Human prostatic gastricsinogen: The precursor of seminal fluid acid proteinase

ARCHIVES OF BWHEMISTRY Vol.

210, No. 1, August,

AND BIOPHYSICS 1981

pp. 14-20,

Human Prostatic Gastricsinogen: The Precursor Seminal Fluid Acid Proteinase LUCIANO

CHIANG,

Departamento

LEDA

de Ciencias

CONTRERAS,

Fisioldgicos, Universidad

JUAN

CHIANG,

Facultad de Ciencias de Conception, Received

AND

of

PETER

H. WARD

Biol&icas y de Recursos Natwales, Chile

Concepei&n,

August

14, 1980

A gastricsinogen-like acid proteinase precursor has been purified by DEAE-cellulose, DEAE-Sephadex A-50, poly-L-lysine-Sepharose 4B, and N-acetyl-L-phenylalanyl-L-tyrosine-Sepharose 4B affinity chromatography from human prostates. The active enzyme hydrolyzes acid-denatured hemoglobin at pH 1.0 and 3.0, while two other active fractions only showed the pH 3.0 activity and resembled cathepsin D (EC 3.4.23.5). The pH optimum, milk-clotting activity, specificity toward synthetic substrates, inhibition by pepstatin, and molecular weight strongly suggest that the prostatic-derived enzyme is identical to seminal fluid and to gastric juice gastricsin.

The existence in human semen of the precursor of an acidic proteinase was first described by Lundquist and Seedorff in 1952 (1). They showed that the seminal proenzyme was converted into a pepsinlike enzyme when the pH was lowered to 3-4 and suggested that the proenzyme was formed in the vesicular glands. Subsequently, it has been shown that human seminal fluid contains two pepsinogen fractions (2) which have the same electrophoretic mobilities and are immunochemically indistinguishable from the group II pepsinogens found in gastric and duodenal mucosae (3). More recently, Ruenwongsa and Chulavatnatol (4) isolated and partially characterized the seminal pepsinogen and concluded that it resembled gastric pepsin rather than gastricsin (5) which, in turn, corresponds to the group II enzymes described by Samloff and Townes (3). Our unpublished observations that human prostatic juice and both unstimulated and pilocarpine-stimulated dog prostatic secretions contain an acid proteinase precursor than on activation gives rise to an acidic proteinase with a pH optimum of 3.0 prompted us to look at the human pros0003-9861/81/090014-07$02.00/O

Copyright0 1981by AcademicPress,Inc. All rights of reproductionin anyform reserved.

tate as the organ most likely to synthesize and secrete this seminal zymogen. This communication deals with the purification and partial characterization of a zymogen from human prostate which on acid activation gives rise to an enzyme which is indistinguishable from that obtained from human semen and from gastric juice gastricsin. MATERIALS

AND

METHODS

Human prostates (250 g). not more than 12 h postmortem and kept frozen at -20°C were homogenized in four times their weight of 0.05 M Tris-HCl buffer, pH 7.2, and centrifuged at 9OOOg for 20 min. The supernatant was fractionated with ammonium sulfate (30-70% saturation). The resulting precipitate was dissolved in a small volume of the Tris-HCl buffer and, after extensive dialysis against the same buffer, was applied to a preequilibrated DEAE-cellulose column (4.5 X 45 cm) which was resolved with a salt gradient. The fractions which were eluted by the gradient and which were active on hemoglobin at pH 1.0 and 3.0 were rechromatographed on a DEAE-Sephadex A-50 column (3.5 X 45 cm) equilibrated with the above buffer but containing 0.2 M NaCl. This column was also eluted with a salt gradient. The proteolytically active fractions were pooled, dialyzed, and absorbed onto a poly-L-lysine-Sepharose 4B column (1 X 10 cm) as described by Nevaldine and Kassell (6). 14

