Purification of 5′-nucleotidase from human seminal plasma

Biochimica et Biophysica Acre, 1075(1991) 20-27 © 1991 ElsevierScience Publishers B.V. 0304-4165/91/$03.50 ADONIS 030441659100218B

Purification of 5'-nucleotidase from human seminal plasma Carlo Fini, Marceilo Coli and Ardesio Floridi Dipartirnento di Medicina Sperimentale e Scienze Biochimiche Unicersitd di Perugia, Perugia (Italy)

(Received 20 December 1990) Key words: 5'-Nucleotidase;Human seminal plasma; Semen; PhosphatidylinositolphospholipaseC; Triton X-114 5'-Nucleotidase from h u m a n seminal plasma was purified to electrophoretic homogeneity and some of its kinetic and molecular properties compared with those of 5'-nucleotidase from bull seminal plasma. The purification of the enzyme was achieved by using the same affinity chromatography media (Con A-Sepharose and AMP-Agarose or ADP-Agarose) previously used for the: purification of bull seminal plasma 5'-nucleotidase (Fini, C., lpata, P.L., Palmerini, C.A. and FIoridi, A. (1983) Biochim. Biophys. Aeta 748, 485-412). However, in the present purification procedure no detergent was used as it had been necessary for the purification of the bovine enzyme. The experimental data reveal some main dif/erences between these two enzymes; first, the h u m a n enzyme seems to be constituted of a single polypeptlde chain of about 71 kDa, while the 5'-nucleotidase of bull seminal plasma, in non denaturing detergent solutions, is a homodimer of about 160 kDa. Another most remarkable difference is that the h u m a n enzyme does not seem to contain a phosphatidylinositol anchoring system like the one present in the bovine enzyme and in 5'-nucleotidase of different sources (Low, M.G. (1987) Biochem. J. 244, 1-13). Finally, the AMPase activity of 5'-nucleotidase from h u m a n seminal plasma is not affected by dithiothreitol which, on the contrary, is a powerful inhibitor of the bovine enzyme causing the dissociation of its subunits which are held together by disulphide bridges (Fini, C., Minelli, A., Camici, M. and FIoridi, A. (1985) Biochem. Biophys. Aeta 827, 403-409). Introduction Mammalian sperm has a very active nucleotide metabolism. The biosynthetic and catabolic processes of ATP are particularly important for the motility of spermatozoa. A T P availability is limited by diffusion and so the adenylate kinase could play an important role in the ATP synthesis at the dynein ATPase sites [1]; on the other hand, a creatin¢ phosphate shuttle has been described in the sea urchin [2]. The interaction of the sperm with the fluids of the male accessory sex glands, which converge in the denominated semen component seminal plasma during semen ejaculation, appears to stimulate the cellular metabolism. However, this phenomenon is quite complex and still little understood [3]. The presence in the seminal plasma of enzymes

Abbreviations: BSP, bull seminal plasma; HSP, human seminal plasma; 5'-N, 5'-nucleotidase; ALP, alkaline phosphatase; AP. acid phosphatase"PI-PLC. phosphatidylinositol-specificphospholipaseC; NTA. nitrilotriacetic acid. Correspondence:Carlo Fini. Dipartimento di MedicinaSperimentale e Scienze Biochimiche, Universitfi di Perugia, Via del Giochetto, 06100-Perugia. Italy.

