©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Properties of a Phosphatidic Acid-preferring Phospholipase A from Bovine Testis
EXAMINATION OF THE MOLECULAR BASIS OF ITS ACTIVATION (*)

(Received for publication, December 26, 1995; and in revised form, February 9, 1996)

Henry N. Higgs John A. Glomset (§)

From the Howard Hughes Medical Institute, Department of Biochemistry, and Regional Primate Research Center, University of Washington, Seattle, Washington 98195-7370

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We recently identified a cytosolic phospholipase A(1) activity in bovine brain and testis that preferentially hydrolyzes phosphatidic acid substrates. We also showed that the enzyme displays sigmoidal kinetics toward phosphatidic acid substrates in a Triton X-100 mixed micelle assay system (Higgs, H. N., and Glomset, J. A.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9574-9578). In the present work we purified the bovine testis enzyme 14,000-fold and used a combination of size exclusion chromatography, labeling with the phospholipase A inhibitor, methyl arachidonyl fluorophosphonate, and SDS-polyacrylamide gel electrophoresis to provide evidence that it is a homotetramer of 110-kDa subunits. Studies of the molecular basis of the enzyme reaction in Triton micelles revealed that (a) a nonhydrolyzable sn-1-alkyl-2-oleoyl analogue of phosphatidic acid activated the enzyme 30-fold in a sigmoidal fashion (Hill coefficient 3.2, EC 4 mol %) without substantially affecting its preference for specific diacyl phosphoglyceride substrates, (b) the activator promoted tight binding of the enzyme to micelles, and (c) the enzyme's activity toward unsaturated phosphatidic acid substrates was affected by the location and nature of the fatty acyl chain double bonds.


INTRODUCTION

Phospholipases of the A(1) type (PLA(1)) (^1)cleave the sn-1 fatty acyl group from diacyl glycero-phospholipids, producing a free fatty acid and an sn-2-lysophospholipid. A variety of PLA(1) activities have been identified. Membrane-bound PLA(1)s are present in platelets, liver, and heart(1, 2, 3) . There also seem to be a number of soluble lysosomal enzymes(4) , some of which can also catalyze transacylation reactions (5) . In addition, cytosolic PLA(1) activities have been identified in heart, brain, and testis(6, 7, 8, 9) . This diversity of PLA(1) activities strongly suggests a diversity of cellular functions. Given the ever expanding roles of phospholipid metabolites in cellular signaling(10) , it is likely that one role of PLA(1) enzymes is to produce or remove such metabolites.

A detailed enzymatic examination of these enzymes would greatly assist the elucidation of their cellular roles. Unfortunately, comparison of the enzymological properties of the PLA(1) enzymes has been hampered by the fact that different investigators have used fundamentally different assay systems. Some systems present the substrate as an undefined aggregate of either individual classes of phospholipid (8, 11) or phospholipids in combination with submicellar concentrations of detergent(7, 12) . In either case, the probability of artefactual results is high, due to the drastically different structures formed by single phospholipids of varying headgroup and acyl chain composition(13) . Interpretation of results obtained with these systems is also hindered by the inability to alter the surface concentration of phospholipid, making comparisons of accepted kinetic parameters impossible.

A number of well-defined systems for the study of other lipolytic enzymes have been developed. One of the most convenient is the mixed micelle system, in which the phospholipid is presented as a low percentage component in detergent micelles(14) . This system has clear advantages over those mentioned above: (a) micelle structure is primarily determined by the detergent, reducing the variability of surface architecture between different lipids, (b) the surface concentration of substrate can be varied, allowing the determination of Michaelis-Menten parameters, and (c) other lipids can be added to the system in a controlled fashion, aiding in the determination of cofactor requirements.

We recently used a Triton X-100 mixed micelle system to study a cytosolic PLA(1) (PA-PLA(1)) that displayed interesting enzymatic properties. First, it preferentially hydrolyzed phosphatidic acid (PA) by 4-fold over phosphatidylinositol (PI), 5-fold over phosphatidylserine (PS), 7.5-fold over phosphatidylethanolamine (PE), and 10-fold over phosphatidylcholine (PC). Second, it hydrolyzed diunsaturated molecular species of PA, such as diarachidonoyl (478 nmol/min/mg) or dioleoyl PA (208 nmol/min/mg) in preference to molecular species with an sn-1 saturated fatty acyl chain, such as sn-1-palmitoyl-2-oleoyl (80 nmol/min/mg) or sn-1-stearoyl-2-arachidonoyl PA (42 nmol/min/mg). Third, it appeared to be cooperatively activated by PA, as each PA substrate displayed sigmoidal kinetics with a Hill coefficient of 3(9) . Fourth, the tissue distribution of PA-PLA(1) activity was limited. High levels were present in bovine testis and brain, and the specific activity of the enzyme was 10-fold higher in mature testis than in immature testis. However, PA-PLA(1) was undetectable in liver, spleen, heart, or blood. Though the enzyme's biological role has yet to be determined, this distribution of enzyme activity suggests that PA-PLA(1) may be involved in cell signaling.

In the current study, we purified the enzyme to the point where a single major band could be seen by silver-stained SDS-PAGE and then addressed the following questions. By what mechanism does PA activate PA-PLA(1), and how does this activation affect the enzyme's preference for PA over other lipids? What is the structural basis for the enzyme's preference for diunsaturated PAs? Does PA-PLA(1) behave similarly to a recently purified brain PLA(1)(7, 12) if assayed under the conditions used for this enzyme, and do results in this assay system agree with those obtained in the Triton micelle system? The results of these studies are described below.


EXPERIMENTAL PROCEDURES

Materials

Mature bovine testes were obtained from a local slaughterhouse. All nonradiolabeled lipids were from Avanti Polar Lipids (Alabaster, Alabama) except for methyl arachidonyl fluorophosphonate (MAFP), which was from Cayman Chemical Co. (Ann Arbor, MI). [-P]ATP (0.1 Ci/mmol, 1 Ci = 37 GBq), sn-1,2-[1-^14C]dioleoyl-PC, sn-1-palmitoyl-2-[1-^14C]arachidonoyl PE, and sn-1-palmitoyl-2-[1-^14C]arachidonsyl PC were from DuPont NEN. sn-1,2-Dioleoyl-3-phosphatidyl[3-^14C]serine and sn-1,2-dioleoyl-3-phosphatidyl[2-^14C]ethanolamine were from Amersham Corp. [^14C]-MAFP was a generous gift from Dr. Zheng Huang of Merck Frosst, Canada. All chromatographic resins, columns, and pumps for protein purification were from Pharmacia Biotech Inc. All organic solvents were from J. T. Baker and were reagent grade or better. Reagents for the bicinchoninic acid protein assay were from Pierce. Silica 60 thin layer chromatography (TLC) plates were from E. M. Merck, and C18 reversed phase plates were from Whatman. Dodecylpoly(ethyleneglycolether)(n) (Thesit), Triton X-100, and Triton X-114 were from Boehringer Mannheim. All other reagents were from Sigma except where indicated.

Purification of PA-PLA(1)

All purification steps were performed at 4 °C. Except where specifically noted, buffers used for purification (PB) contained 25 mM MOPS, pH 7.2, 1 mM EGTA, 1 mM benzamidine, 2 µg/ml leupeptin, and 2 µg/ml antipain. This purification procedure was conducted on two separate occasions with similar results.

