Dipeptidyl peptidase III and alanyl aminopeptidase in the human seminal plasma: Origin and biochemical properties

179

Clinica Chumca Acta. 177 (1988) 179-196 Elsevier

CCA 04281

Dipeptidyl peptidase III and alanyl aminopeptidase in the human seminal plasma: origin and bioche~cal properties Department Key words: Dipeptidyl

Tapani Vanha-Perttula ofAnntom~~, Uniuerst
Kuopio fFin!and)

Enzyme separation;

III; Alanyl aminopeptidase:

Human seminal plasma

Human seminal plasma contained two distinct enzyme activities hydrolysing ArgArgNA. The enzymes were separated by anion exchange chromatography and further purified by gel filtration and/or hydrophobic interaction chromatography. The enzyme eluting at the lower NaCl concentration (0.26 mol/l) displayed an optimum at pH 5.7-6.0 (enzyme A), while the other enzyme eluted at 0.32 mol/l NaCl and showed an optimum at pH 8.5-9.0 (enzyme B). Enzyme A was found to coelute with an a~nopeptidase which hydrolysed various amino acid derivatives as well as dipeptide naphthylamides sequentially. Both enzymes were sensitive to heavy metal ions (Cd, Cu, Hg, Pb) and chelating agents (EDTA, o-phenanthroline) and moderately sensitive to di-isopropylfluorophosphonate (DFP) or phenylmethylsulfonyl~uo~de (PMSF). After EDTA suppression both activities were partially reactivated by divalent metal ions, particularly by Co2+. Enzyme A was highly sensitive to amastatin, bestatin and puromycin, while enzyme B was not markedly influenced. With different substrates the modifier characteristics of enzyme A were equal. High concentrations of some substrates suppressed the hydrolysis rates of both enzymes. Enzyme B was much more sensitive to the thermal treatment than enzyme A. Tentative molecular masses of 110 kD and 80 kD were obtained for enzymes A and B, respectively. Enzyme B was found in all male reproductive tissues (testis, epididymis, vas deferens, ampulla, seminal vesicles, prostate), while enzyme A was only detected in the prostatic homogenate. Thus, ArgArgNA in the human seminal plasma is hydrolysed by dipeptidyl peptidase III, which may originate from different reproductive organs, while the prostate is responsible for the secretion of an aminopeptidase with a wide substrate spectrum including dipeptidyl derivatives.

Correspondence 70211 Kuopio.

~9-S981/88/$03.50

to: Prof. T. Vanha-Perttula, Finland.

Department

0 1988 Elsevier Science Publishers

of Anatomy,

University

B.V. (Biomedi~l

of Kuopio,

Division)

P.O. Box 6.

Introduction Dipeptidyl peptidase III (DPP III; EC 3.4.14.4) has been recognized as a ubiquitous cytosolic exopeptidase in mammalian tissues [l-3]. It prefers ArgArgNA as substrate with optimum at alkaline pH (8.5-9.0). The enzyme has serine at the active center [3]. Moreover, the enzyme is sensitive to thiol reagents (e.g. mercury) and chelating agents (EDTA) with re-activation by thiol compounds and divalent metal ions after suppression [l--5]. In the rat skin [4] and human erythrocytes [6]. however, DPP III is unaffected by EDTA. Partially purified enzyme has been shown to split dipeptide residues from some oligopeptides (Ala,, Ala,, Lys,, Phe,. Val-Leu-Ser-Glu-Gly) [l-4] and biologically active polypeptides (angiotensins, enkephalins) [2-3,7]. Our recent study [S] indicated that high activity of DPP III is present in the epididymis of the mature bull with lower levels in other reproductive tissues. It appears to be hormonally controlled, since negligible levels were encountered in the immature animal. The DPP III levels in the bull seminal plasma were very low in comparison to those of DPP II and DPP IV suggesting a non-secretory nature of the tissue activities. A suppression of the enzyme was obtained with heavy metals (Cd. Cu, Pb, Hg, Ni, Zn) and a serine protease inhibitor (PMSF) but not with EDTA. Human seminal plasma contains a high activity of DPP II with LysAlaNA as substrate, while the thiol-activated DPP I with SerTyrNA as substrate is very low [9]. LysAlaNA was additionally hydrolyzed sequentially by the seminal plasma alanyl aminopeptidase [9]. Recently, evidence has been presented that DPP IV with GlyProNA as substrate is also secreted into the human seminal plasma [lo], although the origin of these enzymes from the human male reproductive organs remains obscure. In this study, two distinct enzymes hydrolysing ArgArgNA have been identified in human seminal plasma and their biochemical properties and origin were analyzed.

