Molecular and Catalytic Properties of Rabbit Testicular Dipeptidyl Carboxypeptidase*

(Received for publication, June 9, 1982)

Hamza A. El-DorryS, Herbert G. Bull§, Kazushi Iwatal, Nancy A. Thornberry§, Eugene H. Cordes& and Richard L. SofferS1 From the Departments of *Biochemistry a n d !Medicine, Cornell University Medical College, New York, New York 10021 a n d SMerck Sharp and Dohme Research Laboratories, Division of Merck and Co., Znc., Rahway, New Jersey 07065

Rabbit testicular dipeptidyl carboxypeptidase activ- ity was purified by a procedure exploiting its affinity for N-a-[l-(S)-carboxy-3-phenylpropyl]-~-lys~l-~- proline. The molecular, catalytic, and immunological properties of the testicular enzyme are presented and compared with the corresponding properties of pul- monary angiotensin-converting enzyme. Although cat- alytically similar and immunologically related to pul- monary dipeptidyl carboxypeptidase, the testicular en- zyme has a molecular weight (100,000) which is lower by a factor of about one-third and differs in its NH2 and COOH termini. Furthermore, we present evidence that the testicular enzyme is not a post-translation product of the pulmonary type enzyme. These data suggest that testicular and pulmonary dipeptidyl carboxypeptidase are two distinct proteins which are catalytically similar and immunologically closely related.

COOH-terminal dipeptidyl carboxypeptidase activity (an- giotensin -converting enzyme, peptidyldipeptide hydrolase, EC 3.4.15.1) is present in the vascular endothelial cells of virtually all mammalian organs (1). The enzyme plays a key role in the control of blood pressure by converting angiotensin I to angiotensin 11, a powerful vasoconstrictor, and by inacti- vating bradykinin, a vasodilator (reviewed in ref. 2). The lung is believed to be the most important physiological site of this conversion (3). Dipeptidyl carboxypeptidase activity in the testis, however, differs from that in the lung, first, because it increases dramatically at puberty (1, 4) and is thus probably hormonally controlled, and second, because antipulmonary enzyme antibodies can discriminate between it and the pul- monary enzyme (5, 6).

As a first step toward establishing the physiological function of the testicular enzyme and the biochemical basis for the structural and regulatory differences between it and the pul- monary enzyme, we have now isolated and characterized the pure testicular enzyme. The purification has been facilitated by a step which takes advantage of its high affinity for N-a- [l-(S)-carboxy-3-phenylpropyl]-~-lysyl-~-proline, a recently described (7) potent inhibitor of pulmonary angiotensin-con- verting enzyme.

* This work was supported by Grant HL21394 from the National Institutes of Health. A preliminary report of some aspects of this study was presented at the 1981 meeting of the American Society of Biological Chemists, St. Louis, MO, June, 1981 (El-Dorry, H., Bull, H., Iwata, K., and Thornberry, N., Fed Proc. 40, 1039). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adver- tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper presents the molecular and catalytic properties of the purified testicular enzyme and their comparison with the properties of the enzyme from lung.

EXPERIMENTAL PROCEDURES

Materials

Pure angiotensin-converting enzyme (90 units/mg) from rabbit lung was prepared as described previously (8). The angiotensin-con- verting enzyme inhibitor, N-~-[l-(S)-carboxy-3-phenylpropyl~-~-ly- syl-L-proline (7) , both unlabeled and containing ''C a t positions 2 and 3 (50 pCi/pmol), was generously provided by Dr. A. Patchett of Merck. Captopril (3-mercapto-2-methyl-~-propanoyl-~-proline) was a gift from Dr. Z. Horovitz of Squibb. 1.4-Butanediol diglycidyl ether was a Sigma product, and Sepharose CL-4B was purchased from Pharmacia. [''SlMethionine (1185 Ci/mmol) was from Amersham Corp. Other products were those described in previous publications (5, 8).

