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The Isolation from Human Seminal Plasma of a New Form of Soluble 5′-Nucleotidase
BIOCHEMICAL AND MOLECULAR MEDICINE ARTICLE NO.
58, 168–175 (1996)
0045
The Isolation from Human Seminal Plasma of a New Form of Soluble 5*-Nucleotidase ALBA MINELLI, MONICA MORONI, NADIA MONACELLI,
AND ISABELLA
MEZZASOMA
Dipartimento di Biologia Cellulare e Molecolare, Sezione di Enzimologia, Universita` degli Studi di Perugia, 06123 Perugia, Italy Received January 2, 1996
of both purine and pyrimidine bases. Among the soluble forms, a further classification is based on the different Km values toward the substrates of the enzyme. A low Km isozyme, derived from the membrane-bound form by phospholipase C cleavage of the glycosyl phosphatidylinositol anchor, shows values of Km’s for AMP and IMP in the micromolar range and, according to the criteria of Truong et al. (7), has a preference for AMP. Truly cytoplasmic types, the high Km isozymes, have Km values for AMP and IMP in the millimolar range and, on the basis of their substrate preference, are subdivided into AMPases and IMPases. However, soluble 5*-nucleotidases are less clearly defined and, at present, data not easily classified into this general scheme are attributed to several not yet well identified factors (6). Recently a rather peculiar form of soluble enzyme was isolated in human seminal plasma (8). The enzyme, a low Km type, does not cross-react with ecto5*nucleotidase antibodies and prefers IMP, and its activity is absolutely Mg 2/-dependent and is affected in different ways by ATP and ADP (9). During the purification of the low Km IMP-specific 5*-nucleotidase (8), the human seminal plasma, chromatographed on AMP–Sepharose 4B, was resolved in two peaks of 5*-nucleotidase activity. In view of the various effects exerted by adenosine on the spermatozoa (10–12), these enzymes are of general interest. The present paper deals with the isolation and the characterization of the form less tightly bound to AMP–Sepharose 4B in order to investigate the properties of this isozyme on the degradation of nucleoside 5-monophosphate and its eventual involvement in the production of adenosine, which may affect the fertilizing capacity of the ejaculate.
Soluble broad spectrum 5*-nucleotidase from human seminal plasma was purified to homogeneity by a combination of (NH4)2SO4 precipitation, affinity chromatography, and gel filtration. The pure enzyme had a specific activity of 4800 nmol min01 mg01. SDS–PAGE of purified enzyme preparation revealed a single polypeptide band of 53 kDa and a tetrameric structure of 203 kDa was proposed for the native enzyme. This form had modest preference for AMP as substrate; Mg 2/ and Mn2/ were activators of the enzyme although its activity was not absolutely dependent on the presence of these exogenous bivalent cations. The enzyme, recovered in the nonsedimentable fraction of human seminal plasma, had a pH optimum of 7.5; ATP and ADP were inhibitors of mixed type, Pi was a potent inhibitor at nonphysiological concentrations, and Con A and adenosine 5-[a,b-methylene]diphosphate had no effect on the enzyme activity. The enzyme described here therefore has some unique properties between truly cytoplasmic and membrane-bound derived forms. q 1996 Academic Press, Inc.
Nucleotidases (5* ribonucleotide phosphohydrolase, EC 3.1.3.5) constitute a large class of heterogeneous isozymes that are widely distributed among animals, plants, and microorganisms (1–5). These isoenzymes catalyze the hydrolysis of monophosphate ester linkages in various 5*-nucleotides, producing inorganic phosphate (Pi) and the corresponding nucleoside. According to Zimmermann (6), the vertebrate tissue 5*-nucleotidase family can be divided into four classes, each characterized by substrate preference, kinetics, cation requirements, and pH optima, although all are active with 5*-nucleotides 168 1077-3150/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Human seminal fluid from healthy volunteers was a gift from Centro di Fisiopatologia della Riproduzione, University of Bologna. ATP, ADP, AMP, p-nitrophenylphosphate, nucleosides, all nucleoside mono-, di-, and triphosphates, and Blue Sepharose CL-6B were from Sigma Chemical Co. Bradford protein dye reagent, high- and low-molecular-weight standards, and the Silver Stain kit were from Bio-Rad. AMP– Sepharose 4B and Sephacryl SW300 were from Pharmacia. All other reagents used were of the highest quality available. Assays The 5*-nucleotidase assay was done as previously described (8,9). Incubations were performed at 377C in medium containing 50 mM Tris–HCl pH 7.4, 2 mM substrate, and other additions, as indicated. The reaction was carried out in a total volume of 100 ml and terminated by heating at 1007C. The amount of Pi liberated was determined according to Itaya and Ui (13). All the experimental values were corrected by subtracting the respective control, i.e., the amount of inorganic phosphate present in parallel assays performed with acid-precipitated samples. The assay was linear with protein and time up to 60 min. One unit is defined as the amount of enzyme which cleaves 1 nmol of substrate in 1 min at 377C. In experiments where the effect of Pi was tested, 1–2 kBq of 2-3H-5*AMP was added to the standard incubation medium. The activity of 5*-nucleotidase was assayed by measuring the production of labeled adenosine according to Truong et al. (7) and Meghji et al. (14). Incubations were conducted at 377C for 30 min in a total volume of 150 ml containing 50 mM Tris–HCl, pH 7.4, 2 mM AMP plus labeled substrate and Pi . The reaction was started by adding the appropriate enzyme so that the overall substrate conversion did not exceed 25%. The reaction was terminated by adding 0.15 mM ZnSO4 and 0.15 mM Ba(OH)2 . Phosphatases were measured by incubating samples in 50 mM Na citrate, pH 4.8, in 50 mM glycine, 0.5 mM MgCl2 , pH 10.5, and in 50 mM Tris–HCl, pH 7.4, for 30 min at 377C, in a total vol of 500 ml containing 5.5 mM pNPP. The reaction was stopped by 2.5 ml of 0.1 M NaOH. The samples, when needed, were clarified by centrifugation and optical density at 410 nm was measured with a U2000 Hitachi Spectrophotometer. Controls were performed as in the 5*-nucleotidase assay.
