Biochimica et Biophysica Acta, 491 (1977) 265-274 © Elsevier/North-Holland Biomedical Press BBA 37609
ISOLATION AND PROPERTIES OF PROPHOSPHOLIPASE A2 AND PHOSPHOLIPASE A2 FROM HORSE PANCREAS AND HORSE PANCREATIC JUICE
A D O L F EVENBERG, H E N D R I K MEYER, H U B E R T U S M. VERHEIJ and G E R A R D H. DE HAAS
Biochemical Laboratory of the State University of Utrecht, University Centre De Uithof, Utrecht (The Netherlands) (Received September 10th, 1976)
SUMMARY
Two phospholipases A 2 ( E C 3.1.1.4) with different isoelectric points have been isolated from horse pancreas in high yield (880 mg/kg tissue). From pancreatic juice the more acidic species was isolated as the sole phospholipase A 2. Upon tryptic activation the zymogens release a hepta- and pentapeptide, respectively from the N-terminal part of the protein giving rise to the formation of one single enzyme with a specific activity higher than that of pancreatic phospholipases A 2 from other mammalian species. Horse phospholipase A 2 differs from the porcine and bovine enzymes with respect to amino acid composition and kinetic properties. The sequence of the first 41 amino acid residues at the N-terminus has been determined by automatic Edman degradation.
INTRODUCTION
From mammalian sources phospholipase A 2 has been isolated in pure form from porcine [1], rat [2], human [3], ox [4] and sheep [4] pancreas. In the pancreas, however, this enzyme is present mainly as an inactive precursor, prophospholipase A2, a single-chain zymogen which is converted by trypsin into the active enzyme. The activation mechanism [5] and amino acid sequence of the porcine enzyme [6, 7] is known and extensive studies on the mechanism of action of this enzyme have been reported [8-11]. Recently the X-ray structure of prophospholipase A2 from pig pancreas has been determined [30]. However, the three-dimensional structure of the active enzyme could not be determined since suitable crystals can not be obtained. The N-terminal region of phospholipase A2 plays a role in the interaction of the enzyme with interfaces [11] and it is therefore of interest to compare N-termini of isoenzymes from related sources. This paper describes a high yield isolation and partial characterization of a phospholipase A2 with high specific activity from horse pancreas. Since horse pancreas
266 is an unusually rich source, crystallisation experiments for further X-ray studies on active enzyme are facilitated. MATERIALS AND METHODS Materials Pancreas was obtained from a nearby slaughterhouse. The material was used as soon as possible after the death of the animal. Connective tissue and fat were carefully removed. Horse pancreatic juice was obtained from a canulated horse over a period of 1 month. The juice was acidified immediately after collection to pH 4.5 with 40 ~ acetic acid. All reagents used, were of reagent-grade purity. Dialyses were carried out in Nojax-28 dialysing tube (Sophyc, Levallois, France). DEAE-cellulose (DE-52) and CM-cellulose (CM-52) were purchased from Whatman and treated according to the manufacturers instructions. Sephadex (G-25, fine, Pharmacia, Uppsala) was prepared for use according to the instructions of the manufacturer. Diisopropylfluorophosphate was purchased from Mann Research Laboratories, N.Y. Phenylisothiocyanate was purchased from Eastman Kodak. Dansyl-chloride, dansylatedamino acids and phenylthiohydantoines of amino acids were products of Sigma, St. Louis, Mo., U.S.A. Silicagel 60 F 254 was obtained from E. Merck, Darmstadt, G.F.R.; polyamide thin-layer plates were obtained from Schleicher and Schfill (Dassel), G.F.R. The following proteolytic enzymes were used: trypsin (Serva) 1180 units/mg 2 × crystallized; Carboxypeptidase A (Sigma) 50 mg/ml. All the materials used in the Beckmann-sequencer were from Beckmann and sequenal grade. Synthetic phospholipids were prepared as described previously [12]. Methods Phospholipase Az activity was routinely assayed using the egg-yolk system [1 ] with some minor modifications: one egg-yolk was suspended in 12 ml of a CaC12 solution (CaC12"2HzO 33.4 g/l) and 100 ml of water. The final test was carried out using 10 ml of this emulsion and 20 ml of a sodium deoxycholate solution (2.56 g/l). Released fatty acids were automatically titrated at pH 8.0 and 40 °C. Activity is expressed as the uptake of alkali in #equiv./min. Kinetic properties of the enzyme were determined using various concentrations of 1,2 dioctanoyl-sn-glycero-3-phosphocholine in a solution containing CaC12 (10 mM) and sodium chloride (100 mM); the final volume was 2 ml. Titration was carried out under a nitrogen atmosphere with 4 mM sodium hydroxyde solution [8]. Starch-gel electrophoresis was carried out on microscopic slides covered with 12 ~ starch in 0.05 M univalent buffer solution of pH 4--8. Samples were subjected to electrophoresis for 2-4 h at 5 V/cm. Disc electrophoresis was carried out on polyacrylamide gel (pH 4-9) using a Shandon disc-electrophoresis apparatus. Gels with 7.5 ~ acrylamide were used. Solutions were prepared according to the Shandon operating instructions. Protein staining was done with a 0.2 ~ solution of Amido Black in 7 ~ acetic acid. High-voltage paper electrophoresis was performed in a Gilson apparatus, using pyridine/acetic acid buffer solutions pH 3.6 and pH 6.5. Peptides were visualised by the ninhydrin-collidine staining [13], with the arginine reagent [14], and the chlorine - KI starch procedure [15].