Methods

Enzyme Assays-Dipeptidyl carboxypeptidase activity was deter- mined using as substrate Hip-His-Leu according to the method of Cushman and Cheung (9). A unit of activity is the amount which catalyzes release of 1.0 pmol of hippuric acid per min at 37 "C under the conditions described by them. Activity on angiotensin I containing a ['4C]leucyl residue at its COOH terminus was quantitated by the release of radioactive His-Leu (8, 10). Products of the enzymatic degradation of bradykinin were identified by paper electrophoresis (11).

Competition Radioimmunoassays-Goat antipulmonary enzyme antibodies were prepared as described previously (5). Antienzyme antibodies were elicited in adult white mice by intraperitoneal injec- tions of pure testicular or pulmonary dipeptidyl Carboxypeptidase (10 pg) contained in 125 p1 of 10 mM Tris-HCI, pH 7.8, and emulsified with an equal volume of complete Freund's adjuvant (Difco). Immu- nizations were given at intervals of 2 weeks, and maximal titers were usually attained after three injections. The antisera were heated a t 56 "C for 30 min. The radioimmunoassays were carried out as described previously (5) except that the secondary antibody was rabbit anti- mouse IgG which had been freed of serum angiotensin-converting enzyme by passage over DEAE-cellulose in 15 mM potassium phos- phate buffer, pH 8.3 (5). The radioiodinated testicular and pulmonary enzymes used as displaceable antigens (1-3 ng per assay) were labeled as described by Hunter (12) and contained, respectively, 2.6 X 10' and 1.8 X lo4 cpm/ng of protein. The final dilutions of mouse antisera against the testicular and lung enzymes were 1:750 and 1:2200, re- spectively.

Preparation of Inhibitor-Sepharose Gel-Sepharose CL-4B was activated and coupled to the amino group of the inhibitor through a bisoxirane spacer arm as described by Sundberg and Porath (13). Washed Sepharose CL-4B (100-ml packed gel, approximately 67 g, suction-dried cake) was stirred for 8 h at 23 "C in a solution made from 100 ml of 1,4-butanediol diglycidyl ether (approximately 70%') and 100 ml of 0.6 M NaOH containing 2 mg of sodium borohydride per ml. The activated Sepharose was then washed on a sintered glass funnel with 10 liters of water, and the filter cake, approximately 67 g, was stirred for 3 days a t 37 "C with 100 ml of 0.5 M KLCO., (pH 11) containing 2.2 mM inhibitor. Residual epoxide was then blocked by

14128

Testicular Dipeptidyl Carboxypeptidase 14129

treatment with 1 M glycine, pH 10, for 12 h at 37 "C. The gel was washed thoroughly with 0.5 M NaCl and water and was finally stored at 4 "C in 0.1 M NaHC0:r (pH 8.0) containing 1 M NaC1. The concentration of covalently bound inhibitor, estimated using the radioactive compound, was 1.0 mM in the packed gel.

Preparation of Radioactive Pulmonary Angiotensin-converting Enzyme-Fresh rabbit lung (4.4 g) was minced and shaken in 6 ml of Ringer bicarbonate (14) which contained 2 mg of glucose per m l , 300 pCi of L-["S]methionine, and 40 p~ of the other L-amino acids. Incubation was for 6 h at 37 "C in an atmosphere of 5% C02 and 958 02, after which the tissue was homogenized and its angiotensin- converting enzyme was solubilized by treatment with Nonidet P-40 (8). The solubilized enzyme was subjected to affinity chromatography as described below for testicular dipeptidyl carboxypeptidase. The resulting eluate (70 munits/pg) contained 1.95 X 10' cpm/pg and exhibited a single radioactive and Coomassie blue-reactive band after slab gel electrophoresis in the reduced denatured state.

Amino Acid Analyses-Amino acid analyses were performed by the method of Spackman et al. (15) on samples of the enzyme which had been hydrolyzed with constant boiling 5.7 N HCI in evacuated sealed tubes for 24 and 72 h in 110 "C. Methionine and half-cystine were estimated as methionine sulfone and cysteic acid on an aliquot of enzyme that had been oxidized with performic acid (16). Trypto- phan was measured after hydrolysis in 4 N methanesulfonic acid (17).