Materials and methods Materials ArgArgNA, AlaNA and other /3-naphthylamide substrates, antipain, leupeptin, N-cu-tosyl-L-lysine-chloromethylketone . HCl (TLCK) and N-p-tosyl-L-phenylalanine-chloromethylketone (TPCK) were obtained from Bachem Feinchemikalien (Bubendorf, Switzerland). Coomassie brilliant blue G-250 and acrylamide were purchased from Eastman Kodak Co. (Rochester, NY, USA), amastatin HCI, acid (EACA), benzamidine HCl, bestatin HCl, di-isoproc-amino-n-caproic N-ethylmaleimide, pepstatin A, opylfluorophosphonate (DFP), leupeptin, phenanthroline (o-Phe), phenylmethylsulfonylfluoride (PMSF), puromycin diHC1, Fast Garnet GBC and tris(hydroxymethyl)-aminomethane (Tris) from Sigma Chemical Co. (St. Louis, MO, USA), the molecular weight standards, Polybuffer 74 and 96 from Pharmacia Fine Chemicals (Uppsala, Sweden) and Triton X-100 from BDH Chemicals (Poole, UK). Metal salts, dithioerythritol (DTE), p-dimethylaminoben-

181

zaldehyde (p-DMAB) and other chemicals and reagents of analytical grade were products of E. Merck AG (Darmstadt, FRG). Human semen samples of normal sperm density (> 20 million/ml) and motility (> 40% motile) were obtained from donors admitted to the Fertility Clinic of Kuopio University Central Hospital. The samples were collected into sterile acidwashed containers by masturbation after 3 days of abstinence. Spermatozoa were separated from seminal plasma by centrifugation at 600 x g for 10 min at 4’ C. Seminal plasma was recentrifuged at 100 000 x g for 2 h at 4’ C. The supernatant was called seminal fluid and the pellet consisted of the prostasomes [9]. Human reproductive tissues were collected at autopsy from 5 adult human males within 3 days after death. The reproductive tract was removed and the accessory glands, testes, and epididymides were dissected free of fat and connective tissue. The epididymis was divided into 6 segments (E,_, in caput, E, in corpus, E,-E, in cauda). Methods Enzyme assays The hydrolysis of the /3-naphthylamide substrates was carried out in a water bath at 37O C. The standard assay mixture contained 100 ~1 of a buffer specified later without or with a modifier, 100 ~1 of the enzyme solution (diluted seminal sample, tissue homogenate or chromatographic fraction) and 100 ~1 of substrate solution (1 mmol/l distilled water). After the incubation for various time periods, the reaction was terminated with 500 ~1 of p-DMAB solution (equal volumes of p-DMAB, 10 g/l in methanol and 1 mol/l sodium acetate buffer, pH 1.4, mixed immediately before use). The absorbances were read at 450 nm in a spectrophotometer. For calibration. a series of /3-naphthylamine dilutions were prepared and the absorbances were measured after the addition of the p-DMAB solution. The protein in the fractionations was followed by absorbance at 280 nm, while in the tissue and seminal samples as well as pooled enzyme preparations the protein concentrations were measured with the Coomassie brilliant blue method [ll]. The unit of enzyme activity is given as pmol /3-napthylamine released in min/mg protein or absorbances at 450 nm after a specified incubation time. Sample preparation The tissue samples were homogenized with a Potter-Elvehjem glass-Teflon homogenizer in 0.025 mol/l imidazole-HCl buffer, pH 7.4, with 0.01% Triton X-100 added, sonicated with a 150 Watt Ultrasonic Disintegrator (MSE Ltd., Crawley, Sussex, UK) and centrifuged at 40000 X g for 1 h. All procedures were carried out at 4O C. The particulate prostasome pellet was similarly handled. The supernatants were used for fractionations or for enzyme assays after proper dilution with distilled water or the 0.025 mol/l imidazole buffer, pH 7.4. Fractionations Anion exchange chromatography (AEC) was carried out on a Q Sepharose Fast Flow (Mono Q FF) column (Pharmacia Fine Chemicals) attached to a high-performance liquid chromatography system (HPLC; Altex, Berkeley, CA, USA). Gradient elution (O-O.4 mol/l NaCl in 0.025 mol/l imidazole-HCl buffer,