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Protein Determination Protein was quantitated by the Bradford assay method with bovine serum albumin as the standard protein (15). Proteins eluted from the columns were monitored by absorbance at 280 nm or by the Bradford method. Purification All procedures were performed at 47C, unless otherwise stated. Human semen, thawed at room temperature, was centrifuged at 700g for 30 min to remove intact spermatozoa. The supernatant was again spun at 30,000g for 30 min and the clear supernatant was referred to as the seminal plasma. Forty milliliters of seminal plasma was further centrifuged at 105,000g for 60 min and solid (NH4)2SO4 was slowly added to the supernatant to give 35% saturation. After 60 min, the precipitate was removed by centrifugation at 17,500g for 30 min. More (NH4)2SO4 was added to the supernatant to give 60% saturation, the solution was centrifuged, and the resulting precipitate was dissolved and dialyzed overnight against 50 mM Tris – HCl, 0.1 M NaCl, pH 7.4. The dialyzed solution (30 ml) was loaded onto an AMP – Sepharose 4B column (8 1 1.5 cm). The column was washed with 150 ml of 50 mM Tris – HCl, 0.1 M NaCl buffer, pH 7.4, and the enzyme was eluted with 0.5 M NaCl in 50 mM Tris – HCl, pH 7.4. Fractions containing 5*-nucleotidase activity (50 ml) were pooled, dialyzed against 50 mM Tris – HCl, pH 7.4, and loaded on a Blue Sepharose column (4 1 1.6 cm) previously equilibrated with 50 mM Tris – HCl, pH 7.4. Non-binding proteins were eluted with the equilibrating buffer containing 0.1 M NaCl. The enzyme (40 ml) was eluted with a 0.5 M NaCl step in 50 mM Tris buffer, pH 7.4, dialyzed against 50 mM Tris– HCl, pH 7.4, lyophilized, dissolved in 50 mM Tris– HCl, 0.5 M NaCl, pH 7.4, applied to a Sephacryl SW 300 column (95 1 1.5 cm) that was preequilibrated in same buffer, and eluted at a flow rate of 7.5 ml/ h. The fractions with high activity were pooled and stored at 47C until use. Molecular Weight Determination The relative molecular mass of the native protein was determined either by gel filtration on a Sephacryl SW300 16/60 column (Pharmacia) or by electrophoresis on polyacrylamide gels. The column was
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TABLE 1 Purification of Soluble 5*-Nucleotidase from Human Seminal Plasma Purification step
Total activity (U)
Total protein (mg)
Specific activity (U/mg)
Purification factor
Yield (%)
Seminal plasma (NH4)2SO4 fractionation First AMP–Sepharose 4B Blue Sepharose CL-6B Sephacryl SW 300
133,200 30,680 2,100 304 72
3520 2050 5.3 0.24 0.015
49 15 396 1266 4800
— 0 26 84 320
— 100 6.8 0.9 0.23
Note. 5* Nucleotidase assays and protein determinations were performed as described under Material and Methods.
run at 12 ml/h and 2-ml fractions were collected. The column was calibrated with thyroglobulin (670 kDa), g-globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa). Polyacrylamide gel electrophoresis under nondenaturing conditions was performed in the presence of 7.5% acrylamide, according to Davis (16), and in the presence of polyacrylamide gels at concentrations ranging from 5 to 12%, according to Thibault et al. (17). From a Ferguson–Hedrick plot, KR , the retardation coefficient, was determined for standard proteins: urease (545 and 272 kDa), bovine serum albumin (132 and 66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), and a-lactalbumin (14.2 kDa) (Sigma). SDS–PAGE was performed according to Weber et al. (18) on a 12% acrylamide gel stained by the Bio-Rad silver stain kit.
by (NH4)2SO4 fractionation, this step was considered the initial step of the purification procedure. The AMP–Sepharose 4B chromatography succeeded in separating alkaline phosphatases and 5*nucleotidase, which was successively 84-fold purified by the Blue Sepharose column. A specific activity of 4800 U/mg was found in the enzyme preparation eluted from Sephacryl SW300, corresponding to a purification factor of 320. An overall purification of greater than 320 can be suggested on the basis that the specific activity of this soluble 5*-nucleotidase is surely smaller than 15, as seminal alkaline phosphatases contribute to this value. The enzyme resisted further purification and was homogeneous by criteria of gel electrophoresis.
Kinetic Calculations
The molecular weight of the native soluble 5*nucleotidase was determined by gel filtration on Sephacryl SW300. The enzyme activity was eluted as a single symmetrical peak with no shoulder, at the position corresponding to an apparent molecular mass of 199 kDa. Gel electrophoresis on polyacrylamide at different concentrations evidenced a single band of 203 kDa. SDS–PAGE electrophoresis resolved the enzyme in a single band of 53 kDa. The enzyme is therefore composed of four identical subunits.
Kinetic data were analyzed by a linear regression with the Enzfitter (Biosoft Elsevier) program by using the equations corresponding to Michaelis–Menten equations. Five triplicates of each point were performed. Goodness of fit was tested according to reduced SD values given by the programs. RESULTS
Molecular Properties
Purification of the Enzyme Soluble 5*-nucleotidase was purified by a combination of (NH4)2SO4 precipitation, affinity chromatography, and gel filtration, as summarized in Table 1. A precise quantitative evaluation of the initial step of this purification is hindered by our inability to distinguish 5*-nucleotidase activity from nonspecific phosphatase in crude preparation. Thus, as the acidic phosphatases were almost entirely removed
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Thermostability and Effect of Temperature The enzyme preparation, kept at 47C, was stable for extended periods. Thermal stability of the enzyme was examined and the results are shown in Fig. 1A. About 80% of the activity was retained after incubation of the enzyme for 30 min at 40–507C, while total inactivation occurred at a temperature of 707C. The influence of temperature on the enzyme
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FIG. 1. Properties of purified soluble 5*-nucleotidase from human seminal plasma. (A) Effect of thermal denaturation on 5*-nucleotidase activity. Aliquots of the enzyme were incubated for 30 min at different temperatures and the effect on activity was determined, as described under Materials and Methods. (B) Effect of temperature on 5*-nucleotidase activity. Activity was measured at different temperatures (0–607C) as described under Materials and Methods. (C) Arrhenius plot for the dephosphorylation of AMP by 5*-nucleotidase. Assays in the presence of various substrate concentrations were run as described under Materials and Methods. (D) Effect of pH on activity and stability of 5*-nucleotidase. The enzyme activity toward AMP (1) and CMP (-r-) was assayed at 377C in 0.2 M Tris– maleate buffer (pH 6.0–8.5), and in 0.2 M Tris–glycine buffer (pH 8.5–9.0). The stability in the presence of AMP (j) and CMP (l) was tested by preincubating the enzyme for 30 min at the indicated pH values in 5 mM citrate–HCl (pH 2.0–6.0), in 5 mM Tris–HCl (pH 7.0–9.0), and in 5 mM glycine–HCl (pH 10–13).
activity is illustrated in Fig. 1B. The enzyme displayed an apparent maximum of activity at 377C. A continuous Arrhenius plot obtained from activity measurements between 0 and 407C gave an activation energy of 14.5 kcal/mol (Fig. 1C). An Ea of 11.7 kcal/mol was obtained in the presence of CMP as substrate. Effect of pH on Activity and Stability The pH dependence of the soluble 5*-nucleotidase is reported in Fig. 1D. The activity against AMP and CMP was measured over a pH range of 6.0–9.0 and two pH optimum values of 7.5 for AMP and 8.0 for CMP were identified. In the presence of these two substrates, preincubation of the enzyme at pH õ7.0 decreased the activity either by forming an improper ionic form or by irreversibly inactivating the enzyme. Preincubation at pH ú7.0 did not negatively affect the enzyme activity.