Carbohydrate Analysis-The content of N-acetylglucosamine and N-acetylgalactosamine was determined on the amino acid analyzer after hydrolysis of the enzyme with 4 N HC1 at 100 "C for 5 h. The amino sugars were separated by elution at a flow rate of 44 ml/h with sodium citrate buffer, pH 5.26 (0.158 N Na') at 70 "C (18). N- Acetylneuraminic acid was estimated after hydrolysis with 0.05 N HzSOi at 80 "C for 1 h as described by Aminoff (19). Other carbohy- drate residues were quantitated by gas-liquid chromatography (20).

Protein Determinations-Protein concentrations were determined by the method of Lowry et al. (21) using bovine serum albumin as the standard.

Isolation of mRNA and Cell-free Protein Synthesis-Male New Zealand White rabbits were killed by air embolism, and total RNA was isolated from the excised testes using the guanidine thiocyanate procedure described by Chirgwin et al. (22). RNA prepared in this manner had an AZ~Y,/AXHII value of 1.8-2.0. Poly(A)-containing RNA was then isolated by chromatography on oligo(dT)-cellulose (Type T-3, Collaborative Research, Waltham, MA) (23) and was estimated by using an A'"?,, value of 25.0. This RNA was translated for 90 min at 30 "C in a micrococcal nuclease-treated rabbit reticulocyte lysate system (24). The assay mixture (180 pl, pH 7.4) contained 60 ~1 of lysate (Bethesda Research Laboratories), 25 mM Hepes,' 10 mM creatine phosphate, 48 mM KC1, 87 mM potassium acetate, 1.2 mM MgCI,, 15 pg/ml of creatine kinase, a mixture of unlabeled amino acids (without methionine), 50 FM each, 200 pl of ['%]methionine (1100 Ci/mmol, New England Nuclear), and 30 pg of poly(A)-contain- ing RNA per ml. After cell-free translation, the reaction mixture was adjusted to 2% SDS, boiled for 3 min, and diluted 4-fold with 2.5% Triton X-100, 190 mM NaCI, 50 mM Tris-HC1, pH 7.5, and 6 mM EDTA. Goat anti-pulmonary angiotensin-converting enzyme antise- rum (15 pl) was then added, followed by 100 p1 of protein A-Sepharose. The total mixture was incubated overnight with end-to-end rotation. The immune complex-protein A-Sepharose was collected by centrif- ugation and washed with 20 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCI, 0.1%' SDS, and 0.5% Nonidet P-40. The antigen was eluted by boiling in 5 mM Tris-HC1, pH 6.8, containing 20% glycerol, 10 mM dithiothreitol, 2% 2-mercaptoethanol, and 5% SDS. The Sepharose was removed by centrifugation, and the super- natant was analyzed by electrophoresis on 7.5% polyacrylamide slab gels in the presence of SDS (25) and by fluorography using E:N.'HANCE (New England Nuclear).

RESULTS

Enzyme Purification-One of three similar preparations which yielded comparable results is described below and is summarized in Table I. All operations were performed at 0-4 "C.

Frozen rabbit testes (100 g) were cut into small pieces and homogenized for 90 s in a chilled Waring Blendor containing 200 ml of 10 mM Tris-HC1, p H 7.5. After passage through two

' The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-pipera- zineethanesulfonic acid; SDS, sodium dodecyl sulfate; Hip, hippuryl.

TABLE I Purification of dipeptidyl carboxypeptidase from lOOg of rabbit

testes

Vol- Total TZ::! Specific Recov- Fraction ume protein ity activity ery

ml mg units units/mp Homogenate 276 5360 911 0.17 100 Nonidet P-40 200 2200 836 0.38 92 Streptomycin superna- 190 2090 669 0.32 73

Affinity eluate (concen- 1.0 1.4 174 124 19

G-200 chromatography 1.0 0.92 154 167 17

tant

trate)