pH 7.4) was programmed, and the remaining protein was eluted with 2 rnol/l NaCl solution. Fractions of 1 ml/min were collected at room temperature. Chromatofocusing (CF) at room temperature was performed on a Mono P column (Pharmacia) attached to the HPLC system. A pH-gradient of pH 7.4 or pH 8.5 was developed with Polybuffer 74 or Polybuffer 96 (Pharmacia) at a flow rate of 1 ml/min. After the gradient, 2 M NaCl was used to elute any remaining activity from the column. Fractions of 1 ml were collected at room temperature. Hydrophophic interaction chromatography (HIC) was carried out on an Octyl-Sepharose CL4B (Pharmacia) column (0.9 x 30 cm) balanced with 1 mol/l ammonium sulphate in 0.02 mol/l Tris-HCl, pH 8.0. containing 0.05 mol/l NaCl. The sample in the same solution was applied to the column and eluted at a flow rate of 0.27 ml/min with a linear gradient of ammonium sulphate (1.0-0.0 mol/l) containing an increasing gradient of Triton X-100 (O-OS%) at 4’C. Fractions of 2.7 ml were collected. Gel filtration (GF) was carried out on Sephacryl S-300 column (1.6 X 89 cm) at 4” C or on a Superose 6 prepacked column (Pharmacia) at room temperature. The columns were eluted with 0.025 mol/l imidazole-HCl buffer. pH 7.4, containing 0.15 mol/l NaCl. Fractions of 2.7 ml and 0.25 ml were collected with Sephacryl S-300 and Superose 6 columns, respectively. The corresponding flow rates were 0.27 and 0.25 ml/min. After fractionations, active fractions were occasionally pooled and concentrated with an Amiconultracentrifugation apparatus with PM 30 membrane. The sample was then eluted in another fractionation.

Electrophoresis The purity of the enzyme preparations was checked by polyacrylamide gel electrophoresis (PAGE) in a 5-15% acrylamide gradient gel with standard proteins (Pharmacia) for calibration. The gels were stained with Coomassie brilliant blue for protein and with AlaNA (1 mmol/l in 0.1 mol/l Tris-HCl, pH 7.0) or ArgArgNA (1 mmol/l in 0.1 mol/l Tris-HCl, pH 7.5) as substrates and Fast Garnet GBC (0.5 mg/ml) as a coupling agent for the demonstration of enzyme activity.

Enzyme characteristics The effect of modifiers and pH on the pooled enzyme fractions was studied as reported earlier [8-91. The hydrolysis rates of amino acid and dipeptidyl naphthylamides were studied at 1 and 0.1 mmol/l final concentrations. Additional studies were carried out with 12 substrate concentrations in the range of 0.01-3.0 mmol/l. The effect of temperature was analyzed by preincubating the pooled enzyme sample for 15 min at the temperatures indicated. After incubation the samples were cooled on ice and the enzyme activity measured in the whole series at the same time. The approximate relative molecular masses ( M,) of the enzymes after GF on Superose 6 column were determined with Blue Dextran 2000 (I$), catalase (M, 232 kD), aldolase (M, 158 kD), bovine serum albumin (BSA; M, 67 kD), ovalbumin (M, 43 kD), chymotrypsinogen A (25 kD) and tyrosine (mol wt 181.2; V,) as standards.

lR3

Results

Initially, a pooled human seminal plasma was used as an enzyme sample to study the hydrolysis of ArgArgNA in a pH-series from pH 3.0 to 9.5. When a seminal

H bd o-o -----

14

Mom

ArgAqNA. pH 60 AlaNA. pH 70 ArgArgNA. pH 90 Protm

A

0 FF

SEMINAL

-A-

PLASMA

A

04 03

7

02

3

01

8 2

I

0

O-0

A-a 02 04 t

O-O

pH 6 0 (Pool A) ArgArgNA. AiaNA. pH 70 (Pool A) pH 90 (Pool 8) ArgArgNA,

ArgArgNA

05

pH 6 0 _ -5 AlaNA, pH 70 GIyRoNA. pH 80 _ .._

H b-c~ 013

_ * _’

Li

7 10

04

i I

*.r _ .,**

”

L”

