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Studies of bovine seminal fluid nucleotide pyrophosphatase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 202, No. 2, July, pp. 396-404, 1980
Studies
of Bovine
Seminal
MATTHEW
E. BUCKON
Department of Biochemistry
Fluid Nucleotide AND BRUCE
Pyrophosphatasel
M. ANDERSON2
and Nutrition, Virginia Polytechnic Institute Blacksburg, Virginia 21061
and State University,
Received February 4, 1980 Nucleotide pyrophosphatase of bovine seminal plasma was demonstrated to catalyze effectively the hydrolysis of NAD, FAD, etheno-NAD, adenosine diphosphoribose, 3aminopyridine adenine dinucleotide, and bis++nitrophenylphosphate. The enzyme was purified SO-fold resulting in a partial separation from 5’-nucleotidase and alkaline phosphatase activities. The SO-fold purified nucleotide pyrophoshatase had a pH optimum of 9.0 and was competitively inhibited by adenosine, AMP, and sodium pyrophosphate. Time-dependent inactivation of the enzyme was observed by thermal denaturation at 62°C and by incubation with EDTA at 4°C. The activity of the enzyme was increased with increasing ionic strength and specific stimulation by MgZ+ was not observed. In the absence of NAD glycohydrolase, the hydrolysis of NAD in bovine seminal plasma was demonstrated through timed product studies to proceed initially through the hydrolysis of the pyrophosphate linkage catalyzed by nucleotide pyrophosphatase. The resulting product, AMP, was further hydrolyzed to adenosine in a reaction catalyzed by 5’nucleotidase. The observed functioning of adenosine diphosphoribose as a substrate for the nucleotide pyrophosphatase provided evidence in support of a second conversion of NAD to adenosine in seminal Dlasma through an initial hydrolytic reaction catalyzed by NAD glycohydrolase.
Earlier studies in this laboratory were concerned with the metabolic fate of NAD in bovine seminal plasma (1). The incubation of NAD with diluted seminal plasma resulted in the formation of adenosine diphosphoribose (ADPR), nicotinamide, and adenosine. The NAD glycohydrolase (NADase)3 of the seminal plasma first reported by Leone and Bonaduce (2) was purified to homogeneity (1, 3) and various properties of the enzyme were determined (3-7). The enzyme-catalyzed hydrolysis of NAD to ADPR and nicotinamide in bovine seminal plasma can be attributed to the presence of the soluble, glycoprotein NADase of 36,300 molecular weight. The
appearance of adenosine which becomes the predominant product after extended periods of NAD incubation with bovine seminal plasma had to result from the functioning of other enzymes in the seminal plasma. The existence of a nucleotide pyrophosphatase in bovine seminal plasma was first reported by Wheat et al. (8). Although several properties of this enzyme were studied (9, lo), the functioning of ADPR as a substrate was not investigated. The presence of 5’-nucleotidase in bovine seminal plasma was reported by Heppel and Hilmoe (11). The combined functioning of nucleotide pyrophosphatase and 5’nucleotidase in seminal plasma would result in the formation of adenosine from NAD. The formation of adenosine from NAD in bovine seminal plasma is of interest if one considers possible interactions of this nucleoside with spermatozoa. Caffeine and theophylline, structurally analogous to adenosine, inhibit cyclic nucleotide phosphodiesterase in bull spermatozoa and in the presence of exogenous substrates,
1 These studies were supported by Research Grant PCM 78 05839 from the National Science Foundation. * To whom correspondence and requests for reprints should be addressed. 3 Abbreviations used: ADPR, adenosine diphosphoribose; etheno-NAD, nicotinamide l,NB-ethenoadenine dinucleotide; AAD, 3-aminopyridine adenine dinucleotide; NADase, NAD glycohydrolase; TEAEcellulose, 0-(triethylaminoethyl)-cellulose. 0003-986Y80/080396-09$02.00/O 396 Copyright AU rights
0 1980 by Academic Press, of reproduction in any form
Inc. reserved.
STUDIES
OF BOVINE
NUCLEOTIDE
markedly stimulate sperm motility and respiration (12). More recently (13) the incubation of bovine epididymal spermatozoa with caffeine, theophylline, cyclic AMP, and N6,02-dibutyryl cyclic AMP was reported to increase the rate of fructolysis and motility. In an earlier study (14) adenosine was shown to stimulate oxygen uptake and motility in bull spermatozoa. In a comparison of epididymal and freshly ejaculated spermatozoa, Bistocchi et al. (15) reported epididymal spermatozoa to be immotile, showing low respiration rates and containing a high level of pyridine nucleotides while ejaculated spermatozoa had higher respiration rates, were motile, and had markedly decreased pyridine nucleotide levels. Leakage of pyridine nucleotides into the seminal plasma was indicated by these (15) and other studies (16). It was therefore of interest to study further the conversion of NAD to adenosine in bovine seminal plasma, to investigate the functioning of adenosine diphosphoribose as a substrate for the nucleotide pyrophosphatase, and to study the properties of enzymes catalyzing the various hydrolytic reactions involved. MATERIALS
AND METHODS
Holstein bull semen was obtained from Select Sires of Columbus, Ohio. Adenosine, 5’-AMP, 5’-ADP, ADPR, 2’-phospho-ADPR, 2’5’-ADP, 3’5’cyclic AMP, 5’-CMP, 5’-GMP, 2’-AMP, 3’-AMP, FAD, FMN, P-glycerophosphate, pancreatic lipase, NAD, nicotinamide 1,N6-ethenoadenine dinucleotide (etheno-NAD), NMN, p-nitrophenylphosphate, bisp-nitrophenylphosphate, riboflavin, concanavalin ASepharose 4B, Sepharose 4B, Sephadex G-200, TEAE-cellulose, Triton X-100, uridine, 5’-UMP, and UDP-Glc were purchased from Sigma. DEAESephadex and blue dextran Sepharose were purchased from Pharmacia. DEAE-cellulose (DE-52) were obtained from Whatman. Carboxymethyl cellulose (Cellex CM) and phosphocellulose (Cellex P) were purchased from Bio-Rad Laboratories. 3Aminopyridine adenine dinucleotide (AAD) was prepared according to Fisher et al. (17). Titrimetric assays of nucleotide pyrophosphatase were performed at 3’7°C in 3-ml reaction mixtures containing 10 mM KCl, 1 mM MgCl,, and substrate. Initial velocities were measured at pH 9.0 recording the utilization of the 100 mM NaOH titrant. The solutions were prepared with CO,-free water and argon was passed over the reaction mixture before
PYROPHOSPHATASE
397
and during the reaction. A Radiometer type TTT II titrator, type SBRSC titragraph, PHM 26C pH meter, type TTA 31 microtitration assembly, and type ABU 12 automatic burette were used in these studies. Units of activity were expressed as micromoles of substrate hydrolyzed per minute calculated on the basis of two protons released per pyrophosphate bond hydrolyzed. Fluorimetric assays of nucleotide pyrophosphatase were performed at 37°C in 3-ml reaction mixtures containing 30 mM Tris-HCl buffer, pH 9.0, 1 mM MgCl*, and substrate. With FAD as substrate, reaction mixtures were excited at 465 nm and the fluorescence emission at 516 nm was recorded. With etheno-NAD as substrate, reaction mixtures were excited at 297 nm and the fluorescence emission at 410 nm was recorded. Units of activity were expressed as fluorescence increase per minute in arbitrary fluorescence units. These values could be converted to international units by direct comparison with concomitant titrimetric assays. Fluorimetric assays were preferred for routine studies, purification studies, and studies of the properties of the nucleotide pyrophosphatase since the titrimetric assay required approximately 60 times as much enzyme per assay. All fluorimetric measurements were performed on an Aminco-Bowman recording spectrophotofluorometer. Protein concentrations were determined according to Lowry et al. (18). Product studies were performed through highperformance liquid chromatography on a Spectra Physics 8000 liquid chromatograph equipped with a ‘770 variable wavelength uv-visible detector. The column (25 x 0.46 cm) employed in these studies was a Whatman Partisil-10 SAX quaternary amine ion exchanger. Only products absorbing at 260 nm were monitored in these studies. RESULTS