Tissue Extraction and Precipitation Steps

Testes were decapsulated, cut into 3-4-cm cubes, and frozen in liquid nitrogen within 1 h of removal from bulls. The frozen tissue was kept at -70 °C until extraction and then placed in a blender along with 3 ml/g extraction buffer (PB + 5 mM EDTA, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF), 6.25 µg/ml leupeptin, 6.25 µg/ml aprotinin, 2 µg/ml pepstatin, 1 mM dithiothreitol (DTT)). It is important to note that DTT was not added until after the blending step. The mixture was blended until no tissue chunks remained (5 times 1-min bursts) and then 1-liter aliquots were homogenized in a Polytron homogenizer for 15 s times 3. The homogenate was centrifuged at 800 times g for 10 min. After being filtered through cheesecloth, the supernatant was centrifuged at 100,000 times g for 60 min. Then 233 mg/ml ammonium sulfate was added with vigorous stirring, and the solution was mixed for 15 min followed by centrifugation at 16,000 times g for 10 min. The resulting pellet was resuspended in PB + 5 mM benzamidine, and 1 mM PMSF was added from a 200 mM stock in isopropyl alcohol. The volume of resuspension buffer was 5-fold less than that of the originally precipitated sample. After the pellet was fully resuspended, DTT was added to 1 mM from a 1 M stock. To the resuspended pellet, 125 mg/ml polyethylene glycol 3350 (PEG) was added with vigorous stirring, and the solution was mixed for 15 min followed by centrifugation at 16,000 times g for 10 min. The same resuspension procedure as for the ammonium sulfate precipitate was followed except that the volume of the resuspension buffer was equal to that of the precipitated sample.

SP-Sepharose Chromatography and Concentration of the Resulting Pool

The resuspended precipitate from the PEG step (600 ml) was diluted with an equal volume of PB + 6 M urea, 40 mM KCl, 4 mM Thesit, and 1 mM DTT. The resulting solution was immediately loaded onto a XK50 column packed with 400 ml of SP-Sepharose Fast Flow and pre-equilibrated with PB + 3 M urea, 20 mM KCl, 2 mM Thesit, and 1 mM DTT. The column was then washed with 1 volume of equilibration buffer followed by 2 volumes of PB + 20 mM KCl, 0.2 mM Thesit, and 1 mM DTT. The activity was eluted with PB + 100 mM KCl, 0.2 mM Thesit, and 1 mM DTT. The column flow rate was kept constant at 10 ml/min using a P-1 pump, and fractions were collected manually by following the A of the eluate. The active pool (300 ml) was supplemented with 1 mM PMSF from a 200 mM stock in isopropyl alcohol and then concentrated using an Amicon RC800 fitted with a low protein binding 300,000 NMWL membrane (Millipore Corp.) until the volume of the concentrated pool was 45 ml.

Mono Q Chromatography and Concentration of the Resulting Pool

The KCl concentration in the concentrated SP pool was raised to 200 mM by the addition of a 2.5 M KCl stock. The pool was then loaded onto a Mono Q 10/10 column connected to a fast protein liquid chromatography apparatus (Pharmacia) and pre-equilibrated in PB + 200 mM KCl, 0.2 mM Thesit, 1 mM DTT. The column was washed with 20 ml of equilibration buffer followed by 20 ml of the same buffer containing 235 mM KCl. The enzyme was eluted with an 80-ml gradient from 235 to 300 mM KCl, followed by a 20-ml gradient to 400 mM KCl and a 30-ml wash with 1 M KCl. The flow rate was 2 ml/min, and 1-min fractions were collected. The active pool (26 ml) was supplemented with 1 mM PMSF from a 200 mM stock in isopropyl alcohol and then concentrated to 4 ml using an Amicon model 52 fitted with a low protein binding 100,000 NMWL membrane (Millipore). The sample was further concentrated to approximately 1 ml in two Centricon 100 concentrators (Amicon).

Superdex 200 Chromatography

The concentrated Mono Q pool was loaded onto a Superdex 200 16/60 column connected to a fast protein liquid chromatography apparatus and equilibrated with PB + 500 mM NaCl, 0.1 mM Thesit, and 1 mM DTT, but with no benzamidine, leupeptin, or antipain. The flow rate was 0.5 ml/min and 2-min fractions were collected.

Phenyl-Superose Chromatography

The concentration of NaCl in the Superdex 200 pool was adjusted to 1.4 M from a 5 M stock. This solution was loaded onto a phenyl-Superose 5/5 column connected to a fast protein liquid chromatography apparatus and equilibrated with PB + 1.4 M NaCl and 1 mM DTT. The column was washed with 10 ml of the same buffer, then PA-PLA(1) was eluted by reducing the NaCl concentration to 0.9 M. The flow rate was 0.5 ml/min and 1-min fractions were collected.

Phenyl-CL-4B Chromatography

The pool from phenyl-Superose chromatography was adjusted to 300 mM NaCl by diluting with PB. This solution was loaded onto 0.5 ml of phenyl-CL-4B packed into a Pierce Econocolumn and equilibrated with PB + 300 mM NaCl. The column was washed with 5 ml of the same buffer, and then PA-PLA(1) activity was eluted with 2 ml of PB. The column was run under gravity flow.

Synthesis of P-Labeled PA from Diacylglycerol (DAG) by Diacylglycerol Kinase

[P]PA was synthesized as described (9) except that an extra purification step was added after the Bond-Elut PRS column (Varian). The anhydrous eluate from this column was resuspended in 250 µl of 1:1 CHCl(3):MeOH and spotted onto a C18 reversed phase TLC plate (Whatman). This plate was developed in 98:2 MeOH, 4 M ammonium formate for 30 min. The PA band was visualized by autoradiography and scraped. PA was eluted from the resin by 2 extractions with 6 ml of 1:1 CHCl(3):MeOH. The solvent was evaporated, and the PA was resuspended in 2 ml of 9:1 dichloroethane:isopropyl alcohol. This extra purification reduced background lyso-PA in the assay from 1 to 0.05% of the total PA counts.

PLA Assays

During enzyme purification, enzyme activity was monitored by the following assay. P-labeled dioleoyl PA was evaporated under argon and resuspended in 80 mM Triton X-100 (critical micelle concentration = 0.3 mM) in H(2)O. This solution was added to buffer, and assays were started by the addition of enzyme. Final assay conditions, in a total volume of 100 µl, were 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 1 mM EGTA, and 8 mM Triton X-100 with 0.5 mol % [P]PA. Assays were conducted at 25 °C for 20 min and terminated by the addition of 25 µl of carrier (1.25 mg/ml of sn-1-18:1-lyso-PA, egg PA and egg PC in 1:1 CHCl(3):ethyl acetate) followed by 900 µl of 1:1 CHCl(3):MeOH + 1% trifluoroacetic acid. After thorough mixing, two phases were formed by the addition of 650 µl of 9:1 H(2)O:MeOH. The mixture was vortexed and centrifuged briefly, and the upper phase was removed by suction. The lower phase was evaporated under vacuum and resuspended in 50 µl of 1:1 CHCl(3):MeOH. From this mixture, 25 µl were spotted onto a C18 reversed phase TLC plate, which was developed in 98:2 MeOH, 4 M ammonium formate for 15 min. Radioactive product lyso-PA bands, which co-migrated with authentic standards, were visualized by autoradiography, scraped, and quantitated by scintillation counting in Ecolume (ICN).

Characterization of the enzyme, purified through the Superdex 200 step, was carried out under different assay conditions. The final buffer concentrations in the 100-µl assay were 50 mM MOPS, pH 7.2, 100 mM KCl, 1 mM EGTA, and 16 mM total micellar lipid, with the bulk detergent being a 1:1 molar mix of Triton X-100 to Triton X-114. Assays included 0.25 mol % of [P]PA, with the rest of the substrate PA being nonradiolabeled. All subsequent steps in the assay were the same as those outlined above.

When PLA activity toward other phospholipid classes was assayed, conditions were the same as those described above. Lyso-PS, lyso-PI, and lyso-PE generation by the enzyme were measured by the same reversed phase TLC technique as described for PA. PC breakdown was measured by the release of ^14C-oleic acid, which was separated from PC and lyso-PC by TLC on Silica 60 plates developed in 6:8:2:2 CHCl(3):acetone:acetic acid:water.

Under all conditions used, assays were linear for time and protein concentration. All assays were carried out in borosilicate glass tubes siliconized with dichlorodimethylsilane. For each lipid class tested, the fraction of lyso-lipid in the upper phase during extraction was measured and was factored into calculations. Calculation of kinetic parameters was carried out following previously established methods for micellar systems(14) . The program, GraFit (Erithacus Software Ltd., London), was used for making graphs of kinetic data.

