analysis of the cdna for phospholipase a2 from honeybee venom glands

7/28/2019 Analysis of the cDNA for Phospholipase A2 From Honeybee Venom Glands

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E u r J Riocheni. 184, 249-254 (1989)( b h B S 1 9 8 9

Analysis of the cDNA for phospholipase A2 from honeybee venom glandsThe deduced amino acid sequence reveals homology to the correspond ing vertebrate enzym esKarl K U C l l L E R , Mi chae l G MA C H L ,Manfred J . SIPPL a n d G u n t h e r K R E I LInstitute of Molecular Biology, Austrian Academ y of Sciences, SalzburgInstitute for Biology, Biochemistry and Biophysics, University of Salzburg(Received March 6/June 7, 1989) ~ E JB 89 0272

A cDNA expression l ibrary was constructed from worker bee venom glands and screened with an ant ibodyagainst phos pho l ipase A2. The nucleot ide sequence of a posit ive clone w ith the largest insert showed an openreading frame tha t codes for part of the s ignal pept ide, the pro-region an d the ent i re ma ture enzym e of the beevenom phosphol ipase A 2 precursor. This sequence differs in the central region from the on e determined byShipolini et al. [FEBSL e t t . 17, 39-40 (1971)] in showing, am ong o ther exchanges, two ad di t ional cysteines . Therevised sequence of bee venom phospholipase is similar to the pancre at ic enzyme in the spacing of cysteines andthe presence of several amino acids known to be part of the act ive s i te or the Ca2+-binding egion in ident icalposi t ions. M oreover, these parts of the bee protein can be f i tted into the three-dimensional s t ructure determinedfor the bo vine pancreat ic phosphol ipase A 2 [Dijkstra et a1.(1981) Nature 28Y, 604- 6061. Contrary to earliersuggest ions, we therefore conclude that the bee venom enzyme shows some hom ology to ph osphol ipases frommam mal ian pancreas and snake venoms

Phosphol ipase A 2 catalyzes the specific h ydrolysis of esterbonds at the C2 posi t ion of 1 ,2-diacyl-3- sn-glycerophospho-lipids [l]. Secreted forms of this enzyme have been isolatedfrom different sources , mainly from m amm alian pancreas andfrom snake venoms. In the pancreas , the enzyme is present asa zymogen which is act ivated upon secret ion into the duo-denum by t rypt ic cleavage of a smal l pro-pcpt ide from theamino-term inus [2]. Com parisons of the am ino acid sequencesof these enzymes ha ve revealed a close relationship typical forproteins with a com mo n evolut ionary origin [ l , 31. Conservedresidues include a histidine- spart ic acid pair which formspar t o f the active site [4], several residues inv olved in t hebinding of C a 2 ' ions, as well as it numb er of disdf ide bridges.The similarity between these enzymes of different origin hasbeen corroborated by an analysis of the three dimensionals t ructure of phosphol ipase A 2 from bovine pancreas [ 5 , 61and from the venom of the rat t lesnake Crotulus atrox [7].More recent ly, the s t ructure of several prepro-phopho l ipasesfrom mammalian pancreas as wel l as snake venom has beendetermined using re com binant D N A techniques [X - 11. In-terestingly, the s t ructure of the pro-region a nd hence the mech-anism of act ivat ion is di fferent in the mam malian and rept i lianenzymes.A phosphol ipase A 2 is one of the main com ponents of thevenom of the honeybee, Apis rnellifera [12]. It is a glyco-protein containing one asparagine-l inked ol igosaccharide [I 3,141. Determ ination of the am ino acid sequ ence of this enzym e[33, 151 as well as the assignment of the disulfide bridges [Ih ],led to the conclusion that the bee phosphol ipase A2 wasunrelated to the vertebrate enzymes [I] . However, aphosphol ipase A 2 from the venoni of a Mexican lizard was

C'orrr.sporzdenc,eo G . Kreil, Inst i tut fur Molekularbiologie, Bill-En;ymr. Phospholipase Az (EC 3.2.2.4).rothstrasse 1 1 , A-5020 Salzburg, Austria

shown by Sosa et al . [17] to possess an amino-terminal se-quence s imilar to the bee venom enzym e.In previous work, the s t ructure of the precursors of thetwo bee venom pept ides mel i t t in an d secapin were determined[18, 191 through the use of cDNA l ibraries from queen beevenom glands. As phosphol ipase A 2 is vi r tual ly absent fromthe venom of this caste [20], we have co nstructed a c D N Alibrary from worke r bee venom g lands an d screened this witha phosphol ipase A 2 ant ibody. Fr om the nucleot ide sequenceof positive clones, the amino acid sequence of the bee venomphosphol ipase A 2 could be deduc ed. The sequence and theanalysis presented here indicate homology to the vertebrateenzymes.

