International Journal of Andrology, 1992, 15, pages 394-406

Purification and characterization of phospholipase A2 from bovine prostate S . RONKKO Department of Anatomy, University of Kuopio, Finland Summary Phospholipase A2 (PLA2) was purified from bovine prostate by ammonium sulphate precipitation and fractionation by anion exchange chromatography, chromatofocusing and gel filtration. The purified enzyme was Ca2+-dependent and had a pH-optimum of 8.0. Ba2+, Fez+, Hg2+, Mg2+, Pb2+, Sr2+ and Zn2+ as well as lysophosphatidylcholine, 1ysophosphatidylethanolamine and p -bromophenacyl bromide @-BPB) inhibited the enzyme strongly. The enzyme had an estimated molecular weight of 12 000 f 1000 daltons on SDS-PAGE. Isoelectric focusing showed one PLA2 activity-containing band at pl5.3. The purified enzyme hydrolysed linoleic acid at the sn-2 position of phosphatidylcholine and phosphatidylethanolamine with high selectivity, compared to arachidonic acid. Keywords: bovine prostate, CaZf-dependence, phospholipase A2. Introduction Phospholipase A2 (PLA2; EC 3.1.1.4) is a group of enzymes that catalyse the hydrolysis of an acyl group at the sn-2 position of phospholipids. PLA2 activity is present extracellulary, for example, in snake and bee venoms (Verheij et al., 1981), in the pancreas from man (Nishijima et al., 1983) and animals (de Haas et al., 1970; Dutilh et al., 1975; Evenberg et al., 1977; Ono et al., 1984) and also in synovial fluid (Hara et al., 1989). Extracellular PLA2 is one of the best characterized enzymes, and the amino acid sequence of these enzymes from many species are known (Verheij et al., 1981). The purpose of this paper is to report the purification and characterization of PLA2 present in bovine prostate. This enzyme was chosen for study because little is known about the biochemical properties and function of this enzyme in the male accessory sex glands. Most studies on this topic have dealt with its presence in spermatozoa (On0 et al., 1982; Thakkar et al., 1983; Hinkovska et al., 1987; Antaki et a f . ,1989) and seminal plasma (Kunze et al., 1974; Wurl & Kunze, 1985) as well as the role of PLA2 in the acrosome reaction (Llanos et al., 1982). Materials and methods Chemicals p -Bromophenacyl bromide (p-BPB), bovine serum albumin (BSA), L-alysophosphatidylcholine, L-a-lysophosphatidylethanolamine, and diisopropylfluorophosphate were obtained from Sigma Chemical Co. (St. Louis, MO, Correspondence: Dr S. Ronkko, Department of Anatomy, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland.

394

Purification of phospholipase A2 395 U.S.A.). Coomassie Brillant Blue G-250 was from Serva (Heidelberg, Germany), Q Sepharose Fast Flow, Mono P, Polybuffer 74, and heparin-Sepharose CLdB were from Pharmacia Fine Chemicals (Uppsala, Sweden), and Spherogel-TSK 2000SW was from Altex Scientific Inc. (Berkeley, California, U.S.A.). OptiPhase 'HiSafe' I1 scintillation liquid was from LKB-Wallac (Turku, Finland) and isopropanol was purchased from Carlo Erba (Milano, Italy). All other chemicals used were of analytical grade from E. Merck AG (Darmstadt, Germany).

