Dafin maciji yana ƙunshe da enzymes waɗanda ke sanya haɗin gwiwar carbonyl ester.Abubuwan da ake amfani da su don hydrolysis sune phospholipids, acetylcholine da acetate aromatic.Wadannan enzymes sun hada da nau'i uku: phospholipase, acetylcholinesterase da aromatic esterase.Arginine esterase a cikin dafin maciji kuma yana iya yin amfani da arginine na roba ko lysine, amma galibi yana hydrolyzes furotin peptide bond a yanayi, don haka nasa ne na protease.Enzymes da aka tattauna a nan kawai suna aiki akan abubuwan ester kuma ba za su iya aiki akan kowane haɗin peptide ba.Daga cikin waɗannan enzymes, ayyukan nazarin halittu na acetylcholinesterase da phospholipase sun fi mahimmanci kuma an yi nazari sosai.Wasu dafin maciji suna da aikin esterase mai ƙamshi mai ƙarfi, wanda zai iya haɓaka p-nitrophenyl ethyl ester, a - ko P-naphthalene acetate da indole ethyl ester.Har yanzu ba a sani ba ko an samar da wannan aikin ta hanyar enzyme mai zaman kanta ko kuma sanannen sakamako na carboxylesterase, balle mahimmancin ilimin halitta.Lokacin da aka mayar da dafin Agkistrodon halys Japonicus tare da p-nitrophenyl ethyl ester da indole ethyl ester, ba a samo hydrolysates na p-nitrophenol da indole phenol ba;Akasin haka, idan waɗannan esters suka amsa da dafin macizai na Zhoushan dafin macizai na Bungarus multicinctus, za su yi sauri da ruwa.An san cewa waɗannan dafin cobra suna da aiki mai ƙarfi na cholinesterase, wanda zai iya zama alhakin hydrolysis na abubuwan da ke sama.A zahiri, Mclean et al.(1971) ya ruwaito cewa yawancin dafin macizai na dangin Cobra na iya yin amfani da indole ethyl ester, naphthalene ethyl ester da butyl naphthalene ester.Wadannan dafin macizai sun fito ne daga: macijiya, macijiya mai bakin wuya, bakar lebe, macijin zinare, macijin Masar, macijin macijiya, mamba na zinare, bakar mamba da farar leben mamba (D. aw har yanzu ya san macijin rhombola na gabas.
Dafin maciji na iya yin hydrolyze methyl indole ethyl ester, wanda shine sinadari don tantance ayyukan cholinesterase a cikin jini, amma wannan dafin maciji baya nuna ayyukan cholinesterase.Wannan yana nuna cewa akwai wani esterase da ba a san shi ba a cikin dafin cobra, wanda ya bambanta da cholinesterase.Don fahimtar yanayin wannan enzyme, ana buƙatar ƙarin aikin rabuwa.
1. phospholipase A2
(I) Bayani
Phospholipase wani enzyme ne wanda zai iya yin hydrolyze glyceryl phosphate.Akwai nau'ikan phospholipase guda biyar a cikin yanayi, wato phospholipase A2 da phospholipase.
A., phospholipase B, phospholipase C da phospholipase D. Dafin maciji ya ƙunshi phospholipase A2 (PLA2), wasu dafin macizai sun ƙunshi phospholipase B, da sauran phospholipases galibi ana samun su a cikin kyallen jikin dabbobi da ƙwayoyin cuta.Hoto 3-11-4 yana nuna wurin aikin waɗannan phospholipases akan hydrolysis na substrate.
Daga cikin phospholipases, an ƙara nazarin PLA2.Yana iya zama mafi binciken enzyme a cikin dafin maciji.Matsakaicin sa shine haɗin ester akan matsayi na biyu na Sn-3-glycerophosphate.Ana samun wannan enzyme a ko'ina a cikin dafin maciji, dafin kudan zuma, dafin kunama da kyallen dabbobi, kuma PLA2 yana da yawa a cikin dafin macizai na iyali guda huɗu.Saboda wannan enzyme yana karya jajayen ƙwayoyin jini kuma yana haifar da hemolysis, ana kiransa "hemolysin".Wasu mutane kuma suna kiran PLA2 hemolytic lecithinase.