M A T E RI AL S A N D M E T H O D SEnzjwzes and reagents

DNA-modifying enzymes and rest r ict ion endonucleaseswere obtained from New England Biolabs (Schwalbach/Frankfurt , FRG), Bethesda Research Laboratories (Vienna),St ra tagene (Heidelberg , FRG) and Boehr inger Mannheim(Vienna). Al l radiochemicals were purchased from A mersh amInternat ional (Am ersham , GB). O l igonucleotides were kindlysuppli ed by D r G . Hogen auer (Univers ity of Graz). All bio-chemicals and reagents were of highest purity commerciallyavai lable.

R N A and D N A iso la tionTota l RN A w as iso lated f rom the venom g lands of newlyemerged que en bees an d of wo rker bees of different ages usingpublished procedures [I 91 with minor modifications. Poly(A)-r ich R N A was then ob ta ined by chromatography on


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250oligo(dT)-cellulose [21]. Genomic DNA was prepared from GGATCCTTGTTCCTTCTCCTCCTCTCTACCTCTCACGGATGGC~TCAGGGATAGGATCbee larvae essentially as described by Vlasak et al. [I 81.

G S L F L L L L S T S H G W Q I R D R It tc D N A cloning and screening of expression libraries

cDNA was synthesized using reverse transcriptase frommoloney murine leukemia virus [22]. The synthesis of thesecond strand was by the method of Gubler et al. 1231. ThemRNA .cDNA hybrid was incubated with DNA polymeraseI (about 200 units/pg hybrid) at 12C over night. The doublestranded cDNA was blunt ended with T4-DNA polymeraseand Klenov polymerase and ligated with phage /? gtl l arms(Stratagene, Heidelberg, FRG) via EcoRI linkers [24]. For invitro packaging of recombinant phage DNA, a commercialkit (Stratagene) was used as recommended by the supplier.cDNA expression libraries were screened with a polyclonalantibody against bee venom phospholipase A (kindly suppliedby Dr F. G. Prendergast) following the procedure of Younget al. [25]. Immunopositive phages were purified, phage DNAwas isolated and its inserts subcloned into Bluescriptphagemids by standard procedures [26].Northern analysis

For Northern blots, 5-10 pg poly(A)-rich RNA wasfractionated in a 1.2% agarose gel containing 2.2 M formal-dehyde [27]. The RNA was then transferred to nitrocellulosesheets and hybridized with the nick-translated phospholipasecDNA.Primer extension

A radiolabeled single stranded primer containing 16 8 basepairs was isolated from a BamHI - phI restriction fragmentusing a strand separation gel. The primer was annealed todifferent amounts of poly(A)-rich RNA from worker beevenom glands for 8h at 80C in siliconized glass capillaries[27]. The primer was then extended with avian myeloblastosisvirus reverse transcriptase (Stratagene) at 42 "C. The productswere analyzed on a 6 % sequencing gel using a Sangersequencing ladder as size markerSequence analysis

Enzymatic sequencing of DNA [28] was performed withdouble-stranded plasmid DN A using a Sequenase kit (UnitedStates Biochemical Corp., Vienna) as recommended by thesuppliers. For the chemical degradation method [29], DNAwas labeled either at the 3' end using reverse transcriptase orat the 5' end with T4-polynucleotide kinase.