Substrates 1-Palmitoyl-2-[l-'4C]linoleoyl-~-3-phosphatidylcholine (lino-PC; 59 mCi mmol-'), l-palmitoyl-2-[l-'4C]linoleoyl-~-3-phosphatidylethanolamine (lino-PE; 59 mCi mmol-'), l-Stearoyl-2-[1-'4C]arachidonyl-~-3-phosphatidylcholine(ara-PC; 58 mCi mmol-I), 1-acyl-2-[1-'4C]arachidonyl-~-3-pho~phatidylethanolamine (ara-PE; 58 mCi mmol-I), pho~phatidyl[2-~H]inositol 4-phosphate (PIP; 1 Ci mmol-I), phosphatidyl[l ,2-3H]inositol 4,5-bisphosphate (PIP2; 1 Ci mmol-I), 1,2dipalmitoyl-~-3-phosphatidyl[N-methyl3H]-choline([ 14C]PC;76 Ci mmol-I), 1,2dioleoyl-~-3-phosphatidyl[2-'~C]ethano1amine([I4C]PE; 49 mCi), bovine [ N -methyl-'4C]~phing~myelin (58 mCi mmol-'), glycerol tri[l-'4C]oleate (60 mCi mmol-') and 1-[l-'4C]palmitoyl-~-lyso-3phosphatidylcholine (['4C]lyso-PC; 56 mCi mmol-') were purchased from Amersham International (Amersham, England) and l-hexadecyl-2(1-O-[acetyl-3H(N)])-sn-glyseryl-3-phosphorylcholine(2PAF; 10.0 Ci mmol-') was purchased from Du Pont (NEN Research Products, Boston, U.S.A.). p -Nitrophenylphosphorylcholine (p -NPPC), p -nitrophenyl phosphate (p -NPP), l-palmitoyl-2-oleoyl-~-a-phosphatidylethanolamine (PE) and methyl esters of palmitic acid, oleic acid and eicosane were obtained from Sigma. l-acyl-2-[4-pyrenyl]-butyroyl-phosphatidylcholine(PPC) was synthesized according to Boss et al. (1975). Sample preparation Prostatic tissue from adult bulls was obtained from a local abattoir after slaughter of the animals. Samples were prepared as described previously (Ronkko et al., 19917 Supernatant was stored at -80°C and utilized in the purification process. Enzyme assays PLA2 activity was measured with sonicated ara-PC, ara-PE, lino-PC, and lino-PE as substrates, as described earlier (Ronkko et al., 1991). The standard incubation system (300 1.11) contained 0.1 M Tris-HC1 (pH 8.0), 5 mM CaC12, 25 1.11 of enzyme samples or fractions and 2.24 p . sonicated ~ substrate. The incubations were performed for 10-30 min in a water bath at 37°C. Blanks without an enzyme sample were included in all experiments. The hydrolysis of PPC was measured as described previously (Ronkko et al., 1991). Positional specificity of PLA2 was measured by gas chromatography (Ronkko, 1991) using 1-palmitoyl-2-oleoyl-L-a-phosphatidylethanolamine(PE) as substrate. The incubation medium consisted of 0.1 M Tris-HC1 (pH 8.0), 5 mM CaCI2, concentrated enzyme samples from the purification steps 2-4 and sonicated 1 mM