involved in the nucleotide and nucleic acid metabolism has been established for many years [3,4]; see also Mann and Lutwak-Mann [5]. In our laboratory we have purified 5'-nucleotidase (EC 3.1.3.5) from bull seminal plasma and have described some of the biochemical and biophysical properties [7-9]. However, there is still no specific physiological role attributed to the enzyme, which is present in the seminal plasma along with many other enzymatic and structural proteins and organic and inorganic components, for which possible relationships with fertility [10,11] as well as pathological conditions have been described [12]. 5'-Nucleotidases have been studied in various mammalian organs and the activity of ecto- and cytosolic forms of the enzyme have been investigated in the myocardium of humans, rats, rabbits, guinea pigs, pigeons and turtles to identify a role for the ~tosolic enzyme in the production of adenosine ouring ischaemia [13]. Truong et al. [14] have described two forms of cytosolic 5'-nucleotidase in the rat heart of which one shows a preference for A M P and the other for IMP. The results indicated that the quantity of enzyme present in the rat heart were sufficient to produce the level of adenosine observed under anaerobic conditions. In order to obtain information on the role of 5'-

nucleotides in the mammalian seminal plasma, we decided to purify the 5'-nucleotidase from human seminal plasma and to study some of the biophysical and biochemical properties of the enzyme and compare them with those of bull seminal plasma 5'-nucleotidase. The results showed that the two enzymes are present in the respective seminal fluids in different molecular forms: particulate and soluble in the bull seminal plasma, while in the human seminal plasma the enzyme appeared to be in a soluble form. The purified h u m a n 5'-nucleotidase consisted of a single polypeptide chain of about 71 kDa, while the purified bull enzyme was a homodimer of 160 kDa, which, in native bull seminal plasma, is partially associated with particulate material from which it can only be extracted using detergents [6]. Partition experiments carried out in Triton X-114 in the presence of PI-PLC showed that the purified human enzyme does not contain a covalently attached glycosylated phosphatidylinositol membrane anchoring system like that observed in other 5'-nucleotidases [15]. Polyclonal antibodies raised in rabbits using bull seminal plasma 5'-nucleotidase as an immunogen inhibited to 50% purified h u m a n seminal plasma 5'-nucleotidase activity. Materials and Methods

Materials Bull semen was obtained from the artificial insemination centre 'Centro Tori' of Dr. A. Chiacchierini, Perugia, Italy. H u m a n semen was obtained from volunteers at the Department of Experimental Medicine and Biochemical Sciences, Perugia, Italy. Sodium thymolphthalein monophosphate, concanavalin A-Sepharose 4B and adenosine 5'-monophosphate-agarose (C8, spacer: 6 carbon) were obtained from Sigma (Deisenhofen, F.R.G.). Adenosine deaminase (EC 3.5.4.4), nucleotides, Triton X-100, Triton X-II4 and phosphatidylinositol-specific phospholipase C from Bacillus cereus were obtained from Boehringer Mannheim Italia SpA, Milano, Italy.

Methods Human semen liquefaction. H u m a n semen samples freshly ejaculated were incubated at 25 ° C for 20 rain until liquefaction. 5 ml aliquots were cooled in a wat e r / i c e mixture and added with 100/zl of a 0.9% NaCl solution containing 5 m M proteinase inhibitors phenylmethylsulfonyl fluoride (PMSF) and cystatin. Spermatozoa were separated by centrifugation at 600 × g for 10 min at 2 - 4 ° C . The supernatant constituted the seminal plasma us_~d for purifying 5'-nucleotidase. Sephacryl S-300 gel filtration chromatography. 3 ml of bull or 5 ml of h u m a n seminal plasma were dialysed against four changes of 25 volumes of 50 m M Tris-HCI