RESULTSStart ing with poly(A)-rich RNA prepared from the venomglands of about 2000 worker bees of different ages, a cDNAexpression library was constructed in the phage I. gt-31. Thislibrary was screened with a polyclonal antibody against beevenom phospholipase A2. Positive clones were analyzed forthe size of their inserts and the one with the largest wasinvestigated further. The cDNA was subcloned into Bluescriptvectors and then sequenced on both strands. The nucleotideand the deduced amino acid sequence of the single open read-

GGGGATAACGAGTTGGAGGAACGGATAATATATCCAGGAACGTTATGGTGCGGGCATGGT

G D N E L E E R I I Y P G T L W C G H G

AACAAGTCGTCCGGCCCGAACGAGCTAGGTCGGTTCAAGCACACGGATGCATGCTGTCGA

N K S S G P N E L G R F K H T D A C C R

ACCCACGACATGTGCCCGGACGTTATGTCAGCTGGTGAATCGAAGCACGGCCTGACCARC

T H D M C P D V M S A G E S K H G L T N

A C G G C C T C C C A C A C C A G G T T G T C G T G C G A C T G C G A C G A C A

T A S H T R L S C D C D D K F Y D C L K

A R T T C G G C G G AC A C G A T T A G C T CG T A T T T C G T A G G G AA G A T C T G A T A G A C

N S A D T I S S Y F V G K M Y F N L I D

ACGARGTGTTACAAACTGGAGCATCCTGTCACCGGGTGCGGTGAGAGAACCGAGGGTCGT

T K C Y K L E H P V T G C G E R T E G R

C L H Y T V D K S K P K V Y Q W F D L R

AAGTATTGATAAAATTCACGGGGCGGATCTTGAGAGTACCACTTCGAGATCGTTTATTTA

K Y / /Fig. 1. Nucleotide sequence and deduced amino acid sequence of clonedc D N A fo r honeybee venom prepro-phospholipase A Z . T h e amino-ter-minus of th e mature enzyme [I31 is underlined, the most likely cleavagesites for signal peptidase [3t ] are marked by ar rows, stop codons areindicated as (/)

ing frame are shown in Fig. 1. This sequence of 54 0 nucleotidesshows part of a signal peptide; a pro-region probably com-prised of 15 or 17 amino acids terminating with a singlearginine residue, the sequence of the mature enzyme contain-ing 134 residues and part of the 3'-untranslated region.A primer extension analysis was carried out to determinethe length of the 5'-untranslated region of the phospholipasemRNA. Using a radiolabeled BamHI - phl restriction frag-ment of the cloned phospholipase cDNA as primer, a singleextension product containing about 150bp beyond the 5' endof the cloned cDNA was obtained (see Fig. 2) .The nick-translated cDNA was then used for Northernblots, with total mRNA from venom glands. As shown inFig. 3, in the mRNA from worker bee venom glands, a singleband with a size of about 850 nucleotides hybridizes withthe labeled cDNA. Under these conditions, the mRNA forphospholipase A2 is not detectable in poly(A)-rich R N A pre-pared from queen bee venom glands. In a genomic Southernblot with total bee DNA digested with EcoR1, the labeledcDNA hybridizes to one band of about 1600 base-pairs in-


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2515 0

a) -L-T-E-T-A-S- -R-L-S-C-N-N-N-D-_ _ -F-Y- -K-N-S-A-D-b) -L-T-Ci-T-A-S-H-T-R-L-S-C-0-C-I2-D-II-F-Y-~-C-~-K-N-S-A-D-

Fig. 4. Comparison of the the central region of the amino acidsequenc eofb ee venomphospholipase A z takenfrom ( a ) Shipolini et al . [ 13 , I S ]an d ( b ) he present wo rk. Differences are underlined

1 10 2 0

a) A L W Q F N G M I K C K I P S S E P L L D F N N Y G C Y

I I Y P G T L W)

1

* 3 0 40 # * 50 +C G L G G S G T P V D D L D R C C Q T H E N S Y

CPHGNKSSGPNELGRFKHTDA~CRTHIMSP

10 2 0 3 0

6 0 7 0 8 0

Fig. 2. Primer extension experiment. A radioactive single-stranded ' A Iprimer was isolated as described in Methods, annealed to differentamounts of poly(A)-rich RNA from worker bee venom glands, andextended with reverse transcriptase. Products were analyzed on aRNA; lane 2 , 5 pg RNA; lanc 3,lO pg RN A