396 S . Ronkko PE as substrate in a total volume of 300 p1. Blanks were included in all experiments. Lysophospholipase activity was determined by the same extraction method as described previously (Ronkko et al., 1991). Incubations were carried out in a total volume of 300 pl at 37°C for 2 h in the presence of appropriate buffer (0.1 M acetate-HC1, pH 3.0-7.0; 0.1 M imidazole-HC1, pH 6.0-8.0; 0.1 M Tris-HC1, pH 7.0-9.5), 1 mM EDTA or 1mM Ca2+,variable amounts of protein and an aqueous ultrasonic suspension of 2.24 pM ['4C]lyso-PC. Blanks without an enzyme sample were included in all experiments. Platelet-activating factor acetylhydrolase (PAF-acetylhydrolase) activity was assayed by the extraction procedure of Nijssen et al. (1986) to separate released [3H]acetate. The standard incubation system contained 0.1 M Tris-HC1 buffer (pH 8.0), 2.24 pM 2-PAF as substrate with variable amounts of protein in a total volume of 200 pl. Incubations were performed for 1 h at 37°C. The same extraction method was utilized to follow lipase activity (Nijssen et al., 1986). Incubations were performed using a total volume of 200 p1 at 37°C for 2 h in the presence of appropriate buffer (0.1 M acetate-HC1, pH 3.0-7.0; 0.1 M imidazole-HC1, pH 6.0-8.0; 0.1 M Tris-Hcl, pH 7.0-9.5), 1 mM EDTA or 1mM Ca2+, variable amounts of protein and an aqueous suspension of glycerol tri[ l-14C]oleate (2.24 pM) as a substrate. Acid phosphatase activity was measured with p-NPP as a substrate. The incubation mixture contained 3 mMp-NPP, 0.1 M Na-acetate (pH 5.0) buffer and enzyme sample in a total volume of 300 p1. The incubation was stopped after 30 min with 500 p10.1 M NaOH, and released p-nitrophenyl was detected at 410 nm. Sphingomyelinase activity was assayed as reported previously (Vanha-Perttula, 1988). Phospholipase-C activity was detected by following the hydrolysis of [14C]PCand ['4C]PE (Vanha-Perttula & Kasurinen, 1989) and also by measuring the hydrolysis of p -NPPC spectrophotometrically , as described before (VanhaPerttula & Kasurinen, 1986). The method to measure phospholipase C, which hyrolyses phosphatidylinositols, using PIP and PIP2 as substrates was the same as reported previously (Vanha-Perttula & Kasurinen, 1989).

Enzyme purification PLA2 from bovine prostate was purified with sequential chromatographic steps. All the purification steps were performed at room temperature (20°C). Columns were equilibrated with the starting buffers and, prior to each step, the pooled samples were concentrated and desalted using an Amicon-52 apparatus with a PMlO membrane (Amicon Corporation, Lexington, MA, U.S.A.) Columns were attached to a high performance liquid chromatograph (HPLC) which consisted of an Altex 240 microprocessor control unit equipped with two Altex 110 pumps (Altex Scientific Inc., Berkeley, California, U.S.A.) and Kratos SF 7692 variable wavelength absorbance detector (Kratos Analytical Instruments, Ramsey, NJ, U.S.A.). Step 1: Ammonium sulphate precipitation. Homogenate of bovine prostate (100 ml) was saturated up to 40% with ammonium sulphate and stirred for 20 min. The precipitate was centrifuged at 30 000 g for 30 min. The pellet was then suspended in 0.025 M imidazole-HC1 buffer (pH 7.4) and desalted and concentrated to 2 ml.

