Molecular cloning and sequence analysis of cDNA for a fibrinolytic enzyme from the snake Vipera lebetina

questions. hybrid molcwulcs were conattCIcd bctwccn the r- or /I-subtmiI 01’ ths sodiunl pump and The prcscncc 01’ the l’usion <;itcs on surlilcc /I-galitcIosid;tsc of E. c.o/i and cxprcsscd in the yeasI S. twwi.vitw. or intracellular sides ol’ Ihe qcns~ plasma mcmhrane b) detc~tioti of the activity or expression ol’ 111~ rcportet cn;ryme /j-g;tl;tctosidase enables us IO propose ten mcmbr;cne-sp;iniiinr domains for the -s-subuniI and a sin& membrane path Ibr the /j-subunit of the sodium pump. These mcmblane domains and it shucture possibly similar IO the P-loop of Na’ and K ’ channels formed bctwccn Ihc sevenIh and eighth membrane span ol’ the z-subuniI mighI be involved in ion conduction and might rcflcc~ the sIructure that is arrested by palylouin inIo Ihc permanenlly open SIW. Based on this strucIural motlcl. muIants of the sodium pump were produced and expressed in yeast. Yeus~ cells do no1 contuin cndogenoua sodium pumps atid ttrz complctcly inscnsiItve ICI pall Ioxin. They became sensitive IO p;~lytoxitl. however. when ~hcy cxpreb\ed mammalian sodium pumps. and A catalytically active sodium pump IOSI their intr;tccllular K - in cxchangc for atrucellular monov;~lcn~ calions. WIS not rquircd Ibr this phsnomsnott. which can also occur whh mutanI sodium p~mtps incapable ol’ ATP hydrolysis. Mutations within the ““PL PI .““* squcncc from the prcdictctl lilih mcmbranc span of the 2-subunit alWed palytoxin-induced K . eHlux I’ront ycnst ccllx. Simih~r obscrb~lions were tl:ndc wiIh muI;ttiotts of ~hr: r’rLID’X’ scqucnce from Ihc hypoIhctic:tl P-loop ol’ IIIC sodium pump. indicating th;II this ~lructure might ‘11~~) be involved in ion recognition and transport. III agrccmcnl wilh this ~OSIU~~IIC. the muttlnI cnzymcs retained their ability to recognize K ions bttI 111~ I)*“~-+ R‘“‘.-mutant did not atslain u Nit‘-promoted &osphorylation by ATP. These results. and Ihe I’,tct Ihat rtnlvh~xin caused da~clusion of ‘“Rb’ from kidney sodium IXI~~. indicalc that palytoxin consIitutc* ;I novel tool &r ~hr invrstigatton 01‘ rhc ionophore part of’ihc sodi& ptimp. ‘They also show what a P-loop-like strttcturc similar to I~;II ()I’ N;I or K ~~hanncls might hc imolvcd in ion recognition and conduction by 111~ sodium punip.

Venoms consist ol’ a variety 01’ pathogcttic constiIucnts which cuuse hacmorrhagc. ncttrotoxici~y. necrosis. ZIG.. ;mtl in order IO WIIWIC antitcnotns. I~C’W conditions musi bc induced in animal mc~dcl~. The United Kingtlonl Home Office guidclincs to Kc&Ice. Rcpk~cs itnd Reline itnimal cxpcrimoitution led IIS IO dcvclop ZIII nlIcrn;~~ivc assay to the sI;mdnrd rodent skin tcsl. WC prcscni h?re :I shell-ICSS chick anhryo ntodcl libr the c~nltt;~~it~n 01’ ;Inli-h;tcmorrlle~i~ agents. In the slundard hkin test assay. mice arc injected intritdcrnt;tll\ I i.d.1 H iIh ;I minimum hacmorrhapic dose (hIHI>) ol’ vcnont sullicicnt lo GIIISC a Icsion 01’ III mm Ji;mteIcr wilhin 33 111. Anli-hucmc)rrh;t~i~ agents untlcr ICSI arc incuhalcd with lhc MHD ol’vcnom Ihr 30 min at 37 (’ and the ntiuturc injected i.cl. Mice are killed 2-l hr lacr and ~hc Jegrce of hacmorrhugc in rhc skin is rccortlctl. Our ;dIcrn;tti\.r’ ass,ty uses I’ertilc hens’ eggs \hhich have ;I highly vascl.tl;trl/rd >olk sac mcmbrnnc at a vcrj c;trly d~\clol~mcnr;~l 7. liltcr-paper discs imprcgnntcd u-iIh venom ;tntivcn~>m mixtures arc pl;~cal on the yolk WC’ mcmbr;tnc ov’cr ;I blood. vcs~l. T\vo and a hnll hour3 lutcr. the dcgrcc oI’hacmc,rrhitg is csnm~nsd ;md the rcsulIs are photogr;tl~hccl. ‘l’o\icitL may ;IISO h c a~sscd ht 111~ viahiliry 01’ the embryo. (.:sing cq:tin< ;imivcnom. mouse ntonocloii~~l anlihoilic*. pl;uil c’xlrilcIs mid live thlliirsnt venoms. the cfg ;~s*ay llilh prca,;dai clear-cuI results which parnllcl lhc rodent skin ;ISI~. The kincIic> ilnd mode ~~l’;tcIion ~‘l’;ltlli-ll;~cmorrl~;lpic ;I;?~III\ arc currcmly Ixing studied in this model. which ha the atlvan~npc 01’ convcnicnI observation 01’ rhc \itc:tlittc’ ~~scuI;~turc throughouI lhc period 01‘ 111~ cxpcrimcttt The egg ;ISS;I> is n \unsiti\c. simple. ethically ;tcccpt;~bl~ antI very cost-clrcctive I I I pqg or I3 nh~uscl tcrccii Ir)r ;tiili-llarinorrh;t~i~ ;~gcnIs.