Nucleotide pyrophosphatase in crude bovine seminal plasma was assayed using FAD as a substrate. FAD was chosen over NAD as substrate since crude seminal plasma contains high NADase activity and the hydrolysis of FAD could be monitored by the more sensitive fluorescence assay. Reaction mixtures containing 30 mM TrisHCl buffer, pH 9.0, 1 lllM MgC12, 66 pg of seminal fluid protein, and FAD concentrations varying from 1.5-14 PM were inCUbated at 37°C. The initial velocities of FAD hydrolysis were measured by monitoring the fluorescence emission at 516 nm with excitation at 465 nm and the initial velocities were linear for 5 min. Initial velocities as a function of FAD concentration were plotted
398
BUCKONANDANDERSON
according to Lineweaver and Burk (19) and the K, value for FAD calculated from these data was 3.6 FM. At saturating concentration of FAD, initial velocities were proportional to enzyme concentration in the range of 30-250 pg of protein. In order to study NAD and other nucleotides as substrates, the nucleotide pyrophosphatase was separated from the NADase by fractionation on a Sepharose 4B column, resulting in a 20-fold purification of the nucleotide pyrophosphatase activity. Using this preparation, the hydrolysis of NAD was investigated titrimetrically at 37°C and pH 9.0 in 3-ml reaction mixtures containing NAD, 10 mM KCl, 1 lllM MgC&, and 90 pug of protein. Initial velocities obtained at nine concentrations of NAD, plotted according to Lineweaver and Burk (19), were used to determine V and K, values. Under the same conditions, FAD, adenosine diphosphoribose, etheno-NAD, and AAD were studied as substrates, and the K, and V values for the hydrolysis of all substrates are listed in Table I. FAD and etheno-NAD were also studied fluorimetrically under conditions described above. The products of the hydrolysis of NAD and other substrates were identified through high-performance liquid chromatography. Reaction mixtures at 37°C contained 30 mM Tris-HCl buffer, pH 9.0, 1 mM MgC&, 0.5 mM substrate and 11 pg of protein (20-fold purified) in a total volume of 2 ml. At 0, ‘7, 60, and 120 min, 0.5-ml aliquots were TABLE K,
I
AND V VALUESOFZO-FOLDPURIFIED NUCLE~TIDE PYROPHOSPHATASE Assay method
&I (PM)
v (jmolmin-klg-1)
NAD
Titrimetric
8.7
2.2
ADPR
Titrimetric
11.0
0.96
AAD
Titrimetric
7.3
1.4
Substrate
FAD
Titrimetric Fluorometric
4.5 2.9
0.75
Ethenc+NAD
Titrimetric Fluorometric
9.0
1.0
8.7
2
7
1 I
/r---7
3 % u-4 a
i
iRetention
Ttme (min)
’
-125 -105
$j
6
03
FIG. 1. High-performance liquid chromatography of possible 260 nm-absorbing products of NAD hydrolysis. Peaks were identified as: 1, nicotinamide riboside; 2, adenosine; 3, NMN; 4, NAD; 5, AMP; 6, ADPR; 7, ADP. Dashed line represents programmed elution profile.
removed, filtered through Millipore filters to remove protein, and a lo-p1 sample was applied to a Partisil-10 column equilibrated with 5 mM sodium phosphate buffer, pH 4.0. The column was eluted with the same buffer for 15 min after which a linear gradient from 5 mM sodium phosphate, pH 4.0 to 129 mM sodium phosphate, pH 4.0, was applied. A separation of possible products from NAD hydrolysis is shown in Fig. 1. Samples from the enzyme-catalyzed hydrolysis of NAD assayed in this manner revealed the presence of NMN and AMP as primary products at the 7-min incubation time and the appearance and predominance of nicotinamide riboside and adenosine at the later time periods. No ADP or ADPR was observed during the hydrolysis of NAD. The enzyme-catalyzed hydrolysis of ADPR yielded AMP which in time was converted to adenosine and no ADP could be identified at any time interval. FAD hydrolysis yielded initially FMN and AMP which were rapidly hydrolyzed to riboflavin and adenosine. With the indicated presence of 5’-nucleotidase and/or alkaline phosphatase activities, a number of different nucleotides were assayed as substrates. A summary of the product studies with these substrates is shown in Table II. In order to evaluate the properties of the nucleotide pyrophosphatase, further
STUDIES TABLE
OF BOVINE
NUCLEOTIDE
II
SUMMARYOFHIGH-PERFORMANCE LIQUID CHROMATOGRAPHYSTUDIES WITH ENZYMEPREPARATION Products observed
Substrate NAD ADPR 5’-AMP NMN ADP FAD FMN NADP 2’-5’-ADP S’-phosphoADPR 3’-5’-CAMP UDP-Glc 5’-UMP
Nicotinamide-riboside, adenosine, NMN, 5’-AMP Adenosine, 5’-AMP Adenosine Nicotinamide-riboside Adenosine, 5’-AMP Adenosine, riboflavin, 5’-AMP, FMN Riboflavin” Nicotinamide-riboside, adenosine, NMN, 2’-AMP, 2’-5’-ADP Adenosine, 2’-AMP Adenosine, 2’-5’-ADP Adenosine Uridine, 5’-UMP Uridine
n Nonenzymatic hydrolysis.
purification was desirable. Seminal plasma from which spermatozoa were removed by centrifugation at 20009 for 15 min at 4°C was centrifuged at 30,OOOg for 30 min, and the supernatant fraction (Fraction 1) was applied to a Sepharose 4B column. The nucleotide pyrophosphatase eluting in the void volume was concentrated by ultrafiltration through a Diaflo PM-10 filter (Fraction 2). Butanol at -9°C was slowly added to Fraction 2 to a final concentration of 20% (v/v). After stirring for 1 h at 4”C, the aqueous layer was removed after centrifugation at 30,OOOg for 30 min. The butanol treatment was repeated and the resulting TABLE
399
PYROPHOSPHATASE
aqueous layer was dialyzed 24 h against two 5 mM sodium phosphate (pH 7.5) buffer changes of 4 liters to yield Fraction 3. Fraction 3 was adjusted at pH 8.5 to 70% saturation of ammonium sulfate and stirred for 1 h at 4°C. The precipitate was redissolved in 2 ml of 5 mM sodium phosphate buffer, pH 7.5 and applied to a Sepharose 4B column (1.5 x 93 cm). Fractions containing nucleotide pyrophosphatase activity were pooled and concentrated by ultrafiltration to give Fraction 4. At this point an 80-fold purification and 40% recovery of nucleotide-pyrophosphatase activity was observed. A summary of the purification procedure is shown in Table III. It was noted that after butanol treatment the nucleotide pyrophosphatase did not elute from Sepharose 4B in the void volume but later in the fractionation region of the column. Ion-exchange chromatography and affinity chromatography with concanavalin A Sepharose and blue dextran Sepharose did not provide further purification of Fraction 4. Treatment of Fraction 2 with 1% Triton X-100, ammonium sulfate fractionation, incubation with lipase, sonication, incubation with mercaptoethanol, acetone precipitation, and freeze-thaw techniques did not contribute significantly to the following butanol fractionation step. Properties of the partially purified nucleotide pyrophosphatase of Fraction 4 were studied fluorimetrically using FAD as substrate. The K, value for FAD redetermined for this preparation was 6.2 PM. A pH optimum of 9.0 was determined in four different buffers. An ionic strength greater than 20 mM was used in these III
PURIFICATION OFNUCLEOTIDE PYROPHOSPHATASE FROMBULLSEMEN
Fraction
Volume (ml)
Activity (units)
Protein (mg)
Specific activity (units/mg)
Recovery (%)
(1)Seminal plasma (2) Sepharose 4B (3) ButanoI/diaIysis (4) Ammonium sulfate (70%)
3 7 15 6
30,000 21,000 18,000 12,000
200 7.3 3.7 1.0
150 2,900 4,900 12,000
70 60 40
a Unit, relative fluorescent increase-min’.