Purification of phospholipase A2 397 Step 2: Anion exhange chromatography. The sample (2 ml) from step 1 was applied to an anion exhange column (Q Sepharose Fast Flow). Chromatography was performed using a column (HR 10/10; 1.0 X 10 cm) equilibrated with 0.025 M imidazole-HC1 buffer (pH 7.4). The sample was eluted with a linear gradient of 0-0.2 M NaCl for 10 min followed by a 0.2-0.6 M NaC1-gradient for 70 min. Fractions of 1 ml were collected at a flow rate of 1 ml min-'. Step 3: Chromatofocusing. Active fractions 30-65 from anion exhange chromatography were pooled, desalted, concentrated to 2 ml and applied to a Mono-P column (HR 5/30; 0.5 x 30 cm) equilibrated with 0.025 M imidazole-HCl buffer (pH 7.4). The proteins were eluted with a pH-gradient using Polybuffer 74 (pH 7.0-4.0). Fractions of 1 ml were collected at a flow rate of 1 ml min-'. Step 4: Spherogel-TSK 2000SW. The concentrated sample (0.5 ml) from Mono-P (fractions 30-43) was applied to a Spherogel-TSK 2000SW column (0.75 X 30 cm) and eluted with 0.2 M Na2S04 buffer (pH 3.0) containing 10 mM acetic acid. Fractions of 0.5 ml were collected at a flow rate of 0.5 ml min-'. Active fractions 20-30 were pooled and utilized for further analysis. During the purification process the enzyme became extremely labile and glycerol was therefore added into the collecting tubes (purification steps 2-4) to a final concentration of 50% (v/v) for stabilization. Heparin-Sepharose chromatography Heparin-Sepharose CL 6B column (1 X 5 cm) was equilibrated with 0.02 M Tris-HCI buffer (pH 7.5) containing 0.2 M NaCl. The pooled sample from Spherogel-TSK 2000SW chromatography was eluted with the same buffer for 3 h followed by a linear gradient of 0.2-1.0 M NaCl for 10 h. Fractions of 1 ml were collected at a flow rate of 6 ml h-'. Amino acid analysis and amino acid sequence determination A sample containing 200 pg purified protein was hydrolysed in an evacuated sealed tube with 100 p1 6 N HCl at 110°C for 24 h. Amino acid analysis was performed using an LKB Alpha Plus amino acid analyser. Norleucine (500 nmol) was used as an internal standard. Applied Biosystems 477A Pulse Liquid Sequencer with a 120A analyser was used to determine the N-terminal amino acid sequence of the purified protein. Protein assay The protein was measured by the method of Bradford (1976) with bovine serum albumin as a standard. Electrophoresis SDS-PAGE was performed using a PhastSystem electrophoresis apparatus (Pharmacia, Uppsala, Sweden). For SDS-PAGE, the samples were dissolved in 0.1 M Tris-HC1 buffer (pH 8.0) containing 5% 2-mercaptoethanol and 2.5% SDS. The samples were heated at 100°C for 10 min. The PhastGel Gradient Media 8-25 gels for native PAGE and Gradient Media 10-15 gels for SDS-PAGE as well as

398 S.Ronkko PhastGel IEF 3-9 gels for isoelectric focusing were stained with Coomassie Brilliant Blue G-250 (PhastGel Blue R). The microcomputer-based image analysis was used to determine the degree of impurities, molecular weight and isoelectric point of the purified enzyme. The intensity of the bands was quantified by digiting the SDS-PAGE and IEF 3-9 gels by a flat-bed gray-scale scanner (e.g. Apple scanner from Apple) connected to a Macintosh I1 microcomputer (Apple) as described before (Lammi & Tammi, 1991). Picture was saved in TIFF file format, then opened and analysed by image analysis with further calculations on standard spreadsheet programs of the microcomputer.

Results Lipolytic activity in homogenate of bovine prostate The homogenate of bovine prostate hydrolysed ara-PE, ara-PC, lino-PE, lino-PC, 2-PAF, p-NPP substrates and to some extend also p;NPPC, PPC, [14C]PE, PIP, PIP2, and sphingomyelin but not [14C]PC. We were unable to detect any lipase activity using glyceryl tri[l-14C]oleateas a substrate, or lysophospholipase activity with ['4C]lyso-PC as a substrate in the absence or presence of sodium deoxycholate at any pH with 1 mM Ca2+ or 1 mM EDTA. Purification of phospholipase AZ Table 1 summarizes the specific activity and recovery of the enzyme at different steps of purification. An 1100-fold purification was obtained in four steps (Figs 1 and 2) resulting in a yield of 16%. After purification, the enzyme preparation showed only PLA2 activity. No other lipolytic activity present originally in the homogenate of prostate was detected. Positional specificity of PLA2 was measured by gas chromatography using 1palmitoyl-2-oleoyl-~-a-phosphatidylethanolamine (PE) as a substrate. After anion exchange chromatography (Q Sepharose Fast Flow) the release of oleic acid in the sn-2 position of PE was about two times higher than that of palmitic acid in the sn-1 position. After chromatofocusing (Mono P), the enzyme preparation hydrolysed only oleic acid from the sn-2 position; no hydrolysis of palmitic acid occurred in the sn-1 position. Table 1. Purification of phospholipase A2 from the prostate of the adult bull

Purification step

Protein (mg)

PR homogenate Ammonium sulphate precipitate Q Sepharose Mono P TSK 2000SW

628 140 13.7 0.9 0.07

Total activity" (nmol min-') 25 16 16 8 3.1

Specific activity (nmol X min-' x mg protein-')

Purification factor

Yield

(%I

0.04 0.11

1 3

100 64

1.17 8.89 44.29

29 222 1107

64 32 16

"Activity was measured using lino-PE as a substrate in the presence of Ca2+ (5 mM).