Covalent Labeling of PA-PLA(1) with Radiolabeled MAFP and Detection of Labeled Protein

An aliquot of ^14C-MAFP was evaporated from its methylethyl ketone stock solution under argon and resuspended in 500 µM Triton X-100 in water to a concentration of 40 µM MAFP. DTT was removed from the enzyme source by loading 10 µg of Superdex 200 pool onto a 0.25-ml column of phenyl-Sepharose CL-4B equilibrated in 25 mM HEPES, pH 8.0, 300 mM KCl, and 1 mM EGTA. The column was washed with 2 ml of equilibration buffer, and the enzyme was eluted with 4 times 250 µl of 25 mM HEPES, pH 8.0, 1 mM EGTA. The majority of the enzyme was in the second fraction. The inhibition reaction was started by the addition of the MAFP/Triton X-100 mix at a 1:10 dilution to the enzyme. Aliquots were removed at various times and diluted 1:20 in 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 1 mM EGTA, and 10 mM DTT. The diluted samples were used to start enzyme assays. Separate aliquots were diluted 1:1 with 2 times SDS-PAGE sample buffer containing 10 mM DTT, immediately boiled, and then loaded onto a 2.7-14% gradient SDS-PAGE gel. After completion of electrophoresis, the gel was soaked in Enhance (Amersham Corp.) for 30 min, dried under vacuum, and exposed to x-ray film (Amersham) for 1 month. Bands were detected after development of the film.

Synthesis of PA from PC by Phospholipase D Digestion

The solvent from CHCl(3) stocks of defined molecular species of PC (100 mg) was evaporated under argon. The PC was resuspended in 2 ml of diethyl ether, and 100 units of Streptomyces species phospholipase D was added in 1 ml of 50 mM MOPS, pH 7.2, and 50 mM CaCl(2). After 2 h at 25 °C with vigorous stirring, the ether phase was evaporated under argon. To the remaining solution, 1 ml of 500 mM sodium-EDTA, pH 7.2, 2 ml CHCl(3), and 4 ml MeOH were added so that a single phase was obtained. An additional 2 ml of CHCl(3) were added along with 2 ml of water, and the mixture was vortexed and briefly centrifuged. After removal of the upper phase, the lower phase was washed once with 9:1 water:MeOH, and the upper phase was discarded. The lower phase was evaporated under argon, the residue was resuspended in 1 ml CHCl(3), and 4 ml of acetone were added to precipitate the PA. The mixture was centrifuged for 5 min at 800 times g, and the supernatant was discarded. The pellet, containing PA uncontaminated by PC or DAG, was resuspended in 2 ml of CHCl(3).

Synthesis of PS from PC by Phospholipase D

Solvent was evaporated from 50 mg of PC, which was then resuspended in 4 ml of diethyl ether. The reaction was started by the addition of 100 units of Streptomyces species phospholipase D in 2 ml of 3.3 ML-serine, 200 mM sodium acetate, pH 5.6, and 100 mM CaCl(2). After 2 h at 25 °C, the diethyl ether was evaporated, and the lipids were extracted by the method of Folch(15) , using 200 mM sodium acetate, pH 5.6, as the aqueous phase. The lower phase was evaporated under nitrogen, and the residue was resuspended in 2 ml of 98:2 CHCl(3):MeOH with 0.1 M HCl. PS was separated from PA and PC on a 1-ml BondElut PRS column (Varian) equilibrated in the same solvent. After the sample was loaded, the column was washed with 14 ml of solvent followed by 8 ml of 90:10. PS was eluted with 24 ml of 50:50. No trace of PA or PC was observable when 100 µg of PS was run on TLC in 6:8:2:2:1 acetone:CHCl(3):MeOH:acetic acid:water and stained with iodine.

Synthesis of DAG from PC by Phospholipase C Digestion

The solvent from a CHCl(3) stock solution of PC (1.4 µmol) was evaporated under argon. The PC was resuspended in 1 ml of diethyl ether, and 1.5 units of Bacillus cereus phospholipase C was added in 250 µl of 50 mM MOPS, pH 7.2, and 10 mM CaCl(2). After vigorous stirring for 1 h at 25 °C, the ether phase was evaporated under argon, 600 µl MeOH and 250 µl CHCl(3) were added, and the mixture was vortexed until a single phase appeared. Then 250 µl of CHCl(3) and 250 µl of water were added and, after vortexing and a brief centrifugation, the upper phase was removed. The lower phase was washed 1 time with 1 ml of 9:1 water:MeOH, and the upper phase was discarded. The lower phase was evaporated under argon, and the DAG was resuspended in 1 ml of CHCl(3). This procedure regularly gave quantitative yields of DAG, as judged by TLC in 60:50:5:2 hexane:diethyl ether:MeOH:acetic acid.

Synthesis of PI from PA

First, PA was converted to cytidine 5`-diphospho-sn-diacylglycerol (CDP-DAG) chemically, and then CDP-DAG was converted to PI. PA conversion to CDP-DAG was accomplished by the method of Carman and Fischl(16) . Briefly, 20 µM of PA (protonated) were reacted with 30 µM of cytidine 5`-monophospho-morpholidate in 10 ml of pyridine containing 80 µM dimethylaminopyridine for 16 h with stirring at 25 °C. The pyridine was removed under vacuum, and the residue was resuspended in 2 ml of 50:30:7 CHCl(3):pyridine:acetic acid. This mixture was loaded onto a 2-ml silica column equilibrated in the same solvent. The mixture was washed with 10 ml of the same solvent, followed by 10 ml of 9:1 CHCl(3):MeOH, and CDP-DAG was eluted with 8 ml of 50:28:4:8 CHCl(3):MeOH:acetic acid:water.

For conversion of the CDP-DAG to PI, the solvent was evaporated under nitrogen. The CDP-DAG was then resuspended in 4 ml of 50 mM Tris-HCl, pH 8.0, 30 mM Triton X-100, and 50 mM myo-inositol. When the CDP-DAG was fully resuspended, MgCl(2) was added to 16 mM, and solubilized yeast membranes were added to 0.3 mg protein/ml. The yeast membranes were a kind gift of G. M. Carman, supplied as a 3 mg/ml protein stock in 50 mM Tris-HCl, pH 8.0, 30 mM MgCl(2), 10 mM beta-mercaptoethanol, 20% glycerol (w/v), and 16 mM Triton X-100. The reaction was incubated at 25 °C for 16 h with agitation. Lipids were extracted by the method of Folch(15) , and the solvent from the lower phase was evaporated under nitrogen. The residue was resuspended in 1 ml of CHCl(3) and passed over a BakerBond PRS column equilibrated in the same solvent. After washing with 4 ml of CHCl(3), followed by 8 ml 9:1 CHCl(3):MeOH, PI was eluted with 8 ml of 75:25:3 CHCl(3):MeOH:water. The solvent was evaporated under nitrogen, and the pure PI was resuspended in 1 ml of CHCl(3). Purity was judged by TLC in 6:8:2:2:1 CHCl(3):acetone:MeOH:acetic acid:water. When P-labeled dioleoyl PI was synthesized, its radiochemical purity was >96%.

Synthesis of Phosphatidylmethanol from PA

Phosphatidylmethanol was synthesized by the method of Jain and Gelb(17) . Briefly, 20 µM of PA (protonated) were methylated(9) , and dimethyl-PA was purified by passing the mixture (in CHCl(3)) over a 1-ml bed of silica. The solvent was evaporated under nitrogen, and the residue was resuspended in 1 ml of acetone containing 7 mg of LiBr. This mixture was left at 25 °C for 16 h with stirring. The solvent was evaporated under nitrogen. PM was purified by passing the residue, resuspended in 1 ml of CHCl(3), over a 1-ml BakerBond PRS column equilibrated in the same solvent, washing with 6 ml of CHCl(3), and eluting with 6 ml of 9:1 CHCl(3):MeOH. Purity was assessed by TLC in 6:8:2:2:1 CHCl(3):acetone:MeOH:acetic acid:water. When P-labeled PM was synthesized, its radiochemical purity was >98%.

