Ubuhlungu benyoka buqulethe i-enzymes eyenza i-hydrolyze ibhondi ye-carboxyl ester.I-substrates ye-hydrolysis yi-phospholipids, i-acetylcholine kunye ne-acetate enuka kamnandi.Ezi enzymes ziquka iindidi ezintathu: i-phospholipase, i-acetylcholinesterase kunye ne-esterase enuka kamnandi.I-Arginine esterase kubuhlungu benyoka isenako ukwenza i-hydrolyze ye-arginine yokwenziwa okanye i-lysine, kodwa ikakhulu i-hydrolyzes i-protein peptide bond kwindalo, ngoko ke iyeye-protease.I-enzymes ekuthethwa ngayo apha isebenza kuphela kwi-ester substrates kwaye ayikwazi ukwenza nayiphi na ibhondi ye-peptide.Phakathi kwezi enzymes, imisebenzi ye-biological ye-acetylcholinesterase kunye ne-phospholipase ibaluleke kakhulu kwaye ifundwe ngokupheleleyo.Ezinye iityhefu zenyoka zinomsebenzi onamandla we-esterase enevumba elimnandi, enokuthi i-hydrolyze i-p-nitrophenyl ethyl ester, i- okanye i-P-naphthalene acetate kunye ne-indole ethyl ester.Akukaziwa nokuba lo msebenzi uveliswa yi-enzyme ezimeleyo okanye isiphumo esaziwayo se-carboxylesterase, singasathethi ke ngokubaluleka kwebhayoloji.Xa i-venom ye-Agkistrodon halys Japonicus yaphendulwa nge-p-nitrophenyl ethyl ester kunye ne-indole ethyl ester, i-hydrolysates ye-p-nitrophenol kunye ne-indole phenol ayifumanekanga;Ngokuchasene noko, ukuba ezi esters zisabela nge-cobra Zhoushan subspecies i-snake venom kunye ne-Bungarus multicinctus snake venom, ziya kukhawuleza i-hydrolyzed.Kuyaziwa ukuba ezi zihlungu ze-cobra zinomsebenzi oqinileyo we-cholinesterase, onokuthi ube noxanduva lwe-hydrolysis ye-substrates engentla.Enyanisweni, uMclean et al.(1971) yabika ukuba uninzi lwetyhefu yenyoka yosapho lweCobra lunokwenza i-hydrolyze indole ethyl ester, naphthalene ethyl ester kunye nebutyl naphthalene ester.Obu buhlungu benyoka buphuma: iphimpi, iphimpi elimnyama, iphimpi elimilebe elimnyama, iphimpi legolide, iphimpi laseYiputa, i-king cobra, igolden cobra mamba, i-black mamba kunye ne-white lipped mamba (D. aw usayazi ieastern rhombola rattlesnake
Ubuthi benyoka bunokwenza i-hydrolyze i-methyl indole ethyl ester, eyi-substrate yokumisela umsebenzi we-cholinesterase kwi-serum, kodwa le tyhefu yenyoka ayibonisi umsebenzi we-cholinesterase.Oku kubonisa ukuba kukho i-esterase engaziwayo kwi-cobra venom, eyahlukileyo kwi-cholinesterase.Ukuze uqonde ubume bale enzyme, kufuneka umsebenzi owongezelelekileyo wokwahlula.
1, Phospholipase A2
(I) Isishwankathelo
I-Phospholipase yi-enzyme enokuthi i-hydrolyze i-glyceryl phosphate.Kukho iintlobo ezi-5 ze-phospholipase kwindalo, ezizezi, i-phospholipase A2 kunye ne-phospholipase.
A. , i-phospholipase B, i-phospholipase C kunye ne-phospholipase D. Ityhefu yenyoka ikakhulu iqukethe i-phospholipase A2 (PLA2), iintsholongwane ezimbalwa zeenyoka ziqukethe i-phospholipase B, kunye nezinye i-phospholipases zifumaneka ikakhulu kwizicubu zezilwanyana kunye neebhaktheriya.Umzobo 3-11-4 ubonisa indawo yesenzo sale phospholipases kwi-substrate hydrolysis.