Purification of phospholipase A2 399

800. E cm 600.

2 L

100

3

.C 400.. 50

c

B

200. 0,

20

-s

-L

60

80

80

E

60

.-c a c

40

g

4'0

0 100

150

100

50

20

0 0

0

10

20

10

30

40

20 Fraction

50

30

60

0 70

40

Fig. 1. Sequential steps (2-4) of PLA, purification. The sample (2 ml) from ammonium sulphate precipitation (step 1) was applied to a Q Sepharose Fast Flow column (panel A). The sample was eluted with a linear gradient of 0-0.2 M NaCl for 10 min followed by a 0.2-0.6 M NaCIgradient for 70 min. Fractions 30-65 of the eluate (36 ml) were pooled, desalted and concentrated to about 2 ml with a subsequent elution from a Mono-P column (panel B). Fractions 30-43 (14 ml) were pooled and applied to a Spherogel-TSK 2000SW column (panel C). Fractions 20-30 (5.5 ml) were pooled for the biochemical analysis. PLA, activity (pmol min-' ml-') in all fractions was measured with lino-PE as a substrate with 5 mM CaZC, and protein concentration was measured according to Bradford (1976).

Studies on enzyme characteristics The effect of temperature was tested by pre-incubating the enzyme preparation in 0.1 M Tris-HC1 buffer (pH 8.0) containing 5 mM CaC12 for 15 min at the temperature indicated. After pre-incubation, 2.24 VM lino-PE was added and the enzyme was then incubated at 37°C for 30 min. The enzyme activity was then assayed and compared with the control, pre-incubated at 37°C. The enzyme was quite stable up to 45"C, after which it was inactivated rapidly, 50% of the activity remaining at 60°C. The pH optimum of the enzyme was measured in 0.1 M acetate-HC1 (pH 3.0-7.0), 0.1 M imidazole-HC1 (pH 6.0-8.0) and 0.1 M Tris-HC1 (pH 8.0-10.25) buffer series. The enzyme showed hydrolytic activity over a wide range of pH values (6.0-10.25) and the pH-optimum was found to be approximately 8.0 (data not shown). Effects of different agents was tested on the purified PLA2 using lino-PE as a substrate (Table 2). The studies indicated that the purified enzyme had an absolute requirement for Ca2+, as evidenced by complete loss of activity in the presence of

'

400 S. Ronkko

255

0 255

255

61

0 255

62

0 Fig. 2. The electrophoretic pattern of the purified PLA? and standard proteins (top) as well as corresponding image analysis of the patterns (bottom). (a) SDS-PAGE of the purified PLA? in PhastGel 10-15 gradient. An aliquot of 1 pg purified enzyme (A2) was applied together with low molecular weight standards ( A l ) . (b) Isoelectric focusing of the purified (about 20 pg) PLA? (B2) with pl standards (BI) in PhastGel IEF 3-9. The pl standard proteins (Bl) were from top to bottom: trypsinogen (pl 9.3). lentil lectin basic (pl 8.65), middle (pl 8.45) and acidic band (pl 8.15), horse myoglobin basic (pl 7.35) and acidic band (pl6.85), human (pl6.55) and bovine carbonic anhydrase (pl 5.85), P-lactoglobulin A (pl5.2), soybean trypsin inhibitor (pl 4.55) and amyloglucosidase (pl 3.5). All electrophoretic runs were performed according to the instructions provided by the manufacturer (Pharmacia). Bottom: The image analyses of the electrophoretic patterns were run from the top to the bottom of the gel (top on the left and bottom on the right). The intensity of the bands is expressed as average pixel gray levels (0-255).