PA-PLA(1) Binding to Micelles as Judged by Triton X-114 Partitioning

On the basis of the method of Bordier(18) . PA-PLA(1) (6 ng) was added to an iced mixture of 50 mM MOPS, pH 7.2, 100 mM KCl, and 1 mM EGTA containing micelles of Triton X-114 with 0.5 mol % dioleoyl PA and 0-15 mol % sn-1-alkyl-2-acyl PA. The total concentration of micellar lipid in the assay was 16 mM (critical micelle concentration of Triton X-114 = 0.3 mM). The solution was immediately transferred to a water bath at 25 °C, incubated for 5 min and then centrifuged for 5 min at 16,000 times g and 25 °C. The supernatant (80 µl) was transferred to another tube, and the pellet was resuspended in 80 µl of 50 mM MOPS, pH 7.2, 100 mM KCl, and 1 mM EGTA. Both supernatant and pellet were assayed for PA-PLA(1) activity as described above, in micelles containing 0.5 mol % P-labeled dioleoyl PA and 10 mol % sn-1-alkyl-2-oleoyl PA. The efficiency of the Triton X-114 phase partitioning was determined by measuring the amount of Triton (18) and P-labeled dioleoyl PA remaining in the upper phase after centrifugation. Both values were similar for all conditions tested and never exceeded 4% of the total lipid.

Assay of PA-PLA(1) Using a CHAPS/Phospholipid Assay System

Assays were carried out as described in (7) and (12) . Briefly, the solvent was evaporated from ^14C-labeled sn-1-palmitoyl-2-arachidonoyl species of PE or PC (0.5 nmol/assay), and the residue was brought up in 5 µl/assay of either 5 mM or 6.5 mM CHAPS, depending on the individual assay. Assays were started by the addition of 5 µl of this mixture to enzyme/buffer preincubated at 37 °C. Final buffer concentrations were: 50 mM or 100 mM Tris-HCl, pH 7.5 or 9.0, 70% glycerol, 3 or 5 mM MgCl(2), 250 or 325 µM CHAPS, and 1 mM EDTA. Assays were terminated after 8 min with 0.5 ml of 1:1 CHCl(3):MeOH, followed by 0.15 ml of water. Lysophospholipid and phospholipid standards (5 µg each) were added, the phases were allowed to separate, the lower phase was removed, and the solvent evaporated under vacuum. The residue was redissolved in 50 µl of 75:25:2 CHCl(3):MeOH:H(2)O and spotted on a silica TLC plate. The plate was developed in 65:35:5 CHCl(3):MeOH:H(2)O until the solvent front was 10 cm from the top. After evaporation of solvent, the plate was redeveloped in 90:60:4 hexane:diethyl ether:formic acid until the solvent front was 2 cm from the top. Bands were detected by iodine staining, scraped, and counted in 5 ml of Aquamix (ICN) with 0.2 ml of H(2)O.

Other Methods

Protein concentrations were determined by the bicinchoninic acid method(19) . Interfering substances were removed by the trichloroacetic acid precipitation method described previously (20) except that 0.025% Triton X-100 was added to the samples in addition to the deoxycholate. SDS-PAGE was performed by the method of Laemmli (21) on 12 times 14 times 0.075-cm 2.7-14% gradient slab gels. Dilute protein samples were precipitated before electrophoresis (20) . Protein bands were identified by silver staining(22) . Phospholipid concentrations were routinely confirmed by organic phosphate determination(23) .


RESULTS

Purification of PA-PLA(1)

A synopsis of the purification is given in Table 1, and a detailed description of the process can be found under ``Experimental Procedures.'' Aspects of the results that warrant special comment are described below. An important general aspect of the purification was the use of fresh-frozen tissues and protease inhibitors. Fresh testes were immediately decapsulated, cut into small cubes, frozen in liquid nitrogen, and stored at -70 °C. The tissue was quickly thawed during homogenization in the presence of an extensive protease inhibitor mixture. After ammonium sulfate precipitation and through Mono Q column chromatography, a less extensive protease inhibitor mixture was used, although it still included a number of serine protease inhibitors.



The combination of ammonium sulfate and PEG 3350 precipitations greatly increased the capacity of the subsequent SP-Sepharose column for the extract. Interestingly, although the high speed supernatant bound to the SP column in the presence of 20 mM KCl, solubilized PEG precipitates flowed through even in the absence of KCl. However, these PEG precipitates did bind in the presence of 3 M urea and 2 mM Thesit, while the enzyme retained the majority of its activity (>80% after 2 h at 4 °C). Treatment of the PEG precipitate with urea and Thesit probably served to release other proteins that had become tightly bound to the PLA(1) during the precipitations.

The inclusion of the nonionic detergent, Thesit, at concentrations around its critical micelle concentration (0.1 mM) enhanced the stability of PA-PLA(1) during the purification. For example, incubation of the Mono Q-purified fraction for 20 h at 4 °C resulted in a recovery of 25% without detergent, whereas 80% was recovered if 0.2 mM Thesit was included. Other nonionic detergents, such as Triton X-100, Triton X-114, and octyl glucoside, behaved similarly. Thesit was chosen over the others because its low UV absorption did not interfere with the monitoring of chromatographic steps at 280 nm.

The concentration of KCl present during loading of the Mono Q column (Fig. 1A) affected the overall purification obtained by that column. Loading at 200 mM KCl gave a 2-fold increase in enrichment over loading at 100 mM followed by washing with 200 mM. PA-PLA(1) activity eluted at 280 mM KCl. Thus, it appeared that a number of proteins that otherwise would have bound to the column and eluted at the same KCl concentration as PA-PLA(1) were unable to bind to the column at 200 mM KCl.


Figure 1: Chromatographic elution profiles and SDS-PAGE evaluation of PA-PLA(1) purification. Elution profiles from Mono Q (A) and Superdex 200 (B) chromatography are shown. Elution volumes for markers on Superdex 200 (molecular mass in kDa in parentheses) were as follows: blue dextran 2000 (2,000,000), 43 ml; thyroglobulin (669), 47 ml; ferritin (440), 55 ml; catalase (232), 65 ml; aldolase (158), 67 ml; phosphorylase B (97), 68 ml; bovine serum albumin (67), 75 ml; ovalbumin (43), 83 ml; chymotrypsinogen (25), 91 ml. C, silver-stained gradient gel SDS-PAGE of the Superdex 200 fractions (2 µl each). D, silver-stained gradient gel SDS-PAGE of the pools from each of the stages of purification. In lanes 1-7, 1 µg of protein was loaded. In lanes 8 and 9, 50 ng of protein was loaded. Lanes 8 and 9 were stained separately in order to bring out minor bands. Lane 1, ammonium sulfate precipitate; lane 2, PEG precipitate; lane 3, SP pool; lane 4, concentrated SP pool; lane 5, Mono Q pool; lane 6, concentrated Mono Q pool; lane 7, Superdex 200 pool; lane 8, phenyl-Superose pool; lane 9, phenyl-CL-4B pool.



PA-PLA(1) activity eluted as a peak centered at 440 kDa on Superdex 200 size exclusion chromatography (SEC) (Fig. 1B). The active fractions from this column were pooled and passed over two successive phenyl-based hydrophobic resins: phenyl-Superose and phenyl-CL-4B. On each of these columns, the enzyme eluted over a broad range of NaCl when gradients were applied, even though the concentrations of salt needed for elution were very different (900 mM NaCl for phenyl-Superose and <100 mM for phenyl-CL-4B). For this reason, step elutions were applied, with no loss of enrichment by these columns.

When fractions from each step were analyzed by silver-stained SDS-PAGE, a band of 110 kDa was the major band remaining at the end of the purification (Fig. 1D). Furthermore, the elution of this band from SEC paralleled the elution of PA-PLA(1) activity (Fig. 1, B and C). These results suggest that the 110-kDa protein contains the PA-PLA(1) activity. In purifications where less care was taken to inhibit endogenous protease activity, additional major bands of 97 and 85 kDa were also observed, suggesting that these bands were proteolytic products of the 110-kDa band.