Fold purification 19 32 80
400
BUCKON
AND ANDERSON
studies since the enzyme activity was ob50 40 30 20 10 served to be affected by ionic strengths I I I I cc below 10 mM. The ionic strength effects .; 6% _ l eoa obtained by varying the concentration of E Q 3NaCl in reaction mixtures is shown in Fig. 2. Identical ionic strength effects were 3 .E obtained by varying concentrations of E \ sodium phosphate buffer, sodium sulfate, % land other chloride salts such as MgCl,. $ ?I The addition of MgCl, to reaction mix= 5 tures containing 20 mM NaCl had no effect 8 Ea= 14.7 KcaWmole E _ on catalytic activity. In fact, in preliminary :: studies using less purified preparations of 3Ff .2the nucleotide pyrophosphatase, enhanceL ment of enzyme activity by MgC&, thought 1 I I I I I to be related to a specific magnesium 310 320 330 340 350 360 requirement, can be explained on the (‘K i’40’ f basis of ionic strength effects. FIG. 3. Arrhenius plot of the effect of temperature The effect of temperature on the initial on the hydrolysis of FAD catalyzed by nucleotide velocities of FAD hydrolysis was studied pyrophosphatase. Reaction mixtures contained 10 mM from 5 to 55°C in glycine-HCl buffer, pH 9.0. The results plotted according to glycine-HCl buffer, pH 9.0, 20 mM NaCl, and pM FAD in a total volume of 3 ml. Above 2O”C, the Arrhenius equation are shown in Fig. 3. 63 3.2 pg protein (Fraction 4) was added to initiate reacA linear relationship was observed from 5 tions; below 2o”C, 8 I.cgprotein (Fraction 4) was added. to 41°C from which an activation energy Initial velocities were proportional to enzyme added at of 14.7 kcal per mole was calculated. the concentrations used and FAD was at saturating Thermal denaturation was indicated at concentration in all cases. temperatures above 41°C. Incubation of the enzyme at 62°C resulted in a timedependent first-order loss of catalytic activ- phosphatase of Fraction 4 did not increase ity with a tllz of 18.3 min. catalytic activity above effects attributed to The metals, Mg*+, Mn*+, Ca2+, and Zn2+ increasing ionic strength. However, dialysis added as chloride salts to nucleotide pyro- of this enzyme preparation against 10 InM EDTA in 5 111M sodium phosphate buffer, pH 7.5, for 24 h resulted in complete loss of enzyme activity which could not be restored by addition of MgCl,, MnCl,, CaCl,, or ZnCl, or by dialysis for 24 h against 10 lllM concentration of these salts. The loss of nucleotide pyrophosphatase activity by treatment with EDTA was observed to be a time-dependent process. Pseudo first-order rate constants for EDTA inactivation were determined for five conY centrations of EDTA and from the plot of I I I I I 0 4 these data (Fig. 4) a second-order rate con8 12 16 20 Ionic Strength(M) ~10~ stant of 8.8 liters-mol+nin-l was calculated. Substrate competitive inhibition of nuFIG. 2. The effect of ionic strength on the hydrolycleotide pyrophosphatase of Fraction 4 sis of FAD catalyzed by nucleotide pyrophosphatase. was observed with adenosine, AMP, and Reaction mixtures contained 63 pM FAD, 10 mM The Ki values for Tris-HCl buffer, pH 9.0, 4.2 pg protein (Fraction 41, sodium pyrophosphate. all three inhibitors are listed in Table IV. and NaCl varying from 0 to 25 mM in a total volume Enzyme activities of Fraction 4 were of 3 ml.
STUDIES
OF BOVINE
NUCLEOTIDE
PYROPHOSPHATASE
401
strates. A specific activity of 2.5 prnoli min/mg protein was observed in the hydrolysis of the 5’-nucleotides while the specific activity of the remaining substrates was one-tenth this value. Alkaline phosphatase activity was also measured spectrophotometrically according to Lansing et al. (21) using p-nitrophenylphosphate as substrate. Phosphodiesterase activity was investigated using 25 mM bis-pnitrophenylphosphate in reaction mixtures I I I I I I II I1 containing 100 mM Tris-HCl buffer, pH 9.0, 0 2 4 6 8 10 5 mM MgC&, and 17 Fg of Fraction 4 EDTA] (mM) protein in a total volume of 3 ml. Initial FIG. 4. The effect of EDTA concentration on the velocities were measured spectrophotopseudo first-order rate constants of EDTA inactivametrically at 420 nm. The specific activities tion of nucleotide pyrophosphatase. The enyzme was for nucleotide pyrophosphatase, 5’nucleoincubated at 4°C in 5 mM sodium phosphate buffer, tidase, and alkaline phosphatase in Fraction pH 7.5, at a concentration of protein (Fraction 4) 4 were compared to the activities of of 180 pg/ml. Seven micrograms of protein were these enzymes in the crude seminal plasma removed at timed intervals and assayed fluorimetripyrophosphatase tally with FAD as substrate. Pseudo first-order rate (Fraction 1). Nucleotide and phosphodiesterase activities copurified, constants were calculated from half-lives determined both showing an &SO-fold purification. The on semilog plots of activity vs time. 5’-nucleotidase activity was significantly reduced, showing only a 2-fold purification identified through high-performance liquid and alkaline phosphatase activity was chromatography of products formed from partially separated, showing a 14-fold the hydrolysis of NAD, FAD, ADPR, ADP, purification. AMP, UDP-Glc, UMP, and NMN. The The sensitivity of the four enzyme products obtained from the enzyme-cataactivities of Fraction 4 toward thermal lyzed hydrolysis of these substrates were denaturation was studied. First-order rate identical to those listed in Table II. Thereconstants for the inactivation of 5’-nucleofore the enzymes responsible for the tidase at 62°C was measured using 5’catalysis of these reactions were still AMP as substrate. Rates of inactivation present in the SO-fold purified enzyme of alkaline phosphatase and phosphodiesterpreparation. One exception was noted in ase were measured using p-nitrophenylthat no hydrolysis of 3’,5’-cyclic AMP phosphate and his?-nitrophenylphosphate was observed with the 80-fold purified as substrates, respectively. The rate preparation. The product study indicated constants for thermal inactivation of nuthe presence in the Fraction 4 enzyme cleotide pyrophosphatase and phosphopreparation of nucleotide pyrophosphatase, 5’-nucleotidase and possibly alkaline phosTABLE IV phatase. The 5’-nucleotidase activity was measured at 37°C in reaction mixtures INHIBITION OF NUCLEOTIDE PYROPHOSPHATASE ACTIVITY containing 30 IrIM Tris-HCl buffer, pH 8.5, 1 mM MgCl*, 8 pg of Fraction 4 protein, Ki (mM) Substrate and 5 mM 5’-AMP in a total volume of 2.1 ml. Aliquots (0.2 ml) of the reaction mixInhibitor FAD e-NAD ture were assayed for inorganic phosphate according to Chen et al. (20). This assay Adenosine 2.1 2.7 procedure was also used to evaluate 2’- Adenylic acid 0.034 0.036 AMP, 3’-AMP, 5’-CMP, 5’-UMP, 5’-GMP, Sodium pyrophosphate 0.39 0.41 5’-ADP, and &glycerophosphate as sub-
402
BUCKON
AND
diesterase were identical, 0.04 min-‘. The rate constant for inactivation of 5’-nucleotidase was much lower, 0.018 min-’ and for alkaline phosphatase, much higher, 0.95 min-‘. DISCUSSION
The presence of nucleotide pyrophosphatase in bovine seminal plasma was studied using FAD as substrate. Direct fluorimetric analysis of FAD hydrolysis was observed to be a convenient and sensitive method for studying the purification and properties of this enzyme and for the assay of the enzyme in crude mixtures. Fractionation of seminal plasma on Sepharose 4B resulted in the elution of nucleotide pyrophosphatase activity in the void volume yielding a ZO-fold purification of this activity and allowing a clean separation of this enzyme from the smaller molecular weight NADase (3). In the absence of NADase, a number of dinucleotides including NAD were demonstrated to function as substrates for the pyrophosphatase (Table I). The similar K, and V values obtained for five substrates indicated that the structures of the heterocyclic bases of these dinucleotides could be varied considerably without a great effect on the kinetic parameters of the enzyme. Broad substrate specificities have been reported for nucleotide pyrophosphatases from a number of sources (10,2228). Of special interest was the demonstration that ADPR effectively functioned as a substrate for the bovine seminal plasma nucleotide pyrophosphatase. ADPR, a product of the seminal plasma NADasecatalyzed hydrolysis of NAD can therefore be metabolized further. The ZO-fold purified preparation of nucleotide pyrophosphatase was shown to contain several other enzyme activities (Table II). Timed studies of products formed from NAD hydrolysis established the initial hydrolytic reaction to be catalyzed by nucleotide pyrophosphatase yielding NMN and AMP. The subsequent conversion of AMP to adenosine indicated the presence of 5’-nucleotidase. The same sequence of reactions was indicated in the product studies of FAD and ADPR hydrolysis.