40

Go

2

*_

E

80

O3 8 02

z 5

01

c; E

_O E#

FRACTION NUMBER

Fig. 1. Fractionations during the purification of seminal plasma enzymes. ArgArgNA hydrolysis was carried out at pH 6.0 and 9.0 with an incubation time of 2 h at 37 o C, while AiaNA hydrolysis at pH 7.0 and GlyProNA hydrolysis at pH 8.0 were terminated after an incubation of 5 min at 37’ C. A. AEC of seminal fluid on Mono Q FF column. The active fractions (pools A and B) were combined. B. Pools A and B fractionated separately by GF in Superose 6 column. The elution sites of marker proteins are indicated. C. HIC of pool A on Octyl-Sepharose CL-4B column eluted with combined ammonium sulfate and Triton X-100 gradients.

184

plasma sample was eluted in GF on Sephacryl S-300, a rather wide activity peak was obtained with ArgArgNA as substrate and after pooling an acid (at pH 6) and an alkaline (at pH 8.5) maximum were found in the pH-series (data not shown). Elution of human seminal plasma in AEC on Mono Q FF column resulted in two main hydrolytic peaks A and B when the assays were carried out at pH 6.0 and pH 9.0 as well as a variable activity at the beginning (Fig. 1, upper panel). After homogenization in a buffer containing 1% Triton X-100 and rechromatography on Mono Q FF column, the latter activity eluted at the site of peak A. The peak A eluting at 0.26 mol/l NaCl concentration was much higher when assayed at pH 6.0, while the other peak B eluting at 0.32 mol/l NaCl was higher in the assay at pH 9.0. The pooled peaks resulted in two clearly distinct optima: the former displayed optimum hydrolysis at pH 5.7-6.0 and is called enzyme A, while the latter had an optimum at pH 8.5-9.0 and is designated as enzyme B. With AlaNA as substrate the major activity eluted with peak A (Fig. 1, upper panel). A smaller peak at the beginning of the gradient was also eluted at the same site after solubilization with Triton X-100. An optimum for the AlaNA hydrolysis was obtained at pH 7.0. The pooled enzymes A and B were separately applied on Octyl-Sepharose CL-4B column and eluted with a decreasing ammonium sulphate and increasing Triton X-100 gradient. Pool A resulted in a single activity peak which coeluted with the hydrolysis of AlaNA but was separate from the GlyProNA hydrolysis by DPP IV at pH 8.0 (Fig. 1, lower panel). Pool B in the same fractionation gave a single peak for ArgArgNA hydrolysis with no coincident AlaNA hydrolysis. The yield of the enzyme was, however. low in this fractionation. After HIC fractionation enzyme A was eluted in Superose 6 column as a single peak again coincident with AlaNA hydrolysis (Fig. 1, middle panel). An identical fractionation of enzyme B also gave a single peak, which eluted slightly later than enzyme A, A typical purification procedure of enzymes A and B is summarized in Table I. In PAGE on native gradient gel, the purified enzyme A resulted in a band at about 110 kD, which also gave an enzyme reaction with AlaNA as substrate (Fig. 2). Another band was found close to albumin but it showed no enzyme reaction. The same band appeared even after re-electrophoresis of the active band after its elution from the first PAGE gel. It may thus represent an enzyme subunit dissociated during PAGE. Enzyme B gave a single band at about 80 kD. but no enzyme reaction could be obtained with ArgArgNA as substrate even after a prolonged incubation. A variety of amino acid and dipeptidyl-P-naphthylamides were used at two different concentrations (0.1 and 1 mmol/l) in the analysis of relative hydrolysis rates by the two pooled enzymes (Tables II and III). The results show that enzyme A is able to split readily numerous amino acid and dipeptide derivatives resulting in the release of free P-naphthylamine. On the other hand, enzyme B had the ability to attack only ArgArgNA and less readily some other dipeptide derivatives but not at all the amino acid-P-naphthylamides. The relative hydrolysis rates of dipeptidyl substrates by enzyme B remained rather similar at the two substrate concentrations. while both amino acid and dipeptidyl substrates showed marked differences in the relative hydrolysis rates by enzyme A at the two substrate concentrations. Using a series of substrate concentrations (0.01-3 mmol/l) the enzyme reaction was found

1

Seminal fluid Mono Q FF OctylSephrose Suverose

Purification step

100 65 32 27

9 289.8 6038.4

2 972.7 2 548.5

304.2 35.9

9.0 4.1

330.3 621.6

30.5 168.2 10.8 20.4

1 5.5

Protein

3.1

304.2 5.3

(mg)

(U)

(U/m&

Yield (%)

Total act.