(concentrate)

layers of cheesecloth, the homogenate (276 ml) was centri- fuged at 16,000 X g for 1 h. The resulting pellet was suspended in 200 ml of Tris buffer, and Nonidet P-40 was added to a final concentration of 0.5% (v/v). The suspension was stirred for 4 h and centrifuged a t 16,000 X g for 1 h. T o the superna- tant solution (Nonideet P-40 extract, 200 ml) was added l g of streptomycin sulfate. This treatment was found to be essential for removing nucleic acid which was present in the testicular Nonidet P-40 extract in high quantity and interfered in the subsequent affinity column procedure. After stirring for 20 min, the turbid solution was centrifuged at 16,000 X g, and the streptomycin supernatant (190 ml) was adjusted to 0.1 M Hepes, pH 7.5, 0.3 M KC1, and 0.1 mM ZnC1.. It was then applied at a flow rate of 20 ml per h to an affinity column (1.5 X 60 cm) containing 1 pmol of bound inhibitor per ml of packed volume and equilibrated in the same Hepes buffer. The column was then washed first with 300 ml of buffer containing 0.5% Nonidet P-40 and then with buffer lacking detergent until the absorbance at 280 nm was below 0.01. Enzyme was finally eluted with buffer containing 0.1 mM inhibitor. Fractions were monitored by their absorbance at 280 nm, and those containing protein were pooled, concen- trated by ultrafiltration through a PM-10 filter, and dialyzed for 12-h intervals, first against 3 changes of 4 liters of 10 mM Tris-HC1, p H 7.5, containing 1 mM EDTA, and then against 4 changes of the same buffer lacking EDTA but supplemented with 100 PM ZnC12. After these dialyses, the affinity eluate was concentrated by ultrafiltration to a final volume of 1.0 ml and was subjected to gel filtration through a column (1.0 X 100 cm) of Sephadex G-200 which was equilibrated and de- veloped with 10 mM Tris-HC1, p H 7.5. Fractions were collected at a flow rate of 20 ml per h, and those having a specific activity greater than 160 were combined, concentrated, and stored frozen at -20 "C.

Two protein components were bound by the affinity column and specifically eluted by the inhibitor (Fig. 1). The minor protein (5%) probably corresponds to the pulmonary type of dipeptidyl carboxypeptidase (angiotensin-converting en- zyme), which is found in vascular endothelial cells throughout the body (26, 271, since it migrated identically with that enzyme during gel electrophoresis in the reduced denatured form with a mobility suggesting a molecular weight of 140,000 and since (not shown) both it and the major protein compo- nent were immunospecifically removed from solution by treat- ment with goat antipulmonary enzyme antibodies and donkey antigoat globulin. When radioactive pulmonary enzyme, which had been purified by the same affinity chromatographic procedure used for testicular dipeptidyl carboxypeptidase, was added to a crude testicular homogenate and the preparation was subjected to the standard isolation procedure, the radio- active protein was not converted to a smaller species (Fig. 1). This suggests that the testicular type of dipeptidyl carboxy-

Testicular Dipeptidyl Carboxypeptidase 14130

A

116- r? 94- 0 X 68- - 3

B C D E F

” - 0.4

- - E 0.: Y)

C . _ &

- ._ c z1 w ._ s 02 al

E, C W

0. I

FIG. 1. Slab gel electrophoresis of enzyme fractions. The reduced denatured proteins were subjected to slab gel electrophoresis through a 5-20’76 acrylamide gradient in the presence of 0.1% sodium dodecyl sulfate (25). Lanes A, B, C, and D were stained with Coo- massie blue and contained, respectively, 100 pg of the streptomycin supernatant, 5 pg of the affinity eluate, 7 pg of the pooled Sephadex G-200 eluate, and 5 pg of purified pulmonary angiotensin-converting enzyme. Lanes E and F a r e fluorograms developed after exposure to x-ray film a t -80 “C for 60 h. Lane E contained 2.0 X 10’ cpm of pulmonary angiotensin-converting enzyme that had been labeled with L-[:‘“S]methionine and purified as described under “Experimental Procedures.” Lane Fcontained equivalent counts of this material that had been mixed with crude testicular homogenate (50 pg of purified enzyme/ml of homogenate), taken through the streptomycin fraction- ation step of the isolation procedure, and then incubated a t 4 “C for 48 h. Protein molecular weight markers (positions indicated on the left side) included P-galactosidase, phosphorylase b, and bovine serum albumin.

peptidase does not arise by post-translation proteolytic cleav- age of the larger molecule (see below).