HUMAN PROSTATIC GASTRICSINOGEN

15

showed a broad optimum pH range between 1.0 and 3.5 with a maximum at pH 3.0. This profile is essentially the same as that obtained with gastric juice gastricsin and with the seminal fluid enzyme (Fig. 5). Cathepsin D (Worthington) is inactive at pH 1.0 and has an optimum around pH 3.7. The same was true for enzyme fractions A and A’. The time course of inactivation at pH 7.96 of both the seminal and prostatic gastricsin-like enzymes is shown in Fig. 6. Clearly, both enzymes are rapidly inactivated at the same rate, while the prostatederived zymogen and the cathepsin D-like enzyme are stable at this pH value. The prostatic proteinase shares several other properties of the seminal and gastric juice gastricsin, namely, (a) it does not hydrolyze the synthetic dipeptide APDT which is a good substrate for human and porcine pepsin (12); (b) it is found in the form of its inactive precursor zymogen as shown by lack of milk-clotting activity at pH 5.3; (c) on acid activation the prostatic zymogen undergoes a molecular mass change from 34,000 to 31,000 daltons (Fig. RESULTS ‘7), which agrees well with the published Figure 1 shows the chromatographic value of 32,820 for porcine gastricsin (12). pattern of the ammonium sulfate fraction On the other hand, pepsinogen changes which was applied to the DEAE-cellulose from about 40,000 to 34,644 for porcine column. The first two break-through peaks pepsin (13, 14); (d) the enzyme clots milk (A and A’) were active at pH 3.0 but in- at pH 5.3 and is strongly inhibited by pepactive at pH 1.0 and resembled the prop- statin, as is gastric gastricsin (15); (e) the erties of cathepsin D (11). The third peak pH optima of gastric, semen, and prostate(B) was active at both pH values, and after derived gastricsin differ from that of clasbeing pooled, concentrated, and dialyzed ical pepsin which has an optimum pH it was rechromatographed, first on a of 1.0. DEAE-Sephadex A-50 column (Fig. 2) and then on a poly+lysine-Sepharose 4B colDISCUSSION umn (Fig. 3). Final purification was achieved on the APDT-Sepharose affinity The prostate gland and seminal vesicles column (Fig. 4). have been shown to secrete a wide variety The ammonium sulfate-fractionated of inorganic and organic constituents (14). protein was purified 900-fold (Table I) Their secretions are especially rich in enwith a final specific activity of 78.95 U/mg zymes which are normally associated with of protein; this value is essentially the the interior, rather than the exterior, misame as that of pure gastric gastricsin lieu of cells due to an apocrine, as opposed which, in the best of cases, has a specific to a merocrine, mechanism of secreactivity of 80 U/mg. tion (14). The acid-activated prostatic enzyme The acidic proteinase precursor obtained from human prostate homogenates i Abbreviations used: APDT, N-acetyl-L-phenylthat was eluted by the salt gradient from alanyl+diiodotyrosine; AH, 1,6-diaminohexane.

Final purification was achieved through the use of an original APDT’-Sepharose 4B affinity column (0.9 x 2.5 cm) which was prepared as follows: AH-Sepharose 4B (1 g) obtained from Sigma Chemical Company was hydrated in 10 ml of deionized water and washed with 200 ml of 0.5 M NaCl. The resin was finally suspended in 4 ml of dimethylformamide. APDT (7.5 mg), dissolved in 1 ml of dimethylformamide, was activated with 1.4 mg of Nhydroxysuccinimide (Pierce Chemical Co.) as described by Parikh et al. (7) After 6 h at room temperature and constant shaking, 4 ml of resin and 76.1 mg of l-ethyl-3-(dimethylaminopropyl)carbodiimide (Sigma Chemical Co.) were added and the reaction was allowed to proceed for a further 14 h under the same conditions. The APDT-Sepharose 4B resin so obtained was succesively washed with 100 ml of 0.1 M sodium acetate buffer, pH 3.5, containing 1 M NaCl and 100 ml of 0.1 M phosphate buffer, pH 8.0, also containing1 M NaCI. After packing, the resin was equilibrated with 0.1 M sodium acetate buffer, pH 5.3. Proteolytic activity, using acid-denatured hemoglobin as substrate, was assayed as described by Anson and Mirsky (8) and milk-clotting activity was assayed as previously described (9). Molecular weights of the zymogens and enzymes were estimated by gel filtration as described by Andrews (10).

16

CHIANG

ET AL.

a I

1.00

2.0

0.50

1. 0

a25

20

40

60

80

100

160

180

200

Fraction

220

240

260

280

.z

300

ND

FIG. 1. Elution profile of ammonium sulfate-fractionated human prostate extract. The resuspended and dialyzed fraction (5.2g of protein in 290 ml of buffer) was absorbed onto a microgranular, preswollen, DEAE 50 (Whatman) column (45 X 4.5 cm) equilibrated with 0.05 M TrisHCl buffer, pH 7.2. The column was resolved by a NaCl gradient as shown. Flow rate: 100 ml/h; fraction size: 20 ml.

%

120 10.0 80 6.0 4.0 2.0 1. 2 /

1.0

1 : E c

E c

1.0 -

22 2

0.8 -

53 2 a 0.5

0.6

-

0.4 -

,x ..? ;; Q

0.2

20

40

60

80

100

120

160 Fraction

180

200

220

240

260

280

300

NO

FIG. 2. DEAE-Sephadex A-50 chromatography of pool B. Sample: 1.92 g of protein in 100 ml of 0.03 M Tris-HCl buffer, pH 7.5, containing 0.2 M NaCl was absorbed onto a 3.5 X 45-cm column equilibrated with the same buffer. Flow rate: 40 ml/h; fraction size: 10 ml.