Protein

peptidase

263.3

1755.0 421.2

(U)

Total act.

and dipeptidyl

(mg)

Purification fold

(1 mmol/l)

DPP III Spec. act.

(AAP) with AlaNA

AAP

Summary of the purification of alanyl aminopeptidase as substrates from the human seminal plasma (5 ml)

TABLE

15

100 24

(W)

Yield

III (DPP

_

84.9

5.8 80.0

(U/m@

Spec. act.

III) with ArgArgNA

14.6

1 13.8

Purification fold

(0.06 mmol/l)

- 232 - 440

- 669

-+--A

I3

C

D

E

Fig. 2. PAGE of the purified and concentrated enzymes A and B. Coomassie brilliant blue staining of A. Protein markers I (thyroglobu~n 669 kD, catalase 232 kD, lactate dehydrogenase 140 kD, bovine serum albumin 67 kD). B. Enzyme A. C. Enzyme B. D. Protein markers II (ferritin 440 kD. lactate dehydrogenase 140 kD, phosphorylase B 94 kD, bovine serum albumin 67 kD, ovalbumin 43 kD, carbonic anhydrase 30 kD, soybean trypsin inhibitor 20.1 kD, a-lactalbumin 14.4 kD). E. Enzyme A demonstrated with AIaNA (1 mmol/l) as substrate and Fast Garnet GBC as a coupling agent. TABLE

11

Relative hydrolysis rates of different dipeptidyl-@-naphthylamides (pH 8.5) at 0.1 and 1 mmol/l substrate concentrations Substrate

ArgArgNA AlaArgNA AlaAlaNA LeuAlaNA LysAlaNA LysLysNA SerTyrNA AspArgNA GlyProNA GlyPheNA TosArgNA Cbz-ArtirgNA

by enzyme

Enzyme

Enzyme A

A (pH 6.0) and enzyme

B

0.1 mmol/l

1 mmol/l

0.1 mmol/l

1 mmol/l

100 156 293 237 137 38 225 0 0 0 0 0

100 217 649 507 152 8 94 0 0 0 0 0

loo 54 7 2 1 2 0 0 0 0 0 0

100 50 6 2 2 2 0 0 0 0 0 0

B

187

TABLE

III

Relative mmol/l

hydrolysis rates of different substrate concentrations

amino

Substrate

Enzyme A

AlaNA MetNA LeuNA GlnNA PheNA TyrNA ArgNA LysNA TrpNA ThrNA ValNA y-GluNA a-GluNA AspNA CysNA ProNA PyrNA

100 129 63 44 27 14 18 22 9 6 8 17 3 0 0 0 0

0.1 mmol/l

acid+-naphthylamides

by enzyme

A (pH 7.0) at 0.1 and 1

1 mmol/l 100 84 35 20 29 17 18 12 8 8 4 8 4 0 0 0 0

to be suppressed by some amino acid substrates (MetNA, LeuNA, GlnNA, ArgNA, LysNA, y-GluNA) at concentrations above 0.2 mmol/l and by all dipeptidyl substrates at variable higher concentration levels. The maximum hydrolysis of

H 12

/

H 0-o O-C

pool PCIOI Pool PWI

A A.Co B B.Co

w 08

Y 8 ;

06

$ 04

0’

0

02

04 ArgArgNA

06

08

10

(mM)

Fig. 3. Hydrolysis of ArgArgNA at various concentrations. The purified enzymes A (pH 6.0) and B (pH 9.0) were incubated with or without Co ‘+ (1 mmol/l) at various substrate concentrations for 30 min at 37 o C. The blank values at each concentration were subtracted.

0

10

30

20

TIME

40

50

60

(mln)

Fig. 4. Time-dependence of enzyme activity. The hydrolysis of ArgArgNA (pH 6.0) and AlaNA (pH 7.0) by the purified enzyme A and that of ArgArgNA (pH 9.0) by enzyme B were followed for different time periods. The results are given as absorbance at 450 nm.