Phakathi kwe-phospholipases, i-PLA2 ifundwe ngakumbi.Isenokuba yeyona enzyme ifundwayo kubuhlungu benyoka.I-substrate yayo ibhondi ye-ester kwindawo yesibini ye-Sn-3-glycerophosphate.Le enzyme ifumaneka ngokubanzi kwityhefu yenyoka, i-bee venom, i-scorpion venom kunye nezicubu zezilwanyana, kwaye i-PLA2 ininzi kwiintsholongwane ezine zeenyoka zentsapho.Ngenxa yokuba le enzyme iphula iiseli ezibomvu zegazi kwaye ibangele i-hemolysis, ikwabizwa ngokuba yi "hemolysin".Abanye abantu bakwabiza i-PLA2 hemolytic lecithinase.
U-Ludeeke waqala wafumanisa ukuba ubuthi benyoka bunokuvelisa ikhompawundi ye-hemolytic ngokusebenza kwi-lecithin ngokusebenzisa i-enzymes.Kamva, u-Deelezenne et al.yangqina ukuba xa ubuhlungu be-cobra busebenza kwiserum yehashe okanye i-yolk, yenza into ene-hemolytic.Ngoku ngoku iyaziwa ukuba i-PLA2 inokusebenza ngokuthe ngqo kwi-phospholipids ye-membrane ye-erythrocyte, ukutshabalalisa isakhiwo se-membrane ye-erythrocyte kwaye ibangele i-hemolysis ngqo;Inokuthi isebenze kwiserum okanye i-lecithin eyongeziweyo ukuvelisa i-lecithin ye-hemolytic, esebenza kwiiseli ezibomvu zegazi ukuvelisa i-hemolysis engathanga ngqo.Nangona i-PLA2 ininzi kwiintsapho ezine zetyhefu yenyoka, umxholo we-enzymes kwiityhefu ezahlukeneyo zenyoka wahluke kancinane.Rattlesnake (C
Ubuhlungu benyoka bubonise umsebenzi obuthathaka we-PLA2.Itheyibhile 3-11-11 ibonisa ukuthelekiswa komsebenzi we-PLA2 we-10 enkulu yetyhefu yeenyoka ezinobuhlungu e-China.
Itheyibhile 3-11-11 Ukuthelekiswa kwe-phospholipase VIII imisebenzi ye-10 yenyoka e-China
Ubuhlungu benyoka
Ukukhutshwa kwamafutha
Aliphatic acid,
Cjumol/mg)
Umsebenzi weHemolytic CHU50/^ g * ml)
Ubuhlungu benyoka
Khupha iiasidi ezinamafutha
(^raol/mg)
Umsebenzi weHemolytic “(HU50/ftg * 1111)
Najanaja atra
9. 62
Shumi elinanye
I-Micracephal ophis
amanqaku amahlanu omnye uziro
iikalyspallas
8. 68
amawaka amabini anamakhulu asibhozo
gracilis
V, i-acutus
7. 56
* * #
uOfiyophago hana
Amanqaku amathathu esibhozo amabini
ikhulu elinamashumi amane
Bnugarus fasctatus
7,56
amakhulu amabini anamashumi asibhozo
B. multicinctus
inqaku elinye elithoba ezintandathu
amakhulu amabini anamashumi asibhozo
Inyoka kunye neruselli
amanqaku asixhenxe nguziro wesithathu
T, mucrosquamatus
inqaku elinye isibhozo isihlanu
Siamensis
T. stejnegeri
0. 97
(2) Ukwahlula kunye nokuhlanjululwa
Umxholo we-PLA2 kwi-snake venom mkhulu, kwaye uzinzile ukutshisa, i-asidi, i-alkali kunye ne-denaturant, ukuze kube lula ukuhlanjululwa kunye nokwahlula i-PLA2.Indlela eqhelekileyo kukuqala ukwenza ukuhluzwa kwejeli kwityhefu ekrwada, emva koko wenze i-ion exchange chromatography, kwaye inyathelo elilandelayo linokuphinda liphindwe.Kufuneka kuqatshelwe ukuba ukumiswa komkhenkce kwe-PLA2 emva kwe-ion-exchange chromatography akufanele kubangele ukuhlanganiswa, kuba inkqubo yokumisa i-freeze ihlala inyusa amandla e-ionic kwinkqubo, eyona nto ibalulekileyo ebangela ukuhlanganiswa kwe-PLA2.Ukongeza kwezi ndlela zingasentla, ezi ndlela zilandelayo zamkelwe: ① Wells et al.