A

sequencing gel using a sequencing ladder as size marker. Lane 1, no 40 5 0

5 90 5 + 100 # 110T C N S E N N A C G A F I C N C E R N A A I C F S K V P Y N

T N T A S H T R L S C D C E D K F Y D C L K N S A D T

60 7 0

120

K E H K N L D K K N C

I S S Y F V G K M Y F N L I D T K C Y K L E H P V T G C G

80 90 100

E R T E G R C L H Y T V D K S K P K V Y Q W . F D L R K Y ( b )

110 120 130

Fig. 5. Comparison of the amino acid sequence of phospholipase A 2fro m bovine pancreas ( a ) and honeybee venom ( b ) . The sequences arenumbered above and below, respectively. Common cysteines andresidues known to be part of the active site or the Ca2+-binding regionof the bovine enzyme are underlined. Pairs of cysteines marked withthe same symbol (*, +, 5, #) form disulfide bridges in the bovine

Fig. 3. Northern blot analysis of RN A fr om queen and worker beevenom glunds. Poly(A)-rich RNA from worker bee (lane 1 ) and queenbee (lane 2) venom glands were fractionated in agarose/formaldehydegels (see Methods) and hybridized with the nick-translated phospho-lipase cDNA insert. Size markers were end-labeled A DNA fragments(ST) enzyme


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25 2

A

YS 29

GLY 328-2H I S 31

GLY 3 2

I 0 3

Fig. 6. P ml ia l v i e i c oj ' the X - ru j striic'ture o fhovinephospholipusc A 2 ( taken from [6]). Three segm ents showing similari ty to thc bee eiuym eare shown (numbers in parenthcsies refer to the sequence of the bee enzy me): Cys-29 - Cly-32 (Cys-9 - Gly-12). Asp-42 - Cys-51 (Asp-28 - Cys-37) and I'he-94 ~ Cys-105 (Leu-59 - Cys-70). Disulfide bridges ar e represented by do tted l ines an d the part icipating sulfur atomsby lilled circles. The larger circle represents the CaZ+on. Bonds along the peptide backbone are emphasized by thick l ines. In ( A ) the sidechains of residues that ar e idcntical in the bovine and the bee enzymes are shown (num bers refer to the bovine sequence). Residues that areimpo rtant for catalysis (His-48, Asp-99). ion bind ing (Asp- 49) an d the thrcc disulfide bridges (Cys-29 - Cys-45, Cys-44 - Cys-105 and Cys-51 ~ Cys-98) are conserved in the two structures. In (B), the side chains of all the residues of the bovine enzyme in these segments are replacedby those of the bee sequence. Most of thcsc replacements point away from the active site. Ala-102, Ala-103, and Phe-106 arc replaced by Phc.Tyr, a n d Leu (not shown in the figure) in the bee enzyme. These replacements are not conservative, yct they provide a strong hydrophobicenvironment around the active si tc in the bee enzymedicating tha t a single, rath er sm all gene for this enzym e existsin the genome of A . mel l fera (da ta no t shown) .A comp arison of the ami no acid sequence of bee venomphosphol ipase A 2 determined by Shipol ini et al. [13, 151 a n dthe sequence deduced f rom the c loned cD NA shows tha t thetwo a re ident ical in the am ino- an d carboxy-terminal regions,except that Asn-39 and Asn-92 are both Asp in the clonedcDNA. However, in the middle region ten differences werefound and these parts of the two sequences are compared inFig. 4. After residue 50, the cDNA sequence contains s ixaddi t ional codons including one for cysteine. A secondcysteine codo n was foun d at a posi t ion where Asn w as detect-ed in the publ ished am ino acid sequence.

The revised sequence deduced from the cloned cDNAdemonstrates notable similarities between the insect and thevertebrate enzymes. M oreove r, the extra cysteine residues callinto question the disulfide bridges previously determined a tthe protein level [16]. Rather, by comparison with the aminoacid sequences of vertebrate phosphol ipases A2 , al ternativedisulfide bridges seem to be more likely. The similarity be-tween the insect and vertebrate enzymes is outlined in Fig. 5 .This comparison reveals several common elements in thcvicinity of the active site but also substantial differences (seeDiscussion).In order to assess this similarity further, we tried to fitparts of the amino acid sequence of the bee enzyme into the

three-dimensional s t ructure of the pancreat ic enzyme. Theresul ts of such an at tem pt are show n in Fig. 6. Three segmentswere taken from the known X-ray s t ructure of bovine pancre-at ic phosphol ipase A2 [6] which form the active site and theregion which binds the Ca2' ion. As shown in Fig. 6 A , th eresidues essential for catalytic activity and binding of the C a2ion, a s well as thc disulfide bridges, are conservcd in the beevenom enzyme. In these segm ents , the am ino acid s ide chainsof the bovine enzyme were then replaced with the correspond-ing residues of the bee venom enzyme (see Fig. 6B). Indeed.the bee venom sequence can readily be accommodated in thethree dimensional s t ructure of the bovine enzyme.