ArgArgNA (Fig. 3). SerTyrNA, LysAlaNA and LysLysNA by enzyme A was found already at concentration of 0.04-0.1 mmol/l, while the maximum activity with AlaAlaNA, LeuAlaNA and AlaArgNA was reached at 0.2-0.5 mmol/l substrate concentration. The hydrolysis of ArgArgNA (0.1 mmol/l) by enzyme B at pH 9.0 and AlaNA (1 mmol/l) by enzyme A at pH 7.0 was linear with time but the hydrolysis of ArgArgNA (Fig. 4) and also other dipeptidyl substrates at pH 6.0 by enzyme A was curvilinear. When the pooled enzymes were exposed to various temperatures (37-75°C) for 15 min, the activity of enzyme B was much more sensitive than that of enzyme A (Fig. 5). At 55” C the former enzyme lost all its activity, while the latter still maintained its full activity both with AlaNA and ArgArgNA as substrates.

LO

50

60

TEMPERATURE

70 (‘0

Fig. 5. Thermal inhibition of enzyme activity. The purified enzymes A and B were exposed to different temperatures for 15 mm, after which the hydrolysis of ArgArgNA was carried out at pH 6.0 for enzyme A and at pH 9.0 for enzyme B for 60 mm at 37 o C. The results are given as the percentage of activity remaining.

189

TABLE

IV

Effect of modifying agents on the hydrolysis of AlaNA (pH 7.0) and ArgArgNA (pH 6.0) by enzyme A as well as on the hydrolysis of ArgArgNA (pH 9.0) by enzyme B. The results are given as percentage from the controls without any modifier added (100%) Modifier

Ba Ca co Cd CU Hg Mg Mn Ni Pb Sr Zn EDTA o-Phe Cystein DTE IAA NEM Amastatin Antipain Bestatin Leupeptin Puromycin Pepstatin A TLCK TPCK Benzamidine EACA DFP PMSF bis-p-NPP

Cone (mmol/l) 1 1 1 1 1 0.1 1 1 1 1 1 1 1 1 1 1 1 1 0.001 0.1 0.001 0.1 1 0.1 1 1 1 1 1 1 1

Enzyme

Enzyme

A

AlaNA

ArgArgNA

ArgArgNA

85 106 135 14 6 36 101 97 82 2 102 24 60 3 112 101 84 98 0 91 4 100 6 94 89 16 103 103 75 93 102

110 94 1.51 32 5 16 97 93 85 1 105 35 63 1 87 96 73 100 1 82 2 103 0 84 98 19 98 110 68 88 101

87 88 103 1 1 1 101 19 8 1 93 2 53 2 92 92 76 39 100 90 97 93 76 83 83 9 105 104 47 42 16

B

A series of modifier agents were tested on the two enzyme samples using the optimum pH and substrate concentrations during incubation. (Table IV). The results showed that both enzyme A and B are highly sensitive to heavy metal ions (Cd, Cu, Hg, Pb, Zn) as well as to chelating agents (~-phenanthroline, EDTA). Some divalent metal ions (Mn, Ni) at 1 mmol/I concentration were also suppressive for enzyme B but not for enzyme A. Co*” caused a clear increase of AlaNA and ArgArgNA hydrolysis by enzyme A and ArgArgNA hydrolysis by both enzymes at different substrate concentrations (Fig. 3). After an about 60% suppression of the enzyme activities with EDTA (1 mmol/l), reactivation was tested with Ca2’, Co2’ and Znzf

190

>

c

200

E L 4 5 3

B

100

0

EDTA

001

01

1

1OEDTA

MODIFIER

001

01

1

10

(mM)

Fig. 6. Reactivation after suppression of enzyme activity by EDTA. The purified enzymes A and B were suppressed by EDTA (1 mmol/l) to about 60% of the original activity. Various concentrations of Ca2+. Co2’ and Zn2’ were added and the activities were measured at pH 6.0 and 9.0 for enzymes A and 8. respectively. The results are given as percentage of activity from the values obtained with EDTA alone (100%).

PMSF

3 0

001

01

1

10

MODIFIER

001

0

01

1

10

(mM)

Fig. 7. Suppression by modifiers. The purified enzymes A and B were exposed for 30 nun to various concentrations of DFP, PMSF, bis-p-NPP and EDTA as indicated. This was followed by the incubation with ArgArgNA at 37OC for 60 mm at pH 6.0 for enzyme A and at pH 9.0 for enzyme B. The results are given as percentage of activity from the control (100%) incubated without any modifier.

