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Expression, Purification, and Kinetic Characterization of a Recombinant 80-kDa Intracellular Calcium-independent Phospholipase A2

1996; Elsevier BV; Volume: 271; Issue: 48 Linguagem: Inglês

10.1074/jbc.271.48.30879

1083-351X

Matthew J. Wolf, Richard W. Gross,

Endoplasmic Reticulum Stress and Disease

A CHO cell-derived 80-kDa recombinant polypeptide (GenBank number I15470) putatively encoding a calcium-independent phospholipase A2 was expressed in S. frugiperda cells resulting in over a 15-fold increase in a calcium-independent phospholipase A1/A2 activity which was entirely inhibitable by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. The recombinant polypeptide was purified from cytosol by sequential tandem affinity chromatographies employing ATP-agarose and calmodulin-Sepharose stationary phases. This strategy resulted in the rapid purification (36 h) of recombinant phospholipase A2 activity in 56% overall yield to a single intense 80-kDa protein band on SDS-polyacrylamide gel electrophoresis after silver staining. The purified protein possessed phospholipase A1, phospholipase A2, and lysophospholipase activities. Microbore anion exchange chromatography demonstrated that the 80-kDa protein band was comprised of multiple distinct isoforms including an anionic isoform which possessed over a 5-fold higher specific activity (5 μmol/mg·min) than earlier eluting isoforms. Collectively, these results unambiguously demonstrate that: 1) the 80-kDa polypeptide catalyzes phospholipase A1/A2 and lysophospholipase activities with distinct kinetic parameters; 2) calmodulin and ATP both interact with the catalytic polypeptide independent of regulatory proteins; and 3) distinct isoforms of this polypeptide exist which possess markedly different specific activities. A CHO cell-derived 80-kDa recombinant polypeptide (GenBank number I15470) putatively encoding a calcium-independent phospholipase A2 was expressed in S. frugiperda cells resulting in over a 15-fold increase in a calcium-independent phospholipase A1/A2 activity which was entirely inhibitable by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. The recombinant polypeptide was purified from cytosol by sequential tandem affinity chromatographies employing ATP-agarose and calmodulin-Sepharose stationary phases. This strategy resulted in the rapid purification (36 h) of recombinant phospholipase A2 activity in 56% overall yield to a single intense 80-kDa protein band on SDS-polyacrylamide gel electrophoresis after silver staining. The purified protein possessed phospholipase A1, phospholipase A2, and lysophospholipase activities. Microbore anion exchange chromatography demonstrated that the 80-kDa protein band was comprised of multiple distinct isoforms including an anionic isoform which possessed over a 5-fold higher specific activity (5 μmol/mg·min) than earlier eluting isoforms. Collectively, these results unambiguously demonstrate that: 1) the 80-kDa polypeptide catalyzes phospholipase A1/A2 and lysophospholipase activities with distinct kinetic parameters; 2) calmodulin and ATP both interact with the catalytic polypeptide independent of regulatory proteins; and 3) distinct isoforms of this polypeptide exist which possess markedly different specific activities. INTRODUCTIONThe intracellular phospholipases A2 represent a rapidly expanding class of enzymes which have been categorized based on their calcium dependence into calcium-dependent and calcium-independent subtypes (1Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar). Prior work has unambiguously identified a sequence encoding an 85-kDa intracellular calcium-dependent phospholipase A2 which translocates to the membrane interface in the presence of calcium ion (2Clark J.D. Lin L.L. Kriz R.W. Ramesh C.S. Stultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar, 3Sharp J.D. White D.L. Chiou X.G. Goodson T. Gamboa G.C. McClure D. Burgett S. Hoskins J. Skatrud P.L. Sportsman J.R. Becker G.W. Kang L.H. Roberts E.F. Kramer R.M. J. Biol. Chem. 1991; 266: 14850-14853Abstract Full Text PDF PubMed Google Scholar, 4Clark J.D. Milona N. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7708-7712Crossref PubMed Scopus (420) Google Scholar, 5Channon J.Y. Leslie C.C. J. Biol. Chem. 1990; 265: 5409-5413Abstract Full Text PDF PubMed Google Scholar, 6Glover S. Bayburt T. Jonas M. Chi E. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Recently, a protein putatively catalyzing an intracellular calcium-independent phospholipase A2 activity from CHO 1The abbreviations used are: CHOChinese hamster ovaryPCRpolymerase chain reactionRTreverse transcriptasePAGEpolyacrylamide gel electrophoresiskbkilobase pair(s)BEL(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cells has been described in abstract form (7Jones S.S. Tang J. Kriz R. Shaffer M. Knopf J. Seehra J. FASEB J. 1996; 10 (abstr.): L15Google Scholar), and its sequence determined (GenBank number I15470). Transient expression of this protein in the same cell type from which it was purified (i.e. CHO cells) results in enhanced calcium-independent phospholipase A2 activity. Of course, the assignment of catalytic function to a recombinant protein expressed in the same context from which it was originally isolated requires purification of the protein to homogeneity to unambiguously demonstrate its role as a catalytic entity and not as an activator of an endogenous activity. However, due to intractable technical obstacles, the protein catalyzing this activity has never been purified to homogeneity to unambiguously discriminate between its potential role as an activator of an endogenous phospholipase activity versus a bona fide catalytic entity.Since at least some members in the family of calcium-independent phospholipases A2 exhibit specific high affinity interactions with ATP and/or calmodulin (8Hazen S.L. Stuppy R.J. Gross R.W. J. Biol. Chem. 1990; 265: 10622-10630Abstract Full Text PDF PubMed Google Scholar, 9Hazen S.L. Gross R.W. J. Biol. Chem. 1991; 266: 14526-14534Abstract Full Text PDF PubMed Google Scholar, 10Hazen S.L. Gross R.W. Biochem. J. 1991; 280: 581-587Crossref PubMed Scopus (51) Google Scholar, 11Ackermann E.J. Kempner E.S. Dennis E.A. J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar, 12Ramanadham S. Wolf M.J. Jett P.A. Gross R.W. Turk T. Biochemistry. 1994; 33: 7442-7452Crossref PubMed Scopus (64) Google Scholar, 13Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 20989-20994Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), we sought to exploit potential interactions between these ligands and the expressed 80-kDa recombinant polypeptide to facilitate its purification to homogeneity to unambiguously identify its role in the catalytic process. We now report: 1) the expression of catalytically active recombinant 80-kDa calcium-independent phospholipase A2 in a baculovirus expression system; 2) the rapid purification of the 80-kDa recombinant calcium-independent phospholipase A2 to homogeneity through tandem sequential ATP and calmodulin affinity columns; and 3) the identification of multiple isoforms of this calcium-independent phospholipase A2 which possess markedly different specific activities.RESULTSTo investigate the biochemical characteristics of a recently described putative calcium-independent phospholipase A2 (7Jones S.S. Tang J. Kriz R. Shaffer M. Knopf J. Seehra J. FASEB J. 1996; 10 (abstr.): L15Google Scholar, GenBank accession I15470), RT-PCR was performed to amplify the cDNA encoding this protein from CHO cell total RNA utilizing oligonucleotide primers corresponding to its 5′ and 3′ coding sequence. The 2.2-kb DNA product from the RT-PCR amplification was subcloned into a pCR-II vector and sequenced to authenticate the fidelity of the amplified product. The 2.2-kb DNA product was subcloned into the E. coli expression vector, pET-21a, and competent E. coli were transformed. Although robust amounts of recombinant 80-kDa polypeptide were produced, induced E. coli did not demonstrate additional calcium-independent phospholipase A2 activity in comparison to control cells. We hypothesized that expression of this polypeptide in the context of a mammalian system was necessary for proper post-translational modifications and/or protein folding for expression of catalytic activity. Accordingly, the 2.2-kb product was subcloned into the baculoviral vector, pFasBac, and transformed into competent DH10Bac E. coli cells for helper plasmid-mediated transposition of the recombinant sequence into a bMON14272 bacmid (19Luckow V.A. Lee S.C. Barry G.F. Olins P.O. J. Virol. 