buffer (pH 7.5), containing 0.1 M NaCI. The dialysates were monitored for 5'-nucleotidase and alkaline and acid phosphatase activities and 2 ml of bull or 5 ml of human seminal plasma dialysate were loaded onto a Sephacryl S-300 column (3.4 cm × 65 cm) equilibrated with the buffer used for the dialysis. Elution was carried out at 15 m l / h with the same buffer and 3 ml fractions were collected, The protein concentration was determined in each fraction which was also assayed for alkaline phosphatase, acid phosphatase and AMPase activities. Measurement of alkaline phosphatase actit'ity. Alkaline pbosphatase activity was determined using pnitrophenyl phosphate as the substrate. The reaction mixture contained in a final volume of 1.1 ml, 1 ml of 5.5 mM substrate in 50 mM glycine buffer, 0.5 mM magnesium chloride (pH 10.5), and 0.1 ml of sample or standard. The reaction was carried out for 30 min at 25 ° C and was then stopped with 10 ml of 0.02 M sodium hydroxide. The p-nitrophenol released was measured spectrophotometrically at 405 nm using a molar absorptivity of 18600 M - l cm -1. Measurement of acid phosphatase actit'ity. Acid phosphatase activity was assayed using sodium thymolphthalein monophosphate as the substrate, according to Roy et al. [16], and 0.2 ml of sample or standard were added to 1 mi of buffered substrate (0.1 M citrate, pH 5.95). Reaction was carried out for 30 min at 25 ° C. 5 ml of color developer (2 g of N a O H and 5.3 g of anhydrous NazCO 3 in 1 I of water) were then added and abso:'bance was read at 590 nm. The/.tmol of thymolphthalein formed per rain per mg of protein were calculated by using a molar extinction coefficient of 45000 M - l c m - L

Measurement of 5'-nucleotidase and of AMPase activities. 5'-nucleotidase and AMPase activities were assayed using A M P as the substrate. The reaction was carried out at 25 ° C in the presence of excess adenosine deaminase according to Ipata [17]. The decrease in absorbance associated with the conversion of adenosine into inosine was monitored at 265 nm in a thermostated Perkin-Elmer Lambda 1 spectrophotometer equipped with a module for kinetic analysis. The standard assay mixture, in a final volume of 1 mi, contained 0.1 M NaCI; 0.01 M Hepes buffer (pH 7.5); an amount of 5'-nucleotidase sufficient to produce an absorbance decrement of about 0.020 per rain; and 1 p.g of adenosine deaminase. The reaction was started by the addition of 40 ~1 of 2 m M AMP. A decrease in absorbance of 1 corresponds to the hydrolysis of 123 tzmol of substrate per litre. Enzyme activity is defined as /zmol of substrate hydrolysed at 25 ° C per h per mg of protein. Protein concentration was determined by using the Bio-Rad Protein Assay kit, with bovine y-globulin as standard.

Gel electrophoresis and Western blotting. SDS-polyacrylamide gel electrophoresis was carried out according to I.aemmli [18]. The gel was 10% acrylamide and protein bands were evidenced by Coomassie blue staining. The transfer of separated proteins after SDS slab gel electrophoresis to nitrocellulose sheets was obtained by western blotting according to Towbin et al. [!9]. After blocking with 2% skimmed milk, 5'-nucleotidase was detected by incubation with rabbit antiserum (1 : 500) followed by incubation with anti-rabbit IgG-peroxidase. The immunocomplexes were evidenced by treatment with 3,3'-diaminobenzidine. Preparation of bull seminal plasma 5'-nucleotidase antisera in rabbits. Antisera against purified bull seminal plasma 5'-nucleotidase were obtained by intradermic injection in rabbits of enzyme suspensions obtained by dialysing the protein against 0.07 M N a 2 H P O 4 (pH 7.5), and then by diluting the dialysate with 0.07 M CaCI 2 (1 : 1). 800 p.g of protein were injected over a 6 week period. The IgG fraction of serum was purified according to Corthier et al. [20].