D ISCU SSIO NPhosphol ipase A 2 is a ma jor const i tuent an d the principalallergen [ I 4,301 of worker bee venom const i tut ing 10- 5 %of its dry mass [I 21. The synthesis of this enzyme appe ars tobe caste specific since it is virtually absent fro m the veno m ofqueen bees [20]. Accord ing to ou r cDN A sequence da ta , thebee venom enzym e is derived from a precu rsor s tart ing with

B signal peptide typical for secreted polypeptides. Based onthe predictions derived by von Heijne [31], signal peptidasemost likely cleaves after Ser-11 or Gly-13 of the sequenceshown in Fig. 1 . The pro-region would then comprise 17 o r


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2531.5am ino acids and thu s be more th an twice as long as that ofpancreat ic phosphol ipases [I]. In the form er case, the amino-terminal sequence of bee venom pro-phosp hol ipase A 2, i. e .His-Gly-Trp-Gln-Ile-&g-Asp-Arg-!e-, would in fact showsome similari ty to the am ino-terminal end of the m ature po r-cine pancreat ic enzyme Ala-Leu-Trp-Gln-Phe-Arg-Ser-Met-k- com mon residues a re underl ined) (see [ I , 31). Activat ionof the bee pro-phosphol ipase involves hydrolysis after thesingle arginine residue at the COOH-terminal of the pro-peptide. In this respect, it thus resembles the mammalianrather than the rept i l ian enzymes [I , 2,10, 111. It is notewor thythat the l iberat ion of the bee venom pep t ide secapin from i tsprecursor [I91 also involves cleavage after a single arginineresidue. Conversely, the biosynthesis of meli t t in , the maincomponent of honeybee venom, proceeds via the s tepwisecleavage of dipep t ides f rom the amino end [32]. At least twodifferent act ivat ion m echanisms for pro-polypept ides thus ap-pear to exist in these glands.The sequence of the m ature bee venom enzyme as deducedfrom the cloned cD N A sequence com prises 134 am ino acids .Apa rt from two Asn-Asp replacements , i t i s ident ical t o theamino acid sequence determined almost 20 years ago byShipolini et al. [13, 151 in the amino - an d carboxy-terminalparts. However, as summarized in Fig. 4, several differenceswere detected in th e central region. T he earl ier conclusionsthat n o similarity exists between the ve rtebrate a nd thehoney bee enzyme have to be reconsidered on the basis of o u rrevised structu re. I t is noteworthy that several key features ofthe two cen tral helices surrou ndin g the active site of vertebrateenzymes [ I , 5-71 a r e also present in the bee enzym e. Theseinclude the conserved sequences Cys-Gly-Xaa-Gly, Cys-Cys-Xaa-Xaa-His-Asp-Xaa-Cys and Cys-Xaa-Cys-Asp-Xaa-Xaa-Xaa-Xaa-Xaa-Cys (where Xaa s t ands fo r any of th e 20amino acids) . In bovine pancreat ic phosphol ipase A 2 , six ofthe cysteines present in these fragments are known to formthree disulfide bridges. Moreover, the histidine in the secondand the aspart ic acid residue in the thi rd fragm ent have beenshown to be par t of the active site, while the two glycineresidues in the first and th e aspa rtic acid in the seco nd peptideare involved in binding Ca 2 ions. We th us conside r it l ikelythat the same funct iona l roles can be assigned to the corre-sponding amino acids in the bee venom enzyme. Indeed, asshown in Fig. 6, these parts of the bee enzyme sequence canreadily be accommodated in the central helices previouslydetermined for a pancreat ic phosphol ipase A2 [6]. These simi-lari ties in the f i rs t two segm ents ment ioned above have in factpreviously been noted by Maraganore et al . [33] ,who havealready quest ioned the arrangement of disulfide bridges asdetermined by Shipol ini et al . [I61 and specu la ted abou t apossible similarity between the bee venom and the vertebrateenzymes [33] .Several other am ino acids which are inva riant in the ver-tebrate enzymes are, however, changed or absent in thehoneybee enzyme. Of the proton-relay system proposed forthe act ive s i te of panc reat ic phosphol ipase A 2 [ I , 5 , 61, o n l yHis-48 and Asp-99 ar e present in the bee enzyme i n apparent lyidentical positions. Of the oth er residues i n this structur e, Tyr-52 is replaced by p roline an d no counterpar t s fo r Ala- I , Gln-4, Pro-68 and Tyr-73 are found in the primary sequence of thebee enzyme. On the other hand, of the four amino acidsforming the proposed Ca2+-binding i te, only Tyr-28 of thevertebrate enzymes, which supplies one carbonyl group, isreplaced by tryptophan.Compared to the mammal ian and snake enzymes , thehoneybee enzyme is shorter by 20 amino ac ids a t the amino