267 Amino acid analyses were performed by the method of Spackman et al. [16] using a Beckman Unichrom apparatus. Small amount of amino acids were analysed using a Technicon TSM amino acid analyser. Proteins and peptides were hydrolysed during 24, 48 and 72 h at 110 °C in evacuated sealed tubes containing 6 N HC1. Tryptophan was determined by hydrolysis in the presence of thioglycolic acid, as described by Matsubara and Sasaki [17]. Hexoses and pentoses were determined by the phenol-sulphuric acid method of Dubois et al. [18]. Hexosamines were determined according to Marshall and Gottschalk [19]. Positional and stereospecificity of the enzyme was determined using synthetic rac-l-palmitoyl-2-oleoyl-glycero-3-phosphocholine. Details have been published earlier [4]. The N-terminal amino acid was determined by the dansylation procedure according to Gray [20] and the Edman degradation as modified by Tarr [21]. Carboxypeptidase A digestion at pH 8.0 and 40 °C was done on a S-methylcysteinyl phospholipase Az, prepared by reaction of reduced protein with methyl p-nitrobenzene sulfonate [22]. The SH content of the enzyme was measured according to Ellman's procedure [23]. Automatic Edman degradation was carried out in a Beckman 890 C Sequencer using the standard fast protein program with 0.4/~mol of native phospholipase A2 as well as on 0.4 #mol of a cyanogen bromide fragment which differs from the enzyme by the lack of 8 N-terminal residues. Released thiazolinon derivatives were converted into the phenylthiohydantoin derivatives by heating for 10 min in 1 N HC1 at 80 °C. Phenylthiohydantoin amino acids were analyzed by thin-layer chromatography in the system chloroform/methanol (90:10, v/v). Spots were detected under ultraviolet light (254 nm) and by spraying with 0.1% (w/v) ninhydrin in 96 % ethanol containing 5 ~ (v/v) collidine, followed by heating of the plate at 110 °C for 5 rain. The derivatives of apolar amino acids were also analyzed by gas-liquid chromatography on 10 % SP 400 under conditions as described in the Beckman Sequencer Manual. Finally isoleucine and leucine were determined by amino acid analysis after hydrolysis of the phenylthiohydantoin derivatives according to Smithies et al. [24]. RESULTS
Purification of phospholipase A2 from pancreatic tissue Homogenisation and heat treatment: Defatted tissue was minced through a meat mincer. 1 Kg of this pulp was homogenised in 3 litres 0.15 M NaC1 solution with a high speed mixer. After acidification (12 N HCI) to pH 4 the homogenate was heated at 70 °C for 3 min and, after cooling to 4 °C, it was centrifuged (3000 × g for 5 min). The supernatant was freed from fat by filtration through filter paper. Ammonium sulphate fractionation: Filtered supernatant of the heat treated material was brought to pH 5.0 with conc. ammonia and solid (NH4)zSO4 was added at 4 °C to obtain 0.40 S. After 2-3 h the precipitate was removed by centrifugation at 4 °C and 3000 × g for 15 min. To the supernatant conc. ammonia was added to raise the pH to 5.8. A few drops ofn octanol were added as antifoaming agent, followed by addition of solid (NH4)2504, under efficient stirring, to reach a final concen-
268
I I I
I I I
I r I
6O
I I
--
50,~ 3O ~
~"
°"°t
ELUTION VOLUME
Fig. 1. Elution pattern of horse prophospholipase As from a DEAE-cellulose column at pH 6.4. - - - - , absorbance at 280 nm; . . . . . . , direct phospholipase A2 activity; - - - , potential phospholipase As activity determined after maximal activation with trypsin. The peaks with potential phospholipase A2 activity are designated precursor I and 2. Precursor l elutes before precursor 2. For experimental details see text. tration of 0.75 S. The solution was allowed to stand over night at 4 °C. The crude prophospholipase A2 precipitate was collected by centrifugation at 4 °C for 15 min at 5000 × g. The pellet was dissolved in a minimal amount of distilled water, brought to p H 7.0 and treated for 30 rain. at 4 °C with diisopropylfluorophosphate (5 ~ v/v of a 0.1 M solution in anhydrous isopropanoi). The solution was then exhaustively dialysed against 1 ~ acetic acid. A small amount of precipitate, which usually formed during dialysis, was removed by centrifugation for 10 rain at 3000 × g and the supernatant was lyophilized.