1993; 67: 4566-4579Crossref PubMed Google Scholar). The bMON14272 bacmid was purified from the DH10Bac E. coli and used to infect Sf9 cells for subsequent formation of recombinant baculovirus and expression of recombinant protein as described in detail under "Experimental Procedures."After initial amplification of the recombinant baculovirus harboring the calcium-independent phospholipase A2 (∼1 × 108 plaque-forming units/ml), Sf9 cells were infected for 48 h at a multiplicity of infection of 1.0, harvested, lysed by sonication, and calcium-independent phospholipase A2 activity was quantified. Sf9 cells infected with recombinant 80-kDa polypeptide reproducibly demonstrated 15-fold increases in calcium-independent phospholipase A2 activity (Fig. 1A) with greater than 80% of the recombinant activity partitioning into the cytosolic compartment (Fig. 1B). Furthermore, treatment of Sf9 cell cytosol which contained recombinant calcium-independent phospholipase A2 with the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (21Hazen S.L. Zupan L.A. Weiss R.H. Getman D.P. Gross R.W. J. Biol. Chem. 1991; 266: 7227-7232Abstract Full Text PDF PubMed Google Scholar, 22Zupan L.A. Weiss R.H. Hazen S.L. Parnas B.L. Aston K.W. Lennon P.J. Getman D.P. Gross R.W. J. Med. Chem. 1991; 36: 95-100Crossref Scopus (55) Google Scholar, 23Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) completely ablated the expressed recombinant calcium-independent phospholipase A2 activity (Fig. 1C).To demonstrate that the 80-kDa recombinant polypeptide was responsible for catalyzing calcium-independent phospholipase A2 activity (i.e. the polypeptide was not an activator or cofactor of an endogenous catalytic entity), a strategy was developed to chromatographically purify the recombinant polypeptide to homogeneity. Since the majority of the recombinant phospholipase A2 activity partitioned into the cytosolic fraction, Sf9 cell cytosol was applied to a DEAE anion exchange column and recombinant calcium-independent phospholipase A2 activity was eluted with a linear salt gradient. Recombinant phospholipase A2 activity eluted at ∼200 mM NaCl (Fig. 2). Since at least some calcium-independent phospholipases A2 interact in a highly selective fashion with ATP (8Hazen S.L. Stuppy R.J. Gross R.W. J. Biol. Chem. 1990; 265: 10622-10630Abstract Full Text PDF PubMed Google Scholar, 9Hazen S.L. Gross R.W. J. Biol. Chem. 1991; 266: 14526-14534Abstract Full Text PDF PubMed Google Scholar, 10Hazen S.L. Gross R.W. Biochem. J. 1991; 280: 581-587Crossref PubMed Scopus (51) Google Scholar, 11Ackermann E.J. Kempner E.S. Dennis E.A. J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar, 12Ramanadham S. Wolf M.J. Jett P.A. Gross R.W. Turk T. Biochemistry. 1994; 33: 7442-7452Crossref PubMed Scopus (64) Google Scholar) and calmodulin (13Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 20989-20994Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), we attempted to exploit the power inherent in double sequential affinity chromatographies to effect the rapid and facile purification of this recombinant phospholipase A2 activity to homogeneity. First, fractions containing calcium-independent phospholipase A2 activity from the DEAE eluent were pooled and immediately loaded onto an ATP-agarose affinity column. After loading, the column was sequentially washed with buffer, buffer containing 10 mM AMP, and finally the recombinant phospholipase A2 activity was eluted by the application of buffer containing only 1 mM ATP (Fig. 3A). Over 95% of the calcium-independent phospholipase A2 activity bound to the ATP-agarose column and was quantitatively eluted by the application of 1 mM ATP. Resolution of the proteins from the ATP-agarose column fractions by SDS-PAGE and visualization after silver-staining demonstrated the specificity of the interaction between the 80-kDa recombinant phospholipase A2 and the ATP-agarose (Fig. 3B).Fig. 2DEAE-Sephacel chromatography of recombinant calcium-independent phospholipase A2 from Sf9 cell cytosol. Recombinant calcium-independent phospholipase A2 from Sf9 cell cytosol (100,000 × g supernatant) (37 mg of protein) was loaded onto a DEAE-Sephacel column and washed with buffer B to remove unbound protein as described under "Experimental Procedures." Recombinant phospholipase A2 was eluted by the application of a linear gradient to 1 M NaCl in buffer B, calcium-independent phospholipase A2 activity was assessed utilizing 25-μl aliquots of the column eluents as described under "Experimental Procedures" and is expressed in disintegrations/min of [14C]arachidonic acid released from 5 μM L-α1-palmitoyl-2-[1-14C]arachidonyl phosphatidylcholine.