end . Interestingly, in the sequence of th e first 39 am ino acidsof the phosphol ipase A 2 from the venom of a l izard, 22 a reidentical to the bee venom sequen ce [17]. It w ould cleariy beof som e interest to know whether the other parts of the se-quence of this phosphol ipase are more s imilar to other ver-t ebra te enzymes o r to the bee enzyme.In the middle region, the insect enzyme lacks a segment of21 amino ac ids which fo rms a loop on the su r face o f thethree-dimensional s t ructure of bovine phospho l ipase A2 [6,71.Conversely, the honeybee enzyme has a n insert of six residuesbetween th e region involved in the binding of Ca2+ ions (Cys-Gly-Xaa-Gly) a nd the act ive s i te his t idine, an d ha s a tail of44 addi t ional amino acids at the carboxyl end. This lat terregion also con tains three cysteines, of which one m us t makea disulfide bridge with th e first cysteine in the conserved Cys-Xaa-Cys-Asp sequence.In o ur view, this comp arison of verteb rate with a n invert -ebrate phosphol ipase A 2 reveals com mo n features typical forproteins having a common evolut ionary origin. I t would bemost unlikely that the identical spacing of cysteines and theadjacent essential amino acids as out l ined above could havebeen formed by convergent evolut ion. Rather, we favor theview that these parts in these enzymes are the products ofdivergent evolut ion originat ing f rom a comm on ances to r . Asfor the observed s imilari ty between the amino-terminal se-quences of the bee an d l izard venom phosphol ipases , severalexplanat ions could be proposed. One possibil ity is that , earlyin the evolut ion of the corresponding genes, a duplicationoccurred with independent l ines leading to the bee/ l izard andthe snake/mammalian pancreas types, respect ively. On theother hand, the lizard enzyme may in fact be closely relatedto the other vertebrate enzymes in all par t s bu t the amino-terminal region. Other reasons, l ike exon shuffling may thenaccount for this localized similarity between two enzymesfrom widely different sources.I t should, however, be noted that in some parts of th eprimary sequence the differences between the honeybee andthe typical vertebra te phosphol ipases are substan t ial indeed,much larger than usual ly encountered when comparingcytochromes c, glycolytic enzymes etc. from different phyla[34]. As m ent ioned abo ve, several am ino acids which a re partof the cha rge relay system surrou nding the act ive s i te of ver-tebrate phosphol ipases are ab sent in the s t ructure of the insectenzyme. Curr ent evidence is insufficient to decide wh ether inthe bee enzyme a different active site is present or whetherresidues from other parts , part icularly the carboxy-terminalextension, perform analogou s funct ions.

We t hank D r F. G. Prendergast (Mayo Medical School, Roches-ter, Minnesota) for the antibody against bee venom phospholipase,an d one of the ano nym ous rererecs for calling our at tention to [33]and the possible similari ty between the am ino-termina l sequences ofbee venom prophosphol ipase A2 and the m ature enzyme f rom porcinepancreas. Part of thi s work was suppor ted by grant number S29T4from the Austriun Fonds zu r Fiirderung de r ,vissc~nschuftlicl~enForschung.

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analysis of the cdna for phospholipase a2 from honeybee venom glands

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