② I-analogue ye-substrate ye-PLA2 isetyenziswe njenge-ligand ye-affinity chromatography.Le ligand inokubophelela kwi-PLA2 kwityhefu yenyoka kunye neCa2 +.I-EDTA isetyenziswa kakhulu njenge-eluent.Emva kokuba i-Ca2 + isusiwe, ubudlelwane phakathi kwe-PLA2 kunye ne-ligand buyancipha, kwaye bunokwahlulwa kwi-ligand.Abanye basebenzisa i-30% ye-organic solution okanye i-6mol/L urea njenge-eluent.③ I-Hydrophobic chromatography yenziwe ngePheiiylSephar0SeCL-4B ukususa umkhondo we-PLA2 kwi-cardiotoxin.④ I-anti-PLA2 ye-antibody yasetyenziswa njenge-ligand ukwenza i-affinity chromatography kwi-PLA2.
Ukuza kuthi ga ngoku, inani elikhulu leenyoka ze-PLAZ lihlanjululwe.Tu et al.(1977) idweliswe i-PLA2 ehlanjululwe kwi-snake venom ngaphambi kwe-1975. Kwiminyaka yakutshanje, inani elikhulu lamanqaku malunga nokuhlukana kunye nokucocwa kwe-PLA2 kuye kwabikwa minyaka yonke.Apha, sigxininise ekuhlukaneni kunye nokuhlanjululwa kwe-PLA ngabaphengululi baseTshayina.
Chen Yuancong et al.(I-1981) yahlula iintlobo ezintathu ze-PLA2 kwi-venom ye-Agkistrodon halys Pallas e-Zhejiang, enokuthi ihlulwe ibe yi-acidic, ingathathi hlangothi kunye ne-alkaline ye-PLA2 ngokweendawo zabo ze-isoelectric.Ngokobutyhefu bayo, i-PLA2 engathathi hlangothi inetyhefu ngakumbi, echongwe njenge-presynaptic neurotoxin Agkistrodotoxin.I-alkaline i-PLA2 ayinayo ityhefu encinci, kwaye i-PLA2 ene-acidic phantse ayinayo ityhefu.Wu Xiangfu et al.(I-1984) ithelekisa iimpawu ze-PLA2 ezintathu, kubandakanywa ubunzima be-molecular, ukubunjwa kwe-amino acid, i-N-terminal, i-isoelectric point, i-thermal stability, umsebenzi we-enzyme, ubuthi kunye nomsebenzi we-hemolytic.Iziphumo zibonise ukuba babenobunzima obufanayo bemolekyuli kunye nokuzinza kwe-thermal, kodwa babenolwahlulo olukhulu kwezinye iinkalo.Kwinkalo yomsebenzi we-enzyme, umsebenzi we-enzyme ye-asidi wawuphezulu kunomsebenzi we-alkaline enzyme;Impembelelo ye-hemolytic ye-alkaline enzyme kwiiseli zegazi ezibomvu zegundane yayiyeyona inamandla, ilandelwa yi-enzayimu engathathi hlangothi, kunye ne-asidi ye-enzayimu inqabile ukuba i-hemolyzed.Ngoko ke, kucingelwa ukuba umphumo we-hemolytic we-PLAZ unxulumene nentlawulo ye-molecule ye-PLA2.UZhang Jingkang et al.(1981) benze iikristale zeAgkistrodotoxin.Tu Guangliang et al.(1983) yabika ukuba i-PLA enetyhefu ene-isoelectric point ye-7. I-6 yahlukaniswa kwaye yahlanjululwa kwi-venom ye-Vipera rotundus esuka kwi-Fujian, kunye neempawu zayo zomzimba kunye neekhemikhali, ukubunjwa kwe-amino acid kunye nokulandelelana kwe-22 amino acid eseleyo kwi-N. -itheminali yamiselwa.ULi Yuesheng et al.