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Substrate Specificity As shown in Table 2, the enzyme preferred AMP with a Vmax/Km ratio of 348 1 105. Although Vmax values were almost on the same order of magnitude for the dephosphorylation of AMP and IMP, the ratio Vmax/Km indicated that AMP was the preferred substrate. Vmax values were fairly high in the presence of pyrimidine nucleotides with an efficiency of substrate utilization of 280 1 105. The enzyme therefore preferentially hydrolyzed in the order AMP ú UMP Å CMP ú IMP. It was less active with deoxy-derivatives and had no activity in the presence of ribose-1P, ribose-5P, pNPP, or bglycerophosphate, as expected. Effect of Metal Ions The activity of purified enzyme, measured in the absence of exogenous cations, indicated a nonabso-
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TABLE 2 Substrate Specificity of Human Seminal Plasm Soluble 5*-Nucleotidase Vmax (nmol/min/mg protein)
Substrate IMP AMP UMP CMP GMP dGMP dIMP dAMP Ribose 1-P Ribose 5-P pNPP b-Glycerophosphate
876 { 871 { 1117 { 1150 { 693 { 270 { 245 { 324 { — — — —
12 13 10 11 9 8 4 5
Substrate efficiency (Vmax/Km)
Km (M) 6.3 2.5 4.0 4.1 4.5 3.5 3.6 4.5
{ { { { { { { {
0.5 1 0.3 1 0.2 1 0.4 1 0.4 1 0.9 1 1.2 1 1.0 1 — — — —
1005 1005 1005 1005 1005 1005 1005 1005
139 348 280 279 155 77 69 72
1 1 1 1 1 1 1 1 — — — —
105 105 105 105 105 105 105 105
Note. Determinations were performed in 25 mM Tris–HCl pH 7.4, with at least five different concentrations of substrate (n Å 15), as described under Material and Methods.
lute requirement of metal ions, although Mg 2/ and Mn2/ were shown to be able to modulate the activity of the enzyme. In the presence of MgCl2 , the enzyme activity increased with increasing concentrations up to 10 mM. With MnCl2 as the bivalent cation, optimal activity was obtained at 3 mM. Higher concentrations up to 10 mM did not result in further enzyme activation. The maximal activity with MnCl2 was approximately two times higher than the maximal activity obtained with MgCl2 . In order to examine the specificity of the effect of these stimulatory cations on soluble 5*-nucleotidase, in the experiments reported in Table 3, other divalent cations were used. The data show that all other metal ions tested inhibited the enzyme. Metal chelators EDTA and EGTA caused a concentration-dependent inhibition of the enzyme with I50 values of 0.04 and 1.26 mM, respectively. Removal of metal ions from the enzyme molecule by treatment with 1 mM EDTA and overnight dialysis against 25 mM Tris–HCl, pH 7.4, caused an 88% inactivation of the enzyme activity and the addition of 10 mM bivalent cation did not restore the native activity. Indeed the treatment of the enzyme with 0.01 mM EDTA caused a 25% inhibition and the addition of MgCl2 restored nearly the full activity while MnCl2 still caused a slight stimulation of the enzyme activity. CaCl2 , as expected, was ineffective in restoring the native enzyme activity. Effect of ATP, ADP, and Pi The concentration dependencies of ATP and ADP on soluble 5*-nucleotidase activity are presented in
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Figs. 2A and 2B, in the presence of AMP and IMP as substrate. ATP was an inhibitor that was not as effective as ADP; 1 mM ATP caused 50% inhibition of the enzyme activity, whereas the same level of inhibition was obtained with 0.05 mM ADP. Moreover, the inhibitory effect caused by ADP was more pronounced than that caused by ATP as it caused 82% inhibition of the AMP dephosphorylation against the 60% inhibition caused by ATP. The dephosphorylation of IMP was equally regulated by ATP and ADP. A 33% inhibition of IMP dephosphorylation represented the maximum inhib-
TABLE 3 Effects of Metal Ions and Metal Chelators on Human Seminal Plasm Soluble 5*-Nucleotidase Ion or chelator
Percentage activity
U/ml
None Mg2/ Zn2/ Ca2/ Mn2/ Cu2/ Ni2/ Hg2/
905 { 1440 { 432 { 576 { 2153 { — 78 { 576 {
1 mM EDTA 1 mM EGTA
11 14 7 9 14 4 8
100 160 47 63 237 — 9 63
110 { 5 570 { 9
12 58
Note. The assay was performed in the presence of the indicated divalent cations at a concentration of 10 mM (n Å 10), as described under Material and Methods.
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FIG. 2. Effect of ATP (A) and ADP (B) on soluble 5*-nucleotidase from human seminal plasma. After preincubation of the enzyme with the indicated concentrations of ATP and ADP for 10 min at 377C, the assay was performed as described under Materials and Methods. (l) AMP; (j) IMP. Kinetic parameters of the inhibition exerted by ATP (A*) and ADP (B*) in the presence of AMP as substrate. (l) Control; (j) 0.05 mM ATP/0.01 mM ADP; (n) 0.5 mM ATP/0.05 mM ADP.