ANDERSON
The seminal plasma nucleotide pyrophosphatase was further purified (go-fold) through butanol fractionation, ammonium sulfate precipitation, and gel filtration to a specific activity of 3 units/mg protein which was considerably higher than those reported for the nucleotide pyrophosphatases isolated from rat liver microsomes (25) and rat liver lysosomes (29). The partially purified seminal plasma enzyme showed a pH optimum of 9.0. Increasing the ionic strength up to 20 mM increased the catalytic activity of the enzyme and a comparison of effects by different salts indicated that earlier reports (9) of Mg*+ stimulation of this enzyme activity should more properly be related to ionic strength effects. The catalytic activity of nucleotide pyrophosphatase increased with increasing temperature up to 41”C, beyond which thermal denaturation and inactivation of the enzyme was indicated. A half-life of 18.3 min was determined for the first-order inactivation of the enzyme at 62°C. In comparison, the enzyme in crude seminal plasma, as expected, was more stable requiring approximately 37 min for 50% inactivation at 62°C. Adenosine, 5’-AMP and inorganic pyrophosphate were demonstrated to inhibit nucleotide pyrophosphatase competitively. These compounds were reported previously (30) to inhibit other nucleotide pyrophosphatases as well. The inhibition by purine derivatives would be expected as normal product inhibition if the enzyme was truly a nucleotide pyrophosphatase. These observations were consistent with substrate specificity studies. The 80-fold purified preparation of seminal plasma nucleotide pyrophosphatase was shown to catalyze the hydrolysis of pyrophosphate derivatives, nucleotides, and nonnucleotide phosphate esters. The kinetic studies and product studies confirmed the presence in this partially purified preparation of 5’-nucleotidase and alkaline phosphatase. The activity in the less purified preparation (Fraction 2) responsible for the catalyzed hydrolysis of cyclicAMP was removed in the purification procedure. It was also evident that during the purification procedure, nucleotide pyro-
STUDIES
OF BOVINE
NUCLEOTIDE
phosphatase was partially separated from the ,5’-nucleotidase and alkaline phosphatase since these latter activities showed a lesser degree of purification in the final preparation. Phosphodiesterase activity assayed with the his+-nitrophenylphosphate substrate was considered a second catalytic activity of the nucleotide pyrophosphatase. One must argue that either the phosphodiesterase copurified with the nucleotide pyrophosphatase or more likely, the two activities were catalyzed by the same enzyme. There are several reports supporting the latter argument (22, 2’7, 28, 31-34). The observation that both the nucleotide pyrophosphatase and the phosphodiesterase activities were equally sensitive to thermal denaturation is likewise consistent with the presence of these activities on the same protein. The 5’nucleotidase and alkaline phosphatase activities were distinct from the nucleotide pyrophosphatase since these activities behaved differently through the purification procedure and showed quite different sensitivities to thermal inactivation. The time-dependent loss of nucleotide pyrophosphatase activity upon incubation with EDTA was an unexpected observation. Inhibition by EDTA of nucleotide pyrophosphatases from various sources has been documented; however, first-order rates of inactivation have not been reported. The time-dependent inactivation of the seminal plasma enzyme by EDTA may reflect a slow extraction of metal ion from the enzyme. The resulting apoenzyme appeared not to retain its native conformation since restoration of enzyme activity could not be accomplished by incubation with various metals. The studies presented demonstrated the presence in bovine seminal plasma of enzymes capable of catalyzing the hydrolysis of NAD through AMP to adenosine. Two routes for the conversion of NAD to adenosine in seminal plasma are obvious since ADPR, a product of the NADase-catalyzed hydrolysis of NAD, was demonstrated to serve as a substrate for the nucleotide pyrophosphatase. Therefore, the NADase-catalyzed hydrolysis of NAD does not constitute a competing catabolic reaction but rather pro-
PYROPHOSPHATASE
403
vides a second route by which adenosine can be produced from NAD. A physiological role for adenosine in seminal plasma remains to be established; however, the leakage of NAD into seminal plasma would be expected to result in the accumulation of adenosine. REFERENCES B. M. (1971) J. 1. YUAN, J. H., AND ANDERSON, Biol. Chem. 246, 2111-21115. 2. LEONE, E., AND BONADUCE, L. (1959) Biochim. Biophys. Acta 31, 292-293. L. B., AND ANDERSON, 3. YUAN, J. H., BARNETT, B. M. (1972) J. Biol. Chem. 247, 511-514. J. H., AND ANDERSON, B. M. (1972) 4. YUAN, J. Biol. Chem. 247, 515-520. J. H., AND ANDERSON, B. M. (1972) 5. YUAN, Arch. Biochem. Biophys. 149, 419-422. 6. YUAN, J. H., AND ANDERSON, B. M. (1973) J. Biol. Chem. 248, 417-421. 7. YUAN, J. H., AND ANDERSON, B. M. (1973) Arch. Biochem. Biophys. 156, 328-334. 8. WHEAT, R. W., KRICK, E. B., AND BROWNLEE, S. T. (1960) Fed. Proc. 672, 169. S. T., AND WHEAT, R. W. (1960) 9. BROWNLEE, J. Biol. Chem. 235, 3567-3569. 10. WHEAT, R. W., KRICK, E. B., AND BROWNLEE, S. T. (1960) J. Biol. Chem. 235, 3570-3572. 11. HEPPEL, L. A., AND HILMOE, R. (1953) J. Biol. Chem. 200, 217-226. 12. GARBERS, D. L., LUST, W. D., FIRST, N. L., AND LARDY, H. A. (1972) Biochemistry 10, 1825-1831. 13. HOSKINS, H. D. (1973) J. Biol. Chem. 248, 1135-1140. 14. BOMSTEIN, R. A., AND STEBERL, E. A. (1959) Arch. Biochem. Biophys. 85, 43-52. 15. BISTOCCHI, M., D’ALESSIO, G., AND LEONE, E. (1968) J. Reprod. Feti. 16, 223-231. 16. BROOKS, D. E., AND MANN, T. (1972) Biochem. J. 129, 1023- 1034. 17. FISHER, T. L., VERCELLOTTI, S. V., AND ANDERSON, B. M. (1973) J. Biol. Chem. 248, 4293-4299. 18. LOWRY, O., ROSEBROUGH, N., FARR, A., AND RANDALL, R. (1951) J. Biol. Chem. 193, 265-275. 19. LINEWEAVER, H., AND BURK, D. (1934) J. Amer. Chem. Sot. 658-666. 20. CHEN, P. S., TORIBARA, T. Y., AND WARNER, H. (1956) Anal. Chem. 28, 1756-1758. 21. LANSING, A. I., BELKHODE, M. L., LYNCH, W. E., AND LIEBERMAN, I. (1967) J. Biol. Chem. 242, 1772-1775. 22. HAROZ, R. K., Twu, J. S., AND BRETTHAUER, R. K. (1972) J. Biol. Chem. 247, 1452-1457.