Purification of phospholipase A2 401 Table 2. The effect of various modifiers on the activity of the purified phospholipase A2 from bovine prostate ~

~~

Modifier

Concentration (1.0 mM)

No addition Ba2+ Cd2+ co2+ cu2+ Fe2+ Hg2+ Mg2+

100 24 61

66 75 7 30

28

Mn2+

69 61 26

Ni2+

PB+ Sr2+ Zn’+ Diisopropylfluorophosphate p-Bromophenacyl bromide + Ca2+ (+lmM) p -Bromophenacyl bromide EDTA N-Ethylmaleimide Dithiothreitol Chlorpromazine Trifluoperazine Mepacrine Chloroquine Primaquine Lysophosphatidylethanolamine Lysophosphatidylcholine Palmitate

Remaining activity (%)

33

(5.0 mM) (5.0 mM) (5.0 mM) (5.0 mM) (5.0 mM) (0.01 KIM) (0.01 mM) (0.1 mM)

18 80 60 35 0 106 63 73 50 87 68 7 39 8 87

Purified phospholipase A2 was pre-incubated in a total volume of 250 p1 for 15 min in the standard incubation mixture minus lino-PE (2.24 p ~ and ) Ca2+ (1 mM). The reaction was started by adding lino-PE and Ca2+ and was continued for 30 min at 37°C. The final volume was 300 p1. Results of duplicate determinations are shown as mean per cent activities relative to the control value.

EDTA, Ba2+, Fe2+, HgZf, Mg2+, Pb2+, Sr2+,and Zn2+ as well as lysophosphatidylcholine, lysophosphatidylethanolamine, and p-bromophenacyl bromide (p -BPB) were inhibitory. p -BPB was added in acetone solution into the incubation mixture and pre-incubated for 15 min with or without Ca2+. The enzyme activity was suppressed by detergents. A 0.1 mM concentration of sodium deoxycholate resulted in a 50% inhibition, but Triton X-100 slightly activated the PLA2 at concentrations below 0.25 mM (data not shown). PLA2 showed no affinity toward heparin in the tested conditions. The substrate specificity of the purified enzyme was determined using lino-PE, lino-PC, ara-PE, ara-PC, PPC, and 2-PAF as substrates (Table 3). The purified PLA2 hydrolysed lino-PE followed by lino-PC, ara-PE, and ara-PC but no hydrolysis of PPC nor platelet activating factor (2-PAF) was observed. The response of PLA2 to some polycyclic compounds was studied at different concentrations (Table 2). The enzyme was sensitive to primaquine at concentra-