itory effect by ATP whereas ADP resulted in 78% inhibition. Kinetic investigations of these inhibitory effects (Figs. 2A* and 2B*) indicated that ATP and ADP produced changes in both slopes and intercepts, consistent with mixed-type inhibition (19). In the presence of AMP as substrate (Figs. 2A* and 2B*), Ki and K*i values for ATP were 5.4 1 1005 and 54 1 1005 M. Ki and K*i for ADP were 4.4 1 1006 and 36 1 1006 M, showing an affinity toward the enzyme and its enzyme–substrate complex at least 10 times higher than that of ATP. The same results were obtained in the presence of IMP, as Ki and K*i values were 10 1 1005 and 25 1 1005 M for ATP and 3.3 1 1006 and 12 1 1006 M for ADP. The data indicate that ATP and ADP always show greater affinity to the enzyme than to the enzyme–substrate complex. Inorganic phosphate inhibited enzyme activity at very high, nonphysiological concentrations. It had
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no effect at concentrations up to 10 mM, whereas 50 mM Pi caused a total enzyme inactivation (data not shown). DISCUSSION Soluble 5*-nucleotidase from human seminal plasma was purified to near homogeneity, as judged by silver-stained SDS–PAGE. The main purification step was affinity chromatography on AMP–Sepharose 4B, as reported in the purification of several nucleotidases from human placenta (20), human seminal plasma (8), chicken gizzard (21), human Tand B-lymphoblasts (22), rat liver (23), and bovine liver (24). The apparently homogeneous enzyme was eluted from Sephacryl SW300 as a symmetrical peak with no shoulder at the position corresponding to a molecular mass of 199 kDa. A tetrameric structure of the active enzyme was suggested since the subunit
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molecular mass estimated by SDS–PAGE was about 53 kDa. The purified enzyme had a specific activity of about 4800 U/mg protein, corresponding to a purification factor of 320. The poor recovery was not unexpected since a densitometric scanning of the (NH4)2SO4 precipitate showed that this soluble 5*nucleotidase constituted about 0.5% of the total protein in the sample. This form of enzyme, recovered in the nonsedimentable fraction of the seminal plasma, was not inhibited either by Con A or by adenosine5-[a,b-methylene]diphosphate, specific and powerful inhibitors of the AMP-specific solubilized ecto5*nucleotidase (6,25). Moreover, in agar gel immunodiffusion, no precipitin line was observed between rabbit anti-human spermatozoa ecto5*-nucleotidase antibodies and the enzyme preparation at various purification steps. These antibodies did not inhibit the purified enzyme activity nor did they make any effective immune complex with the enzyme. On the other hand, our enzyme preparation had a Km for AMP of 25 mM, quite in agreement with values reported for ecto and solubilized forms (6). A cytoplasmic AMP-specific 5*-nucleotidase from Saccharomyces ovi formis was originally characterized by Takei (26,27). A Km value of õ0.2 1 1003 M for AMP was determined at pH 6.1, and Co2/ and Ni2/ were activators of the enzyme, while Mg 2/ had no effect at all. The activity, reduced by treatment with EDTA, was fully restored by Co2/ and Ni2/, whereas Mg 2/ or Mn2/ did not reactivate the enzyme. Quite recently a cytosolic IMP-specific hydrolyzing enzyme from Saccharomyces cerevisiae was purified by Itoh (28). This enzyme had virtually no detectable activity with other purine and pyrimidine nucleotides except IMP, and shared some features in common with IMP/GMP 5*-nucleotidase from animals (29,30,6). It was absolutely dependent on bivalent metal salts and S0.5 for IMP was 0.4 mM. The enzyme from the nonsedimentable fraction of human seminal plasma described here had some unique properties. It had little preference for AMP as a substrate; Mg 2/ and Mn2/ were activators of the enzyme, although its activity was not absolutely dependent on the presence of these exogenous bivalent cations. The enzyme was soluble, had a pH optimum of 7.5, and was inhibited by ATP and ADP. These properties are close to those reported for solubilized ecto5*-nucleotidase (6). However, there are some differences in the regulatory characteristics between the enzyme described here and the solubilized
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forms. Indeed it is well known that ATP, ADP, and adenosine 5-[a,b-methylene]diphosphate are highly effective competitive inhibitors of the AMP-specific solubilized form and that the inhibition by Con A is noncompetitive. First, adenosine 5-[a,b-methylene]diphosphate and Con A did not inhibit the purified enzyme; second, ATP and ADP behaved like mixed-type inhibitors and Pi , like cytosolic AMP specific 5*-nucleotidase (7,31–33), was a potent inhibitor at nonphysiological concentrations, whereas it had no effect on nonsolubilized enzyme (6). Our enzyme also differed with respect to substrate specificity. In contrast with the solubilized form, it showed activity with 2-deoxyderivatives, a feature shared with truly cytoplasmic forms (6). The suggested tetrameric structure and its molecular mass were also in contrast with the dimeric structure detected for solubilized enzyme (21,33). According to Zimmermann (6), cytoplasmic forms of 5*-nucleotidase are encoded by genes different from the gene coding the membrane-bound form; therefore it could be expected that, as in the case of alkaline phosphatase, there might be tissue-specific cytoplasmic distinct isoforms. Studies on the physiological effectors of this reported form of soluble 5*nucleotidase may clarify its role in the human seminal production of adenosine. REFERENCES 1. Liu J, Burns DM, Beacham IR. J Bacteriol 165:1002–1010, 1986. 2. Drummond GI, Yamamoto M. The Enzymes. New York/London: Academic Press, 1971, pp 337–354. 3. Ipata PL. Biochemistry 7:507–515, 1968. 4. Evans WH, Gurd JW. Biochem J 133:189–199, 1973. 5. Mallol J, Bozal J. J Neurochem 40:1205–1211, 1983. 6. Zimmerman H. Biochem J 285:345–365, 1992. 7. Truong VL, Collison AR, Lowenstein JM. Biochem J 253:117–121, 1988. 8. Minelli A, Moroni M, Fabiani R, Miscetti P, Mezzasoma I. Biochim Biophys Acta 1080:252–258, 1991. 9. Minelli A, Moroni M, Luzi L, Mezzasoma I. Int J Biochem 25:1203–1207, 1993. 10. Fraser LR, Duncan AE. J Reprod Fertil 98:187–194, 1993. 11. Shen MR, Linden J, Chen SS, Wu SN. Clin Exp Pharmacol Physiol 20:527–534, 1993. 12. Minelli A, Miscetti P, Allegrucci C, Mezzasoma I. Arch Biochem Biophys 322:272–276, 1995. 13. Itaya K, Ui M. Clin Chim Acta 14:361–366, 1966. 14. Meghji P, Middleton KM, Newby AC. Biochem J 249:695– 703, 1988.
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SOLUBLE 5*-NUCLEOTIDASE FROM HUMAN SEMINAL PLASMA 15. Bradford MM. Anal Biochem 72:248–258, 1966. 16. Davis BJ. Ann NY Acad Sci 121:404–427, 1964. 17. Thibault C, Chan JK, Perdue JF, Daughaday WH. J Biol Chem 259:3361–3367, 1984. 18. Weber K, Pringle JR, Osborne M. Methods Enzymol 26:3– 27, 1972. 19. Dixon M, Webb EC. Enzymes, 3rd ed. United Kingdom: Longmann Group Ltd. 1979, pp 339–353. 20. Kaminska, Berry J, Madrid-Marina V, Fox IH. J Biol Chem 261:449–452, 1986. 21. Dieckoff J, Knebel H, Heidemann M, Mannherz HJ. Eur J Biochem 151:377–383, 1985. 22. Madrid-Marina V. Int J Biochem 22:1283–1289, 1990. 23. Spychala J, Madrid-Marina V, Nowak PJ, Fox IH. Am J Physiol 256:E386–E391, 1989.
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24. Zekri M, Harb J, Bernard S, Meflah K. Eur J Biochem 172:93–99, 1988. 25. Skladanowski AC, Newby AC. Biochem J 268:117–122, 1990. 26. Takei S. Agr Biol Chem 31:1251–1255, 1967. 27. Takei S. Agr Biol Chem 30:1215–1220, 1966. 28. Itoh R. Biochem J 298:593–598, 1994. 29. Itoh R. Comp Biochem Physiol B 105:13–19, 1993. 30. Itoh R, Yamada K. Int J Biochem 22:231–238, 1990. 31. Yamazaki Y, Truong VL, Lowenstein JM. Biochemistry 30:1503–1509, 1991. 32. Skladanowski AC, Smolenski RT. Proceedings, International Society for Heart Research, European Section Meeting (Monduzzi, Ed.), pp 27–34. 33. Grondal EJM, Zimmermann H. Biochem J 245:805–810, 1987.