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BUCKONANDANDERSON
23. JACOBSON, B. K., AND KAPLAN, N. 0. (1957) J. Biol. Chem. 226, 427-437. 24. SCHLISELFIELD, L. H., VAN EYS, J., AND TOUSTER, 0. (1965) J. Biol. Chem. 240, 811-818. 25. OGAWA, H., SAWADA, M., AND KAWADA, M. (1966) J. Biochem. 59, 126-134. 26. BACHORIK, P. S., AND DIETRICH, L. S. (1972) J. Biol. Chem. 247, 5071-5078. 27. BISCHOFF, E., TRAN-THI, T., AND DECKER, K. F. A. (1975) Eur. J. Biochem. 51,353-361. 28. DECKER, K. F. A., AND BISCHOFF, E. (1972) FEBS Lett. 21, 95-98.
29. RAGOB, M. H., BRIGHTWELL, R., AND TAPPEL, A. L. (1968) Arch. Biochem. Biophys. 123, 179-185. 30. SKREDE, S. (1973) Eur. J. Biochem. 38.401-407. 31. HAUGEN, H. F., AND SKREDE, S. (1977) Clin. Chem. 23, 1531-1537. 32. EVANS, W. H., HOOD, D. O., AND GURD, J. W. (1973) Biochem. J. 135, 819-826. 33. TOIJSTER, O., ARONSON, N. N., DULANEY, J. T., AND HENDRICKSON, H. (1970) J. Cell. Biol. 47, 604-618. 34. ABNEY, E. R., EVANS, W. H., AND PARKHOUSE, R. M. E. (1976) Biochem. J. 159, 293-299.
Studies
of Bovine
Seminal
MATTHEW
E. BUCKON
Department of Biochemistry
Fluid Nucleotide AND BRUCE
Pyrophosphatasel
M. ANDERSON2
and Nutrition, Virginia Polytechnic Institute Blacksburg, Virginia 21061
and State University,
Received February 4, 1980 Nucleotide pyrophosphatase of bovine seminal plasma was demonstrated to catalyze effectively the hydrolysis of NAD, FAD, etheno-NAD, adenosine diphosphoribose, 3aminopyridine adenine dinucleotide, and bis++nitrophenylphosphate. The enzyme was purified SO-fold resulting in a partial separation from 5’-nucleotidase and alkaline phosphatase activities. The SO-fold purified nucleotide pyrophoshatase had a pH optimum of 9.0 and was competitively inhibited by adenosine, AMP, and sodium pyrophosphate. Time-dependent inactivation of the enzyme was observed by thermal denaturation at 62°C and by incubation with EDTA at 4°C. The activity of the enzyme was increased with increasing ionic strength and specific stimulation by MgZ+ was not observed. In the absence of NAD glycohydrolase, the hydrolysis of NAD in bovine seminal plasma was demonstrated through timed product studies to proceed initially through the hydrolysis of the pyrophosphate linkage catalyzed by nucleotide pyrophosphatase. The resulting product, AMP, was further hydrolyzed to adenosine in a reaction catalyzed by 5’nucleotidase. The observed functioning of adenosine diphosphoribose as a substrate for the nucleotide pyrophosphatase provided evidence in support of a second conversion of NAD to adenosine in seminal Dlasma through an initial hydrolytic reaction catalyzed by NAD glycohydrolase.
Earlier studies in this laboratory were concerned with the metabolic fate of NAD in bovine seminal plasma (1). The incubation of NAD with diluted seminal plasma resulted in the formation of adenosine diphosphoribose (ADPR), nicotinamide, and adenosine. The NAD glycohydrolase (NADase)3 of the seminal plasma first reported by Leone and Bonaduce (2) was purified to homogeneity (1, 3) and various properties of the enzyme were determined (3-7). The enzyme-catalyzed hydrolysis of NAD to ADPR and nicotinamide in bovine seminal plasma can be attributed to the presence of the soluble, glycoprotein NADase of 36,300 molecular weight. The
appearance of adenosine which becomes the predominant product after extended periods of NAD incubation with bovine seminal plasma had to result from the functioning of other enzymes in the seminal plasma. The existence of a nucleotide pyrophosphatase in bovine seminal plasma was first reported by Wheat et al. (8). Although several properties of this enzyme were studied (9, lo), the functioning of ADPR as a substrate was not investigated. The presence of 5’-nucleotidase in bovine seminal plasma was reported by Heppel and Hilmoe (11). The combined functioning of nucleotide pyrophosphatase and 5’nucleotidase in seminal plasma would result in the formation of adenosine from NAD. The formation of adenosine from NAD in bovine seminal plasma is of interest if one considers possible interactions of this nucleoside with spermatozoa. Caffeine and theophylline, structurally analogous to adenosine, inhibit cyclic nucleotide phosphodiesterase in bull spermatozoa and in the presence of exogenous substrates,
1 These studies were supported by Research Grant PCM 78 05839 from the National Science Foundation. * To whom correspondence and requests for reprints should be addressed. 3 Abbreviations used: ADPR, adenosine diphosphoribose; etheno-NAD, nicotinamide l,NB-ethenoadenine dinucleotide; AAD, 3-aminopyridine adenine dinucleotide; NADase, NAD glycohydrolase; TEAEcellulose, 0-(triethylaminoethyl)-cellulose. 0003-986Y80/080396-09$02.00/O 396 Copyright AU rights
0 1980 by Academic Press, of reproduction in any form
Inc. reserved.