(1985) yodwa kwaye yahlambulula enye i-PLA2 kwi-venom ye-Viper rotundus e-Fujian.I-subunit ye-PLA2 * yi-13 800, indawo ye-isoelectric yi-10.4, kwaye umsebenzi othile ngu-35 / xnioI / miri mg I-LD5 itofwa ngomthambo kwiimpuku.Yi-0.5 ± 0.12mg/kg.Le enzyme ineempembelelo ezicacileyo ze-anticoagulant kunye ne-hemolytic.Imolekyuli eyityhefu yePLA2 ineentsalela ezili-123 zeentlobo ezili-18 zeeamino acid.I-molecule icebile kwi-cysteine (14), i-aspartic acid (14) kunye ne-glycine (12), kodwa iqulethe i-methionine enye, kunye ne-N-terminal yayo i-serine residue.Xa kuthelekiswa ne-PLA2 eyodwa yi-Tuguang, ubunzima be-molecular kunye nenani leentsalela ze-amino acid ze-isoenzymes ezimbini zifana kakhulu, kwaye ukubunjwa kwe-amino acid kufana kakhulu, kodwa inani le-aspartic acid kunye ne-proline residues lihluke ngandlela-thile.I-Guangxi king cobra yenyoka inetyhefu ye-PLA2 etyebileyo.Shu Yuyan et al.(I-1989) ihlukanise i-PLA2 kwi-venom, enomsebenzi othile ngamaxesha angama-3.6 aphezulu kune-venom yasekuqaleni, ubunzima be-molecular 13000, ukubunjwa kwe-122 amino acid residues, i-isoelectric point ye-8.9, kunye nokuzinza okuhle kwe-thermal.Ngokujonga kwi-electron microscope yesiphumo se-PLA2 esisiseko kwiiseli ezibomvu zegazi, kunokubonwa ukuba inefuthe elicacileyo kwinwebu yeseli ebomvu yomntu, kodwa ayinasiphumo esicacileyo kwiiseli zegazi ezibomvu zebhokhwe.Le PLA2 inefuthe elicacileyo lokulibaziseka kwisantya se-electrophoretic seeseli ezibomvu zegazi ebantwini, iibhokhwe, imivundla kunye neehagu zeGuinea.UChen et al.Le enzyme inokuthintela ukuhlanganiswa kweplatelet eyenziwa yi-ADP, i-collagen kunye ne-sodium arachidonic acid.Xa ugxininiso lwe-PLA2 luyi-10/xg/ml~lOOjug/ml, ukuhlanganiswa kweplatelet kuthintelwe ngokupheleleyo.Ukuba iiplatelet ezihlanjiweyo zisetyenziswe njengezinto eziphathekayo, i-PLA2 ayinakunqanda ukuhlanganisana kwi-concentration ye-20Mg / ml.I-Aspirin yi-inhibitor ye-cyclooxygenase, enokuthintela umphumo we-PLA2 kwiiplatelet.I-PLA2 inokuthintela ukuhlanganiswa kweplatelet nge-hydrolyzing arachidonic acid ukuze idibanise i-thromboxane A2.Ukulungiswa kwesisombululo se-PLA2 eveliswe yi-Agkistrodon halys Pallas venom kwiPhondo laseZhejiang yaphononongwa nge-circular dichroism, fluorescence kunye nokufunxwa kwe-UV.Iziphumo zovavanyo zibonise ukuba ikhonkco eliphambili lokudibanisa le enzyme lalifana nolo lolo hlobo lwe-enzyme oluvela kwezinye iintlobo kunye nejenara, i-skeleton conformation yayinokumelana nokushisa okulungileyo, kwaye utshintsho lwesakhiwo kwindawo ye-asidi yayinokuguqulwa.Ukudibanisa kwe-activator Ca2 + kunye ne-enzyme ayichaphazeli indawo ye-tryptophan eseleyo, ngelixa i-inhibitor Zn2 + yenza okuchaseneyo.Indlela apho ixabiso le-pH lesisombululo lichaphazela umsebenzi we-enzyme lihluke kwii-reagents ezingentla.
