel containing 8 M urea, under reducing conditions in the presence of 8 mM dithiothreitol. The gel was stained with Coomasie blue after electrophoresis. Numbers a t left indicate the molecular weights of the myoglobin-derived standard proteins (see Materials and Methods).

20 pg of each of the lyophilized proteins in 0.0625 M Tris HC1 (pH 6.81, 10% glycerol, 2.5% sodium dodecyl sulfate (SDS), 0.001% bromophenol blue, and, when present, 8 mM dithiothreitol. Analytical SDS-polyacrylamide gel electrophoresis (SDS-PAGE)was carried out under both reducing and nonreducing conditions (Adekunle et al., 1987).For the lower M, proteins A and B, 15% acrylamide gels containing 8 M urea were used with M, standards derived from myoglobin (LKBPharmacia) of known sequence with values ranging from 2,512 to 16,949. For protein Cnb, 12.5% gels containing 8 M urea were used. The M, standards were purified proteins ranging from 14 kDa for a-lactalbumin to 94 kDa for phosphorylase b (LKB-Pharmacia). The gels were stained with 0.1% Coomasie brilliant blue in 45% methanol and 10% acetic acid for 45 min, then destained first in 9% acetic acid and 45% methanol for 4 hr and subsequently with 7.5% acetic acid and 5% methanol.

Fig. 6. Analysis of purified inhibitor fraction Cnb by SDS-PAGE. Samples containing 10 and 20 kg protein were separated on a 12.5% polyacrylamide gel containing 8 M urea in the presence of 8 mM dithiothreitol. The gel was stained with Coomasie blue after electrophoresis. Numbers left indicate the molecular weights in kDa of the protein standards (see Materials and Methods).

molecular-weight inhibitory fraction into three separate components was obtained with cation exchange chromatography on SP-Sephadex (Fig. 2). The three peaks of inhibitory activity, labeled A, B, and C, correlated loosely with the peaks of absorbance at 280 nm. The peaks of inhibitory activity were each pooled separately and individually subjected to anion exchange chromatography on QAE-Sephadex.This purification step removed further contaminants and brought inhibitory activity and protein content, as estimated by absorbance at 280 nm, into a tighter correlation for peaks A and B (Fig. 3). Peak C did not bind to the QAE column and so was designated Cnb for nonbinding. A final purification with Sephadex G-50 superfine column resulted in a still tighter correlation between protein as assessed by absorbance and inhibRESULTS itory activity (Fig. 4). Enzymatic inhibition assays of the column fractions A summary of the inhibitory activity recovered at after gel filtration on Sephadex G-100 resulted in the each stage of purification after Sephadex G-50 chromaidentification of a single, broad elution peak of trypsin tography is shown in Table 1. The result shows that inhibitory activity in the low molecular-weight fraction 22.4%,35.0%, and 3.3% of total inhibitory activity was (Fig. 1). Further removal of low-molecular-weight recovered after Sephadex G-50 and that a final purificontaminants was achieved by gel filtration using cation of 73-, 64-, and 83-fold was achieved for compoSephadex G-50 (not shown). Resolution of the low- nents A, B, and Cnb, respectively.