Covalent Labeling of PA-PLA(1) by MAFP

Although only one major band of 110 kDa remained in the active fraction after 14,000-fold enrichment, additional evidence linking this band to PA-PLA(1) was needed. For this reason, we sought to covalently modify the enzyme with a radiolabeled inhibitor of its activity and then to correlate the inhibition of PA-PLA(1) activity over time with the labeling of a band on SDS-PAGE. Fluorinated phosphorous or sulfur compounds have long been used as covalent inhibitors of a variety of esterases, and labeling of esterases with radiolabeled compounds of this kind has been used as a way of confirming the identity of the purified activities(24) . We tested a number of such compounds for inhibition of PA-PLA(1). Neither diisopropyl fluorophosphate nor PMSF inhibited the enzyme when incubated at 0.5 mM for 30 min at 25 °C (data not shown). In contrast, MAFP, an inhibitor of certain phospholipase A(2) enzymes, (^2)proved to be a potent inhibitor of PA-PLA(1), with 4 µM MAFP inhibiting the enzyme fully at 30 min (Fig. 2A). When radiolabeled MAFP was used, the time-dependent inhibition of PA-PLA(1) (purified to the Superdex 200 step) was paralleled by labeling of the 110-kDa band (Fig. 2C). Furthermore, the 110-kDa band was the only band labeled, even though several other minor bands could be seen by silver stain (compare Fig. 2, B and C). This provides strong evidence that the 110-kDa band corresponds to the PA-PLA(1) activity. When enzyme preparations that contained additional bands of 97 and 85 kDa were treated with ^14C-MAFP, these bands also were labeled (data not shown), providing further evidence that they were proteolytic fragments of the 110-kDa band.


Figure 2: MAFP inhibition and labeling of PA-PLA(1). PA-PLA(1) (0.3 µg of the Superdex 200 pool) was incubated with 50 µM Triton X-100 or with 4 µM^14C-labeled MAFP in 50 µM Triton X-100. At various times, aliquots were removed, diluted in buffer containing 10 mM DTT, and tested for PA-PLA activity while other aliquots were removed, boiled in SDS-PAGE loading buffer containing 10 mM DTT, and run on 2.7-14% gradient SDS-PAGE. The radioactive bands on the gel were observed by autoradiography. A, inhibition curve for ^14C-MAFP. B, silver-stained SDS-PAGE of samples incubated for the indicated times with ^14C-MAFP. This gel was heavily overstained in order to show the minor bands present in the fractions. C, autoradiogram of SDS-PAGE showing time-dependent labeling of the 110-kDa band with ^14C-MAFP. The 110-kDa band was labeled with ^14C-MAFP on two separate occasions.



Although PA-PLA(1) migrated as a 110-kDa band on SDS-PAGE (Fig. 1D), it eluted from Superdex 200 SEC with an apparent molecular mass of 440 kDa (Fig. 1B). The enzyme also migrated at 440 kDa on a silica-based, Toso Haas 3000 column (data not shown). One possible explanation for this difference is that the enzyme exists as a homotetramer in solution.

Enzyme Activation by Hydrolyzable and Nonhydrolyzable PA

As with partially purified PA-PLA(1)(9) , the purified enzyme displayed sigmoidal kinetics (Hill coefficient of 2.8) when assayed in the presence of increasing surface concentrations of dioleoyl PA in Triton micelles (Fig. 3A). To study the basis for this activation, we synthesized a nonhydrolyzable analogue, sn-1-alkyl-2-oleoyl PA (AO-PA), in the hope that it would mimic PA activation but not compete with sn-1,2-diacyl PA as a substrate. As predicted, AO-PA was not a substrate for PA-PLA(1) at concentrations of up to 20 mol % (data not shown). When added to assays containing 0.5 mol % dioleoyl PA as substrate, however, AO-PA stimulated PA-PLA(1)(9) activity over 30-fold in a sigmoidal fashion (Hill coefficient of 3.5) with an EC of 4 mol % (Fig. 3B). Thus, both hydrolyzable and nonhydrolyzable PA could activate the enzyme.


Figure 3: Activation of PA-PLA(1) by alkyl-oleoyl-phospholipids. A, hydrolysis of varying concentrations of dioleoyl-[P]PA by PA-PLA(1) (0.6 ng) in micelles of 1:1 (mol:mol) Triton X-100:Triton X-114 in the absence of AO-PA (open circles), presence of 10 mol % AO-PA (closed circles), and presence of variable AO-PA such that the concentration of total PA (dioleoyl PA + AO-PA) remained at 10 mol % (open squares). There was 16 mM total micellar lipid. B, effect of varying concentrations of AO-phospholipids on PA-PLA(1) hydrolysis of 0.5 mol % dioleoyl-[P]PA in Triton micelles (same conditions as above). Closed circles, AO-PA; open circles, AO-PS; closed triangles, AO-phosphatidylmethanol; closed squares, AO-PC. Each experiment was conducted in duplicate on two separate occasions with similar results.



When PA-PLA(1) activity toward increasing mole percentages of dioleoyl PA was retested in the presence of 10 mol % AO-PA, the kinetics were hyperbolic (Fig. 3A). Under these conditions, the enzyme displayed an apparent K(m) (K(m)) of 5.22 mol % and a V(max) of 392 µmol/min/mg (Table 2). When the total mole percentage of PA (dioleoyl PA + AO-PA) was kept at 10 while the ratio of AO-PA to dioleoyl PA was varied from 19 to 0, the results mimicked those observed at 10 mol % AO-PA. Thus, it appeared that AO-PA did not compete significantly with hydrolyzable PA for binding to the enzyme's active site.



Other anionic lipids also could stimulate PA-PLA(1). AO-phosphatidylmethanol stimulated dioleoyl PA hydrolysis to an extent similar to that stimulated by AO-PA (Hill coefficient of 2.7) but with an EC of 9 mol % (Fig. 3B). Similarly, AO-PS cooperatively activated the enzyme but at higher concentrations than for AO-PA. At 20 mol % AO-PS, dioleoyl PA hydrolysis had not fully plateaued. Higher concentrations of AO-PS were not tried for fear of severely altering the state of the micelles. In contrast to the stimulatory effect of these anionic lipids, AO-PC had no effect on the hydrolysis of PA (Fig. 3B).

Effect of Activator on Binding of PA-PLA(1) to Micelles

AO-PA activation of PA-PLA(1) might occur via a number of possible mechanisms. For example, AO-PA might induce an allosteric change in the enzyme, or it might increase the enzyme's affinity for the micelle surface, thus eliminating one step of the enzyme's catalytic cycle(14) . We tested the latter possibility using mixed micelles of Triton X-114 that contained 0.5 mol % dioleoyl PA and variable amounts of AO-PA. At temperatures of >25 °C, these mixed micelles aggregate and can be sedimented by low speed centrifugation. Proteins tightly bound to the micelle are found in the pellet, whereas those not bound remain in the supernatant(18) . With an increasing mol % of AO-PA, increasing amounts of PA-PLA(1) pelleted. The increase in pelleted PA-PLA1 was sigmoidal and paralleled the AO-PA-induced activation of PA-PLA(1) (Fig. 4). Thus, one component of PA-PLA(1) activation is likely to be an increase in surface affinity.


Figure 4: Influence of AO-PA on micelle binding by PA-PLA(1). The enzyme (6 ng) was incubated in 100 µl of assay buffer containing Triton X-114 micelles with 0.5 mol % dioleoyl PA and varying amounts of AO-PA (16 mM total micellar lipid) for 5 min at 25 °C, followed by a 5-min centrifugation at 16,000 times g at the same temperature. 80 µl of supernatant were removed, and the pellet was resuspended in 80 µl of assay buffer. Both pellet and supernatant were assayed in Triton X-100:Triton X-114 micelles containing 0.5 mol % dioleoyl-[P]PA and 10 mol % AO-PA (16 mM total micellar lipid). In separate experiments, the increase in PA-PLA(1) (0.6 ng) activity in response to increasing AO-PA in Triton X-100:Triton X-114 micelles containing 0.5 mol % dioleoyl-[P]PA was tested. Similar results were obtained in two separate experiments conducted in duplicate.