During affinity chromatography, approximately 25% of the applied enzyme activity was recovered. The loss of activity was almost certainly not due to residual inhibitor in the eluted enzyme since the specific activity was so high and since pilot experiments indicated that radioactive inhibitor could be com- pletely removed by the dialysis procedure described above. Furthermore, increasing the concentration of inhibitor 5-fold during the elution procedure did not augment the recovery of activity. The affinity column, which had a capacity of 7 units/ ml of packed volume, could be reused after extensive washing with 0.1 M Hepes, pH 7.5, and 1.0 M KC1 and then reequili- brating in the same buffer but with a concentration of 0.3 M KCl.

Physiochemical Properties-The testicular dipeptidyl car- boxypeptidase exhibited a mobility corresponding to a molec- ular weight of 100,000 after slab gel electrophoresis in the reduced denatured state (Fig. 1). Under the same conditions, the molecular weight of the pulmonary enzyme was calculated to be 140,000. The molecular weight of native pulmonary and testicular dipeptidyl carboxypeptidases was estimated by cen- trifugation of a mixture of the two enzymes on a glycerol gradient using the method of Martin and Ames (28) (Fig. 2). Molecular weight values of 145,000 and 94,000 were calculated

A 0 C + + + -

- 0.

- 0.

- 0.:

- 1

)P

FIG. 2. Density gradient centrifugation of purified testicular dipeptidyl carboxypeptidase. After determining its approximate sedimentation properties in the absence of other proteins, a sample (0.5 unit) of purified testicular enzyme was mixed with 0.7 unit of purified pulmonary angiotensin-converting enzyme and 0.5 mg of bovine liver catalase in a final volume of 0.2 ml. The mixture was subjected to centrifugation for 18 h at 39,000 rpm a t 2 O C through a 5-20% glycerol gradient (13 ml) in 10 mM Tris-HCI, pH 7.5. using an SW 41 rotor. The tube was then punctured at the bottom, and 26 fractions (0.5 ml) were collected and monitored for enzyme activity with Hip-His-Leu as substrate (0) and for absorbance at 404 nm (0). A, B, and C denote, respectively, the position of catalase, pul- monary angiotensin-converting enzyme, and testicular dipeptidyl car- boxypeptidase.

for the native pulmonary and testicular enzymes, respectively. Arginine (0.6 mol/mol of enzyme) was identified as the

NHp-terminal residue of testicular dipeptidyl carboxypepti- dase by the Edman procedure (29) followed by acid hydrolysis of the anilinothiazolinone (30). Serine (0.6 mol/mol of enzyme) was identified as the COOH-terminal residue by hydra- zinolysis (29). Analysis also yielded the expected correspond- ing threonyl and alanyl residues from pulmonary angiotensin- converting enzyme, confirming results obtained previously by the dansylation technique and by digestion with carboxypep- tidase A (8).

The amino acid compositions of the testicular and pulmo- nary enzymes are presented in Table 11. The higher content of tryptophanyl residue (68 pg/mg) probably accounts for the extraordinarily high absorbance value a t 280 nm of 2.9 dis- played by a solution containing 1.0 mg of enzyme protein per ml.

The carbohydrate component of the testicular enzyme (Ta- ble 11) accounted for about 20% of the weight of its combined aminoacyl and sugar residues. The composition differed from that of the pulmonary glycoprotein particularly in its rela- tively large content of galactosamine and in the absence of fucose.