Purification of human seminal plasma 5'-nucleotidase. H u m a n seminal plasma was obtained by centrifugation of sperm at 6 0 0 x g for 10 rain at 2 - 4 ° C . Purification of 5'-nucleotidase was carried out through the following steps: 1. Sephacryl S-300 gel filtration. Aliquots of 5 - 1 0 ml of h u m a n seminal plasma were dialysed against four changes of 25 volumes of 50 m M Tris-HCi buffer (pH 7.4), containing 0.1 M NaCI. 5 ml of dialysate were loaded into a Sephacryl S-300 column (3.4 cm × 65 cm) equilibrated with the Tris buffer. The column w a s eluted with the equilibration buffer at a rate of 15 m l / h and 3 ml fractions were collected. The absorbance, the alkaline and acid phosphatase and the AMPase activities were determined. The fractions containing the highest AMPase activity were collected and concentrated to 2 ml through an Amicon YM 30 membrane. The solution was then diluted and concentrated in the Amicon cell several times with the buffer used for the equilibration in the next step. 2. Concanavalin A-Sepharose affinity chromatography. The ultradialysed enzymatic solution was loaded onto a Con A-Sepharose 4B column (1 × 20 cm) equilibrated in 50 m M Tris-HCI buffer (pH 7.5), containing 0.5 M NaCI and 1 m M CaCI 2 and MnCI2, respectively. The column was washed with the equilibration buffer until the absorbance at 280 nm reached the lowest value. A first elution of the absorbed glycoproteins was carried out with 2% methyl a-D-glucopyranoside added to the same buffer. The column was washed until the acid phosphatase activity was almost absent. At this point, final elution of 5'-nucleotidase activity was carried out with the equilibration buffer containing 6% methyl a-o-mannoside. The active fractions were combined, concentrated to about 2 ml and dialysed against four

changes of 25 volumes of the equilibration buffer of the AMP-Agarose affinity medium. 3. AMP-Agarose affinity chromatography. The dialysate from the previous step was applied to an AMPAgarose column (1 × 10 cm) equilibrated with I0 m M imidazole (pH 6.8), containing 75 m M NaCI, 1 m M sodium /~-glycerophosphate and 1 m M NaN a. The colu m n was washed with the buffer and elution was carried out with 10 m M A M P in the same buffer. The active fractions were combined, concentrated through the YM30 membrane and dialysed against four changes of 25 volumes of imidazole buffer. The enzyme was stored at - 20 ° C. Results

Fractionation of AMPase activity of human and bull seminal plasma on Sephacryl S-300 gel filtration chromatography Fig. 1A shows that, in HSP the AMPase activity is eluted into an asymmetrical peak corresponding to molecular mass of about 100 kDa. Fig. 1B, on the other hand, shows that, in agreement with our previous o h A

Activity 2O0O

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140

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Fraction Number Fig, 1. Sephacv/I S-300 gel filtration of human ( A ) and bull (B) seminal plasma. The chromatographic conditions were those d e scribed in the Materials and Methods section. Absorbance at 280 nm

(o); AMPase activity (o); acid phosphatase activity (A); alkaline phosphatase activity( A ).

servations [6], the BSP AMPase activity is eluted into at least two peaks of which the first corresponds to the V0 of the column and the second to a molecular mass of about 160 kDa. Chromatography carried out in the presence of detergents, such as sodium cholate, Triton X-100 or Lubrol, results in the elution of the heterogeneous AMPase activity in BSP into a single peak of molecular mass of about i60 kDa, whilst the presence of detergents does not affect the elution pattern of AMPase activity of HSP (data not shown).

Purification of 5'-nucletidase from human seminal plasma The observation that the elution of the AMPase activity in the gel filtration chromatography is not influenced by the presence of detergents, prompted us to avoid the use of detergents in the purification of HSP 5'-nucleotidase. Moreover, with their use, we noticed a significant worsening in the final recovery and yield of the enzyme activity. Table I summarizes the protocol for the purification of 5'-N from HSP. As can be seen, more than 90% of the acid phosphate activity (most probably of prostatic origin) is eluted from the Con A column by 2% of methyl a-o-glucopyranoside; while 5'-nucleotidase is eluted by 6% of methyl a-o-mannoside, thus indicating a different carbohydrate composition for the two enzymes. The final enzyme preparation showed a purification factor of 978-fold and only one protein band of about 71 kDa on SDS-PAGE (Fig. 2).