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58, 168–175 (1996)
0045
The Isolation from Human Seminal Plasma of a New Form of Soluble 5*-Nucleotidase ALBA MINELLI, MONICA MORONI, NADIA MONACELLI,
AND ISABELLA
MEZZASOMA
Dipartimento di Biologia Cellulare e Molecolare, Sezione di Enzimologia, Universita` degli Studi di Perugia, 06123 Perugia, Italy Received January 2, 1996
of both purine and pyrimidine bases. Among the soluble forms, a further classification is based on the different Km values toward the substrates of the enzyme. A low Km isozyme, derived from the membrane-bound form by phospholipase C cleavage of the glycosyl phosphatidylinositol anchor, shows values of Km’s for AMP and IMP in the micromolar range and, according to the criteria of Truong et al. (7), has a preference for AMP. Truly cytoplasmic types, the high Km isozymes, have Km values for AMP and IMP in the millimolar range and, on the basis of their substrate preference, are subdivided into AMPases and IMPases. However, soluble 5*-nucleotidases are less clearly defined and, at present, data not easily classified into this general scheme are attributed to several not yet well identified factors (6). Recently a rather peculiar form of soluble enzyme was isolated in human seminal plasma (8). The enzyme, a low Km type, does not cross-react with ecto5*nucleotidase antibodies and prefers IMP, and its activity is absolutely Mg 2/-dependent and is affected in different ways by ATP and ADP (9). During the purification of the low Km IMP-specific 5*-nucleotidase (8), the human seminal plasma, chromatographed on AMP–Sepharose 4B, was resolved in two peaks of 5*-nucleotidase activity. In view of the various effects exerted by adenosine on the spermatozoa (10–12), these enzymes are of general interest. The present paper deals with the isolation and the characterization of the form less tightly bound to AMP–Sepharose 4B in order to investigate the properties of this isozyme on the degradation of nucleoside 5-monophosphate and its eventual involvement in the production of adenosine, which may affect the fertilizing capacity of the ejaculate.
Soluble broad spectrum 5*-nucleotidase from human seminal plasma was purified to homogeneity by a combination of (NH4)2SO4 precipitation, affinity chromatography, and gel filtration. The pure enzyme had a specific activity of 4800 nmol min01 mg01. SDS–PAGE of purified enzyme preparation revealed a single polypeptide band of 53 kDa and a tetrameric structure of 203 kDa was proposed for the native enzyme. This form had modest preference for AMP as substrate; Mg 2/ and Mn2/ were activators of the enzyme although its activity was not absolutely dependent on the presence of these exogenous bivalent cations. The enzyme, recovered in the nonsedimentable fraction of human seminal plasma, had a pH optimum of 7.5; ATP and ADP were inhibitors of mixed type, Pi was a potent inhibitor at nonphysiological concentrations, and Con A and adenosine 5-[a,b-methylene]diphosphate had no effect on the enzyme activity. The enzyme described here therefore has some unique properties between truly cytoplasmic and membrane-bound derived forms. q 1996 Academic Press, Inc.
Nucleotidases (5* ribonucleotide phosphohydrolase, EC 3.1.3.5) constitute a large class of heterogeneous isozymes that are widely distributed among animals, plants, and microorganisms (1–5). These isoenzymes catalyze the hydrolysis of monophosphate ester linkages in various 5*-nucleotides, producing inorganic phosphate (Pi) and the corresponding nucleoside. According to Zimmermann (6), the vertebrate tissue 5*-nucleotidase family can be divided into four classes, each characterized by substrate preference, kinetics, cation requirements, and pH optima, although all are active with 5*-nucleotides 168 1077-3150/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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SOLUBLE 5*-NUCLEOTIDASE FROM HUMAN SEMINAL PLASMA
MATERIALS AND METHODS Human seminal fluid from healthy volunteers was a gift from Centro di Fisiopatologia della Riproduzione, University of Bologna. ATP, ADP, AMP, p-nitrophenylphosphate, nucleosides, all nucleoside mono-, di-, and triphosphates, and Blue Sepharose CL-6B were from Sigma Chemical Co. Bradford protein dye reagent, high- and low-molecular-weight standards, and the Silver Stain kit were from Bio-Rad. AMP– Sepharose 4B and Sephacryl SW300 were from Pharmacia. All other reagents used were of the highest quality available. Assays The 5*-nucleotidase assay was done as previously described (8,9). Incubations were performed at 377C in medium containing 50 mM Tris–HCl pH 7.4, 2 mM substrate, and other additions, as indicated. The reaction was carried out in a total volume of 100 ml and terminated by heating at 1007C. The amount of Pi liberated was determined according to Itaya and Ui (13). All the experimental values were corrected by subtracting the respective control, i.e., the amount of inorganic phosphate present in parallel assays performed with acid-precipitated samples. The assay was linear with protein and time up to 60 min. One unit is defined as the amount of enzyme which cleaves 1 nmol of substrate in 1 min at 377C. In experiments where the effect of Pi was tested, 1–2 kBq of 2-3H-5*AMP was added to the standard incubation medium. The activity of 5*-nucleotidase was assayed by measuring the production of labeled adenosine according to Truong et al. (7) and Meghji et al. (14). Incubations were conducted at 377C for 30 min in a total volume of 150 ml containing 50 mM Tris–HCl, pH 7.4, 2 mM AMP plus labeled substrate and Pi . The reaction was started by adding the appropriate enzyme so that the overall substrate conversion did not exceed 25%. The reaction was terminated by adding 0.15 mM ZnSO4 and 0.15 mM Ba(OH)2 . Phosphatases were measured by incubating samples in 50 mM Na citrate, pH 4.8, in 50 mM glycine, 0.5 mM MgCl2 , pH 10.5, and in 50 mM Tris–HCl, pH 7.4, for 30 min at 377C, in a total vol of 500 ml containing 5.5 mM pNPP. The reaction was stopped by 2.5 ml of 0.1 M NaOH. The samples, when needed, were clarified by centrifugation and optical density at 410 nm was measured with a U2000 Hitachi Spectrophotometer. Controls were performed as in the 5*-nucleotidase assay.