Ion exchange chromatography For separation on ion exchange columns 100 ml of ion exchange material was used for each gram of protein to be purified. Using D E A E cellulose the column was run in 5 mM sodium acetate p H 6.4 using a linear salt gradient from 0 to 0.1 m NaCI to elute the desired protein. A typical elution pattern is shown in Fig. 1. The peaks containing potential phospholipase A2 activity were purified to homogeneity on CM-cellulose equilibrated with 5 m M sodium acetate p H 4.8 using a linear salt gradient from 0 to 0.25 M NaC1. Starch gel electrophoresis as well as electrophoresis on polyacrylamide disc gel at p H 8.5 revealed for both zymogens only one band. Table I summarizes yields and specific activities (after maximal activation) of the prophospholipase Az at various stages of the purification.
Purification of prophospholipase Az from pancreaticjuice Just after collection the juice was acidified (pH 4.5) with 40 ~ acetic acid in order to avoid conversion of zymogen into active enzyme. The first purification step is a heat treatment: the juice was brought to pH 4.0 with conc. HCI and heated at 70 °C for 3 min, cooled and centrifuged as described before. The supernatant was lyophilized and a m m o n i u m sulfate fractionation was carried out on a 5 times concentrated solution as described for the material obtained from pancreatic tissue. Also the ion exchange chromatographic steps were essentially the same as described in the foregoing section. A precursor with chromatographic properties identical to those of peak 2 (Fig. 1) and the same specific activity was obtained in 81 ~ yield, thus 46 mg pure protein were obtained per liter. Precursor 1 is virtually absent in this purification.
269 TABLE 1 PURIFICATION OF PROSPHOSPHOLIPASE Az 1000 g of defatted pancreatic tissue were homogenised at 4 °C in 3000 ml of 0.15 M NaC1 solution and purification was carried out as described in the text. The figures in the table are mean values of four purification experiments. Step
Activity (/~equiv.min-1)
Activity after activation with trypsin (,uequiv. rain -1)
Specificactivity Yield (ftequiv. min-~.mg-1) (%)
1 Crude homogenate 2 Heat treatment and filtration 3 (NH4)SO4 fractionation and dialysis 4 DEAE-cellulose chromatography pH 6.4 5 CM-cellulose chromatography pH 4.8
3- 104
--
--
2- 104
2.3.106
58
100
4" 104
2.15. II36
800
94
5' 104
2.05. 106*
1950
89
4' 103
1.95. 106*
2200
85
--
Pure zymogen (precursor 1 and 2) isolated (g/kg): 0.88. * These numbers are the sum of precursors 1 and 2.
Activation of the prophospholipase by trypsin; isolation of phospholipase Az Zymogens (10 mg/ml) were converted into active enzyme with the aid o f trypsin (20 #g/ml) in a 1 m M CaCI2 solution at p H 8. The solution was kept in ice and the course o f the activation waas followed with the egg-yolk test. W h e n maximal activity was reached the solution was made 10 -3 M in diisopropylfluorophosphate. Final purification o f phospholipase A2 was achieved by ion-exchange chromatography on CM-cellulose (precurser 2) or DEAE-cellulose (precursor 1) under the conditions that have been described for the isolation o f the precursors. The enzyme was obtained in 90-95 % yield, had specific activity o f 2200 and showed one band on starch gel or disc-gel electrophoresis at various p H values. Enzyme obtained from precursor 1 or 2 (from tissue or juice) could not be distinguished by: amino acid composition, electrophoretic behaviour on starch or polyacrylamide gel (pH 4 to 8.5), sugar content, the identity o f the first 2 N-terminal amino acids or specific activity. Finally a 1 : 1 mixture o f phospholipase Az obtained from precursors 1 and 2 yielded only a single peak on C M - or DEAE-cellulose columns. Therefore it is concluded that both fractions are identical and hence only one phospholipase Az can be isolated from horse pancreas.