402 S. Ronkko Table 3. Substrate specificity of the purified enzyme Substrate

nmoI x min-' x mg protein-'

~~~~

Lino-PE Lino-PC Ara-PE Ara-PC PPC 2-PAF

15.7 7.0 1 .o

1 .o ND ND

The hydrolysis rate (nmol X min- x mg protein-') of four phosholipids with ''C-labelled fatty acids (linoleic acid or arachidonic acid) at the sn-2 position (lino-PE, ara-PE, lino-PC, ara-PC) and that of l-acyl-2-[4-pyrenyl]-butyroyl-phosphatidylcholine (PPC) and l-hexadecyl-2(1-O-[acetyl-"H(N)])-sn-glyseryl-3-phosphorylcholine (2-PAF) by the purified PLAz from the prostate of the adult bull. ND is not detectable.

tions above 1mM and was less sensitive to other tested compounds. Mepacrine and chlorpromazine had almost no effect on the PLA2 activity. SDS-PAGE showed only one protein band and the molecular weight of the purified protein was extrapolated by microcomputer-based image analysis to be about 12000 f 1000 (Fig. 2a) and, in non-denaturing conditions, a similar value was obtained in a native PAGE gradient (8-25%) gel (data not shown). The isoelectric point of the enzyme was about 5.3 as analysed by chromatofocusing (purification step 3) and isoelectric focusing. When the IEF 3-9 gel was overloaded (Fig.2b), it resulted in two bands with pl values of about 6.2 (minor band) and 5.3 (major band), In a separate experiment (results not shown) the isoelectric focusing gel was cut into small pieces and the activity of PLA2 in these pieces was measured using the standard procedure with lino-PE as a substrate. The activity was located only in the major band with a pl of about 5.3. The application point of the isoelectric focusing run was seen in the image analysis (Fig. 2) of pl standards (Bl) and purified enzyme (B2) between pl values of 7.0 and 6.85. The amino acid composition, computed for a molecular weight of 12000, is presented in Table 4. Amino acid analysis revealed high contents of asparagine, aspartic acid, glysine, and alanine. The cysteine content was slightly lower than the reported values (12-14) for phospholipases A2 (Verheij et al., 1981). The N terminal amino acid sequence of the purified enzyme did not react on sequencing and we concluded that the N-terminus is blocked. Discussion Reverse-phase high-performance liquid chromatography has been shown to be a powerful technique for purification of PLA2. In pilot experiments, attempts to purify bovine prostatic PLAz by reverse-phase high-performance liquid chromatography were unsuccessful, as no activity could be eluted with acetonitrile using the same conditions as described before (Hara et al., 1989). Therefore we were obliged to use other chromatographic techniques. During ammonium sulphate

Purification of phospholipase A2 403 Table 4. Amino acid composition of bovine prostatic phospholipase A2

Amino acid Asx Thr Ser Gix Pro GIY

Ala CYS Val

mol mol-’ protein“ 31.4 (31) 11.4 (11) 9.0 (9) 5.3 (5) 5.0 (5) 13.5 (14) 13.1 (13) 9.0 (9) 1.0 (1)

Amino acid Met Ile Leu TYr Phe TrP LYS His A% Total amino acids

moI mo1-l protein” ND 1.0 (1) 3.0 (3) 3.0 (3) 1.9 (2) 0.7 (1) 9.4 (9) ND ND 117

“The amino acid composition is expressed as moles of residue per mole of purified enzyme and is computed for a molecular weight of 12000. ND is not detectable.

precipitation, ion exhange chromatography and chromatofocusing, large amounts of contaminating proteins, such as phospholipase C, phosphoinositide-specific phospholipase C and sphingomyelinase activity could be discarded. An efficient step in the purification was, however, Spherogel-TSK 2000SW chromatography because after that step we were unable to detect contaminating platelet activating acetylhydrolase and acid phosphatase activities. The whole purification procedure, including concentrations and time-consuming enzymic assays, can be completed in 4 days. Although the purification process was rather rapid, the purification coefficient (1100-fold) was low compared with up to about 100 000-fold purification reported for human synovial fluid (Hara et af., 1989) and bovine platelet (Kim el af., 1991) forms of PLA2. This seems to be, at least partly, due to the instability of the active enzyme. The loss of the activity could be overcome to a certain extent by adding glycerol and freezing immediately at -80°C. The present study is the first report to describe some biochemical properties of a purified PLA2 from bovine prostate. Earlier investigations have shown that human seminal plasma (Kunze et al., 1974), spermatozoa (Antaki et af., 1989), rat testis (Ellis et a f . , 1981), and epididymis (Bjerve et al., 1978) contain Ca2+-dependent PLA2 activities. Our previous report showed that, in