Snake venoms 01’ C‘roI;tliJas ;mrl Vlpcrid;ls liuniliss cabntnin librinolyiic cn~ymss. which hytlr~~l~\c librin rind librinogcn directly withoui an? ,pl;lsminogcn ;Ictiv;ttion. ‘1’11~ lihrinolyiir cn~yntc Inamcd Icbctaxc) l’ronl I .i;wr:r /c,k,rir~r v~om has been idenultcd an:1 purilicd to homogcneit). It is ;I Gnglcchnir t~~ct;~llopmtsit~;ts~ uith ;II~ apparctil mol. wl 01’ 23.700. I .~hct;isc rcndily cle;tvcs I.hc 4r-chain and nitm2 slowI> the H/&zhniii 01’ lihrinogcn. In librin. ~hc same chains arc ;ttt;tckctl. Oli~~~nttclcotldcs dcsipnrd on ihe basis ol’ the internal pspt~dc squsnur 01’ Ichct~ (the N-lcrmm;d 01‘ Ichstac ix hlo&d) and 01‘ the highly co~~sc~~vcd domains scq~~cncc nl’ ;II~;I~O~CUI~ mctallopn~leini~ses terc used its priniCrr in the polymcriisc chain reactIon to iimplil’y :I ~l)“\I,4 I’rqmcnt I’rom the I ‘. /cWi~cr venom gland cl)Nt\ library. ‘I’hc rcsultinp cDKA li;tgmcnt W:I~ sub~~lonctl. scqttcttccd ;mhl\ lihr;lr! prepared I’rom ~ltc vc’nom gI;~nds. TIIC dcd LICL’~ ~otnl protein scqucncc consists ol’an I b iimino Xid?; sigiinl pcp!idc. ;I /ymogcn propeptitle 01’ l7i aminr~ .Icitl\ and ;I miilurc protein tbl’ 20-l imlino acids. :III~ ;I cill+OS~ Iermin;tl scgmctil 01’ XI amino ocid3. I.zhsl;lrc ii tl;insl;ttc~l it\ ii precursor protcin. which m;l) hc pro025sc.l posttransl;tIion;tlly. The W~UI*IICS or ICI~CI~W cuhihith :I high tlcgrec ol’scyucncc identity v.ith other sn;lkc \cnom

Report

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Absiracr~

817

fibrinol~ 1ic enzynes: fihrolaae from A,yLi.s/rncio~~ c.o~/rwt~i\ co~/w/ri\and atroxase from I:‘,-• /U/U.\ tr!r’o Y venom. The sequence homology oflehetase IS also relah\ely high with low mol. wt haemorrhagic Illetallopro~einases. The hqhly conserved cvaeine residues allow the prcdicllon of the same paltern of three disulfide bonds for lehetase as determined for fibrolase (Randolph r/ tri.. lYY2). ‘The sequence homology between lebetase and other members of the matrix metallopro1einase family is mobt remarkable in the region which binds the calalytic zinc atom. Removal of zinc eliminates all activity of lebetasc. Lebetare is the first fihrinolytic enzyme to he sequenced from Viperidae snake venom.

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