Properties of the isolated zymogens and phospholipase Az Carbohydrate content. Determination o f the carbohydrate content of the horse (pro) phospholipase showed that the proteins contained only trace amounts o f carbohydrates (less than 7 mol %). Like pig, ox and sheep phospholipase [1, 4] these enzymes are not glycoproteins. Molar absorbance. The molar absorption coefficient o f zymogens and phospholipase Az was determined by measuring the absorbance at 280 n m of solutions of k n o w n concentrations o f protein (biuret method using phospholipase A2 from porcine
270 pancreas as a standard). The values of E~°/% obtained for the precursors and the enzyme were 11.8 and 12.3, respectively. Determination of free SH groups. No free SH groups could be detected under denaturing conditions (0.2 ~ sodium dodecyl sulfate or 8 M urea). Carboxypeptidase digestion. Digestion of S-methylcysteinyl protein with carboxypeptidase A released S-methylcysteine and alanine in that order, lysine was liberated much more slowly. Therefore we conclude that the C-terminus of the protein is: -Lys.Ala.1/2 Cys and since no free SH groups are present this half-cystine must be present in a disulfide bridge. lsoelectric points of zymogens and phospholipase A2. The isoelectric points of the horse pancreatic prophospholipases and the active enzyme were determined by isoelectric focussing in polyacrylamide gels [25]. The values obtained were: 5.5 (precursor 1), 5.4 (precursor 2) and 5.5 (phospholipase A2). Positional specificity. Using rac-l-palmitoyl-2-oleoylglycero-3-phosphocholine as a substrate, horse phospholipase released exclusively oleic acid, whereas palmitic acid was the sole fatty acid in the lysophosphatidylcholine. In agreement with the stereospecific properties of phospholipase A2 the enzyme hydrolyzed the racemic substrate for only 50 ~ . Kinetic properties. Using 1,2 dioctanoyl-sn-glycero-3-phosphocholine Km (app.) and V values were determined at pH 6 and 50 °C. This temperature was chosen since no straight lines were obtained in Lineweaver-Burke plots when working with the horse phospholipase Az at a temperature below this value. Probably the phase separation which is known [26] to occur in the range of 40-45 °C yields two different interfaces to which the enzyme binds not equally well. Above the phase separation straight Lineweaver-Burke plots were obtained. For reasons of comparison the enzymes from cow and pig pancreas were also tested at 50 °C. Measurements were carried out at a CaC12 concentration of 10 mM which value is at least 5 times the Kca values of these enzymes. The V values obtained were: 12 000 (horse), 4000 (pig) and 2500 (cow)/~equiv. min- ~. mg- 1, whereas the apparent Km values were 5 mM (horse), 1 mM (pig) and 1 mM (cow). Amino acid composition. The amino acid composition of horse pancreatic phospholipase A2 is given in Table II. Values are means of 6 hydrolysates; the values of Thr and Ser are extrapolated to zero time. For reasons of comparison the values for pig and ox phospholipase Az are included. The amino acid compositions of the zymogens (not shown in the table) suggest that the two zymogens and phospholipase A2 differ only by a small activation peptide. These differences were confirmed by amino acid analysis of the activation peptides (see also Table III).
Isolation and sequence of the activation peptides Activation of the precursors was carried out as usual. After maximal activity had been reached the pH was brought to 3 with 0.1 M HC1 and the mixture was applied to a Sephadex-G25 (fine) column (1.4 × 200 cm). Elution was performed with 10 -3 M HC1. Absorbance was monitored at 280 nm and 206 nm. After the appearence of the active enzyme which eluted in the void volume, only one main peak was registered, absorbing exclusively at 206 rim. The fractions corresponding with the peaks were pooled and lyophilized. Purity of the peptides was checked by high-voltage electrophoresis at pH 3.5. It appeared that both activation peptides gave a positive
271 TABLE II AMINO ACID COMPOSITION OF PHOSPHOLIPASE Az ISOLATED FROM PANCREATIC TISSUE OF HORSE, PIG [6] AND OX [4] Amino acid
Source Horse
Asp Thr Ser Glu Pro Gly Ala Cys Val Met lie Leu Tyr Phe Trp Lys His Arg Total number of amino acids
Pig
Ox
19 6 11 11 6 6 9 14 5 1 4 6 7 6 I 8 1 4
23 6 10 7 5 6 8 12 2 2 5 7 8 5 1 9 3 4
23 4 10 9 5 7 6 12 4 1 5 7 7 4 1 11 2 2
125
123
120
nin h y d ri n staining, which distinguishes these peptides f r o m the activation peptides f r o m pig, ox an d sheep z y m o g e n s since the latter ones co n t ai n p y r o g l u t a m i c acid as N - t e r m i n a l residue [4]. T h e a m i n o acid sequence o f the activation peptides f r o m the horse pancreatic zymogens was d e t e r m i n e d by m a n u a l E d m a n and E d m a n - d a n s y l techniques [21, 20]. Results are s u m m a r i z e d in T a b le III.