the reproductive tissues of the adult bull, the highest level of PLA2 activity was in the prostate (Ronkko et al., 1991). In this paper we have studied in detail the biochemical properties of this enzyme which has an absolute requirement for Ca2+-ions. The maximum activity was obtained at a concentration of 5 mM Ca2+. The final preparation showed a single protein band, but unfortunately due to our limited methodology (no lower standard than 14000 was used) we could not determine exactly the molecular weight of the enzyme. Microcomputer-based image analysis gave a value of 12000 _+ 1000, which is near the range (12000-15000) reported typically for Ca2+dependent phospholipase A2 forms from other tissues (van den Bosch, 1980). We wanted to estimate possible impurities in the enzyme preparation due to the low purification coefficient and therefore we loaded an isoelectric focusing gel with a

404 S . Ronkko high amount of protein (about 20 pg). Results showed that the purified enzyme contained contaminating proteins at about 7%, measured by microcomputer-based image analysis and these impurities did not contain PLA2 activity. Considering the natural limitations of our methodology we could not measure the amount of contaminating proteins which had the same molecular weight and isoelectric point as the purified PLA2 from bovine prostatic tissue. The purified PLA2 was able to hydrolyse fatty acid from the sn-2 position but we were unable to detect any hydrolysis in the sn-1 position by gas chromatographic analysis using l-palmitoyl-2oleoyl-L-a-phosphatidylethanolamineas a substrate. This result suggests that the purified enzyme preparation does not contain any phospholipase A l activity. p-Bromophenacyl bromide (p-BPB), which is known to be an active sitedirected histidine reagent for a large number of phospholipase A2 forms (Volwerk et af., 1974), leading to inactivation of the enzyme, inhibits PLA2 activity in human seminal plasma (Wurl & Kunze, 1985) as well as in human (Thakkar et al., 1984) and mouse (Thakkar et al., 1983) spermatozoa and in bull seminal plasma and seminal vesicle fluid (Ronkko et af., 1991). This haloketone also suppressed PLA2 activity in the bovine prostate. This result indicates that the prostatic enzyme may contain a histidine residue at the active site, although we were unable to detect any histidine by amino acid analysis. The activity was only inhibited partially in the presence of Ca2+, indicating that Ca2+mayoccupy the active site. Furthermore, the serine-esterase inhibitor diisopropylfluorophosphate had almost no effect on the prostatic enzyme. This result is in agreement with an earlier report showing that this compound is a potent inhibitor of Ca2+-independentplatelet activating factor acetylhydrolase but has no effect on PLA2 activity (Nijssen et af., 1986). Phospholipase A2 from human synovial fluid (Hara et af., 1989), rat peritoneal fluid (Chang et af.,1987), and secreted from rabbit platelets (Mizushima et al., 1989) typically show high affinity towards heparin. PLA2 from the bull prostate differs from these enzymes in this respect because it showed no affinity towards heparin under the test conditions. Phospholipae A2 enzymes have an obvious function in metabolism as catabolic enzymes that hydrolyse the phospholipid components of membranes. Pancreatic PLA2 is responsible for the initial digestion of the phospholipids in dietary fat and PLA2 in snake venoms promote cell lysis and membrane disruption (Dennis, 1983). In addition to degradative functions, phospholipase A2 may have a role in biosynthetic processes. It is widely assumed that, in the human reproductive system, PLA2 has the ability to produce arachidonic acid for the generation of prostaglandins (Kunze et af., 1974). Bovine prostatic PLA2 may not have such a function, because it hydrolysed linoleic acid at the sn-2 position of phospholipids with high selectivity compared to arachidonic acid. This enzyme may be involved in phospholipid remodelling functions, such as has been proposed for PLA2 coupled with specific acyl-CoA: lysophospholipid acyltransferases (van den Bosch, 1980). This cycle provides a mechanism by which the fatty acid content of the sn-2 position of the phospholipid backbone can be altered quite readily by a deacylationreacylation mechanism. However, the physiological role of bovine prostatic PLA2 remains to be shown. In order to better understand the biochemical and physiological role of this prostatic enzyme, it is important to elucidate its precise

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Received 25 November 1991; accepted 27 March 1992