191

at certain (Fig. 6). In both cases Co2’ was highly effective in the reactivation had negligible effect and at the higher concentrations, while Ca2 + and Zn” concentrations a decline ensued. Both enzyme preparations showed an identical response to EDTA at various concentrations (Fig. 7). A clear difference between the enzymes was found in their response to amastatin (1 ,nmol/l), ‘oestatin (1 ~mol/l and puromycin (1 mmol/l). Enzyme A was highly sensitive both with AlaNA and ArgArgNA as substrate to these modifiers, while enzyme B was not influenced at all (Table IV). Higher levels of the serine protease inhibitors DFP and PMSF as well as bis-p-nitrophenyl phosphate (bis-p-NPP) suppressed both enzymes; enzyme B, however, was more sensitive in each case (Fig. 7). The relative molecular masses were estimated by gel filtration on Superose 6 column with appropriate standards (Fig. 1) The tentative values obtained were 110 kD and 80 kD for enzymes A and B, respectively. The semen samples of 10 donors were used to separate the spermatozoa (SZ) and seminal plasma (SP) by centrifugation at 600 x g for 10 min. An aliquot of the seminal plasma was recentrifuged at 100000 X g for 2 h to obtain the seminal fluid (SF) and prostasome (PS) fractions. After homogenization and sonication of SZ and PS samples, specific activities of enzymes A and B were assayed in all four samples without and with Co’+ (1 mmol/l) as well as after heating the samples at 55OC for 15 min. The results with ArgArgNA (0.1 mmol/l) as substrate showed that both enzyme A and B activities are stimulated by Co*+, while heating at 55 ‘C markedly suppressed only enzyme B activity (Fig. 8). Enzyme A was highly enriched in the PS

&ArgNA,pH6.0

sz

SP

SF

1

PS

Fig. 8. Enzyme activities in semen and its constituents. The hydrolysis of ArgArgNA at pH 6.0 and 4.0 was measured in 10 samples of human spermatozoa (SZ), seminal plasma (SP), seminal fluid (SP) and prostasomes (F’S) with and without Co’+ (1 mmoI/I) as well as after pretreatment at 55 QC for 15 min as indicated.

192

0

TE

E,

Ez

Ej

E‘

Es

Eg

VD

AM

VS

PR

Fig. 9. Enzyme activities in the human reproductive tissues. The hydrolysis of AlaNA at pH 7.0, ArgArgNA with Co” at pH 6.0 and 9.0 was measured from homogenates (n = 5) of human testis (TE), six segments (El-E6) of the epididymis. vas deferens (VT)), ampulla (AM). vesicuia seminalis (VS) and prostate

(PR).

fraction, while enzyme B seemed to be mainly confined to the SF. Both activities were present in the SZ fraction. but the reaction may be due to contamination by the SF enzymes, since further washing of SZ with 0.9% NaCl before homogenization markedly reduced both enzyme reactions. With AlaNA as substrate at pH 7.0 the dist~bution followed closely that of ArgArgNA hydrolysis at pH 6 and neither this activity was influenced by heat exposure at 55 o C. The specific activity of enzyme A with AlaNA and ArgArgNA (pH 6.0 with Co’+ at 1 mmol/l) and enzyme B with ArgArgNA (pH 9.0 with Co” at 1 mmol/l) was determined in homogenates of various male reproductive tissues (n = 5) without and with prior thermal treatment of the samples at 55 ’ C for 15 mm (Fig. 9). The level of enzyme A with both substrates was highest in the prostatic homogenate and it was aImost totally resitant to thermal treatment. In other tissues, the heat exposure totally abolished the activity, which indicated the absence of the heat-stable enzyme A. Testicular and epididymal tissues were about equally active in the hydrolysis of ArgArgNA at pH 9.0, while the other tissues displayed about half the specific activity. The heat treatment almost totally suppressed these activities, which suggests the contribution of enzyme B to the reaction. AEC of the tissue homogenates confirmed that enzyme B was always present at the same elution site (0.32 mol/l NaCI), while only the prostatic homogenate resulted in a separate enzyme A peak at 0.26 M NaCl concentration (Fig. 10). This tissue also displayed a coincident hydrolysis of AlaNA.

Discussion The present study disclosed in the human seminal plasma.

two clearly distinct enzymes hydrolyzing ArgArgNA One of them (enzyme B) was closely similar to the

193

16.

14.