TABLE 11I THE AMINO ACID SEQUENCES OF THE ACTIVATION PEPTIDES OF PROPHOSPHOLIPASES A2 FROM VARIOUS SOURCES These peptides are released from the N terminal part of the prophospholipase Az during the activation process. Source
Sequence
Pig Ox Sheep 1 Sheep 2 Horse 1 Horse 2
< Glu-Glu-Gly-Ile- Ser- Ser- Arg < Glu-Ala- Gly-Leu-Asn-Ser- Arg < Glu-Ala- Gly-Leu-Asn-Ser- Arg Asn-Ser- Arg Gly-Iie- Ser- Pro-Arg Glu-Asn-Gly-Ile- Ser- Pro-Arg
ISOLATION AND PROPERTIES OF PROPHOSPHOLIPASE A2 AND PHOSPHOLIPASE A2 FROM HORSE PANCREAS AND HORSE PANCREATIC JUICE
A D O L F EVENBERG, H E N D R I K MEYER, H U B E R T U S M. VERHEIJ and G E R A R D H. DE HAAS
Biochemical Laboratory of the State University of Utrecht, University Centre De Uithof, Utrecht (The Netherlands) (Received September 10th, 1976)
SUMMARY
Two phospholipases A 2 ( E C 3.1.1.4) with different isoelectric points have been isolated from horse pancreas in high yield (880 mg/kg tissue). From pancreatic juice the more acidic species was isolated as the sole phospholipase A 2. Upon tryptic activation the zymogens release a hepta- and pentapeptide, respectively from the N-terminal part of the protein giving rise to the formation of one single enzyme with a specific activity higher than that of pancreatic phospholipases A 2 from other mammalian species. Horse phospholipase A 2 differs from the porcine and bovine enzymes with respect to amino acid composition and kinetic properties. The sequence of the first 41 amino acid residues at the N-terminus has been determined by automatic Edman degradation.
INTRODUCTION
From mammalian sources phospholipase A 2 has been isolated in pure form from porcine [1], rat [2], human [3], ox [4] and sheep [4] pancreas. In the pancreas, however, this enzyme is present mainly as an inactive precursor, prophospholipase A2, a single-chain zymogen which is converted by trypsin into the active enzyme. The activation mechanism [5] and amino acid sequence of the porcine enzyme [6, 7] is known and extensive studies on the mechanism of action of this enzyme have been reported [8-11]. Recently the X-ray structure of prophospholipase A2 from pig pancreas has been determined [30]. However, the three-dimensional structure of the active enzyme could not be determined since suitable crystals can not be obtained. The N-terminal region of phospholipase A2 plays a role in the interaction of the enzyme with interfaces [11] and it is therefore of interest to compare N-termini of isoenzymes from related sources. This paper describes a high yield isolation and partial characterization of a phospholipase A2 with high specific activity from horse pancreas. Since horse pancreas
266 is an unusually rich source, crystallisation experiments for further X-ray studies on active enzyme are facilitated. MATERIALS AND METHODS Materials Pancreas was obtained from a nearby slaughterhouse. The material was used as soon as possible after the death of the animal. Connective tissue and fat were carefully removed. Horse pancreatic juice was obtained from a canulated horse over a period of 1 month. The juice was acidified immediately after collection to pH 4.5 with 40 ~ acetic acid. All reagents used, were of reagent-grade purity. Dialyses were carried out in Nojax-28 dialysing tube (Sophyc, Levallois, France). DEAE-cellulose (DE-52) and CM-cellulose (CM-52) were purchased from Whatman and treated according to the manufacturers instructions. Sephadex (G-25, fine, Pharmacia, Uppsala) was prepared for use according to the instructions of the manufacturer. Diisopropylfluorophosphate was purchased from Mann Research Laboratories, N.Y. Phenylisothiocyanate was purchased from Eastman Kodak. Dansyl-chloride, dansylatedamino acids and phenylthiohydantoines of amino acids were products of Sigma, St. Louis, Mo., U.S.A. Silicagel 60 F 254 was obtained from E. Merck, Darmstadt, G.F.R.; polyamide thin-layer plates were obtained from Schleicher and Schfill (Dassel), G.F.R. The following proteolytic enzymes were used: trypsin (Serva) 1180 units/mg 2 × crystallized; Carboxypeptidase A (Sigma) 50 mg/ml. All the materials used in the Beckmann-sequencer were from Beckmann and sequenal grade. Synthetic phospholipids were prepared as described previously [12]. Methods Phospholipase Az activity was routinely assayed using the egg-yolk system [1 ] with some minor modifications: one egg-yolk was suspended in 12 ml of a CaC12 solution (CaC12"2HzO 33.4 g/l) and 100 ml of water. The final test was carried out using 10 ml of this emulsion and 20 ml of a sodium deoxycholate solution (2.56 g/l). Released fatty acids were automatically titrated at pH 8.0 and 40 °C. Activity is expressed as the uptake of alkali in #equiv./min. Kinetic properties of the enzyme were determined using various concentrations of 1,2 dioctanoyl-sn-glycero-3-phosphocholine in a solution containing CaC12 (10 mM) and sodium chloride (100 mM); the final volume was 2 ml. Titration was carried out under a nitrogen atmosphere with 4 mM sodium hydroxyde solution [8]. Starch-gel electrophoresis was carried out on microscopic slides covered with 12 ~ starch in 0.05 M univalent buffer solution of pH 4--8. Samples were subjected to electrophoresis for 2-4 h at 5 V/cm. Disc electrophoresis was carried out on polyacrylamide gel (pH 4-9) using a Shandon disc-electrophoresis apparatus. Gels with 7.5 ~ acrylamide were used. Solutions were prepared according to the Shandon operating instructions. Protein staining was done with a 0.2 ~ solution of Amido Black in 7 ~ acetic acid. High-voltage paper electrophoresis was performed in a Gilson apparatus, using pyridine/acetic acid buffer solutions pH 3.6 and pH 6.5. Peptides were visualised by the ninhydrin-collidine staining [13], with the arginine reagent [14], and the chlorine - KI starch procedure [15].