STUDIES
OF BOVINE
NUCLEOTIDE
markedly stimulate sperm motility and respiration (12). More recently (13) the incubation of bovine epididymal spermatozoa with caffeine, theophylline, cyclic AMP, and N6,02-dibutyryl cyclic AMP was reported to increase the rate of fructolysis and motility. In an earlier study (14) adenosine was shown to stimulate oxygen uptake and motility in bull spermatozoa. In a comparison of epididymal and freshly ejaculated spermatozoa, Bistocchi et al. (15) reported epididymal spermatozoa to be immotile, showing low respiration rates and containing a high level of pyridine nucleotides while ejaculated spermatozoa had higher respiration rates, were motile, and had markedly decreased pyridine nucleotide levels. Leakage of pyridine nucleotides into the seminal plasma was indicated by these (15) and other studies (16). It was therefore of interest to study further the conversion of NAD to adenosine in bovine seminal plasma, to investigate the functioning of adenosine diphosphoribose as a substrate for the nucleotide pyrophosphatase, and to study the properties of enzymes catalyzing the various hydrolytic reactions involved. MATERIALS
AND METHODS
Holstein bull semen was obtained from Select Sires of Columbus, Ohio. Adenosine, 5’-AMP, 5’-ADP, ADPR, 2’-phospho-ADPR, 2’5’-ADP, 3’5’cyclic AMP, 5’-CMP, 5’-GMP, 2’-AMP, 3’-AMP, FAD, FMN, P-glycerophosphate, pancreatic lipase, NAD, nicotinamide 1,N6-ethenoadenine dinucleotide (etheno-NAD), NMN, p-nitrophenylphosphate, bisp-nitrophenylphosphate, riboflavin, concanavalin ASepharose 4B, Sepharose 4B, Sephadex G-200, TEAE-cellulose, Triton X-100, uridine, 5’-UMP, and UDP-Glc were purchased from Sigma. DEAESephadex and blue dextran Sepharose were purchased from Pharmacia. DEAE-cellulose (DE-52) were obtained from Whatman. Carboxymethyl cellulose (Cellex CM) and phosphocellulose (Cellex P) were purchased from Bio-Rad Laboratories. 3Aminopyridine adenine dinucleotide (AAD) was prepared according to Fisher et al. (17). Titrimetric assays of nucleotide pyrophosphatase were performed at 3’7°C in 3-ml reaction mixtures containing 10 mM KCl, 1 mM MgCl,, and substrate. Initial velocities were measured at pH 9.0 recording the utilization of the 100 mM NaOH titrant. The solutions were prepared with CO,-free water and argon was passed over the reaction mixture before
PYROPHOSPHATASE
397
and during the reaction. A Radiometer type TTT II titrator, type SBRSC titragraph, PHM 26C pH meter, type TTA 31 microtitration assembly, and type ABU 12 automatic burette were used in these studies. Units of activity were expressed as micromoles of substrate hydrolyzed per minute calculated on the basis of two protons released per pyrophosphate bond hydrolyzed. Fluorimetric assays of nucleotide pyrophosphatase were performed at 37°C in 3-ml reaction mixtures containing 30 mM Tris-HCl buffer, pH 9.0, 1 mM MgCl*, and substrate. With FAD as substrate, reaction mixtures were excited at 465 nm and the fluorescence emission at 516 nm was recorded. With etheno-NAD as substrate, reaction mixtures were excited at 297 nm and the fluorescence emission at 410 nm was recorded. Units of activity were expressed as fluorescence increase per minute in arbitrary fluorescence units. These values could be converted to international units by direct comparison with concomitant titrimetric assays. Fluorimetric assays were preferred for routine studies, purification studies, and studies of the properties of the nucleotide pyrophosphatase since the titrimetric assay required approximately 60 times as much enzyme per assay. All fluorimetric measurements were performed on an Aminco-Bowman recording spectrophotofluorometer. Protein concentrations were determined according to Lowry et al. (18). Product studies were performed through highperformance liquid chromatography on a Spectra Physics 8000 liquid chromatograph equipped with a ‘770 variable wavelength uv-visible detector. The column (25 x 0.46 cm) employed in these studies was a Whatman Partisil-10 SAX quaternary amine ion exchanger. Only products absorbing at 260 nm were monitored in these studies. RESULTS
Nucleotide pyrophosphatase in crude bovine seminal plasma was assayed using FAD as a substrate. FAD was chosen over NAD as substrate since crude seminal plasma contains high NADase activity and the hydrolysis of FAD could be monitored by the more sensitive fluorescence assay. Reaction mixtures containing 30 mM TrisHCl buffer, pH 9.0, 1 lllM MgC12, 66 pg of seminal fluid protein, and FAD concentrations varying from 1.5-14 PM were inCUbated at 37°C. The initial velocities of FAD hydrolysis were measured by monitoring the fluorescence emission at 516 nm with excitation at 465 nm and the initial velocities were linear for 5 min. Initial velocities as a function of FAD concentration were plotted
398
BUCKONANDANDERSON
according to Lineweaver and Burk (19) and the K, value for FAD calculated from these data was 3.6 FM. At saturating concentration of FAD, initial velocities were proportional to enzyme concentration in the range of 30-250 pg of protein. In order to study NAD and other nucleotides as substrates, the nucleotide pyrophosphatase was separated from the NADase by fractionation on a Sepharose 4B column, resulting in a 20-fold purification of the nucleotide pyrophosphatase activity. Using this preparation, the hydrolysis of NAD was investigated titrimetrically at 37°C and pH 9.0 in 3-ml reaction mixtures containing NAD, 10 mM KCl, 1 lllM MgC&, and 90 pug of protein. Initial velocities obtained at nine concentrations of NAD, plotted according to Lineweaver and Burk (19), were used to determine V and K, values. Under the same conditions, FAD, adenosine diphosphoribose, etheno-NAD, and AAD were studied as substrates, and the K, and V values for the hydrolysis of all substrates are listed in Table I. FAD and etheno-NAD were also studied fluorimetrically under conditions described above. The products of the hydrolysis of NAD and other substrates were identified through high-performance liquid chromatography. Reaction mixtures at 37°C contained 30 mM Tris-HCl buffer, pH 9.0, 1 mM MgC&, 0.5 mM substrate and 11 pg of protein (20-fold purified) in a total volume of 2 ml. At 0, ‘7, 60, and 120 min, 0.5-ml aliquots were TABLE K,
I
AND V VALUESOFZO-FOLDPURIFIED NUCLE~TIDE PYROPHOSPHATASE Assay method
&I (PM)
v (jmolmin-klg-1)
NAD
Titrimetric
8.7
2.2
ADPR
Titrimetric
11.0
0.96
AAD
Titrimetric
7.3
1.4
Substrate
FAD
Titrimetric Fluorometric
4.5 2.9
0.75
Ethenc+NAD
Titrimetric Fluorometric
9.0
1.0
8.7
2
7
1 I
/r---7
3 % u-4 a
i
iRetention
Ttme (min)
’
-125 -105
$j
6
03
FIG. 1. High-performance liquid chromatography of possible 260 nm-absorbing products of NAD hydrolysis. Peaks were identified as: 1, nicotinamide riboside; 2, adenosine; 3, NMN; 4, NAD; 5, AMP; 6, ADPR; 7, ADP. Dashed line represents programmed elution profile.
removed, filtered through Millipore filters to remove protein, and a lo-p1 sample was applied to a Partisil-10 column equilibrated with 5 mM sodium phosphate buffer, pH 4.0. The column was eluted with the same buffer for 15 min after which a linear gradient from 5 mM sodium phosphate, pH 4.0 to 129 mM sodium phosphate, pH 4.0, was applied. A separation of possible products from NAD hydrolysis is shown in Fig. 1. Samples from the enzyme-catalyzed hydrolysis of NAD assayed in this manner revealed the presence of NMN and AMP as primary products at the 7-min incubation time and the appearance and predominance of nicotinamide riboside and adenosine at the later time periods. No ADP or ADPR was observed during the hydrolysis of NAD. The enzyme-catalyzed hydrolysis of ADPR yielded AMP which in time was converted to adenosine and no ADP could be identified at any time interval. FAD hydrolysis yielded initially FMN and AMP which were rapidly hydrolyzed to riboflavin and adenosine. With the indicated presence of 5’-nucleotidase and/or alkaline phosphatase activities, a number of different nucleotides were assayed as substrates. A summary of the product studies with these substrates is shown in Table II. In order to evaluate the properties of the nucleotide pyrophosphatase, further
STUDIES TABLE
OF BOVINE
NUCLEOTIDE
II
SUMMARYOFHIGH-PERFORMANCE LIQUID CHROMATOGRAPHYSTUDIES WITH ENZYMEPREPARATION Products observed
Substrate NAD ADPR 5’-AMP NMN ADP FAD FMN NADP 2’-5’-ADP S’-phosphoADPR 3’-5’-CAMP UDP-Glc 5’-UMP
Nicotinamide-riboside, adenosine, NMN, 5’-AMP Adenosine, 5’-AMP Adenosine Nicotinamide-riboside Adenosine, 5’-AMP Adenosine, riboflavin, 5’-AMP, FMN Riboflavin” Nicotinamide-riboside, adenosine, NMN, 2’-AMP, 2’-5’-ADP Adenosine, 2’-AMP Adenosine, 2’-5’-ADP Adenosine Uridine, 5’-UMP Uridine
n Nonenzymatic hydrolysis.