The effect of calcium ions on both PA-PLA(1) activity and surface binding in response to activator further suggested that the activator functions by enhancing the interaction of PA-PLA(1) with the surface. With no AO-PA in the assay, CaCl(2) had a slightly stimulatory effect on PA-PLA(1) activity (Fig. 5; see legend). In the presence of 10 mol % AO-PA, however, CaCl(2) inhibited activity, with an IC50 of 0.6 mM. This inhibition was paralleled by a decrease in PA-PLA(1) binding to Triton X-114 micelles (Fig. 5). These results suggest that calcium ions compete with the enzyme for binding to the activator, causing less surface binding and, as a consequence, less PA-PLA(1) activity. Magnesium ions also stimulated the enzyme in the absence of activator (Fig. 5; see legend) but inhibited when 10 mol % AO-PA was included (Fig. 5). However, activator-containing micelles precipitated in the presence of magnesium ions, making interpretation of this ion's effect difficult.


Figure 5: Effect of divalent cations on PA-PLA(1) activity/binding. Both binding (6 ng enzyme) and activity (0.6 ng enzyme) measurements used micelles of 16 mM total micellar lipid containing 10 mol % AO-PA and 0.5 mol % dioleoyl PA. Binding measurements were conducted in Triton X-114, whereas activity measurements were conducted in 1:1 (mol:mol) Triton X-100:Triton X-114. Free calcium ion concentrations were varied in the presence of 2 mM nitrilotriacetic acid, using established binding constants(32) . The binding or activity in the absence of CaCl(2) was set at 100%. In the absence of AO-PA, PA-PLA(1) activity in the presence of calcium was as follows: 130% at 0.1 mM, 150% at 0.5 mM, and 206% at 2 mM. For the MgCl(2) study, conditions were the same as above. Precipitates were observed at all concentrations of magnesium but grew more pronounced at higher concentrations. In the absence of AO-PA, PA-PLA(1) activity in the presence of magnesium was 108% at 0.5 mM, 121% at 2 mM, 148% at 5 mM, and 177% at 10 mM. Similar results were obtained in two separate experiments conducted in duplicate.



The importance of surface binding to PA-PLA(1) activity was further tested by varying the concentration of micelles in the assay. If the sole effect of AO-PA were to increase the fraction of enzyme bound to micelles, then in the absence of AO-PA the activity of PA-PLA(1) should increase as the total concentration of micelles increases, but in the presence of AO-PA the enzyme's activity should be fairly independent of this parameter. However, we found that the enzyme's activity was independent of surface concentration under both experimental conditions when the concentration of total micellar lipid exceeded 12 mM (Fig. 6). These results seem to contradict the micelle binding experiments, which were conducted at 16 mM total micellar lipid. A possible explanation is that the enzyme can bind to surfaces with different degrees of affinity (see ``Discussion'').


Figure 6: Surface dilution properties of unactivated and fully activated PA-PLA(1). Micelles of 1:1 (mol:mol) Triton X-100:Triton X-114 containing either 0.5 mol % dioleoyl-[P]PA or 0.5 mol % dioleoyl-[P]PA, 10 mol % AO-PA were assayed for PA-PLA(1) (0.6 ng) activity at various total micelle concentrations for 20 min at 25 °C. Similar results were obtained in two separate experiments conducted in duplicate.



Headgroup Preference of PA-PLA(1) in the Presence of Activator

The potent activating effects of both hydrolyzable and nonhydrolyzable PA on PA-PLA(1) brought into question the earlier observations (9) of the enzyme's substrate preference for PA. If nonhydrolyzable PA were substituted for hydrolyzable PA as the activator, might the enzyme's preference for PA over other phospholipids be negated? To address this question, we re-examined the headgroup preference of PA-PLA(1) in the presence of maximally activating concentrations of AO-PA. The enzyme showed barely detectable activity toward dioleoyl molecular species of PI, PE, PC, or PS in the absence of activator lipid (not shown), but when 10 mol % AO-PA was added, activity toward PI, PE, and PC was measurable and hyperbolic (Fig. 7). Nevertheless, these substrates were 6.1-, 7.5-, and 15-fold less well utilized, respectively, than was dioleoyl-PA, in terms of k/K(m) (Table 2). PS was an even poorer substrate under these conditions (Fig. 7), although kinetic parameters could not be calculated because of the slight sigmoidal nature of the curve (Hill coefficient 1.4). Thus, even when fully activated, PA-PLA(1) preferred PA as a substrate to other phospholipids.


Figure 7: Headgroup preference of PA-PLA(1). Dioleoyl molecular species of phospholipids were compared as substrates for PA-PLA(1) (0.6 ng) in the presence of 10 mol % AO-PA in micelles of 1:1 (mol:mol) Triton X-100:Triton X-114 (16 mM total micellar lipid). Closed circles, PA; open circles, PI; closed squares, PE; open squares, PC; closed triangles, PS. Similar results were obtained in two separate experiments conducted in duplicate.



Molecular Species Preference of PA-PLA(1) in the Presence of Activator

The partially purified enzyme displayed a preference for diunsaturated PAs over those containing an sn-1-saturated fatty acyl group(9) . This preference still existed in the presence of maximally stimulating activator, as the enzyme hydrolyzed molecular species of PA in the following order: diarachidonoyl, dilinolenoyl, dilinoleoyl > dioleoyl > sn-1-stearoyl-2-arachidonoyl, sn-1-palmitoyl-2-oleoyl, in terms of k/K(m) (Fig. 8A, Table 2). To test the possibility that the enzyme might have been sensitive to the character of the double bonds in the diunsaturated substrates, we assayed the enzyme's activity toward two unnatural isomers of dioleoyl PA: dielaidoyl-PA (di18:1-Delta9trans) and dipetroselinoyl-PA (di18:1-Delta6cis). In the presence of 10 mol % AO-PA, both substrates were significantly less well hydrolyzed than was dioleoyl PA (Fig. 8B, Table 2).


Figure 8: Acyl chain preference of PA-PLA(1). A, dioleoyl PA (closed circles) is compared with diarachidonoyl PA (closed squares), dilinolenoyl PA (open squares), dilinoleoyl PA (crosses), stearoyl-arachidonoyl PA (closed triangles), and palmitoyl-oleoyl PA (open triangles) in micelles of 1:1 (mol:mol) Triton X-100:Triton X-114 containing 10 mol % AO-PA (16 mM total micellar lipid) as substrates for PA-PLA(1). B, dioleoyl PA (closed circles) is compared with dielaidoyl PA (closed squares) and dipetroselinoyl PA (open squares) as substrates for PA-PLA(1) under the same conditions as in A. Similar results were obtained for each compound in two separate experiments conducted in duplicate.



Comparison of PA-PLA(1) with a Previously Identified PLA(1) Activity

A soluble PLA(1) activity was recently purified from bovine brain(7) . It had an apparent molecular mass of 365 kDa on SEC but displayed two bands, of 112 and 95 kDa, on SDS-PAGE. When assayed in a system containing 70% glycerol, submicellar concentrations of the detergent CHAPS, and the absence of NaCl or KCl, the PLA(1) displayed a confusing set of enzymatic properties(7, 12) . The enzyme hydrolyzed sn-1-palmitoyl-2-arachidonoyl PE 13-fold better than it did sn-1-palmitoyl-2-arachidonoyl PC at pH 7.5 but, when 1 mM Triton X-100 was added, activity toward PE was reduced >90%. MgCl(2) stimulated the enzyme's activity toward PE, with much greater stimulation at pH 9 than at pH 7.5. PC hydrolysis was stimulated greatly by the presence of bovine brain PS, but only at pH values above 8.0. Few insights into the enzyme's properties can be drawn from these data because of the undefined nature of the lipid particles present.