267 Amino acid analyses were performed by the method of Spackman et al. [16] using a Beckman Unichrom apparatus. Small amount of amino acids were analysed using a Technicon TSM amino acid analyser. Proteins and peptides were hydrolysed during 24, 48 and 72 h at 110 °C in evacuated sealed tubes containing 6 N HC1. Tryptophan was determined by hydrolysis in the presence of thioglycolic acid, as described by Matsubara and Sasaki [17]. Hexoses and pentoses were determined by the phenol-sulphuric acid method of Dubois et al. [18]. Hexosamines were determined according to Marshall and Gottschalk [19]. Positional and stereospecificity of the enzyme was determined using synthetic rac-l-palmitoyl-2-oleoyl-glycero-3-phosphocholine. Details have been published earlier [4]. The N-terminal amino acid was determined by the dansylation procedure according to Gray [20] and the Edman degradation as modified by Tarr [21]. Carboxypeptidase A digestion at pH 8.0 and 40 °C was done on a S-methylcysteinyl phospholipase Az, prepared by reaction of reduced protein with methyl p-nitrobenzene sulfonate [22]. The SH content of the enzyme was measured according to Ellman's procedure [23]. Automatic Edman degradation was carried out in a Beckman 890 C Sequencer using the standard fast protein program with 0.4/~mol of native phospholipase A2 as well as on 0.4 #mol of a cyanogen bromide fragment which differs from the enzyme by the lack of 8 N-terminal residues. Released thiazolinon derivatives were converted into the phenylthiohydantoin derivatives by heating for 10 min in 1 N HC1 at 80 °C. Phenylthiohydantoin amino acids were analyzed by thin-layer chromatography in the system chloroform/methanol (90:10, v/v). Spots were detected under ultraviolet light (254 nm) and by spraying with 0.1% (w/v) ninhydrin in 96 % ethanol containing 5 ~ (v/v) collidine, followed by heating of the plate at 110 °C for 5 rain. The derivatives of apolar amino acids were also analyzed by gas-liquid chromatography on 10 % SP 400 under conditions as described in the Beckman Sequencer Manual. Finally isoleucine and leucine were determined by amino acid analysis after hydrolysis of the phenylthiohydantoin derivatives according to Smithies et al. [24]. RESULTS
Purification of phospholipase A2 from pancreatic tissue Homogenisation and heat treatment: Defatted tissue was minced through a meat mincer. 1 Kg of this pulp was homogenised in 3 litres 0.15 M NaC1 solution with a high speed mixer. After acidification (12 N HCI) to pH 4 the homogenate was heated at 70 °C for 3 min and, after cooling to 4 °C, it was centrifuged (3000 × g for 5 min). The supernatant was freed from fat by filtration through filter paper. Ammonium sulphate fractionation: Filtered supernatant of the heat treated material was brought to pH 5.0 with conc. ammonia and solid (NH4)zSO4 was added at 4 °C to obtain 0.40 S. After 2-3 h the precipitate was removed by centrifugation at 4 °C and 3000 × g for 15 min. To the supernatant conc. ammonia was added to raise the pH to 5.8. A few drops ofn octanol were added as antifoaming agent, followed by addition of solid (NH4)2504, under efficient stirring, to reach a final concen-
268
I I I
I I I
I r I
6O
I I
--
50,~ 3O ~
~"
°"°t
ELUTION VOLUME
Fig. 1. Elution pattern of horse prophospholipase As from a DEAE-cellulose column at pH 6.4. - - - - , absorbance at 280 nm; . . . . . . , direct phospholipase A2 activity; - - - , potential phospholipase As activity determined after maximal activation with trypsin. The peaks with potential phospholipase A2 activity are designated precursor I and 2. Precursor l elutes before precursor 2. For experimental details see text. tration of 0.75 S. The solution was allowed to stand over night at 4 °C. The crude prophospholipase A2 precipitate was collected by centrifugation at 4 °C for 15 min at 5000 × g. The pellet was dissolved in a minimal amount of distilled water, brought to p H 7.0 and treated for 30 rain. at 4 °C with diisopropylfluorophosphate (5 ~ v/v of a 0.1 M solution in anhydrous isopropanoi). The solution was then exhaustively dialysed against 1 ~ acetic acid. A small amount of precipitate, which usually formed during dialysis, was removed by centrifugation for 10 rain at 3000 × g and the supernatant was lyophilized.