TESTICULAR PROTEINASE INHIBITORS

35

the mouse testis by immunofluorescence (Poirier and Nicholson, 1984). A useful method for the determination of the functional M, of protein inhibitors of this type is inhibition of the hydrolysis of MUGB, and active site titrant of trypsin, utilizing the Easson-Stedman plot (Easson and Stedman, 1936-1937) to analyze the data. This method has the advantage that the M, obtained may be compared with that obtained by SDS-PAGE and so act as a check of the purity of the protein. It also provides a 10 15 0 5 20 value for the inhibition constant, Ki. Easson-Stedman plots obtained for the three protein inhibitors are 4 shown in Figure 7. Good linear plots were obtained with inhibitors A and Cnb (Figs. 7A and 7C). The M, values calculated from the Y axis intercepts were 11.2 and 17.0 kDa, respectively, for the two proteins, in close agreement with the values obtained from SDS gels (Figs. 5 and 6). The plot obtained with inhibitor B showed more scatter from linearity (Fig. 7B), and the value of M, calculated from the intercept was 10.5 kDa, more than twice that obtained by SDS-PAGE (Fig. 5 ) . The molecular weights determined from the EassonStedman plots and from the SDS-gel electrophoresis are listed for comparison in Table 2. Double reciprocal plots for the inhibition of trypsincatalyzed hydrolysis of CBZ-Arg-AFC by increasing concentrations of the three inhibitors are shown in Figure 8. The proteins A and Cnb showed competitive inhibition, whereas protein B showed noncompetitive inhibition. Values for the inhibition constants, Ki, were calculated according to Segel (19761, using the plot of KM(app)/Vmax vs. [I] for A and Cnb, and the plot of l/Vmaxvs. [I] for B. Values of the kinetic parameters were obtained from the plots of Figure 8 and listed in Table 3. Conversion of inhibitor concentration from 0 1 2 3 4 5 kg/ml to nM utilized the molecular weights obtained from the Easson-Stedman plots in Figure 6, since those 1 4) values are the functional ones obtained under assay conditions essentially identical to those used in the Fig. 7. Easson-Stedman plots for the different purified proteinase kinetic determinations of Ki. Also listed in Table 3 are inhibitors A, B, and Cnb. The regression line was calculated to be A: the values of Ki obtained from the slopes of the linear y = 0.90407 + 8.8600e - 2x (R2= 0.986) for protein A; B: y = 1.3822 + 0.11407~(R2= 0.868) for protein B; C: y = 1.3807 + 2.3062~(R2= Easson-Stedman plots (Fig. 71, again using the M, 0.995) for protein Cnb. They intercept is E,, according to the equation values obtained from those plots. The Ki values so I,/i = KJl - i) + E,. I, is the inhibitor concentration that is equivalent to the release of 81.0 pmole of MUB. The molecular weight of the determined were lower than those obtained by the proteinase inhibitors were calculated from the equation M, = (IJy/E0. double reciprocal plots by approximately twofold. A source for this apparent systematic difference could not be found. The values obtained with the two methods are sufficiently close, however, that one may conclude that The purification procedure yielded single, somewhat proteins A and B are relatively high-affinity inhibitors broad bands after SDS-electrophoresis in 8 M urea for compared with protein Cnb. When tested at the highest concentrations used in each of the three components, as shown in Figure 5 . All three were of low M,, with protein A showing a band the inhibition assays of Figure 8, none of the three centered at 11 kDa and protein B showing a band protein trypsin inhibitors had any effect on the chycentered at 4 kDa (Fig. 51, whereas protein Cnb showed motrypsin-catalyzed hydrolysis of Glu-Tyr-AFC, an a band centered at 19 kDa (Fig. 6). No difference in M, active substrate for this protease. This result confirms values was observed under nonreducing conditions in the specificity of this group of testis-derived inhibitors the absence of dithiothreitol. The M, of 4 kDa for for trypsin-like proteinases. The possibility that these protein B is consistent with that isolated by Poirier and proteinase inhibitors were not testicular and were due Jackson (1981)from the mouse epididymis and found in to serum components trapped by homogenization was

/(I

36

E.A.O. FALASE ET AL. TABLE 2 . Molecular Weight Determination by Active Site Titration and by SDS-PAGE Method Active site titration with MUGB SDS-PAGE

Protein A

M, (kDa) Protein B

11.2

10.5

11

4

ruled out by the fact that gel filtration of whole serum or serum diluted with 1 mM HC1 failed to reveal any inhibitory components with M, less than 45,000.

DISCUSSION This study demonstrates that the guinea pig testis contains three proteinase inhibitors, apparently specific for trypsin and trypsin-like enzymes, which are similar to the inhibitors isolated from mammalian seminal vesicles and seminal fluid. The purification methods used here are akin to those used by others to isolate this type of inhibitor (Poirier and Jackson, 1981; Fink and Fritz, 1976; Cechova and Jonakov, 19811, with slight modifications. The molecular weights of the proteinase inhibitors A and Cnb, determined by both active site titration and SDS-PAGE (Table 31, compare favorably, but the two methods give different values for protein B. The reason for the discrepancy in the molecular weights does not appear to be the result of cleavage of a higher-molecular-weight protein, since only one band is observed when analyzed by SDS-PAGE (Fig. 51, rather than the expected multiple bands associated with this sort of breakdown, Glycosylation of a protein can affect the molecular weight determination by gel electrophoresis, and broadening of the band, indicative of carbohydrate moieties, is observed. However, it is difficult to visualize how glycosylation could so compact a protein in 8 M urea that its apparent M, would be halved. The apparent M, of all three proteins remained the same under both reducing and nonreducing conditions, suggesting that disulfide-linked subunits are not the cause of the anomaly. The most probable source of the discrepancy in M, values is that protein B in its undenatured state exists as a noncovalently associated dimer or trimer and is active only in the native oligomeric form. For this reason, we believe that the M, derived from the MUGB titration (Fig. 7B) for protein B is the correct one to use in determining the inhibitor constants Ki (Table 2). The values of Ki calculated for the three protein inhibitors with trypsin show slightly lower affinity for this enzyme than do the two guinea pig seminal plasma inhibitors reported by Fink and Fritz (1976),which had Ki values of 2 and 3 nM. They are quite similar to the bull seminal plasma inhibitors BUSI I and BUSI 11, which had Ki values of 8 nM and 500 nM, respectively, with trypsin (Cechova and Jonakova, 1981). Insufficient data from other species are available at present to