Kwinkqubo ye-PLA2 yokuhlanjululwa kwetyhefu yenyoka, into ecacileyo kukuba ubuthi benyoka iqulethe iincopho ezimbini okanye ngaphezulu ze-PLA2 elution.Lo mcimbi unokuchazwa ngolu hlobo lulandelayo: ① ngenxa yobukho bee-isozymes;② Olunye uhlobo lwe-PLA2 lufakwe kwi-polymerized kwiindidi ze-PLA2 zemixube ezinobunzima beemolekyuli ezahlukeneyo, uninzi lwayo olukuluhlu lwe-9 000 ~ 40 000;③ Indibaniselwano yePLA2 kunye namanye amacandelo etyhefu yenyoka yenza nzima iPLA2;④ Ngenxa yokuba i-amide bond kwi-PLA2 i-hydrolyzed, intlawulo iyatshintsha.① Kwaye ② zixhaphakile, ngaphandle nje okumbalwa, okufana ne-PLA2 kwi-CrWa/w yetyhefu yenyoka
Kukho iimeko ezimbini: ① kunye ②.Imeko yesithathu ifunyenwe kwi-PLA2 kwityhefu yezi nyoka zilandelayo: i-Oxyranus scutellatus, iParademansia microlepidota, i-Bothrops a ^>er, inyoka yasePalestina, i-sand viper, kunye ne-rattlesnake eyoyikekayo km.
Isiphumo setyala ④ yenza isantya sokufuduka se-PLA2 sitshintshe ngexesha le-electrophoresis, kodwa ukwakheka kwe-amino acid akutshintshi.Iipeptides zinokophulwa yi-hydrolysis, kodwa ngokubanzi zisabotshwa kunye ngamabhondi e-disulfide.Ubuhlungu be-eastern pit rattlesnake iqulethe iindlela ezimbini ze-PLA2, ebizwa ngokuba yi-type a kunye nohlobo lwe-p PLA2 ngokulandelelanayo.Umahluko phakathi kwezi ntlobo zimbini ze-PLA2 yi-amino acid enye kuphela, oko kukuthi, i-glutamine kwi-molecule ye-PLA2 ithathelwa indawo yi-glutamic acid kwenye i-molecule ye-PLA2.Nangona esona sizathu salo mahluko singacacanga, ngokuqhelekileyo kukholelwa ukuba sinxulumene ne-deamination ye-PLA2.Ukuba i-PLA2 kwi-Palestinian viper venom igcinwa ishushu kunye netyhefu ekrwada, amaqela okugqibela kwiimolekyuli ze-enzyme aya kuba ngaphezulu kunangaphambili.Ukusuka kwi-C PLA2 eyodwa ukusuka kwityhefu yenyoka ine-N-terminal ezimbini ezahlukeneyo, kwaye ubunzima bayo bemolekyuli yi-30000. Le nto inokubangelwa yi-asymmetric dimer ye-PLA2, efana ne-symmetric dimer eyenziwe yi-PLA2 kwityhefu ye-diamondback rattlesnake yasempumalanga. kunye nentshona diamondback rattlesnake.I-Asian cobra yenziwe ngeentlobo ezininzi, ezinye zazo ezingaqinisekanga kakhulu kuhlelo.Umzekelo, into ebikade ibizwa ngokuba yiCobra Outer Caspian subspecies iyaqatshelwa ngoku