Immunological studies indicate a high degree of similarity between the two protein chains despite the structural differ- ences described above. In the case of studies using antipul- monary enzyme antibodies, immune preparations from both goat and mouse were employed and yielded essentially iden- tical results. The data obtained with mouse preparations (Figs. 3 and 4) were chosen for presentation since that species was the only one immunized with the pure rabbit testicular en- zyme. In a competition radioimmunoassay using antipulmon- ary enzyme antibodies and radioiodinated pulmonary enzyme,

Testicular Dipeptidyl Carboxypeptidase 14131

TABLE I1 Amino acid and carbohydrate composition of rabbit testicular and

pulmonary dipeptidyl carboxypeptidase

Amino acid or carbohydrate Protein"

Testis Lung Testis Lung

w / m g rnol/molh Lysine Histidine Arginine Aspartic acid Threonine' Serine" Glutamic acid Proline Glycine Alanine Half-cystine" Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan' Fucose Mannose Galactose N-Acetylglucosamine' N-Acetylgalactosamine' N-Acetylneuraminic

acid

53.2 41.3 60.1

101.0 57.9 42.4

126.8 29.4 25.5 49.1 10.3 49.7 34.9 41.4 81.2 42.6 49.9 67.6

47.2 65.5 69.7 31.5 32.7

t l

37.7 34.2 31.4 24.8 61.5 31.8 83.1 72.6 36.0 47.2 34.3 40.2

106.1 81.2 52.0 25.0 27.2 36.9 40.4 57.1 8.0 8.2

38.4 41.3 22.0 22.0 32.7 30.2 78.9 59.3 55.0 21.6 50.5 27.9 49.0 30.0 5.7

65.0 77.0

109.0

55.0

37.9 29.3 51.0 93.3 45.6 51.0

106.4 69.5 61.8 73.8 5.4

50.0 21.7 37.0 90.0 43.4 44.5 33.6

~. . ~ ~~

'' All values are the average of two separate runs. Based on a molecular weight of 140,000 and 100,000 for pulmonary

"Corrected for decomposition by extrapolation of 24- and 72-h

" Determined as cysteic acid. e Determined after hydrolysis with 4-N-methanesulfonic acid (16). 'Determined as galactosamine and glucosamine after acid hydrol-

ysis and verified to be derived from N-acetylgalactosamine and N- acetylglucosamine, respectively, by gas-liquid chromatography (20).

and testicular enzyme, respectively.

values to zero time.

\ , I

$ 1 1.0 IO 100 1000

Competing antigen (m units) FIG. 3. Competition radioimmunoassays. The assays were car-

ried out as described under "Experimental Procedures." Results pre- sented in A were obtained using the radioiodinated pulmonary ell- zyme as displaceable antigen and antipulmonary enzyme antiserum (1:2200). Those in B represent experiments using the radioiodinated testicular enzyme as the displaceable antigen and antitesticular en- zyme antiserum (1:750). Competing antigens were the purified testic- ular (0) and pulmonary (0) enzymes.

the pure pulmonary glycoprotein was much more effective than the testicular enzyme as a competing antigen (Fig. 3A). As expected, in the control experiment carried out identically except that the testicular protein was used as displaceable antigen, the pulmonary and the testicular preparations yielded superimposable displacement curves (not shown). These re- sults indicated that the antipulmonary enzyme antibodies recognized determinants on the pulmonary protein which

- 2 4 6 8 1 0

Ant isero ( P I ) FIG. 4. Inhibition of testicular and pulmonary dipeptidyl

carboxypeptidase by antilung enzyme antibodies. Samples (5.0 milliunits) of purified testicular dipeptidyl carboxypeptidase (0) or pulmonary angiotensin-converting enzyme (0) were preincubated at 37 "C in 150 pl of 10 mM Tris-HC1, pH 7.5, containing 0.15 M NaCl, 1 mg of bovine serum albumin per ml, and the indicated amounts of heat-inactivated (56 "C, 30 min) antiserum from a mouse which had been immunized with the pure pulmonary enzyme. After 1 h of preincubation, residual enzyme activity was determined in the stan- dard assay (9) by addition of 100 pl of a solution containing 12.5 mM NaCl and incubation for an additional 30 min.