Protein Cnb 17.0 19

make meaningful general comparisons of Ki values for these inhibitors with regard to their source in the male reproductive tract, but results to date indicate that this class of protein may be common to the male tract of all mammalian species. To obtain enough protein for purification and characterization of the low-molecular-weight proteinase inhibitors, it was necessary to use a bulk source: in this case, guinea pig testicular acetone powder. The cellular location of the inhibitors in the testis is therefore as yet unknown. The methods described in this report yield sufficient purified inhibitor of each M, to generate antibodies for immunocytochemical localization. A question fundamental to the functionality of proteinase inhibitors in the guinea pig testis is raised by the finding that there are not one but three low-molecularweight inhibitors of trypsin-like enzymes. It is conceivable that one is cosynthesized and packaged in the forming acrosome with proacrosin. This hypothesis is testable by immunocytochemistry. A function for one or more of these proteins, not directly related to the assayable trypsin inhibitor activity, is presented here as a hypothesis, based on the analogy of blockage of cell adhesion in mouse sperm by this class of inhibitors. Poirier and Nicholson (1984) showed that the inhibitor from the mouse seminal vesicle blocked binding of mouse sperm to mouse zonae. Saling (1981) demonstrated that trypsin inhibitors as a group blocked the binding of mouse sperm to zonae. Benau and Storey (1987)found that the sperm binding site affected by the trypsin inhibitors assayed specifically for ester hydrolase activity with guanidinobenzoate esters and had no trypsin-like activity. These observations suggest that one or more low-molecular-weight inhibitors could potentially play a role in blocking cell-to-cell adhesion. In the guinea pig testis, this would be inhibition of binding of sperm cell to sperm cell, such that sperm stacking and rouleaux formation would not occur in the testis, as observed (Tung et al., 1980).Disappearance of the inhibitor during epididymal transit would allow rouleaux formation at the appropriate location in the epididymis. Tung et al. (1980) found that monovalent Fab antibodies, isolated from guinea pigs immunized either with their own epididymal spermatozoa or with homogenates of their own testes, dispersed sperm rouleaux from the cauda epididymis. This result indicates the presence of sperm plasma membrane surface antigens which may act as specific receptors in

TESTICULAR PROTEINASE INHIBITORS

- 0 02

- 0 00

0 02

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.o.oo

0.02

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37

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8-

6-

4-

2-

0 - 0 02

I

Fig. 8. Double reciprocal plots for the inhibition of trypsin-catalyzed hydrolysis of CBZ-Arg. AFC at pH 8.0 by the purified proteinase inhibitors A, B, and Cnb. The units of liv and lis are (pmolesi min - mg protein)-' and (pM)-l, respectively. The reciprocals (liv)of the rate of hydrolysis at different substrate concentrations (S) were

determined at different concentrations of the proteinase inhibitors. For inhibitor A, the concentrations, in pgiml, were 0.32 (U), 0.48 @I, 0.63 ( A ) , 0.95 ( + ). For inhibitor B, these were 0.35 (W, 0.53 @), 0.70 (A),and 1.05 (+). For inhibitor C, these were 0.07 (Uj, 0.10 W, 0.13 (A),and 0.20 ( + 1. No inhibitor (0).

rouleaux formation. The rouleaux in the guinea pig epididymis are precisely aligned, and their surface shows the presence of anionic sites (Enders and Friend, 1985). The sequence of dispersal in the testis and rouleaux formation during epididymal transit appears to be essential in the maturation of guinea pig sperm (Tung et al., 1980). Our hypothesis predicts that one of these low-molecular-weight inhibitors in the testis mediates dispersion of the sperm in that location and suggests that adding that particular inhibitor to

stacked epididymal sperm may result in their dispersion, in the same manner as did the Fab antibodies of Tung et al. (1980). Their protocol may be used to test this prediction of the hypothesis. The hypothesis further predicts the presence of this inhibitor in the seminiferous tubule lumen and its progressive loss from caput to cauda in the epididymal lumen, concomitant with sperm rouleaux formation. This prediction is accessible to experimental testing through immunocytochemistry .