Effect of rabbit anti-bull seminal plasma 5'-nucleotidase antisera on human and on bull seminal plasma 5'nucleotidase Fig. 3 refers to the transfer by Western blotting of human and bull seminal plasma 5'-nucleotidase onto a cellulose nitrate sheet, which was then incubated in a solution of antisera obtained from rabbits which had previously been immunized using BSP 5'-nucleotidase as an antigen. As can be seen, the polyclonal anti-BSP 5'-nucleotidase antibodies are also able to recognize

the 5"-nucleotidase from human seminal plasma. Moreover, a spectrophotometric test of 5'-nucleotidase activity in the presence of the antisera (see Fig. 4) shows that a 50% inhibition of AMPase activity is achieved in the presence of 3.5 ~g and 18 p.g of IgGs in the assay mixture for bull and human seminal plasma 5'-nucleotidase respectively.

Effect of some metal ions on 5'-nucleotidase from human and bull seminal plasma The AMPase activity of the two purified enzymes is practically unaffected by the presence of Ca z+, Mg 2+ and Mn -~+ ions to the level of 5 mM (not shown). However, as can be seen in Fig. 5, a complex behaviour pattern is demonstrated in the presence of zinc ions. At concentrations of 0.1 to 4 mM, the activity is strongly inhibited, whilst higher concentrations result in a significant activation of the enzyme. Fig. 5 also shows that cobalt, copper and iron are all powerful inhibitors of the two 5'-nucleotidases precisely in that range of concentration where zinc proves to be such a significant activator.

Enzymatic properties of 5"-nucleotidase from human seminal plasma Some of the enzymatic properties of HSP 5'-N are reported in Table I1. As can be noted, the enzyme shows a Km value for 5'-AMP of 8 t~M, which is quite similar to that of BSP 5'-N that is 11 ~,M [6] and, moreover, the human enzyme is also inhibited by nueleoside di- and triphosphates, as previously observed for the bovine enzyme [21]. It must be pointed out that the human 5'-N is not affected by DTI', which, on the contrary proved to be a very powerful inhibitor of BSP 5'-N [71.

Partition of liSP and BSP 5'-N in Triton X-114 sohaions The characteristic of the detergent Triton X-114 aqueous solutions to yield homogeneous solutions at 0 ° C and to separate into two phases at over 20 o C, of which the lower localizes the detergent and the upper the water, has been exploited to recognize the pres-

TABLE 1

Purification schemeof 5'-nucleotidase from haman seminalplasma Step

Total protein (mg)

5'-N total act. (units)

ALP total act, (units)

AP total act. (units)

1. Humanseminalplasma 2. Sephacr~lS-300 3. Con A-Sepharose 2% Glucoside 6% Mannoside 4. AMP-Agarose

175 21.8

962 667

7

41.8 410 269

-

15.5 5,0 0.05

15200 125~)

Specific activity (units/rag protein) 5.5 30.6

Purification (5'-N) (fold) I 5.6

10500 580 -

2.7 82 5 380

14.9 978

Values are representative of seven preparationsstarted with 5 ml of human seminal plasma.

Yield (%) 100 69 43 28

A

e n c e o f h y d r o p h o b i c moieties in p r o t e i n s [22]. In p a r ticular, e x p e r i m e n t s of p a r t i t i o n i n g in T r i t o n X - 1 1 4 a q u e o u s solutions c o m b i n e d with t r e a t m e n t with PIPLC from various sources have allowed the recognition o f a specific p h o s p h a t i d y l i n o s i t o l a n c h o r i n g s y s t e m in 5 ' - N o f d i f f e r e n t s o u r c e s [15]. T h e e x p e r i m e n t s s u m m a rized in T a b l e III s h o w t h a t B S P 5 ' - N c a n b e r e c o v e r e d in the a q u e o u s p h a s e only a f t e r t r e a t m e n t with P I - P L C f r o m B. cereus. O n the c o n t r a r y , p u r i f i e d H S P 5 ' - N c a n

/..,,

kDa 97.4

i

WW .