TABLE I11 Catalytic properties ofpulmonary and testicular dipeptidyl

carboxypeptidase Pulmonary Testicular

-

K," Hip-His-Leu (mM) 2.6 2.6 Angiotensin I (mM) 0.09 0.09

VmaX Hip-His-Leu (pmol/min/mg) 141 222 Angiotensin I (pmol/min/mg) 6.3 9.5

K,.," Hip-His-Leu (mol/min/mol) 18,120 18,500 Angiotensin I (mol/min/mol) 810 792

' I Based on a molecular weight of 140,000 and 100,OOO and a weight of 1.08 and 1.21 mg of enzyme per mg of protein measured by the Lowry procedure for pulmonary and testicular enzyme, respectively.

were not present in the testicular polypeptide. In striking contrast (Fig. 3B), when the radioimmunoassay was per- formed using antitesticular enzyme antibodies and radioiodi- nated testicular enzyme, identical displacement curves were generated with the preparations from lung and testis, ie. the antitesticular enzyme antibodies did not recognize any deter- minants of the testicular protein which were not also present in the pulmonary species. These data suggest that the enzyme from testis is immunologically closely related to the pulmo- nary polypeptide.

Catalytic Properties-The basic catalytic properties of the pulmonary and the testicular enzyme are presented in Table 111. Although the kinetic parameters of the pulmonary enzyme have been reported previously (8), they were redetermined in this study so that meaningful comparisons of the two enzymes, assayed under identical conditions, could be made. The data obtained for both enzymes plotted according to the method of Lineweaver and Burk are presented in Fig. 5. Both enzymes were found to have the same K , for Hip-His-Leu and angio- tensin I. Although the testicular enzyme displayed much higher V,,, values for both substrates, the corresponding turnover numbers, based on a molecular weight of 100,000 and a weight of 1.21 mg of enzyme per mg of protein measured by the Lowry procedure, are quite comparable to the pulmonary glycoprotein (see Table 111). Braydkinin (80 nmol) was com- pletely degraded by incubation with 20 milliunits of enzyme at 37 "C for 1 h. Phe-Arg and Ser-Pro, the COOH-terminal and penultimate dipeptidyl residues, were identified as reac- tion products. Activity of the enzyme with 5 mM Hip-His-Leu

14132 Testicular Dipeptidyl Carboxypeptidase

A - B 3 0.05- e

32 0.5 - c- " ._ 7 E- 0.04 a:

- ._ I .;

E: - 0.03-

. 3 & 0.02 - > o

\ -

1 I I I I , I , , # #

0.4 0.0 1.2 1.6 2.0 2.4 2 6 10 14

14132

I A

Testicular Dipeptidyl Carboxypeptidase

- I B 3 0.05- e 0.5 - 32 c- " ._ 7 E- 0.04 a:

- ._ I .;

E: - 0.03-

. 3 & 0.02 - > o

\ -

1 I I I I , I , , # #

0.4 0.0 1.2 1.6 2.0 2.4 2 6 10 14

1/Hip-His-Leu (mM")

was reduced more than 90% by omission of NaCl or by addition of 0.1 mM EDTA and 6.6 milliunits of enzyme activity and was inhibited 50% by the presence of 5.1 x 10"" M N-a- [ l-(S)-carboxy-3-phenylpropylJ-~-lysyl-~-proline (7) or 3.7 X 10"" captopril(31). In addition, the testicular and pulmonary enzymes were inhibited comparably by antipulmonary en- zyme antibodies (Fig. 4).

Cell- free Synthesis of Testicular Dipeptidyl Carhoxypepti- dase-Polyadenylated mRNA isolated from mature rabbits was translated in a mRNA-dependent rabbit reticulocyte ly- sate system in the presence of [""S]methionine, and the prod- ucts were immunoprecipitated using antiserum raised against pulmonary angiotensin-converting enzyme (shown in Fig. 3 to cross-react with testicular enzyme). The immunoprecipitate was analyzed by electrophoresis on polyacrylamide gels in the presence of SDS, and the protein pattern was visualized using fluorography. The immunospecific protein was observed to migrate faster than the corresponding authentic enzyme (Fig. 6) due to the absence of a glycosylation system in the in vitro protein system (32. 33). An apparent M , of 83,000 was calcu- lated for the testicular enzyme synthesized in uitro. This M , is 17,000 less than the value determined using the authentic glycosylated enzyme. The immunospecific radioactive band was found to be competitively eliminated when excess au- thentic unlabeled testicular enzyme was added to the trans- lation mixture prior to immunoprecipitation. In addition, when serum from nonimmune goat was used for immunopre- cipitation of the in vitro translation mixture, no specific bands were observed (data not shown).