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Protein Determination Protein was quantitated by the Bradford assay method with bovine serum albumin as the standard protein (15). Proteins eluted from the columns were monitored by absorbance at 280 nm or by the Bradford method. Purification All procedures were performed at 47C, unless otherwise stated. Human semen, thawed at room temperature, was centrifuged at 700g for 30 min to remove intact spermatozoa. The supernatant was again spun at 30,000g for 30 min and the clear supernatant was referred to as the seminal plasma. Forty milliliters of seminal plasma was further centrifuged at 105,000g for 60 min and solid (NH4)2SO4 was slowly added to the supernatant to give 35% saturation. After 60 min, the precipitate was removed by centrifugation at 17,500g for 30 min. More (NH4)2SO4 was added to the supernatant to give 60% saturation, the solution was centrifuged, and the resulting precipitate was dissolved and dialyzed overnight against 50 mM Tris – HCl, 0.1 M NaCl, pH 7.4. The dialyzed solution (30 ml) was loaded onto an AMP – Sepharose 4B column (8 1 1.5 cm). The column was washed with 150 ml of 50 mM Tris – HCl, 0.1 M NaCl buffer, pH 7.4, and the enzyme was eluted with 0.5 M NaCl in 50 mM Tris – HCl, pH 7.4. Fractions containing 5*-nucleotidase activity (50 ml) were pooled, dialyzed against 50 mM Tris – HCl, pH 7.4, and loaded on a Blue Sepharose column (4 1 1.6 cm) previously equilibrated with 50 mM Tris – HCl, pH 7.4. Non-binding proteins were eluted with the equilibrating buffer containing 0.1 M NaCl. The enzyme (40 ml) was eluted with a 0.5 M NaCl step in 50 mM Tris buffer, pH 7.4, dialyzed against 50 mM Tris– HCl, pH 7.4, lyophilized, dissolved in 50 mM Tris– HCl, 0.5 M NaCl, pH 7.4, applied to a Sephacryl SW 300 column (95 1 1.5 cm) that was preequilibrated in same buffer, and eluted at a flow rate of 7.5 ml/ h. The fractions with high activity were pooled and stored at 47C until use. Molecular Weight Determination The relative molecular mass of the native protein was determined either by gel filtration on a Sephacryl SW300 16/60 column (Pharmacia) or by electrophoresis on polyacrylamide gels. The column was
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TABLE 1 Purification of Soluble 5*-Nucleotidase from Human Seminal Plasma Purification step
Total activity (U)
Total protein (mg)
Specific activity (U/mg)
Purification factor
Yield (%)
Seminal plasma (NH4)2SO4 fractionation First AMP–Sepharose 4B Blue Sepharose CL-6B Sephacryl SW 300
133,200 30,680 2,100 304 72
3520 2050 5.3 0.24 0.015
49 15 396 1266 4800
— 0 26 84 320
— 100 6.8 0.9 0.23
Note. 5* Nucleotidase assays and protein determinations were performed as described under Material and Methods.
run at 12 ml/h and 2-ml fractions were collected. The column was calibrated with thyroglobulin (670 kDa), g-globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa). Polyacrylamide gel electrophoresis under nondenaturing conditions was performed in the presence of 7.5% acrylamide, according to Davis (16), and in the presence of polyacrylamide gels at concentrations ranging from 5 to 12%, according to Thibault et al. (17). From a Ferguson–Hedrick plot, KR , the retardation coefficient, was determined for standard proteins: urease (545 and 272 kDa), bovine serum albumin (132 and 66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), and a-lactalbumin (14.2 kDa) (Sigma). SDS–PAGE was performed according to Weber et al. (18) on a 12% acrylamide gel stained by the Bio-Rad silver stain kit.
by (NH4)2SO4 fractionation, this step was considered the initial step of the purification procedure. The AMP–Sepharose 4B chromatography succeeded in separating alkaline phosphatases and 5*nucleotidase, which was successively 84-fold purified by the Blue Sepharose column. A specific activity of 4800 U/mg was found in the enzyme preparation eluted from Sephacryl SW300, corresponding to a purification factor of 320. An overall purification of greater than 320 can be suggested on the basis that the specific activity of this soluble 5*-nucleotidase is surely smaller than 15, as seminal alkaline phosphatases contribute to this value. The enzyme resisted further purification and was homogeneous by criteria of gel electrophoresis.
Kinetic Calculations
The molecular weight of the native soluble 5*nucleotidase was determined by gel filtration on Sephacryl SW300. The enzyme activity was eluted as a single symmetrical peak with no shoulder, at the position corresponding to an apparent molecular mass of 199 kDa. Gel electrophoresis on polyacrylamide at different concentrations evidenced a single band of 203 kDa. SDS–PAGE electrophoresis resolved the enzyme in a single band of 53 kDa. The enzyme is therefore composed of four identical subunits.
Kinetic data were analyzed by a linear regression with the Enzfitter (Biosoft Elsevier) program by using the equations corresponding to Michaelis–Menten equations. Five triplicates of each point were performed. Goodness of fit was tested according to reduced SD values given by the programs. RESULTS
Molecular Properties
Purification of the Enzyme Soluble 5*-nucleotidase was purified by a combination of (NH4)2SO4 precipitation, affinity chromatography, and gel filtration, as summarized in Table 1. A precise quantitative evaluation of the initial step of this purification is hindered by our inability to distinguish 5*-nucleotidase activity from nonspecific phosphatase in crude preparation. Thus, as the acidic phosphatases were almost entirely removed
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Thermostability and Effect of Temperature The enzyme preparation, kept at 47C, was stable for extended periods. Thermal stability of the enzyme was examined and the results are shown in Fig. 1A. About 80% of the activity was retained after incubation of the enzyme for 30 min at 40–507C, while total inactivation occurred at a temperature of 707C. The influence of temperature on the enzyme
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FIG. 1. Properties of purified soluble 5*-nucleotidase from human seminal plasma. (A) Effect of thermal denaturation on 5*-nucleotidase activity. Aliquots of the enzyme were incubated for 30 min at different temperatures and the effect on activity was determined, as described under Materials and Methods. (B) Effect of temperature on 5*-nucleotidase activity. Activity was measured at different temperatures (0–607C) as described under Materials and Methods. (C) Arrhenius plot for the dephosphorylation of AMP by 5*-nucleotidase. Assays in the presence of various substrate concentrations were run as described under Materials and Methods. (D) Effect of pH on activity and stability of 5*-nucleotidase. The enzyme activity toward AMP (1) and CMP (-r-) was assayed at 377C in 0.2 M Tris– maleate buffer (pH 6.0–8.5), and in 0.2 M Tris–glycine buffer (pH 8.5–9.0). The stability in the presence of AMP (j) and CMP (l) was tested by preincubating the enzyme for 30 min at the indicated pH values in 5 mM citrate–HCl (pH 2.0–6.0), in 5 mM Tris–HCl (pH 7.0–9.0), and in 5 mM glycine–HCl (pH 10–13).
activity is illustrated in Fig. 1B. The enzyme displayed an apparent maximum of activity at 377C. A continuous Arrhenius plot obtained from activity measurements between 0 and 407C gave an activation energy of 14.5 kcal/mol (Fig. 1C). An Ea of 11.7 kcal/mol was obtained in the presence of CMP as substrate. Effect of pH on Activity and Stability The pH dependence of the soluble 5*-nucleotidase is reported in Fig. 1D. The activity against AMP and CMP was measured over a pH range of 6.0–9.0 and two pH optimum values of 7.5 for AMP and 8.0 for CMP were identified. In the presence of these two substrates, preincubation of the enzyme at pH õ7.0 decreased the activity either by forming an improper ionic form or by irreversibly inactivating the enzyme. Preincubation at pH ú7.0 did not negatively affect the enzyme activity.