Kufuneka kubangelwe kwi-Outer Caspian Sea Cobra.Njengoko kukho ezininzi i-subspecies kwaye zixutywe kunye, ukubunjwa kwetyhefu yenyoka kuyahluka kakhulu ngenxa yemithombo eyahlukeneyo, kwaye umxholo we-PLA2 isozymes nawo uphezulu.Umzekelo, i-cobra venom
Ubuncinci zisi-9 iintlobo ze-PLA2 isozymes zohlobo lwe-r ^ ll zafunyanwa kuzo, kunye neentlobo ezisi-7 ze-PLA2 isozymes zafunyanwa kwityhefu ye-cobra subspecies iCaspian.Durkin et al.(I-1981) ifunde umxholo we-PLA2 kunye nenani le-isozymes kwiintsholongwane zenyoka ezahlukeneyo, kuquka i-18 ye-cobra venoms, i-3 ye-mamba venoms, i-5 ye-viper venoms, i-16 ye-rattlesnake venoms kunye ne-3 yenyoka elwandle.Ngokubanzi, umsebenzi we-PLA2 we-cobra venom uphezulu, kunye ne-isozymes ezininzi.Umsebenzi we-PLA2 kunye ne-isozymes ye-viper venom ziphakathi.Umsebenzi we-PLA2 we-mamba venom kunye ne-rattlesnake venom uphantsi kakhulu okanye akukho msebenzi we-PLA2.Umsebenzi we-PLA2 wetyhefu yenyoka yolwandle nawo uphantsi.
Kwiminyaka yakutshanje, akuzange kuxelwe ukuba i-PLA2 kubuhlungu benyoka ikhona ngendlela ye-dimer esebenzayo, njenge-eastern rhombophora rattlesnake (i-C. i-snake venom iqulethe uhlobo a kunye nohlobo lwe-PLA2, zombini eziqulunqwe ngamacandelo amabini afanayo. , kwaye yi-dimerase kuphela enayo
Umsebenzi.Shen et al.Kwakhona kwacebisa ukuba kuphela i-dimer ye-PLA2 yetyhefu yenyoka yindlela esebenzayo ye-enzyme.Uphononongo lobume besithuba lukwangqina ukuba i-PLA2 ye-diamondback rattlesnake yasentshona ikhona ngohlobo lwe-dimer.Ikhompawundi yePiscivorous
Kukho ezimbini ezahlukeneyo zePLA ^ Ei kunye ne-E2 yetyhefu yenyoka, apho i-仏 ikhona ngendlela ye-dimer, i-dimer iyasebenza, kwaye i-monomer yayo edibeneyo ayisebenzi.ULu Yinghua et al.(1980) wafunda ngakumbi iimpawu zomzimba kunye neekhemikhali kunye ne-reaction kinetics ye-E. Jayanthi et al.(1989) yakhetha i-PLA2 esisiseko (VRVPL-V) kwityhefu yerhamba.Ubunzima be-molecular of monomer PLA2 yi-10000, enobungozi, i-anticoagulant kunye nemiphumo ye-edema.I-enzyme inokwenza iipolymers ezinobunzima obuhlukeneyo beemolekyuli phantsi kwemeko ye-PH 4.8, kunye neqondo le-polymerization kunye ne-molecular weight of polymers yanda ngokunyuka kweqondo lokushisa.Ubunzima bemolekyuli bepolymer obuveliswe kwi-96 ° C yi-53 100, kwaye umsebenzi we-PLA2 wale polima unyuka ngamabini amabini.
Ixesha lokuposa: Nov-18-2022