B

66.2

)"

42.7

"

31.0

D.-

.

Fig. 3. Western blot of purified human (lane A) and bull (lane B) seminal plasma 5'-mlcleotidase. The enzymes were localized as described in the Materials and Methods section. The arrows indicate the position of the molecular mass markers from top to bottom: rabbit muscle pbosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa, hen egg white ovalbumin, 42.7 kDa and bovine carbonic anhydrase, 31 kDa.

Q qp I

b e r e c o v e r e d in t h e a q u e o u s p h a s e o f T r i t o n X - 1 1 4 solutions without PI-PLC treatment, thus indicating t h a t the p u r i f i e d h u m a n e n z y m e is l a c k i n g in p h o s p h o lipid moieties. Discussion

Fig. 2. SDS-polyacrylamide gel electrophoresis of purified 5'-nucleotidase from human seminal plasma. A total of 36 /~g of molecular weight standards (lane A) and 8/zg of purified enzyme (lane B) were processed as described in the Materials and Methods section. The standards used were from tnn to bottom: rabbit muscle phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; hen egg white ovalbumin, 42.7 kDa; bovine carbonic anhydrase, 31 kDa and soybean tripsin inhibitor, 21.5 kDa.

A f f i n i t y m e d i a like l e c t i n - b o u n d S e p h a r o s e a n d n u c l e o t i d e - b o u n d a g a r o s e have, for m a n y y e a r s , b e e n u s e d f o r t h e p u r i f i c a t i o n o f 5 ' - n u c l e o t i d a s e f r o m vario u s s o u r c e s [ 7 , 2 3 - 2 5 ] a n d t h e y h a v e also p r o v e d to b e m o s t effective f o r t h e 5 ' - n u c l e o t i d a s e o f h u m a n s e m i n a l p l a s m a . U n l i k e o t h e r 5 ' - n u c l e o t i d a s e s , this e n z y m e d o e s n o t r e q u i r e d e t e r g e n t s like T r i t o n , s o d i u m c h o l a t e

or C H A P S for purification. Moreover, their use resulted in a lower recover, f the enzymatic activity at almost every stage of the purification procedure. The hydrophilic n a t u r e of the protein was particularly evident from the experiments of partition in Triton X-114 in which more than 90% of the A M P a s e activity associated with the protein was recovered in the a q u e o u s phase. This constitutes a significant difference from the 5'-nucleotidase of bull seminal p l a s m a which was recovered at about 20% u n d e r the same conditions. F u r t h e r m o r e , our previous study [6] showed that 5'-nucieotidase is present in BSP in h e t e r o g e n e o u s forms. T h r e e different forms of the enzyme were recognized of which: the first c o n t a i n e d 5'-nucleotidase activity s e d i m e n t a b l e at 1 0 5 0 0 0 × g and was memb r a n e bound; the second r e p r e s e n t e d m o l e c u l a r aggregation forms of the third form, which in turn corres p o n d e d to the d e t e r g e n t purified 5'-nucleotidase with a m o l e c u l a r mass of 160 kDa. O n the contrary, the Sephacryl S-300 gel filtration c h r o m a t o g r a p h y shown in Fig. IA, and the centrifugation at 1 0 5 0 0 0 x g (see below) do not s e e m to indicate a similar betrogeneity in the m o l e c u l a r forms of the 5'-nucleotidase of HSP: in fact, the enzyme a p p e a r s quite soluble and is recov-

30o Activity (% of c o n t r o l ) A

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Fig. 5. Effect of metal ions on purified human and bull seminal plasma 5'-nucleotidase. 0.1 pg of human (panel A) or bull (panel B) 5'-nucleotidase were incubated for 5 rain at 25°C in the reaction medium in the presence of concentrations of the metal ions investigated as indicated. Reaction was started by the addition of adenosine deamiuase and AMP in quantities as indicated in the Materials and Methods section. Cobalt chloride (•)" nickel chloride (zx): iron (ID chloride (o) and zinc chloride (e). 0