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3ATP-agarose chromatography of recombinant calcium-independent phospholipase A2. A, the fractions from the DEAE-Sephacel eluent which contained calcium-independent phospholipase A2 activity were pooled (11 mg of protein) and immediately loaded onto a 1-ml column of ATP-agarose which was previously equilibrated in buffer B. After loading, the column was washed sequentially with 10 ml of buffer B, 10 ml of buffer B containing 10 mM AMP, 10 ml of buffer B, and finally 10 ml of buffer B containing 1 mM ATP. Calcium-independent phospholipase A2 activities in the load (lane 1), void (lane 2), AMP fraction (lane 3), ATP fraction 1 (lane 4), and ATP fraction 2 (lane 5) were quantified as described above and expressed as disintegrations/min of [14C]arachidonic acid released from phospholipid substrate. B, the proteins in 25-μl aliquots of the fractions from ATP-agarose chromatography were resolved on 10% polyacrylamide gels and stained with silver as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since at least one calcium-independent phospholipase A2 interacts with calmodulin in a calcium sensitive fashion, we attempted to exploit the power of ternary complex affinity chromatography employing a calmodulin-Sepharose stationary phase (13Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 20989-20994Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The fractions containing recombinant calcium-independent phospholipase A2 activity from the ATP-agarose column were pooled, adjusted to 5 mM calcium, and directly loaded onto a calmodulin-Sepharose column as described under "Experimental Procedures." Recombinant calcium-independent phospholipase A2 activity quantitatively bound to calmodulin-Sepharose in the presence of calcium ion and was quantitatively eluted by dispersal of the ternary complex with application of buffer containing EGTA (Fig. 4A). SDS-PAGE of fractions from calmodulin-Sepharose chromatography displayed only a single intense band after silver staining demonstrating that recombinant calcium-independent phospholipase A2 activity copurified with the 80-kDa protein (Fig. 4B). Moreover, the resolving power of ternary complex affinity chromatography employing the calmodulin-Sepharose stationary phase was demonstrated by additional experiments in which crude Sf9 cytosol was loaded directly onto a calmodulin-Sepharose affinity column in the presence of calcium. Phospholipase A2 activity was quantitatively bound to calmodulin-Sepharose in the presence of calcium ion and was quantitatively eluted by washing with EGTA resulting in a 10-fold purification step (Fig. 4, panels C and D).Fig. 4Calmodulin-Sepharose chromatography of recombinant calcium-independent phospholipase A2. A, the fractions from the ATP-agarose column containing calcium-independent phospholipase A2 activity were pooled, adjusted to 5 mM calcium, and loaded onto a 0.5-ml calmodulin-Sepharose column as described under "Experimental Procedures." The column was washed with 10 bed volumes of a buffer containing 25 mM imidazole (pH 8.0) and 500 μM calcium. Calcium-independent phospholipase A2 was eluted by the application of buffer containing 25 mM imidazole (pH 8.0) and 4 mM EGTA. Calcium-independent phospholipase A2 activity in the calmodulin-Sepharose load (lane 1), void (lane 2), EGTA fraction 1 (lane 3), EGTA fraction 2 (lane 4), and EGTA fraction 3 (lane 5) was quantitated as described above and is expressed in disintegrations/min of [14C]arachidonic acid released from 5 μM L-α1-palmitoyl-2-[1-14C]arachidonyl phosphatidylcholine substrate. B, the proteins in 25-μl aliquots