MonoCi H o-0

ArgArgNAsd’60 AqAqNA, PH 9.0

---

protein

PROSTATE

FF

FFKTION

NUMBER

Fig. 10. Anion exchange chromatography of homogenates of prostate and seminal vesicle. The distribution of the hydrolysis of ArgArgNA was measured at pH 6.0 and 9.0 for 2 h in fractions of anion exchange chromatography in Mono Q FF coh~mn eluted by a NaCl gradient as indicated.

widely distributed cytosolic enzyme first described in the bovine pituitary [I]. It had an optimum around pH 8.5, was suppressed by chelating agents and serine protease inhibitors as well as by heavy metal ions and disclosed a molecular weight of 80 kD. Similar properties have been reported for DPP III in many tissues [l-7,12-14]. Such an enzyme has also been found in the bull seminal plasma, but at very low concentration [8]. In the bull reproductive tissues [8] as well as in the rat skin [4], however, the enzyme has been resistant to EDTA. The other enzyme in human seminal plasma active on ArgArgNA was characterized by an optimum around pH 6. It also differed from enzyme B in its higher molecular weight (110 kD) and thermal stability. After extensive purification of enzyme A, it was obvious that this activity is actually an aminopeptidase, which has a wide substrate spectrum and sequentially hydrolyzed also many dipeptide derivatives including ArgArgNA. Due to its preference of AlaNA as substrate it can be

194

regarded as alanyl aminopeptidase (EC 3.4.11.2). Its substrate spectrum was rather similar to that of the alanyl aminopeptidase in human serum [16]. Both were also highly sensitive to amastatin, bestatin and puromycin. The structural identity of the enzymes would require. however, immunological comparisons. The thermal stability of enzyme A at 55°C could be utilized to separately quantitate enzymes A and B in the seminal plasma and tissue homogenates. Such an analysis revealed that enzyme A is only present in the human prostatic tissue. while enzyme B is widely distributed in all human male reproductive organs, particularly in the testis and epididymis. This result was confirmed by the fractionation methods. Moreover, in the seminal plasma enzyme A was enriched in the particulate fraction, which contains membrane-bound particles presumably derived from the prostate [15]. These particles designated as prostasomes [15] have been found to contain numerous other enzyme activities including Mg’+, Ca’+-dependent ATPase [ 171, protein kinase [ 181, Zr?+-dependent endopeptidase [19], angiotensin converting enzyme [20] and DPP II [9]. A rather similar particulate material in the bovine seminal plasma originates mainly from the seminal vesicles and is called vesiculosome [21]. It also contains numerous hydrolytic enzymes, eg Mg’+. Ca’+-dependent ATPase, alanyl aminopeptidase, aminopeptidase A, DPP II and DPP IV. Vesiculosomes, however, do not contain any Zn’+ -dependent endopeptidase or angiotensin converting enzyme [21] and in this respect they differ from the prostasomes. The alanyl aminopeptidase of the bovine vesiculosomes appears to have also rather similar properties with those of enzyme A [22], Enzyme A can be regarded as an additional secretory marker for the human prostatic tissue similar to the tartrate-sensitive acid phosphatase. The secretory mechanism of these two enzymes may be different. however, since acid phosphatase is not confined to the prostasome particles. We do not know. whether the secretion of the prostasomes and the fluid is differently regulated in the human prostate, but enzyme A can possibly be utilized to monitor the secretion of the prostasomes. It is also interesting to find out. whether enzyme A is elevated in serum of patients with metastatic cancer of the prostate. DPP III in other tissues have been shown to readily hydrolyse angiotensins and enkephalins [2-3,7]. The same appears to be true also for the neutral dipeptidyl peptidases of the monkey brain [23-241. Such compounds may eventually be present in the human male reproductive tissues, but also other biologically active polypeptides may serve as natural substrates for these two enzymes. The highly active alanyl aminopeptidase of the human prostate secretion may contribute to the processing of the same peptides and proteins as DPP III by sequential hydrolysis from the NH,-terminus. The wide substrate spectrum, however, indicates that alanyl aminopeptidase is less specific in its function with regard to the natural substrates present in the human seminal plasma. Acknowledgements The technical assistance of Miss Eija Kettunen and the secretarial help of Mrs. Arja Hoff& and Mrs. Pirjo Pitkanen are highly appreciated. This work has been supported by the Medical Research Council of The Academy of Finland.

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