purification was desirable. Seminal plasma from which spermatozoa were removed by centrifugation at 20009 for 15 min at 4°C was centrifuged at 30,OOOg for 30 min, and the supernatant fraction (Fraction 1) was applied to a Sepharose 4B column. The nucleotide pyrophosphatase eluting in the void volume was concentrated by ultrafiltration through a Diaflo PM-10 filter (Fraction 2). Butanol at -9°C was slowly added to Fraction 2 to a final concentration of 20% (v/v). After stirring for 1 h at 4”C, the aqueous layer was removed after centrifugation at 30,OOOg for 30 min. The butanol treatment was repeated and the resulting TABLE
399
PYROPHOSPHATASE
aqueous layer was dialyzed 24 h against two 5 mM sodium phosphate (pH 7.5) buffer changes of 4 liters to yield Fraction 3. Fraction 3 was adjusted at pH 8.5 to 70% saturation of ammonium sulfate and stirred for 1 h at 4°C. The precipitate was redissolved in 2 ml of 5 mM sodium phosphate buffer, pH 7.5 and applied to a Sepharose 4B column (1.5 x 93 cm). Fractions containing nucleotide pyrophosphatase activity were pooled and concentrated by ultrafiltration to give Fraction 4. At this point an 80-fold purification and 40% recovery of nucleotide-pyrophosphatase activity was observed. A summary of the purification procedure is shown in Table III. It was noted that after butanol treatment the nucleotide pyrophosphatase did not elute from Sepharose 4B in the void volume but later in the fractionation region of the column. Ion-exchange chromatography and affinity chromatography with concanavalin A Sepharose and blue dextran Sepharose did not provide further purification of Fraction 4. Treatment of Fraction 2 with 1% Triton X-100, ammonium sulfate fractionation, incubation with lipase, sonication, incubation with mercaptoethanol, acetone precipitation, and freeze-thaw techniques did not contribute significantly to the following butanol fractionation step. Properties of the partially purified nucleotide pyrophosphatase of Fraction 4 were studied fluorimetrically using FAD as substrate. The K, value for FAD redetermined for this preparation was 6.2 PM. A pH optimum of 9.0 was determined in four different buffers. An ionic strength greater than 20 mM was used in these III
PURIFICATION OFNUCLEOTIDE PYROPHOSPHATASE FROMBULLSEMEN
Fraction
Volume (ml)
Activity (units)
Protein (mg)
Specific activity (units/mg)
Recovery (%)
(1)Seminal plasma (2) Sepharose 4B (3) ButanoI/diaIysis (4) Ammonium sulfate (70%)
3 7 15 6
30,000 21,000 18,000 12,000
200 7.3 3.7 1.0
150 2,900 4,900 12,000
70 60 40
a Unit, relative fluorescent increase-min’.
Fold purification 19 32 80
400
BUCKON
AND ANDERSON
studies since the enzyme activity was ob50 40 30 20 10 served to be affected by ionic strengths I I I I cc below 10 mM. The ionic strength effects .; 6% _ l eoa obtained by varying the concentration of E Q 3NaCl in reaction mixtures is shown in Fig. 2. Identical ionic strength effects were 3 .E obtained by varying concentrations of E \ sodium phosphate buffer, sodium sulfate, % land other chloride salts such as MgCl,. $ ?I The addition of MgCl, to reaction mix= 5 tures containing 20 mM NaCl had no effect 8 Ea= 14.7 KcaWmole E _ on catalytic activity. In fact, in preliminary :: studies using less purified preparations of 3Ff .2the nucleotide pyrophosphatase, enhanceL ment of enzyme activity by MgC&, thought 1 I I I I I to be related to a specific magnesium 310 320 330 340 350 360 requirement, can be explained on the (‘K i’40’ f basis of ionic strength effects. FIG. 3. Arrhenius plot of the effect of temperature The effect of temperature on the initial on the hydrolysis of FAD catalyzed by nucleotide velocities of FAD hydrolysis was studied pyrophosphatase. Reaction mixtures contained 10 mM from 5 to 55°C in glycine-HCl buffer, pH 9.0. The results plotted according to glycine-HCl buffer, pH 9.0, 20 mM NaCl, and pM FAD in a total volume of 3 ml. Above 2O”C, the Arrhenius equation are shown in Fig. 3. 63 3.2 pg protein (Fraction 4) was added to initiate reacA linear relationship was observed from 5 tions; below 2o”C, 8 I.cgprotein (Fraction 4) was added. to 41°C from which an activation energy Initial velocities were proportional to enzyme added at of 14.7 kcal per mole was calculated. the concentrations used and FAD was at saturating Thermal denaturation was indicated at concentration in all cases. temperatures above 41°C. Incubation of the enzyme at 62°C resulted in a timedependent first-order loss of catalytic activ- phosphatase of Fraction 4 did not increase ity with a tllz of 18.3 min. catalytic activity above effects attributed to The metals, Mg*+, Mn*+, Ca2+, and Zn2+ increasing ionic strength. However, dialysis added as chloride salts to nucleotide pyro- of this enzyme preparation against 10 InM EDTA in 5 111M sodium phosphate buffer, pH 7.5, for 24 h resulted in complete loss of enzyme activity which could not be restored by addition of MgCl,, MnCl,, CaCl,, or ZnCl, or by dialysis for 24 h against 10 lllM concentration of these salts. The loss of nucleotide pyrophosphatase activity by treatment with EDTA was observed to be a time-dependent process. Pseudo first-order rate constants for EDTA inactivation were determined for five conY centrations of EDTA and from the plot of I I I I I 0 4 these data (Fig. 4) a second-order rate con8 12 16 20 Ionic Strength(M) ~10~ stant of 8.8 liters-mol+nin-l was calculated. Substrate competitive inhibition of nuFIG. 2. The effect of ionic strength on the hydrolycleotide pyrophosphatase of Fraction 4 sis of FAD catalyzed by nucleotide pyrophosphatase. was observed with adenosine, AMP, and Reaction mixtures contained 63 pM FAD, 10 mM The Ki values for Tris-HCl buffer, pH 9.0, 4.2 pg protein (Fraction 41, sodium pyrophosphate. all three inhibitors are listed in Table IV. and NaCl varying from 0 to 25 mM in a total volume Enzyme activities of Fraction 4 were of 3 ml.