from individual fractions of the calmodulin-Sepharose column were resolved on a 10% polyacrylamide gel and stained with silver. C, Sf9 cell cytosol which contained recombinant calcium-independent phospholipase A2 was adjusted to 5 mM calcium and loaded onto a 0.5-ml calmodulin-Sepharose column and chromatographed as described above. Calcium-independent phospholipase A2 activity in the load (lane 1), void (lane 2), EGTA fraction 1 (lane 3), and EGTA fraction 2 (lane 4) was quantitated and expressed as described above. D, the proteins in 25-μl aliquots from individual fractions of the calmodulin-Sepharose column were resolved on a 10% polyacrylamide gel and stained with silver.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mono-Q chromatography of the calmodulin-Sepharose eluent demonstrated an elution profile identifying the presence of multiple isoforms of the 80-kDa polypeptide which each cochromatographed with phospholipase A2 enzymic activity (Fig. 5). The majority of recombinant phospholipase A2 activity eluted at ∼50 mM NaCl with a specific activity of 1 μmol/mg·min while a second, more anionic, peak eluted at ∼120 mM NaCl with a specific activity of 5 μmol/mg·min. Each of three early eluting peaks as well as the late eluting peak contained calcium-independent phospholipase A1, phospholipase A2, and lysophospholipase activities in similar ratios. Peaks I and II were individually pooled, diluted, and subsequently rechromatographed on a re-equilibrated Mono-Q stationary phase demonstrating that each peak chromatographed according to its elution profile during the initial chromatography (i.e. rechromatography of peak I resulted in a single peak eluting at 50 mM NaCl while rechromatography of peak II resulted in a single peak eluting at 120 mM NaCl) (Fig. 6). These results demonstrate that the isoforms are long-lived entities and not the result of a rapidly equilibrating mixture.Fig. 5Mono-Q chromatography of recombinant calcium-independent phospholipase A2. A, recombinant calcium-independent phospholipase A2 activity from the calmodulin-Sepharose column eluent (10 μg of protein) was loaded onto a PC1.6/5 Mono-Q column previously equilibrated with buffer B. After washing, recombinant calcium-independent phospholipase A2 activity was eluted by the application of a linear gradient of NaCl in buffer B. UV absorption at 280 nm (-) and NaCl concentration (- - -). B, fractions from the Mono-Q column elute were dried in the presence of 10% SDS, reconstituted in 50 μl of 50 mM Tris-HCl (pH 6.8), 5% glycerol, and 0.5%β-mercaptoethanol and the proteins were resolved by SDS-PAGE on 10% polyacrylamide gels and stained with silver as described under "Experimental Procedures." The calcium-independent phospholipase A2 activity in each fraction was measured as described under "Experimental Procedures" and is expressed in dintegrations/min of [14C]arachidonic acid released from 5 μM L-α1-palmitoyl-2-[1-14C]arachidonyl phosphatidylcholine and shown on the top of the gel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Rechromatography of previously resolved recombinant calcium-independent phospholipase A2 isoforms on a Mono-Q stationary phase. A, purified recombinant calcium-independent phospholipase A2 from the calmodulin-Sepharose eluent (5 μg of protein) was purified on a Mono-Q column and resolved into earlier eluting (peak I) and later eluting (peak II) peaks as described under "Experimental Procedures." B, peak I from the Mono-Q chromatography in A was diluted 3-fold in buffer B to reduce the NaCl concentration and reapplied to Mono-Q resin and rechromatographed employing identical conditions as above. C, peak II from three iterative preparations similar to those represented in A was diluted 3-fold in buffer B to reduce the NaCl concentration and resubjected to Mono-Q chromatography as described above.