,

,

,

,

,

10

20

30

40

,50

60

Fig. 4. Inhibition of human and seminal plasma 5'-nucleotidase by increasing amounts of rabbit anli-BSP 5'-nucleotidase polyclonal antibodies. 0.1 y.g of purified human (Q) or bull (o) seminal plasma 5'-nucleotidase were incubated with the antibodies at the indicated concentrations for 20 rain at 25°C. AMPase activity was then measured as described in the Materials and Methods section. AMPase activity of purified human and bull seminal plasma 5'-uucleotisade incubated in the presence of unspecific rabbit IgG under identical experimental conditions repr: sented 100% of activity.

erable from the gel filtration column at a Ve corresponding to a m o l e c u l a r mass of about 100 kDa, The characterization of the enzyme had shown that the 5'-nucleotidase from bull seminal p l a s m a was a h o m o d i m e r i c protein whose subunits were held tog e t h e r by disulphide bridges [7]. On the o t h e r hand, the centrifugation at 1 0 5 0 0 0 x g of h u m a n seminal plasma did not result in the s e d i m e n t a t i o n of 5'nucleotidase activity and the purified enzyme showed on S D S - P A G E a m o l e c u l a r mass of about 71 kDa, In

26 TABLE 11 Prop,,rtia~ of p,rified 5'-nucleotidase from human seminal plasma

g m and K i values are means+S.D. (n = 7); Mr values represent means+ ranges of five experiments; values for effect of NTA; dilhiothreitol and IgGs values are representative of seven experiments. Property

Value

K m (AMP) Vm~X Competitive inhibitors

8/zM 22" 10- 6 mol/min, mg ADP K i = 0.7+0.08/zM ATP K i = 2+0.2 ~tM

Molecular mass determined by: SDS-PAGE S-300 chromatography: in the absence of detergent in the presence of 2% lubrol or 50 mM sodium cholate Residual activity in the presence of 0.35 mM NTA Residual AMPase activity in the presence of 0.1-5 mM dithiothreitol (1.5 mM dithiothreitol inhibits BSP 5'-N up to about 80% [7]) Residual AMPasc activity after incubation with 20 ~g of purified anti-BSP 5'-N IgGs from rat blood serum (under the same conditions, BSP 5"-N is inhibited up to about 90%)

71 _+2 kDa 80+_4 kDa 80+4 kDa 50% 100%

50%

a d d i t i o n , t h e p r o t e i n m o l e c u l e did n o t s e e m to b e o r g a n i z e d into a h o m o d i m e r i e s t r u c t u r e like t h a t f o u n d in bull s e m i n a l p l a s m a [6], c h i c k e n g i z z a r d [25] o r r a t h e a r t [23]. TABLE Ill Partition of purified human and bull seminal plasma 5'-nucleotidase in Triton )(-;14 solution

1 p.g of purified 5'-N from human (sample 1) or bull (sample 2) seminal plasma in 0.05 M Tris-HCI buffer (pH 7.4), 1% Triton X-II4, final volume 0.2 ml, were incubated for 10 mln at 0*C in Eppendorf microfuge tubes. The tubes were then incubated at 30 °C for 3 min and centrifuged at 3000xg for 3 min at room temperature. Supernatants were reextracted with Triton X-114 and, after the second eentrifugation, AMPase activity was determined in the aqueous phases. 0.1 ml of samples 3 and 4 containing l u.g of 5'-nucleotidase from human and bull seminal plasma, respectively, were incubated with 0.2 units of PI-PLC from B. cereus (Boheringer Mannheim Italia Spa, Milano, Italy) at 37°C for 30 rain. After incubation, the samples were mixed with an equal volume of 0.05 M Tris-HCI buffer (pH 7.4), containing 1% Triton X-II4 and processed as described for samples 1 and 2. Enzyme activity measured before phase separation was taken as 100%. Sample