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Substrate Specificity As shown in Table 2, the enzyme preferred AMP with a Vmax/Km ratio of 348 1 105. Although Vmax values were almost on the same order of magnitude for the dephosphorylation of AMP and IMP, the ratio Vmax/Km indicated that AMP was the preferred substrate. Vmax values were fairly high in the presence of pyrimidine nucleotides with an efficiency of substrate utilization of 280 1 105. The enzyme therefore preferentially hydrolyzed in the order AMP ú UMP Å CMP ú IMP. It was less active with deoxy-derivatives and had no activity in the presence of ribose-1P, ribose-5P, pNPP, or bglycerophosphate, as expected. Effect of Metal Ions The activity of purified enzyme, measured in the absence of exogenous cations, indicated a nonabso-
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TABLE 2 Substrate Specificity of Human Seminal Plasm Soluble 5*-Nucleotidase Vmax (nmol/min/mg protein)
Substrate IMP AMP UMP CMP GMP dGMP dIMP dAMP Ribose 1-P Ribose 5-P pNPP b-Glycerophosphate
876 { 871 { 1117 { 1150 { 693 { 270 { 245 { 324 { — — — —
12 13 10 11 9 8 4 5
Substrate efficiency (Vmax/Km)
Km (M) 6.3 2.5 4.0 4.1 4.5 3.5 3.6 4.5
{ { { { { { { {
0.5 1 0.3 1 0.2 1 0.4 1 0.4 1 0.9 1 1.2 1 1.0 1 — — — —
1005 1005 1005 1005 1005 1005 1005 1005
139 348 280 279 155 77 69 72
1 1 1 1 1 1 1 1 — — — —
105 105 105 105 105 105 105 105
Note. Determinations were performed in 25 mM Tris–HCl pH 7.4, with at least five different concentrations of substrate (n Å 15), as described under Material and Methods.
lute requirement of metal ions, although Mg 2/ and Mn2/ were shown to be able to modulate the activity of the enzyme. In the presence of MgCl2 , the enzyme activity increased with increasing concentrations up to 10 mM. With MnCl2 as the bivalent cation, optimal activity was obtained at 3 mM. Higher concentrations up to 10 mM did not result in further enzyme activation. The maximal activity with MnCl2 was approximately two times higher than the maximal activity obtained with MgCl2 . In order to examine the specificity of the effect of these stimulatory cations on soluble 5*-nucleotidase, in the experiments reported in Table 3, other divalent cations were used. The data show that all other metal ions tested inhibited the enzyme. Metal chelators EDTA and EGTA caused a concentration-dependent inhibition of the enzyme with I50 values of 0.04 and 1.26 mM, respectively. Removal of metal ions from the enzyme molecule by treatment with 1 mM EDTA and overnight dialysis against 25 mM Tris–HCl, pH 7.4, caused an 88% inactivation of the enzyme activity and the addition of 10 mM bivalent cation did not restore the native activity. Indeed the treatment of the enzyme with 0.01 mM EDTA caused a 25% inhibition and the addition of MgCl2 restored nearly the full activity while MnCl2 still caused a slight stimulation of the enzyme activity. CaCl2 , as expected, was ineffective in restoring the native enzyme activity. Effect of ATP, ADP, and Pi The concentration dependencies of ATP and ADP on soluble 5*-nucleotidase activity are presented in
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Figs. 2A and 2B, in the presence of AMP and IMP as substrate. ATP was an inhibitor that was not as effective as ADP; 1 mM ATP caused 50% inhibition of the enzyme activity, whereas the same level of inhibition was obtained with 0.05 mM ADP. Moreover, the inhibitory effect caused by ADP was more pronounced than that caused by ATP as it caused 82% inhibition of the AMP dephosphorylation against the 60% inhibition caused by ATP. The dephosphorylation of IMP was equally regulated by ATP and ADP. A 33% inhibition of IMP dephosphorylation represented the maximum inhib-
TABLE 3 Effects of Metal Ions and Metal Chelators on Human Seminal Plasm Soluble 5*-Nucleotidase Ion or chelator
Percentage activity
U/ml
None Mg2/ Zn2/ Ca2/ Mn2/ Cu2/ Ni2/ Hg2/
905 { 1440 { 432 { 576 { 2153 { — 78 { 576 {
1 mM EDTA 1 mM EGTA
11 14 7 9 14 4 8
100 160 47 63 237 — 9 63
110 { 5 570 { 9
12 58
Note. The assay was performed in the presence of the indicated divalent cations at a concentration of 10 mM (n Å 10), as described under Material and Methods.
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FIG. 2. Effect of ATP (A) and ADP (B) on soluble 5*-nucleotidase from human seminal plasma. After preincubation of the enzyme with the indicated concentrations of ATP and ADP for 10 min at 377C, the assay was performed as described under Materials and Methods. (l) AMP; (j) IMP. Kinetic parameters of the inhibition exerted by ATP (A*) and ADP (B*) in the presence of AMP as substrate. (l) Control; (j) 0.05 mM ATP/0.01 mM ADP; (n) 0.5 mM ATP/0.05 mM ADP.