To test the possibility that the testis PA-PLA(1) might be related to the brain PLA(1), we assayed the activity of PA-PLA(1) under conditions similar to those used for the brain enzyme. Under these conditions, PA-PLA(1) behaved similarly to the brain enzyme in all properties tested (Table 3). We then asked whether PA was a preferred substrate in this system. Under the exact conditions of the brain assay system, dioleoyl PA was 8-fold less well utilized than was sn-1-palmitoyl-2-arachidonoyl PE. However, the buffer in this system contained a superphysiological concentration of MgCl(2), which we had previously shown to be inhibitory to AO-PA-stimulated PA hydrolysis by PA-PLA(1) in the Triton assay system (Fig. 5). When MgCl(2) was removed from the brain system, PA-PLA(1) activity toward PA increased over 8-fold, making PA a slightly better substrate than PE (Fig. 9). Thus, the catalytic properties of PA-PLA(1) in the brain PLA(1) assay system are very similar to those of the brain enzyme and are in conflict with those obtained in the Triton micelle system. However, when the brain assay system was made more similar to our system by removal of magnesium ions, PA was again found to be the preferred substrate.




Figure 9: Effect of MgCl(2) on PA-PLA(1) activity in the brain PLA(1) assay system. PA-PLA(1) (0.4 ng) was assayed in 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 70% glycerol, and 325 µM CHAPS, in the absence or presence of 3 mM MgCl(2), for the hydrolysis of 10 µM dioleoyl [P]PA, sn-1-palmitoyl-2-[^14C]arachidonoyl PE, or sn-1-palmitoyl-2-[^14C]arachidonoyl PC. For details of the assay procedure, see ``Experimental Procedures.'' Assays were conducted in duplicate on two separate occasions.




DISCUSSION

We purified bovine testis PA-PLA(1) 14,000-fold and used a Triton mixed micelle assay system to examine its properties. The purified enzyme had an apparent molecular mass of 440 kDa as determined by SEC but corresponded to a major band of 110 kDa as determined by SDS-PAGE and labeling with the phospholipase inhibitor, MAFP. This strongly suggests that PA-PLA(1) exists as a homotetramer of 110-kDa subunits in solution. However, definitive proof of the enzyme's identity will have to await cloning of the cDNA for the 110-kDa protein and evidence that the cellular expression of this cDNA is associated with a parallel increase in PA-PLA(1) activity.

Proteins of lower molecular weight (97 and sometimes 85 kDa), observed in earlier purification trials, appeared to be proteolytic products of the 110-kDa polypeptide. They were not observed in later purifications, where proteolysis was reduced by freezing fresh cubes of tissue in liquid nitrogen immediately after removing testes from bulls, rapid thawing of the tissue cubes during homogenization, and adding an extensive protease inhibitor mixture to all purification buffers. Furthermore, when an earlier enzyme preparation containing the 110-, 97-, and 85-kDa bands was treated with radiolabeled MAFP and analyzed by SDS-PAGE, all three bands incorporated the label. (^3)

Pete et al. recently purified a soluble PLA(1) from bovine brain that displayed bands of 112 and 95 kDa on SDS-PAGE (7) . Though further studies were not done to determine whether either of these polypeptides corresponded to the enzyme, analysis of the native protein by SEC revealed active material of 365 kDa, suggesting that this protein might also be a tetramer. The protease inhibitors included in buffers during this purification were similar to those we used when three bands appeared on SDS-PAGE, suggesting that the 95-kDa band might have been a proteolytic product of the 112-kDa band. A phospholipase A(2) from macrophages also appeared to be a homotetramer of 80-kDa subunits in solution(25, 26) . These similarities are intriguing, but their functional significance is unknown.

It would be interesting to know whether the tetrameric nature of these enzymes provides a means for their regulation. We observed sigmoidal kinetics in our enzyme assay experiments with mixed micelles of Triton containing either increasing concentrations of PA substrates or a fixed concentration of PA substrate and increasing concentrations of AO-PA (Fig. 3, A and B). In addition, our studies of PA-PLA(1) binding to mixed micelles of Triton X-114 revealed an activator-dependent, sigmoidal increase in surface binding that paralleled the activator-dependent increase in enzyme activity (Fig. 4). These sigmoidal effects may reflect cooperative interactions between the subunits of the tetramer, but could also be due to interactions involving multiple sites within subunits. A number of monomeric proteins display sigmoidal binding to lipid surfaces. For example, small basic peptides, protein kinase C, and annexins bind with apparent cooperativity to negatively charged surfaces(27, 28, 29) . Therefore, mechanisms other than those involving the tetrameric state of PA-PLA(1) could be responsible for the observed sigmoidal effects.

The parallel sigmoidal effects of AO-PA on the enzyme's binding and activity do, however, suggest that an increase in some type of binding may be responsible for enzyme activation. In our current working model, each enzyme subunit contains two different types of binding site, a single catalytic site that preferentially binds diacyl PA and at least one activator-binding site that binds diacyl PA, AO-PA, or another anionic lipid. In support of this model, AO-PA activated the enzyme without noticeably inhibiting the hydrolysis of diacyl PA (Fig. 3, A and B). In addition, AO-PS effectively activated the enzyme even though diacyl PS was a very poor substrate (Fig. 3B and Fig. 7). Furthermore, the fact that AO-PA increased the enzyme's ability to hydrolyze PE and PC, both poor substrates compared with diacyl PA, suggests that the enzyme first binds to AO-PA via noncatalytic sites and then interacts with diacyl phosphoglyceride substrates via its catalytic site.

The model might also explain the apparently contradictory results of our surface dilution studies, which revealed that the enzyme's activity was not affected by changes in micelle concentration in excess of 12 mM, even in the absence of AO-PA (Fig. 6). This suggested that all of the enzyme was bound to the surface even in the absence of the activator. However, several states of enzyme activity and binding might exist if the PA-PLA(1) contains multiple activator-binding sites and if the enzyme's activity and strength of binding to micelles increase in parallel with increasing occupancy of these sites, as illustrated in .

where E is unbound enzyme, M is micelle surface, E-M is the enzyme-micelle complex, PA^1, PA^2, and PA^n are sequentially binding activators, and E*^1-M, E*^2-M, and E*^n-M are the increasing affinities of E-M binding due to increasing activator concentration. Following this model, the enzyme might bind weakly to the micelle surface in the activator's absence and dissociate during the centrifugation step of the Triton X-114 binding assay. Conversely, in the presence of activator, PA-PLA(1) might bind more strongly to the surface and pellet with the micelles upon centrifugation. Stronger binding might also bring all four catalytic sites into proximity with substrates, leading to maximal enzyme activity.

The AO-PA-activated enzyme catalyzed the hydrolysis of diacyl PA at a much greater rate than it did the other diacyl phosphoglycerides tested. This strongly suggests that the catalytic site preferentially interacts with the phosphate headgroup over those containing phosphodiesters. In addition, the fully activated enzyme catalyzed the hydrolysis of diunsaturated molecular species of diacyl PA at greater rates than it did sn-1-saturated-2-unsaturated molecular species. The molecular basis of this preference has yet to be determined, but both dielaidoyl (di18:1Delta9trans) and dipetroselinoyl PA (di18:1Delta6cis) were appreciably poorer substrates than was dioleoyl-PA (di18:1Delta9cis). The former two species would be predicted to be more ordered than dioleoyl PA, based on comparisons of the phase transition temperatures of the corresponding molecular species of PC and PE(30, 31) . Moreover, diunsaturated molecular species of PC and PE species are less ordered than sn-1-saturated-2-unsaturated species, based on similar comparisons. The degree of substrate acyl chain order could conceivably affect the enzyme's ability to bind substrate or transfer substrate from the surface of a micelle into its substrate-binding pocket.

Whereas our experiments with mixed micelles seemed to yield important new information about the enzyme's properties, our experiments with the assay system used in the study of a brain PLA(1) yielded very different results, as PA-PLA(1) mimicked the brain enzyme in a number of characteristics (Table 3). These results raise issues concerning both the enzyme's identity and its properties. First, the similarity of enzymatic properties between the two enzymes, coupled with similarities in molecular mass (see ``Results''), suggests that they may be closely related. Studies of the molecular biology of bovine testis PA-PLA(1) are in progress in our laboratory and may help to clarify this point.