DISCUSSION

Dipeptidyl carboxypeptidase activity in the male reproduc- tive system, unlike that elsewhere in the body, appears to be hormonally regulated (1, 4). The catalytic properties of a partially purified fraction of this exopeptidase from semen were found to be similar to those of pulmonary angiotensin- converting enzyme (34). This observation provided the ration- ale for adapting the recently developed competitive inhibitor of converting enzyme, N-a-[ l-(S)-carboxy-3-phenylpropyl]-~- lysyl-L-proline (7), to purification of the testicular dipeptidyl carboxypeptidase, and this strategy has enabled us to obtain the enzyme in pure form.

The polypeptide responsible for dipeptidyl carboxypepti- dase activity in most organs and body fluids is structurally and catalytically similar to pulmonary angiotensin-converting enzyme (2). However, our results indicate that the responsible testicular polypeptide is much shorter and contains different NH2- and COOH-terminal residues than the pulmonary en- zyme. In view of these structural differences and because there is no known function of angiotensin I1 in the male reproductive system, it seems appropriate to refer to this enzyme as testic- ular dipeptidyl carboxypeptidase rather than angiotensin-con-

FIG. 5. Double reciprocal Line- weaver-Burk plots. Pulmonary (0) and testicular (0) dipeptidyl carhoxy- peptidase were assayed as described un- der "Experimental I'rocedures" with varying levels of Hip-His-Leu ( A ) and angiotensin I ( B ) .

l/ongiotensin I (mM"1

1 2

rr) 116-

I 94-

3 x 68-

2 45-

FIG. 6. SDS-polyacrylamide gel electrophoresis of protein synthesized in translation mixtures programmed with RNA from mature testis. Lane 1 represents a mixture of radioiodinated authentic rabbit pulmonary and testicular enzymes. Lane 2 represents an immunoprecipitate obtained from a translation mixture directed with poly(A)-containing RNA from rahhit testis (see under "Experimental Procedures").

verting enzyme. Despite these structural differences, our data obtained with

antibodies against the two pure glycoproteins indicate that they are closely related. Thus their active sites are probably very similar, since antipulmonary enzyme antibodies inhibit the activity of both enzymes identically and since they exhibit indistinguishable requirements, substrate specificities, kinetic properties, and inhibitor profiles. That antipulmonary enzyme antibodies can recognize determinants of the pulmonary pro- tein which are not present in the testicular enzyme is to be expected since the pulmonary polypeptide is so much larger. However, antitesticular enzyme antibodies failed to identify determinants on the testicular enzyme which are absent from the pulmonary glycoprotein. The data from the in vitro trans- lation experiment suggests that the testicular enzyme is not generated post-translationally by proteolysis of pulmonary type angiotensin-converting enzyme. Furthermore, mRNA isolated from lung and translated in the same reticulocyte lysate system used for the in vitro synthesis of the testicular enzyme, produced an immunoprecipitable translation product with an M , of 129,000 (35). Thus it is likely that the difference in size of the two enzymes may be attributed to the existence of two related genes, a difference in the transcription of the same gene or a difference in the processing of the RNA transcript. It will be especially interesting to determine whether the biosynthetic mechanism responsible for the dif- ference in structure of the testicular dipeptidyl carboxypepti- dase also mediates its atypical regulatory properties.

Testicular Dipeptidyl Carboxypeptidase 14133

Acknowledgments-We are grateful to Anthony Tolvo and Dr. B. Saxena for performing the gas-liquid chromatographic analyses of sugar residues. We also thank Dr. C. Y. Lai for helpful advice concerning the Edman and hydrazinolysis procedures. We are grateful to I>rs. B. L. Horecker and dohn S. MacGregor for their helpful discussions.

1.

2.

3. 4. 5. 6.

7.

8. 9.

10.

11.

12.

13. 14.

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