itory effect by ATP whereas ADP resulted in 78% inhibition. Kinetic investigations of these inhibitory effects (Figs. 2A* and 2B*) indicated that ATP and ADP produced changes in both slopes and intercepts, consistent with mixed-type inhibition (19). In the presence of AMP as substrate (Figs. 2A* and 2B*), Ki and K*i values for ATP were 5.4 1 1005 and 54 1 1005 M. Ki and K*i for ADP were 4.4 1 1006 and 36 1 1006 M, showing an affinity toward the enzyme and its enzyme–substrate complex at least 10 times higher than that of ATP. The same results were obtained in the presence of IMP, as Ki and K*i values were 10 1 1005 and 25 1 1005 M for ATP and 3.3 1 1006 and 12 1 1006 M for ADP. The data indicate that ATP and ADP always show greater affinity to the enzyme than to the enzyme–substrate complex. Inorganic phosphate inhibited enzyme activity at very high, nonphysiological concentrations. It had
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no effect at concentrations up to 10 mM, whereas 50 mM Pi caused a total enzyme inactivation (data not shown). DISCUSSION Soluble 5*-nucleotidase from human seminal plasma was purified to near homogeneity, as judged by silver-stained SDS–PAGE. The main purification step was affinity chromatography on AMP–Sepharose 4B, as reported in the purification of several nucleotidases from human placenta (20), human seminal plasma (8), chicken gizzard (21), human Tand B-lymphoblasts (22), rat liver (23), and bovine liver (24). The apparently homogeneous enzyme was eluted from Sephacryl SW300 as a symmetrical peak with no shoulder at the position corresponding to a molecular mass of 199 kDa. A tetrameric structure of the active enzyme was suggested since the subunit
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molecular mass estimated by SDS–PAGE was about 53 kDa. The purified enzyme had a specific activity of about 4800 U/mg protein, corresponding to a purification factor of 320. The poor recovery was not unexpected since a densitometric scanning of the (NH4)2SO4 precipitate showed that this soluble 5*nucleotidase constituted about 0.5% of the total protein in the sample. This form of enzyme, recovered in the nonsedimentable fraction of the seminal plasma, was not inhibited either by Con A or by adenosine5-[a,b-methylene]diphosphate, specific and powerful inhibitors of the AMP-specific solubilized ecto5*nucleotidase (6,25). Moreover, in agar gel immunodiffusion, no precipitin line was observed between rabbit anti-human spermatozoa ecto5*-nucleotidase antibodies and the enzyme preparation at various purification steps. These antibodies did not inhibit the purified enzyme activity nor did they make any effective immune complex with the enzyme. On the other hand, our enzyme preparation had a Km for AMP of 25 mM, quite in agreement with values reported for ecto and solubilized forms (6). A cytoplasmic AMP-specific 5*-nucleotidase from Saccharomyces ovi formis was originally characterized by Takei (26,27). A Km value of õ0.2 1 1003 M for AMP was determined at pH 6.1, and Co2/ and Ni2/ were activators of the enzyme, while Mg 2/ had no effect at all. The activity, reduced by treatment with EDTA, was fully restored by Co2/ and Ni2/, whereas Mg 2/ or Mn2/ did not reactivate the enzyme. Quite recently a cytosolic IMP-specific hydrolyzing enzyme from Saccharomyces cerevisiae was purified by Itoh (28). This enzyme had virtually no detectable activity with other purine and pyrimidine nucleotides except IMP, and shared some features in common with IMP/GMP 5*-nucleotidase from animals (29,30,6). It was absolutely dependent on bivalent metal salts and S0.5 for IMP was 0.4 mM. The enzyme from the nonsedimentable fraction of human seminal plasma described here had some unique properties. It had little preference for AMP as a substrate; Mg 2/ and Mn2/ were activators of the enzyme, although its activity was not absolutely dependent on the presence of these exogenous bivalent cations. The enzyme was soluble, had a pH optimum of 7.5, and was inhibited by ATP and ADP. These properties are close to those reported for solubilized ecto5*-nucleotidase (6). However, there are some differences in the regulatory characteristics between the enzyme described here and the solubilized
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forms. Indeed it is well known that ATP, ADP, and adenosine 5-[a,b-methylene]diphosphate are highly effective competitive inhibitors of the AMP-specific solubilized form and that the inhibition by Con A is noncompetitive. First, adenosine 5-[a,b-methylene]diphosphate and Con A did not inhibit the purified enzyme; second, ATP and ADP behaved like mixed-type inhibitors and Pi , like cytosolic AMP specific 5*-nucleotidase (7,31–33), was a potent inhibitor at nonphysiological concentrations, whereas it had no effect on nonsolubilized enzyme (6). Our enzyme also differed with respect to substrate specificity. In contrast with the solubilized form, it showed activity with 2-deoxyderivatives, a feature shared with truly cytoplasmic forms (6). The suggested tetrameric structure and its molecular mass were also in contrast with the dimeric structure detected for solubilized enzyme (21,33). According to Zimmermann (6), cytoplasmic forms of 5*-nucleotidase are encoded by genes different from the gene coding the membrane-bound form; therefore it could be expected that, as in the case of alkaline phosphatase, there might be tissue-specific cytoplasmic distinct isoforms. Studies on the physiological effectors of this reported form of soluble 5*nucleotidase may clarify its role in the human seminal production of adenosine. REFERENCES 1. Liu J, Burns DM, Beacham IR. J Bacteriol 165:1002–1010, 1986. 2. Drummond GI, Yamamoto M. The Enzymes. New York/London: Academic Press, 1971, pp 337–354. 3. Ipata PL. Biochemistry 7:507–515, 1968. 4. Evans WH, Gurd JW. Biochem J 133:189–199, 1973. 5. Mallol J, Bozal J. J Neurochem 40:1205–1211, 1983. 6. Zimmerman H. Biochem J 285:345–365, 1992. 7. Truong VL, Collison AR, Lowenstein JM. Biochem J 253:117–121, 1988. 8. Minelli A, Moroni M, Fabiani R, Miscetti P, Mezzasoma I. Biochim Biophys Acta 1080:252–258, 1991. 9. Minelli A, Moroni M, Luzi L, Mezzasoma I. Int J Biochem 25:1203–1207, 1993. 10. Fraser LR, Duncan AE. J Reprod Fertil 98:187–194, 1993. 11. Shen MR, Linden J, Chen SS, Wu SN. Clin Exp Pharmacol Physiol 20:527–534, 1993. 12. Minelli A, Miscetti P, Allegrucci C, Mezzasoma I. Arch Biochem Biophys 322:272–276, 1995. 13. Itaya K, Ui M. Clin Chim Acta 14:361–366, 1966. 14. Meghji P, Middleton KM, Newby AC. Biochem J 249:695– 703, 1988.
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SOLUBLE 5*-NUCLEOTIDASE FROM HUMAN SEMINAL PLASMA 15. Bradford MM. Anal Biochem 72:248–258, 1966. 16. Davis BJ. Ann NY Acad Sci 121:404–427, 1964. 17. Thibault C, Chan JK, Perdue JF, Daughaday WH. J Biol Chem 259:3361–3367, 1984. 18. Weber K, Pringle JR, Osborne M. Methods Enzymol 26:3– 27, 1972. 19. Dixon M, Webb EC. Enzymes, 3rd ed. United Kingdom: Longmann Group Ltd. 1979, pp 339–353. 20. Kaminska, Berry J, Madrid-Marina V, Fox IH. J Biol Chem 261:449–452, 1986. 21. Dieckoff J, Knebel H, Heidemann M, Mannherz HJ. Eur J Biochem 151:377–383, 1985. 22. Madrid-Marina V. Int J Biochem 22:1283–1289, 1990. 23. Spychala J, Madrid-Marina V, Nowak PJ, Fox IH. Am J Physiol 256:E386–E391, 1989.
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24. Zekri M, Harb J, Bernard S, Meflah K. Eur J Biochem 172:93–99, 1988. 25. Skladanowski AC, Newby AC. Biochem J 268:117–122, 1990. 26. Takei S. Agr Biol Chem 31:1251–1255, 1967. 27. Takei S. Agr Biol Chem 30:1215–1220, 1966. 28. Itoh R. Biochem J 298:593–598, 1994. 29. Itoh R. Comp Biochem Physiol B 105:13–19, 1993. 30. Itoh R, Yamada K. Int J Biochem 22:231–238, 1990. 31. Yamazaki Y, Truong VL, Lowenstein JM. Biochemistry 30:1503–1509, 1991. 32. Skladanowski AC, Smolenski RT. Proceedings, International Society for Heart Research, European Section Meeting (Monduzzi, Ed.), pp 27–34. 33. Grondal EJM, Zimmermann H. Biochem J 245:805–810, 1987.
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