1. HSP 5'-N 2. BSP 5'-N 3. PIPLC-treated HSP 5'-N 4. PIPLC-treated BSP 5'-N

AMPase activity released in the aqueous phase (% of control) 90 20 105 102

A s yet, we h a v e n o definite p r o o f for t h e existence o f i s o e n z y m a t i c f o r m s o f 5 ' - N f o r e i t h e r bull o r h u m a n s e m i n a l p l a s m a ; h o w e v e r , given the v a r i e t y o f isoenzymes r e p o r t e d o n we h a v e b e e n d i r e c t i n g o u r r e s e a r c h t o w a r d s this e n d . Kinetic parameters of human seminal plasma 5'-N s u c h as K m f o r A M P , p H o p t i m u m , K i for t h e inhibition b y n u c l e o s i d e di- a n d t r i p h o s p h a t e s a r e similar to t h o s e f o u n d f o r 5 ' - N o f B S P [6,21]. F u r t h e r m o r e , t h e e f f e c t o f s o m e divalent m e t a l s is very similar f o r b o t h e n z y m e s a n d t h a t o f zinc ions in p a r t i c u l a r , s t i m u l a t e s f u r t h e r c o n s i d e r a t i o n . T h e c u r v e d e s c r i b i n g this a c t i o n is complex. A t low zinc c h l o r i d e c o n c e n t r a t i o n s , u p to 0.01 m M , A M P a s e activity is u n a f f e c t e d ; zinc c h l o r i d e c o n c e n t r a t i o n s r a n g i n g f r o m 0.4 to 3 m M ( H S P ) a n d f r o m 0.3 to 1 m M (BSP) inhibit t h e A M P a s e activity a l m o s t totally a n d h i g h e r c o n c e n t r a t i o n s s t i m u l a t e t h e e n z y m a t i c activity. Our atomic absorption spectrometry measurements s h o w e d t h a t t h e zinc c o n t e n t in bull s e m i n a l p l a s m a w a s 2 m g p e r 100 ml a n d t h a t o f h u m a n s e m i n a l p l a s m a 8 m g p e r 100 ml. T h e s e results s e e m to i n d i c a t e t h a t t h e c o n c e n t r a t i o n o f zinc n o r m a l l y f o u n d b o t h in bull a n d h u m a n s e m i n a l p l a s m a w o u l d b e sufficient to inhibit t h e 5 ' - n u c l e o t i d a s e activity u p to a n d o v e r 9 0 % . T h e r e f o r e , it m a y b e possible t h a t also in vivo 5 ' nueleotidase would be completely inactivated unless sonte o t h e r , as yet u n k n o w n , f a c t o r s w e r e to i n t e r v e n e . It h a s a l r e a d y b e e n e s t a b l i s h e d t h a t t h e d e p h o s p h o r y l a t i o n o f A M P h a s a r e l e v a n t f u n c t i o n in r a t h e a r t [14] a n d p i g e o n h e a r t [13] i s c h a e m i a ; h o w e v e r , w e still d o n o t k n o w if s u c h a r o l e c o u l d b e a t t r i b u t e d to a d e n o sine in fertility. E x p e r i m e n t s a r e u n d e r w a y to e s t a b l i s h w h e t h e r t h e p e n e t r a b i l i t y o f t h e v a g i n a l m u c u s by s p e r m a t o z o a is in s o m e w a y i n f l u e n c e d by t h e a d e n o s i n e levels a n d if t h e c o n c e n t r a t i o n o f zinc m a y h a v e a role in this p r o c e s s .

Acknowledgement T h e a u t h o r s w o u l d like to e x p r e s s t h e i r g r a t i t u d e to Ms. Sally M c Gill f o r h e r helpful s u g g e s t i o n s .

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