The second issue concerns the validity of the enzymatic data collected with these different assay systems. Whereas our studies with Triton micelle assay systems identified PA as the major substrate, PE was a better substrate in the brain enzyme assay system. One reason for this was that the superphysiological levels of MgCl(2) present in the buffer specifically inhibited PA hydrolysis (Fig. 9). When the brain PLA(1) assay system was modified by removing magnesium ions, PA was slightly preferred over PE as a substrate. The reason why the enzyme showed a higher relative activity toward PE in this assay than it did in the Triton micelle assay remains to be explained. However, PE is known to have special physical properties. For example, liposomes can be made from pure suspensions of PC but not from pure suspensions of PE. Furthermore, some molecular species of PE readily form nonbilayer structures and adopt inverted micellar conformations(13, 31) . Such conformations cause a greater exposure of acyl chains than do continuous bilayer structures, and the low dielectric constant of the brain PLA(1) assay buffer (70% glycerol, no NaCl or KCl) might further favor acyl chain exposure. PA-PLA(1) might interact well with the exposed acyl chains of PE because it binds strongly to hydrophobic chromatographic resins. In contrast, PE acyl chains are probably not significantly exposed in the Triton micelle assay system. Thus, the difference in relative activity toward PE in the two assay systems might be due to the fundamentally different conformations adopted by this lipid in the respective systems.

What is needed at this point is an assay system that presents substrates in well defined lipid bilayers. Such a system could be used to investigate in more detail the properties elucidated in this study as well as to reveal other as yet undetected properties. Experiments with unilamellar liposomes are planned and would seem a logical next step in our efforts to learn about how testis PA-PLA(1) functions in intact cells.


FOOTNOTES

*
This work was supported by the Howard Hughes Medical Institute and by National Institutes of Health Grant RR00166 to the Regional Primate Research Center at the University of Washington. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Howard Hughes Medical Institute, Box 357370, University of Washington, Seattle, WA 98195-7370. Tel: 206-685-2503; Fax: 206-543-0858.

(^1)
The abbreviations used are: PLA(1), phospholipase A(1); PA, phosphatidic acid; PA-PLA(1), PA-preferring PLA(1); AO-PA, sn-1-alkyl-2-oleoyl phosphatidic acid; CDP-DAG, cytidine 5`-diphospho-sn-glycerol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DAG, diacylglycerol; DTT, dithiothreitol; K(m), apparent K; MAFP, methyl arachidonyl fluorophosphonate; MeOH, methanol; MOPS, 3-(N-morpholino)propanesulfonic acid; PB, purification buffer; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEG, polyethylene glycol 3350; PI, phosphatidylinositol; PMSF, phenylmethylsulfonyl fluoride; PS, phosphatidylserine; SEC, size exclusion chromatography; PAGE, polyacrylamide gel electrophoresis; Thesit, dodecylpoly(ethyleneglycolether) where the average n is 9.

(^2)
Z. Huang, personal communication.

(^3)
H. N. Higgs and J. A. Glomset, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Guy Johnson for conducting some of the MAFP inhibition tests and to Wendy Thomas, Dr. Michael Gelb, and Dr. David Teller for helpful comments.


REFERENCES

  1. Inoue, M., Okuyama, H. (1984) J. Biol. Chem. 259, 5083-5086 [Abstract/Free Full Text]
  2. Waite, M., Rao, R. H., Griffin, H., Franson, R., Miller, C., Sisson, P., and Frye, J. (1981) Methods Enzymol. 71, 674-689 [Medline] [Order article via Infotrieve]
  3. Badiani, K., Xiaoli, L., and Arthur, G. (1992) Biochem. J. 288, 965-968 [Medline] [Order article via Infotrieve]
  4. Hostetler, K. Y., Gardner, M. F., and Giordano, J. R. (1986) Biochemistry 25, 6456-6461 [Medline] [Order article via Infotrieve]
  5. Huterer, S. J., Hostetler, K. Y., Gardner, M. F., and Wherrett, J. R. (1993) Biochim. Biophys. Acta 1167, 204-210 [Medline] [Order article via Infotrieve]
  6. Nalbone, G., and Hostetler, K. Y. (1985) J. Lipid Res. 26, 104-114 [Abstract]
  7. Pete, M. J., Ross, A. H., and Exton, J. H. (1994) J. Biol. Chem. 269, 19494-19500 [Abstract/Free Full Text]
  8. Ueda, H., Kobayashi, T., Kishimoto, M., Tsutsumi, T., Watanabe, S., and Okuyama, H. (1993) Biochem. Biophys. Res. Commun. 195, 1272-1279 [CrossRef][Medline] [Order article via Infotrieve]
  9. Higgs, H. N., and Glomset, J. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9574-9578 [Abstract/Free Full Text]
  10. Exton, J. H. (1994) Biochim. Biophys. Acta 1212, 26-42 [Medline] [Order article via Infotrieve]
  11. Ueda, H., Kobayashi, T., Kishimoto, M., Tsutsumi, T., and Okuyama, H. (1993) J. Neurochem. 61, 1874-1881 [Medline] [Order article via Infotrieve]
  12. Pete, M. J., and Exton, J. H. (1995) Biochim. Biophys. Acta 1256, 367-373 [Medline] [Order article via Infotrieve]
  13. Tate, M. W., Eikenberry, E. F., Turner, D. C., Shyamsunder, E., and Gruner, S. M. (1991) Chem. Phys. Lipids 57, 147-164 [CrossRef][Medline] [Order article via Infotrieve]
  14. Carman, G. M., Deems, R. A., and Dennis, E. A. (1995) J. Biol. Chem. 270, 18711-18714 [Free Full Text]
  15. Folch, J., Lees, M., and Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226, 497-501 [Free Full Text]
  16. Carman, G. M., and Fischl, A. S. (1980) J. Food Biochem. 4, 53-59
  17. Jain, M. K., and Gelb, M. H. (1991) Methods Enzymol. 197, 112-125 [Medline] [Order article via Infotrieve]
  18. Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607 [Abstract/Free Full Text]
  19. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. O., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85 [Medline] [Order article via Infotrieve]
  20. Brown, R. E., Jarvis, K. L., and Hyland, K. J. (1989) Anal. Biochem. 180, 136-139 [Medline] [Order article via Infotrieve]
  21. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  22. Morissey, J. H. (1981) Anal. Biochem. 117, 307-310 [Medline] [Order article via Infotrieve]
  23. van Veldhoven, P. P., and Mannaerts, G. P. (1987) Anal. Biochem. 161, 45-48 [Medline] [Order article via Infotrieve]
  24. Hattori, M., Arai, H., and Inoue, K. (1993) J. Biol. Chem. 268, 18748-18753 [Abstract/Free Full Text]
  25. Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233 [Abstract/Free Full Text]
  26. Ackerman, E. J., and Dennis, E. A. (1995) Biochim. Biophys. Acta 1259, 125-136 [Medline] [Order article via Infotrieve]
  27. Mosior, M., and McLaughlin, S. (1992) Biochemistry 31, 1767-1773 [Medline] [Order article via Infotrieve]
  28. Bazzi, M. D., and Nelsestuen, G. L. (1993) Cell Signalling 5, 357-365 [CrossRef][Medline] [Order article via Infotrieve]
  29. Orr, J. W., and Newton, A. C. (1992) Biochemistry 31, 4661-4667 [Medline] [Order article via Infotrieve]
  30. Marsh, D. (1990) CRC Handbook of Lipid Bilayers , CRC Press, Inc., Boston
  31. Koynova, R., and Caffrey, M. (1994) Chem. Phys. Lipids 69, 1-34 [CrossRef][Medline] [Order article via Infotrieve]
  32. Martell, A. E., and Smith, R. M. (1974) Critical Stability Constants , Vol. 1, p. 139, Plenum Publishing Corp., New York

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.