Ion exchange chromatography For separation on ion exchange columns 100 ml of ion exchange material was used for each gram of protein to be purified. Using D E A E cellulose the column was run in 5 mM sodium acetate p H 6.4 using a linear salt gradient from 0 to 0.1 m NaCI to elute the desired protein. A typical elution pattern is shown in Fig. 1. The peaks containing potential phospholipase A2 activity were purified to homogeneity on CM-cellulose equilibrated with 5 m M sodium acetate p H 4.8 using a linear salt gradient from 0 to 0.25 M NaC1. Starch gel electrophoresis as well as electrophoresis on polyacrylamide disc gel at p H 8.5 revealed for both zymogens only one band. Table I summarizes yields and specific activities (after maximal activation) of the prophospholipase Az at various stages of the purification.
Purification of prophospholipase Az from pancreaticjuice Just after collection the juice was acidified (pH 4.5) with 40 ~ acetic acid in order to avoid conversion of zymogen into active enzyme. The first purification step is a heat treatment: the juice was brought to pH 4.0 with conc. HCI and heated at 70 °C for 3 min, cooled and centrifuged as described before. The supernatant was lyophilized and a m m o n i u m sulfate fractionation was carried out on a 5 times concentrated solution as described for the material obtained from pancreatic tissue. Also the ion exchange chromatographic steps were essentially the same as described in the foregoing section. A precursor with chromatographic properties identical to those of peak 2 (Fig. 1) and the same specific activity was obtained in 81 ~ yield, thus 46 mg pure protein were obtained per liter. Precursor 1 is virtually absent in this purification.
269 TABLE 1 PURIFICATION OF PROSPHOSPHOLIPASE Az 1000 g of defatted pancreatic tissue were homogenised at 4 °C in 3000 ml of 0.15 M NaC1 solution and purification was carried out as described in the text. The figures in the table are mean values of four purification experiments. Step
Activity (/~equiv.min-1)
Activity after activation with trypsin (,uequiv. rain -1)
Specificactivity Yield (ftequiv. min-~.mg-1) (%)
1 Crude homogenate 2 Heat treatment and filtration 3 (NH4)SO4 fractionation and dialysis 4 DEAE-cellulose chromatography pH 6.4 5 CM-cellulose chromatography pH 4.8
3- 104
--
--
2- 104
2.3.106
58
100
4" 104
2.15. II36
800
94
5' 104
2.05. 106*
1950
89
4' 103
1.95. 106*
2200
85
--
Pure zymogen (precursor 1 and 2) isolated (g/kg): 0.88. * These numbers are the sum of precursors 1 and 2.
Activation of the prophospholipase by trypsin; isolation of phospholipase Az Zymogens (10 mg/ml) were converted into active enzyme with the aid o f trypsin (20 #g/ml) in a 1 m M CaCI2 solution at p H 8. The solution was kept in ice and the course o f the activation waas followed with the egg-yolk test. W h e n maximal activity was reached the solution was made 10 -3 M in diisopropylfluorophosphate. Final purification o f phospholipase A2 was achieved by ion-exchange chromatography on CM-cellulose (precurser 2) or DEAE-cellulose (precursor 1) under the conditions that have been described for the isolation o f the precursors. The enzyme was obtained in 90-95 % yield, had specific activity o f 2200 and showed one band on starch gel or disc-gel electrophoresis at various p H values. Enzyme obtained from precursor 1 or 2 (from tissue or juice) could not be distinguished by: amino acid composition, electrophoretic behaviour on starch or polyacrylamide gel (pH 4 to 8.5), sugar content, the identity o f the first 2 N-terminal amino acids or specific activity. Finally a 1 : 1 mixture o f phospholipase Az obtained from precursors 1 and 2 yielded only a single peak on C M - or DEAE-cellulose columns. Therefore it is concluded that both fractions are identical and hence only one phospholipase Az can be isolated from horse pancreas.