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To examine the kinetic characteristics of the phospholipase A1, phospholipase A2, and lysophospholipase activities catalyzed by the recombinant 80-kDa polypeptide, substrate-activity profiles were compared. First, phospholipase A1, phospholipase A2, and lysophospholipase activities were linear with respect to time over the incubation times utilized (30 s). Second, each of the activities displayed saturation kinetics (Fig. 7, A and B). Third, phospholipase A1 activity (as assessed by the production of sn-2 labeled lysophospholipid from the substrate L-α1-palmitoyl-2-[1-14C]arachidonyl phosphatidylcholine) demonstrated an apparent maximum velocity of 1.2 μmol/mg·min with an apparent Km of 3.3 μM. Fatty acid production catalyzed by the recombinant 80-kDa polypeptide (which reflects both direct phospholipase A2 catalyzed release of the sn-2 radiolabeled arachidonic acid moiety as well as fatty acid generated through sequential phospholipase A1 and lysophospholipase activities) demonstrated an apparent maximum velocity of 0.9 μmol/mg·min and an apparent Km of 1.6 μM. We point out that a substantial amount of fatty acid release could result from sequential phospholipase A1 and lysophospholipase activities. Incubation of enzyme with L-α1-O-hexadecyl-2-[3H]arachidonyl phosphatidylcholine demonstrated the expressed 80-kDa polypeptide possessed a phospholipase A2 activity of 0.5 μmol/mg·min. Since the sn-1 alkyl ether linkage is not susceptible to hydrolysis by esterolytic processes, these results unambiguously demonstrate phospholipase A2 activity as an inherent catalytic function of the expressed polypeptide. Fourth, kinetic analysis of lysophospholipase activities demonstrated that the enzyme rapidly catalyzes hydrolysis of monomeric lysophosphatidylcholine (critical micellular concentration of palmitoyl lysophosphatidylcholine = 7 μM (24Stafford R.E. Fanni T. Dennis E.A. Biochemistry. 1989; 28: 5113-5120Crossref PubMed Scopus (159) Google Scholar)) with only modest increases of enzymic activity at supramicellar concentrations of substrate.Fig. 7Purified recombinant 80-kDa polypeptide catalyzes calcium-independent phospholipase A1/A2 and lysophospholipase activities. A, calcium-independent phospholipase A1/A2 activities were measured from 120 ng of highly purified recombinant 80-kDa polypeptide (i.e. calmodulin-Sepharose eluent) by quantitating the release of radiolabeled arachidonic acid (+) and sn-2 radiolabeled lysophospholipid (⋄) from the indicated concentrations of sn-2 radiolabeled L-α1-palmitoyl-2-[1-14C]arachidonyl phosphatidylcholine after incubation at 37°C for 30 s as described under "Experimental Procedures." The production of radiolabeled lysophospholipid from L-α1-palmitoyl-2-[1-14C]arachidonyl phosphatidylcholine represents phospholipase A1 activity while the production of radiolabeled free fatty acid represents combined phospholipase A2 activity as well as sequential phospholipase A1 and lysophospholipase activities. B, calcium-independent lysophospholipase activity was measured from 120 ng of highly purified recombinant 80-kDa polypeptide (i.e. calmodulin-Sepharose eluent) by measuring the release of radiolabeled [14C]palmitic acid from the indicated concentrations of sn-1 radiolabeled palmitoyl lysophosphatidylcholine after incubation at 37°C for 30 s as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe present results demonstrate that the recombinant chromatographically pure 80-kDa polypeptide catalyzes phospholipase A1/A2 and lysophospholipase activities when expressed in a baculoviral expression system. Moreover, individual isoforms of the 80-kDa polypeptide can be chromatographically resolved and calcium-independent phospholipase A1/A2 and lysophospholipase activities comigrate with each peak of 80-kDa protein mass. Collectively, these results identify the 80-kDa polypeptide as a catalytic entity mediating phospholipolysis. Furthermore, they underscore the necessity of expression of this protein in the context of a mammalian cell since E. coli expressed robust quantities of recombinant protein which did not possess either inherent or inducible (in our hands) catalytic activity.The recombinant protein expressed calcium-independent phospholipase A1/A2 and lysophospholipase activities in similar amounts. Potentially, two mechanisms can be responsible for the sn-2 fatty acid release from choline glycerophospholipids including: 1) the sequential hydrolysis of the sn-1 acyl group followed by lysophospholipase activity; or 2) direct hydrolysis of the sn-2 acyl group and the concomitant