STUDIES
OF BOVINE
NUCLEOTIDE
PYROPHOSPHATASE
401
strates. A specific activity of 2.5 prnoli min/mg protein was observed in the hydrolysis of the 5’-nucleotides while the specific activity of the remaining substrates was one-tenth this value. Alkaline phosphatase activity was also measured spectrophotometrically according to Lansing et al. (21) using p-nitrophenylphosphate as substrate. Phosphodiesterase activity was investigated using 25 mM bis-pnitrophenylphosphate in reaction mixtures I I I I I I II I1 containing 100 mM Tris-HCl buffer, pH 9.0, 0 2 4 6 8 10 5 mM MgC&, and 17 Fg of Fraction 4 EDTA] (mM) protein in a total volume of 3 ml. Initial FIG. 4. The effect of EDTA concentration on the velocities were measured spectrophotopseudo first-order rate constants of EDTA inactivametrically at 420 nm. The specific activities tion of nucleotide pyrophosphatase. The enyzme was for nucleotide pyrophosphatase, 5’nucleoincubated at 4°C in 5 mM sodium phosphate buffer, tidase, and alkaline phosphatase in Fraction pH 7.5, at a concentration of protein (Fraction 4) 4 were compared to the activities of of 180 pg/ml. Seven micrograms of protein were these enzymes in the crude seminal plasma removed at timed intervals and assayed fluorimetripyrophosphatase tally with FAD as substrate. Pseudo first-order rate (Fraction 1). Nucleotide and phosphodiesterase activities copurified, constants were calculated from half-lives determined both showing an &SO-fold purification. The on semilog plots of activity vs time. 5’-nucleotidase activity was significantly reduced, showing only a 2-fold purification identified through high-performance liquid and alkaline phosphatase activity was chromatography of products formed from partially separated, showing a 14-fold the hydrolysis of NAD, FAD, ADPR, ADP, purification. AMP, UDP-Glc, UMP, and NMN. The The sensitivity of the four enzyme products obtained from the enzyme-cataactivities of Fraction 4 toward thermal lyzed hydrolysis of these substrates were denaturation was studied. First-order rate identical to those listed in Table II. Thereconstants for the inactivation of 5’-nucleofore the enzymes responsible for the tidase at 62°C was measured using 5’catalysis of these reactions were still AMP as substrate. Rates of inactivation present in the SO-fold purified enzyme of alkaline phosphatase and phosphodiesterpreparation. One exception was noted in ase were measured using p-nitrophenylthat no hydrolysis of 3’,5’-cyclic AMP phosphate and his?-nitrophenylphosphate was observed with the 80-fold purified as substrates, respectively. The rate preparation. The product study indicated constants for thermal inactivation of nuthe presence in the Fraction 4 enzyme cleotide pyrophosphatase and phosphopreparation of nucleotide pyrophosphatase, 5’-nucleotidase and possibly alkaline phosTABLE IV phatase. The 5’-nucleotidase activity was measured at 37°C in reaction mixtures INHIBITION OF NUCLEOTIDE PYROPHOSPHATASE ACTIVITY containing 30 IrIM Tris-HCl buffer, pH 8.5, 1 mM MgCl*, 8 pg of Fraction 4 protein, Ki (mM) Substrate and 5 mM 5’-AMP in a total volume of 2.1 ml. Aliquots (0.2 ml) of the reaction mixInhibitor FAD e-NAD ture were assayed for inorganic phosphate according to Chen et al. (20). This assay Adenosine 2.1 2.7 procedure was also used to evaluate 2’- Adenylic acid 0.034 0.036 AMP, 3’-AMP, 5’-CMP, 5’-UMP, 5’-GMP, Sodium pyrophosphate 0.39 0.41 5’-ADP, and &glycerophosphate as sub-
402
BUCKON
AND
diesterase were identical, 0.04 min-‘. The rate constant for inactivation of 5’-nucleotidase was much lower, 0.018 min-’ and for alkaline phosphatase, much higher, 0.95 min-‘. DISCUSSION
The presence of nucleotide pyrophosphatase in bovine seminal plasma was studied using FAD as substrate. Direct fluorimetric analysis of FAD hydrolysis was observed to be a convenient and sensitive method for studying the purification and properties of this enzyme and for the assay of the enzyme in crude mixtures. Fractionation of seminal plasma on Sepharose 4B resulted in the elution of nucleotide pyrophosphatase activity in the void volume yielding a ZO-fold purification of this activity and allowing a clean separation of this enzyme from the smaller molecular weight NADase (3). In the absence of NADase, a number of dinucleotides including NAD were demonstrated to function as substrates for the pyrophosphatase (Table I). The similar K, and V values obtained for five substrates indicated that the structures of the heterocyclic bases of these dinucleotides could be varied considerably without a great effect on the kinetic parameters of the enzyme. Broad substrate specificities have been reported for nucleotide pyrophosphatases from a number of sources (10,2228). Of special interest was the demonstration that ADPR effectively functioned as a substrate for the bovine seminal plasma nucleotide pyrophosphatase. ADPR, a product of the seminal plasma NADasecatalyzed hydrolysis of NAD can therefore be metabolized further. The ZO-fold purified preparation of nucleotide pyrophosphatase was shown to contain several other enzyme activities (Table II). Timed studies of products formed from NAD hydrolysis established the initial hydrolytic reaction to be catalyzed by nucleotide pyrophosphatase yielding NMN and AMP. The subsequent conversion of AMP to adenosine indicated the presence of 5’-nucleotidase. The same sequence of reactions was indicated in the product studies of FAD and ADPR hydrolysis.
ANDERSON
The seminal plasma nucleotide pyrophosphatase was further purified (go-fold) through butanol fractionation, ammonium sulfate precipitation, and gel filtration to a specific activity of 3 units/mg protein which was considerably higher than those reported for the nucleotide pyrophosphatases isolated from rat liver microsomes (25) and rat liver lysosomes (29). The partially purified seminal plasma enzyme showed a pH optimum of 9.0. Increasing the ionic strength up to 20 mM increased the catalytic activity of the enzyme and a comparison of effects by different salts indicated that earlier reports (9) of Mg*+ stimulation of this enzyme activity should more properly be related to ionic strength effects. The catalytic activity of nucleotide pyrophosphatase increased with increasing temperature up to 41”C, beyond which thermal denaturation and inactivation of the enzyme was indicated. A half-life of 18.3 min was determined for the first-order inactivation of the enzyme at 62°C. In comparison, the enzyme in crude seminal plasma, as expected, was more stable requiring approximately 37 min for 50% inactivation at 62°C. Adenosine, 5’-AMP and inorganic pyrophosphate were demonstrated to inhibit nucleotide pyrophosphatase competitively. These compounds were reported previously (30) to inhibit other nucleotide pyrophosphatases as well. The inhibition by purine derivatives would be expected as normal product inhibition if the enzyme was truly a nucleotide pyrophosphatase. These observations were consistent with substrate specificity studies. The 80-fold purified preparation of seminal plasma nucleotide pyrophosphatase was shown to catalyze the hydrolysis of pyrophosphate derivatives, nucleotides, and nonnucleotide phosphate esters. The kinetic studies and product studies confirmed the presence in this partially purified preparation of 5’-nucleotidase and alkaline phosphatase. The activity in the less purified preparation (Fraction 2) responsible for the catalyzed hydrolysis of cyclicAMP was removed in the purification procedure. It was also evident that during the purification procedure, nucleotide pyro-
STUDIES
OF BOVINE
NUCLEOTIDE
phosphatase was partially separated from the ,5’-nucleotidase and alkaline phosphatase since these latter activities showed a lesser degree of purification in the final preparation. Phosphodiesterase activity assayed with the his+-nitrophenylphosphate substrate was considered a second catalytic activity of the nucleotide pyrophosphatase. One must argue that either the phosphodiesterase copurified with the nucleotide pyrophosphatase or more likely, the two activities were catalyzed by the same enzyme. There are several reports supporting the latter argument (22, 2’7, 28, 31-34). The observation that both the nucleotide pyrophosphatase and the phosphodiesterase activities were equally sensitive to thermal denaturation is likewise consistent with the presence of these activities on the same protein. The 5’nucleotidase and alkaline phosphatase activities were distinct from the nucleotide pyrophosphatase since these activities behaved differently through the purification procedure and showed quite different sensitivities to thermal inactivation. The time-dependent loss of nucleotide pyrophosphatase activity upon incubation with EDTA was an unexpected observation. Inhibition by EDTA of nucleotide pyrophosphatases from various sources has been documented; however, first-order rates of inactivation have not been reported. The time-dependent inactivation of the seminal plasma enzyme by EDTA may reflect a slow extraction of metal ion from the enzyme. The resulting apoenzyme appeared not to retain its native conformation since restoration of enzyme activity could not be accomplished by incubation with various metals. The studies presented demonstrated the presence in bovine seminal plasma of enzymes capable of catalyzing the hydrolysis of NAD through AMP to adenosine. Two routes for the conversion of NAD to adenosine in seminal plasma are obvious since ADPR, a product of the NADase-catalyzed hydrolysis of NAD, was demonstrated to serve as a substrate for the nucleotide pyrophosphatase. Therefore, the NADase-catalyzed hydrolysis of NAD does not constitute a competing catabolic reaction but rather pro-
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