38

E.A.O. FALASE ET AL. TABLE 3. Inhibitor Constants, Ki, Calculated From the Inhibition of Typsin-Catalyzed Hydrolysis of CBZ-Arg-AFC and MUGB

Haendle H, Fritz H, Trautschold I, Werle E (1965): Uber einen hormonabhangigen Inhibitor fur proteolytische Enzyme in mannlichen accessorischen Geschlechtsdrusen und im Sperma. Hoppe_Seyler’s Z Physiol Chem 3433855188. Ki (MI Irwin M. Nicholson N. Havwood JT, Poirier GR (1983): Immunofluorescent localization of murine seminal vesicle proteinase inhibSubstrate Protein A Protein B Protein Cnb itor. Biol Reprod 28:1202-1206. CBZ-Arg-AFCa 1.5 X lop8 1.5 X 2.2 X lop7 Jameson GW, Roberst DV, Adams RW, Kyle WSA, Elmore DT (1973): MUGB 7.7 x 10-9 6.7 x 10-9 1.4 x 10-7 Determination of the operational molarity of solutions of bovine alpha-chymoptrypsin, trypsin, thrombin, and factor Xa by spectro“Ki calculated u s i n g molecular w e i g h t s d e r i v e d f r o m active fluorimetric titration. Biochem J 131:107-117. site titration. Jonakova V, Cechova D (1985): Demonstration of an anionic inhibitor in spermatozoa, epididymal fluid, and seminal plasma of the boar. Andrologia 17:466471. Jonakova V, Cechova D, Meloun B, Veselesky L (1988): An acrosin inhibitor from boar seminal vesicle fluid immunologically related to Although the mammalian male reproductive tract the trypsin-kallikrein inhibitor (Kunitz). Biol Chem Hoppe-Seyler appears to contain low-molecular-weight proteinase 369[Suppll:43-49. inhibitors in all species, the various types found at the Layne E (1957): Spectrophotometric and turbidometric methods for measuring proteins. Methods Enzymol 3:447454. different locations appear to be species specific. This RA, Williams WL (1974): Biochemistry of mammalian ferconclusion is reinforced by the results reported here. McRorie tilization. Annu Rev Biochem 43:777-803. This raises the possibility that the differences in these Miyamoto H, Chang MC (1973): The importance of serum albumin and proteins reflect the differences in sperm processing metabolic intermediates for capacitation of spermatozoa and fertilization of mouse eggs in vitro. J Reprod Fertil 32:193-205. during maturation in the different species and thus N, Irwin M, Poirier GR (1983): Immunofluorescent localcould provide another approach to the study of the Nicholson ization of a seminal vesicle proteinase inhibitor in the female reactions involved in the sperm maturation process. reproductive tract of naturally inseminated mice. J Exp Zool 225:481-487. ACKNOWLEDGMENTS Poirier GR, Jackson J (1981): Isolation and characterization of two proteinase inhibitors from the male reproductive tract of mice. The authors thank Ms. Vanessa Johnson for secreGamete Res 4: 555-569. tarial assistance. This research was supported by NIH Poirier GR, Nicholson N (1984): Distribution of a proteinase inhibitor grants HD-06274 and HD-21926 and by a grant from of epididymal origin in the tissues and secretions of the male reproductive tract of mice. J Exp Zool 230:465-471. the Rockefeller Foundation. E.A.O.F. is the recipient of Poirier GR, Robinson R, Richardson R, Hinds K, Clayton D (1986): a Rockefeller Foundation Postdoctoral Fellowship. Evidence for a binding site on the sperm plasma membrane which recognizes the murine zona pellucida. Gamete Res 14:235-243. REFERENCES Polakoski KL, Williams WL (1974): Isolation of proteinase inhibitors Aarons D, Speake U, Poirier GR (1984): Evidence for a proteinase from boar sperm acrosomes and boar seminal plasma and effect on inhibitor binding component associated with murine spermatozoa. fertilization. In H Fritz, H Tschesche, W Greene, E Truscheit (eds): “Proteinase Inhibitors-Second International Research Conference. Biol Reprod 31911-817. Adekunle AO, Arboleda CE, Zervos PH, Gerton GL, Teuscher C Bayer Symposium V.” New York: Springer Verlag, pp 156-163. 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a

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Zaneveld LJD, Robertson RT, Kesseler M, Williams WL (1971): Inhibition of fertilization in vivo by pancreatic and seminal plasma trypsin inhibitors. J Reprod Fertil 25387-392. Zaneveld LJD, Srivastava PN, Williams WL (1969): Relationship of a trypsin-like enzyme in rabbit spermatozoa to capacitation. J Reprod Fertil 20:337-339. Zimmerman RE, Burch PJ (1978): The loss of a low molecular weight Exp acrosin inhibitor from acrosomes during capacitation. Proc SOC Biol Med 158:491495.