generation of sn-1 acyl-lysophospholipids. Both mechanisms likely contribute to the production of radiolabeled free fatty acid from specifically radiolabeled sn-2 L-α1-palmitoyl-2-[1-14C]arachidonyl phosphatidylcholine. We point out the potential possibility that all of the observed release of free fatty acid could result from sequential phospholipase A1 and lysophospholipase activities since the recently generated sn-2 labeled lysophospholipid is present at the active site of the enzyme and could serve as the preferred substrate for a second round of hydrolysis. In this paradigm, the relative fractional percentage of radiolabeled fatty acid to lysophospholipid generated reflects the relative rates of a second round of enzymatic cleavage in comparison to the rate of release of the radiolabeled lysophospholipid bound at the active site. However, the recombinant protein has substantive amounts of phospholipase A2 activity since L-α1-O-hexadecyl-2-[3H]arachidonyl phosphatidylcholine (where the sn-2 acyl group is the only acyl group which can be hydrolyzed by this enzyme) is a good substrate. Finally, the rate of lysophospholipase activity was similar to that of phospholipase A2 activity, suggesting the similar interactions of the carboxyl moiety destined for hydrolysis with critical amino acids at the active site are present (i.e. the activation energies are similar for the hydrolysis of both substrates).There are several features of the purification strategy which merit consideration. First, the utilization of tandem sequential affinity columns facilitates the procurement of geometric increases in the high purification factors typically obtained through affinity chromatographic approaches. Second, ternary complex affinity chromatography with calmodulin-Sepharose resin facilitates the discrimination not only between entities which can recognize calmodulin but also between those proteins which possess an obligatory requirement for binding to the calcium-calmodulin complex. Third, the ATP-agarose affinity chromatography exploited the molecular recognition of ATP by the protein in the context of an anionic stationary phase. Since the proteins had been previously selected for binding to a cationic stationary phase (DEAE-Sephacel resin), the subsequent forced affinity binding to a negatively charged affinity column further amplified the power and selectivity of ATP affinity chromatography. The integration of these three key principles in the purification strategy allowed the complete purification of this polypeptide in 36 h in >56% overall yield (Table I) while prior attempts employing conventional chromatographic strategies have resulted in technically demanding approaches accompanied by poor yields of inhomogenous preparations.Table IPurification table of recombinant calcium-independent phospholipase A2ProteinTotal activitySpecific activityYieldPurificationmgnmol/minnmol/mg·min%-foldCytosol17.0462.71001DEAE-Sephacel7.8314.0671.5ATP-agarose0.0327900.059333.3Calmodulin-Sepharose0.02726963.056357.0 Open table in a new tab The separation of the 80-kDa phospholipase A2 into distinct chromatographically resolvable isoforms with differing specific activities was unanticipated. Rechromatography of constituent isoforms eluting with their original chromatographic profiles demonstrated that these isoforms are isolatable entities (on a laboratory time scale) and are not the result of a dynamic interchange of an equilibrium mixture. It is intriguing to speculate that the higher specific activity isoform of this polypeptide has a higher phosphorylation state giving rise to an increased retention time on an anionic exchange resin and a higher specific activity. Whatever covalent modifications are eventually determined to be responsible for these effects, the results suggest a potential biochemical mechanism for agonist-induced increases in calcium-independent phospholipase A2 activity whereby a high specific activity isoform can be generated from a lower specific activity pool during cellular stimulation.Recently, studies of the crystal structure of phospholipase Cδ identified the unanticipated finding of an EF-hand calcium binding motif (residues 133-281) which shares substantial homology with calmodulin (25Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (513) Google Scholar). The present results demonstrate a direct interaction between calmodulin and the recombinant 80-kDa phospholipase A2. Accordingly, it is intriguing to speculate that the phospholipase Cδ structure recapitulates the bimolecular calmodulin-phospholipase A2 complex demonstrated herein, suggesting an ancestral relationship between these two regiospecific phospholipases. Although comparisons of the primary sequences of phospholipase Cδ and calcium-independent phospholipase A2 have not revealed sequence homology, we anticipate that comparisons of the three-dimensional structure of the recombinant phospholipase A2-calmodulin complex and phospholipase Cδ could show regions of homology and provide insights into the structure of an evolutionarily distant ancestral polypeptide from which both phospholipases are derived. INTRODUCTIONThe intracellular phospholipases A2 represent a rapidly expanding class of enzymes which have been categorized based on their calcium dependence into calcium-dependent and calcium-independent subtypes (1Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar). Prior work has unambiguously identified a sequence encoding an 85-kDa intracellular calcium-dependent phospholipase A2 which translocates to the membrane interface in the presence of calcium ion (2Clark J.D. Lin L.L. Kriz R.W. Ramesh C.S. Stultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar, 3Sharp J.D. White D.L. Chiou X.G. Goodson T. Gamboa G.C. McClure D. Burgett S. Hoskins J. Skatrud P.L. Sportsman J.R. Becker G.W. Kang L.H. Roberts E.F. Kramer R.M. J. Biol. Chem. 1991; 266: 14850-14853Abstract Full Text PDF PubMed Google Scholar, 4Clark J.D. Milona N. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7708-7712Crossref PubMed Scopus (420) Google Scholar, 5Channon J.Y. Leslie C.C. J. Biol. Chem. 1990; 265: 5409-5413Abstract Full Text PDF PubMed Google Scholar, 6Glover S. Bayburt T. Jonas M. Chi E. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Recently, a protein putatively catalyzing an intracellular calcium-independent phospholipase A2 activity from CHO 1The abbreviations used are: CHOChinese hamster ovaryPCRpolymerase chain reactionRTreverse transcriptasePAGEpolyacrylamide gel electrophoresiskbkilobase pair(s)BEL(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cells has been described in abstract form (7Jones S.S. Tang J. Kriz R. Shaffer M. Knopf J. Seehra J. FASEB J. 1996; 10 (abstr.): L15Google Scholar), and its sequence determined (GenBank number I15470). Transient expression of this protein in the same cell type from which it was purified (i.e. CHO cells) results in enhanced calcium-independent phospholipase A2 activity. Of course, the assignment of catalytic function to a recombinant protein expressed in the same context from which it was originally isolated requires purification of the protein to homogeneity to unambiguously demonstrate its role as a catalytic entity and not as an activator of an endogenous activity. However, due to intractable technical obstacles, the protein catalyzing this activity has never been purified to homogeneity to unambiguously discriminate between its potential role as an activator of an endogenous phospholipase activity versus a bona fide catalytic entity.Since at least some members in the family of calcium-independent phospholipases A2 exhibit specific high affinity interactions with ATP and/or calmodulin (8Hazen S.L. Stuppy R.J. Gross R.W. J. Biol. Chem. 1990; 265: 10622-10630Abstract Full Text PDF PubMed Google Scholar, 9Hazen S.L. Gross R.W. J. Biol. Chem. 1991; 266: 14526-14534Abstract Full Text PDF PubMed Google Scholar, 10Hazen S.L. Gross R.W. Biochem. 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We now report: 1) the expression of catalytically active recombinant 80-kDa calcium-independent phospholipase A2 in a baculovirus expression system; 2) the rapid purification of the 80-kDa recombinant calcium-independent phospholipase A2 to homogeneity through tandem sequential ATP and calmodulin affinity columns; and 3) the identification of multiple isoforms of this calcium-independent phospholipase A2 which possess markedly different specific activities.