0.2

: I I

,

’ ’

I I ,

N a

’ I I I

% E c z

1

! ,

I 1

;

I

0.1

10

20

30

40 Fraction

50 No

60

70

60

90

FIG. 3. El&on profile of human prostatic gastricsinogen from a poly-L-lysine-Sepharose 4B affinity column. The column was loaded with 47 ml of sample (containing 35 mg of protein) and was eluted with a salt gradient as shown. Flow rate: 15 ml/h; fraction size: 3 ml.

.6

30

40 Fraction

50

60

70

80

90

No

WC. 4. APDT-Sepharose 4B affinity chromatography. The pooled fractions obtained from the polylysine column were equilibrated with 0.1 M sodium acetate buffer, pH 5.3. The sample was loaded (30 ml containing 7.8 mg of protein) onto a 0.9 X 2.5-cm column. Proteins were eluted by a stepwise increase in NaCl. Flow rate: 15 ml/h; fraction size: 3 ml; final yield: 950 pg of gastricsinogen.

18

CHIANG

ET AL.

TABLE PURIFICATION

OF PROSTATIC

Total protein bd

Step Ammonium sulfate fractionation DEAE-cellulose DEAE-Sephadex Poly-L-LysineSepharose 4B APDT-Sepharose 4B

a

ZYMOGEN

Total activity UJ)

Specific activity UJhd

Purification (n-fold)

5.200.00 1.920.06 35.00

460 335 194

0.088 0.174 5.540

1.00 1.98 63.52

(1W 72.8 42.2

7.80

87

11.154

126.75

18.9

0.95

75

78.947

897.12

16.3

the DEAE-cellulose column and that was subsequently further purified shares the same properties as the zymogen obtained from seminal plasma. The possibility that seminal “pepsinogen” is secreted by the prostate, as opposed to the seminal vesicles or other male accessory organs, arose from the following two unpublished observations: first, both human and dog prostatic secretions contain an inactive zymogen which on activation is active on

E c 0 2 a

I

GASTRICSINOGEN-LIKE

Yield (%I

hemoglobin and has a pH optimum of 3.0, and second, prostates from patients that underwent surgery for benign prostatic hypertrophy had little or no pH 1.0 activity compared to normal prostates. The presence of androgen-dependent secretory granules in rat prostatic tissue (17) also supports the hypothesis that this zymogen is synthesized and secreted by the prostate gland in a manner similar to that of the gastric zymogens (18). Studies are in prog-

040

0.30

0.20

I

I

310

5.0

PH

FIG. 5. Optimum pH profiles of enzymes obtained from: prostate (0, 10 pg/O.l ml); gastric gastricsin (0,8.2 rg/O.l ml); seminal plasma (A, 12 pg10.1 ml); and cathepsin D from pool A (A). For the first three curves, protein dilutions were made that would result in a AAm of about 0.600 when incubated at pH 3.0 for 10 min at 37°C. The cathepsin D curve has been amplified (88 wg gave a AAm of 0.130 at pH 3.5 when incubated for 2 h at the same temperature).

HUMAN

PROSTATIC

19

GASTRICSINOGEN

‘0°,

60 -

I 10

1 20 TIME

I 30 (min)

1 40

bo 60

FIG. 6. Stability of seminal fluid and prostatic gastricsin, gastricsinogen, and cathepsin D. Incubations were carried out in 0.2 M Tris-HCl buffer, pH 7.96, at 25°C. Aliquots were taken at the indicated times and quickly acidified and measured for proteolytic activity. Prostate gastricsin (0); seminal fluid gastricsin (A); prostatic cathepsin D (0); prostate gastricsinogen (0).

ress in order to evaluate if the simple determination of seminal plasma gastricsin reflects the functional status of the gland and if it can be correlated with other markers which have been described (16). Serum acid phosphatase activity is used as such a marker in the diagnosis of metastatic carcinoma of the prostate (19, 20).

5 I

FIG. 7. Molecular mass of human prostatic symogen and enzyme estimated by gel filtration on a 1.2 X 200-cm column of Sephadex G-75 calibrated as shown.