Properties of the isolated zymogens and phospholipase Az Carbohydrate content. Determination o f the carbohydrate content of the horse (pro) phospholipase showed that the proteins contained only trace amounts o f carbohydrates (less than 7 mol %). Like pig, ox and sheep phospholipase [1, 4] these enzymes are not glycoproteins. Molar absorbance. The molar absorption coefficient o f zymogens and phospholipase Az was determined by measuring the absorbance at 280 n m of solutions of k n o w n concentrations o f protein (biuret method using phospholipase A2 from porcine
270 pancreas as a standard). The values of E~°/% obtained for the precursors and the enzyme were 11.8 and 12.3, respectively. Determination of free SH groups. No free SH groups could be detected under denaturing conditions (0.2 ~ sodium dodecyl sulfate or 8 M urea). Carboxypeptidase digestion. Digestion of S-methylcysteinyl protein with carboxypeptidase A released S-methylcysteine and alanine in that order, lysine was liberated much more slowly. Therefore we conclude that the C-terminus of the protein is: -Lys.Ala.1/2 Cys and since no free SH groups are present this half-cystine must be present in a disulfide bridge. lsoelectric points of zymogens and phospholipase A2. The isoelectric points of the horse pancreatic prophospholipases and the active enzyme were determined by isoelectric focussing in polyacrylamide gels [25]. The values obtained were: 5.5 (precursor 1), 5.4 (precursor 2) and 5.5 (phospholipase A2). Positional specificity. Using rac-l-palmitoyl-2-oleoylglycero-3-phosphocholine as a substrate, horse phospholipase released exclusively oleic acid, whereas palmitic acid was the sole fatty acid in the lysophosphatidylcholine. In agreement with the stereospecific properties of phospholipase A2 the enzyme hydrolyzed the racemic substrate for only 50 ~ . Kinetic properties. Using 1,2 dioctanoyl-sn-glycero-3-phosphocholine Km (app.) and V values were determined at pH 6 and 50 °C. This temperature was chosen since no straight lines were obtained in Lineweaver-Burke plots when working with the horse phospholipase Az at a temperature below this value. Probably the phase separation which is known [26] to occur in the range of 40-45 °C yields two different interfaces to which the enzyme binds not equally well. Above the phase separation straight Lineweaver-Burke plots were obtained. For reasons of comparison the enzymes from cow and pig pancreas were also tested at 50 °C. Measurements were carried out at a CaC12 concentration of 10 mM which value is at least 5 times the Kca values of these enzymes. The V values obtained were: 12 000 (horse), 4000 (pig) and 2500 (cow)/~equiv. min- ~. mg- 1, whereas the apparent Km values were 5 mM (horse), 1 mM (pig) and 1 mM (cow). Amino acid composition. The amino acid composition of horse pancreatic phospholipase A2 is given in Table II. Values are means of 6 hydrolysates; the values of Thr and Ser are extrapolated to zero time. For reasons of comparison the values for pig and ox phospholipase Az are included. The amino acid compositions of the zymogens (not shown in the table) suggest that the two zymogens and phospholipase A2 differ only by a small activation peptide. These differences were confirmed by amino acid analysis of the activation peptides (see also Table III).
Isolation and sequence of the activation peptides Activation of the precursors was carried out as usual. After maximal activity had been reached the pH was brought to 3 with 0.1 M HC1 and the mixture was applied to a Sephadex-G25 (fine) column (1.4 × 200 cm). Elution was performed with 10 -3 M HC1. Absorbance was monitored at 280 nm and 206 nm. After the appearence of the active enzyme which eluted in the void volume, only one main peak was registered, absorbing exclusively at 206 rim. The fractions corresponding with the peaks were pooled and lyophilized. Purity of the peptides was checked by high-voltage electrophoresis at pH 3.5. It appeared that both activation peptides gave a positive
271 TABLE II AMINO ACID COMPOSITION OF PHOSPHOLIPASE Az ISOLATED FROM PANCREATIC TISSUE OF HORSE, PIG [6] AND OX [4] Amino acid
Source Horse
Asp Thr Ser Glu Pro Gly Ala Cys Val Met lie Leu Tyr Phe Trp Lys His Arg Total number of amino acids
Pig
Ox
19 6 11 11 6 6 9 14 5 1 4 6 7 6 I 8 1 4
23 6 10 7 5 6 8 12 2 2 5 7 8 5 1 9 3 4
23 4 10 9 5 7 6 12 4 1 5 7 7 4 1 11 2 2
125
123
120
nin h y d ri n staining, which distinguishes these peptides f r o m the activation peptides f r o m pig, ox an d sheep z y m o g e n s since the latter ones co n t ai n p y r o g l u t a m i c acid as N - t e r m i n a l residue [4]. T h e a m i n o acid sequence o f the activation peptides f r o m the horse pancreatic zymogens was d e t e r m i n e d by m a n u a l E d m a n and E d m a n - d a n s y l techniques [21, 20]. Results are s u m m a r i z e d in T a b le III.
TABLE 11I THE AMINO ACID SEQUENCES OF THE ACTIVATION PEPTIDES OF PROPHOSPHOLIPASES A2 FROM VARIOUS SOURCES These peptides are released from the N terminal part of the prophospholipase Az during the activation process. Source
Sequence
Pig Ox Sheep 1 Sheep 2 Horse 1 Horse 2
< Glu-Glu-Gly-Ile- Ser- Ser- Arg < Glu-Ala- Gly-Leu-Asn-Ser- Arg < Glu-Ala- Gly-Leu-Asn-Ser- Arg Asn-Ser- Arg Gly-Iie- Ser- Pro-Arg Glu-Asn-Gly-Ile- Ser- Pro-Arg