The active fractions that are not retained by the ion-exchange resin (Fig. 1) differed markedly in their pH stability profiles from the gastricsin-like proteinases. They are irreversibly inactivated at pH 1.0 (Fig. 5), are stable at pH 8.0 (Fig. 6.0), and present all the properties of lysosomal cathepsin D which is involved in intracellular protein turnover (21). The gastricsin-like proteinase, on the other hand, is highly active at pH 1.0 (Fig. 5), but is rapidly inactivated at pH 8.0 (Fig. 6) while the zymogen is stable; in fact, it was partially purified at pH 7.5 (Fig. 2) without loss of activity. The differential stability of the two enzymes at pH 1.0 has led us to the development of a simple assay for their estimation in tissue homogenates (22). Gastric juice gastricsin (5) and gastric mucosa gastricsinogen (23) have been shown to differ from classical pepsin and pepsinogen. Instead, they seem to be identical to the seminal and prostatic enzymes and their precursors. Samloff and Liebman (2) established the immunological

20

CHIANG

connection between seminal gastricsinogen and group II gastric pepsinogens. The molecular weights of the prostatic and seminal enzymes and zymogens also resemble those of gastric gastricsinogen (23) and gastricsin (24) and differ quite markedly from the molecular weights of gastric pepsinogen (40,000) and pepsin (34,600). From the above we feel that it can be safely concluded that prostate gastricsinogen is equivalent to gastric and seminal gastricsinogen and that the latter is synthesized and secreted by the human prostate gland. ACKNOWLEDGMENTS This work Vice Rectoria Conception.

was supported by Grant 299.52 de Investigaciones, Universidad

from de

REFERENCES 1. LUNDQUIST, F., AND SEEDORFF, H. H. (1952) Nuture (London) 170, 1115-1116. 2. SAMLOFF, I. M., AND LIEBMAN, W. M. (1972) Cl&. Exp. Immunol. 11.405-414. 3. SAMLOFF, I. M., AND TOWNES, P. L. (1970) Gas-

troenterology 4. RUENWONGSA,

58.462-469. P., AND CHIJLAVATNATOL,

M. (1975)

J. Biol. Chem. 250, 7574-7578. 5. TANG, J., WOLF, S., CAPUTTO, R., AND TRUCCO, R. E. (1959) J. Biol. Chem. 234, 1174-1178. 6. NEVALDINE,

B., AND KASSELL,

B. (1971)

Biochim.

Biqphys. Acta 250,207-209. 7. PARIKH, (1974)

I., MARCH, in Methods

S., AND CUATRECASAS, P. in Enzymology (Jakoby,

ET AL. W. B., and Wilchek, M., eds.), Vol. 34, pp. 77102, Academic Press, New York. A. E. (1932) J. Gen. 8. ANSON, M. L., AND MIRSKY, Physiol. 16, 59-63. B. (1968) J. Biol. 9. CHOW, R. B., AND KASSELL, Chem. 243, 1718-1724. P. (1964) Biochem. J. 91. 222-233. 10. ANDREW& 11. BARRETT, A. J. (1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A. J., ed.), pp. 209-248, North-Holland, Amsterdam. L., SANCHEZ-CHIANG, L., MILLS, J. N., 12. CHIANG, AND TANG, J. (1967) J. Biol. Chem. 242, 30983102. 13. WILLIAMS, R. C., JR., AND RAJAGOPALAN, (1966) J. Biol. Chem. 241, 4951-4954.

T. G.

14. TANG, J., SEP~LVEDA, P., MARCINISZYN, J., JR., CHEN, K. C. S., HUANG, W. Y., TAO, N., LIU, D., AND LANIER, J. P. (1973) Proc. Nat. Acad

Sci. USA 70,3437-3439. 15. AOYAGI, T., KUNIMOTO, S., MORISHIMA, H., TAKEUCHI, T., AND UMEZAWA, H. (1971) J. Antibiot. 24.687-694. 16. MANN, T. (1974) J. Reprod. Fert. 37,179-188. 17. THOMPSON, S. A., ROWLEY, D. R., AND HEIDGER, P. M., JR. (1979) Invest. Ural. 17,83-89. 18. LILLIBRIDGE, C. B. (1964) Gastroenterology 47,

2694290. YAMN, L. T. (1974) Amer. J. Med. 56,604-616. VIHKO, P. (1979) Invest. Ural. 16,349-352. KAY, J. (1978) B&hem. Sot. Trans. 64, 789-797. WARD, P. H., CONTRERAS, L., MALDONADO, M., ANDCHIANG, L. (1980) IRCS Med Sci-Biochem. 8, 830-831. V. K., AND CHIANG, L. 23. WARD, P. H., NEUMANN, (1978) Comp. Biochem. Physiol. B 61,491-498. 24. TANG, J., MILLS, J., CHIANG, L., AND DE CHIANG, L. (1967) Ann. N. Y. Acad. Sci. 140, 688-696.

19. 20. 21. 22.