Ludeeke da farko ya gano cewa dafin maciji na iya samar da sinadarin hemolytic ta hanyar yin aiki da lecithin ta hanyar enzymes.Daga baya, Delezenne et al.ya tabbatar da cewa idan dafin kumbura ya yi aiki akan maganin doki ko gwaiduwa, yana samar da sinadarin hemolytic.Yanzu an san cewa PLA2 na iya yin aiki kai tsaye a kan phospholipids na erythrocyte membrane, lalata tsarin erythrocyte membrane kuma haifar da hemolysis kai tsaye;Hakanan yana iya yin aiki akan magani ko ƙara lecithin don samar da lecithin hemolytic, wanda ke aiki akan jajayen ƙwayoyin jini don samar da hemolysis na kai tsaye.Kodayake PLA2 yana da yawa a cikin iyalai huɗu na dafin maciji, abubuwan da ke cikin enzymes a cikin dafin maciji daban-daban sun ɗan bambanta.Rattlesnake (C
Dafin maciji ya nuna raunin aikin PLA2 kawai.Shafin 3-11-11 yana kwatanta kwatankwacin ayyukan PLA2 na manyan dafin macizai guda 10 a kasar Sin.
Table 3-11-11 Kwatanta ayyukan phospholipase VIII na dafin maciji 10 a kasar Sin
Dafin maciji
Sakin mai
Aliphatic acid,
Cjumol/mg)
Ayyukan Hemolytic CHU50/^ g * ml)
Dafin maciji
Saki fatty acids
(^raol/mg)
Ayyukan Hemolytic" (HU50/ftg * 1111)
Najaja atra
9.62
goma sha daya
Microcephalo ophis
maki biyar sifili daya
kalyspallas
8.68
dubu biyu da dari takwas
gracilis
V, akutu
7.56
**
Ophiophagus hannah
maki uku takwas biyu
dari da arba'in
Bnugarus fasctatus
7,56
dari biyu da tamanin
B. multicinctus
maki daya tara shida
dari biyu da tamanin
Viper da russelli
maki bakwai sifili uku
T, mucrosquamatus
maki daya takwas da biyar
Siamensis
T. stejnegeri
0.97
(2) Rabuwa da tsarkakewa
Abin da ke cikin PLA2 a cikin dafin maciji yana da girma, kuma yana da ƙarfi ga zafi, acid, alkali da denaturant, don sauƙin tsarkakewa da raba PLA2.Hanyar gama gari ita ce fara aiwatar da tacewar gel akan dafin dafin, sannan aiwatar da chromatography musayar ion, kuma ana iya maimaita mataki na gaba.Ya kamata a lura cewa daskarewa-bushewa na PLA2 bayan ion-exchange chromatography bai kamata ya haifar da haɗuwa ba, saboda tsarin bushewa daskarewa sau da yawa yana ƙara ƙarfin ionic a cikin tsarin, wanda shine muhimmin mahimmanci wanda ke haifar da tarawa na PLA2.Baya ga hanyoyin gabaɗayan da ke sama, an kuma ɗauki waɗannan hanyoyin: ① Wells et al.② Ana amfani da analog ɗin substrate na PLA2 azaman ligand don chromatography na alaƙa.Wannan ligand na iya ɗaure zuwa PLA2 a cikin dafin maciji tare da Ca2+.Ana amfani da EDTA mafi yawa azaman eluent.Bayan an cire Ca2 +, alaƙar da ke tsakanin PLA2 da ligand tana raguwa, kuma ana iya rabuwa da ligand.Wasu suna amfani da maganin 30% na halitta ko 6mol/L urea azaman eluent.③ An yi chromatography na hydrophobic tare da PheiiylSephar0SeCL-4B don cire alamar PLA2 a cikin cardiotoxin.④ Anti PLA2 antibody an yi amfani dashi azaman ligand don yin chromatography na alaƙa akan PLA2.
Ya zuwa yanzu, an tsarkake ɗimbin dafin maciji PLAZ.Tu et al.(1977) da aka jera PLA2 da aka tsarkake daga dafin maciji kafin 1975. A cikin 'yan shekarun nan, an ba da rahoton babban adadin labarai game da rabuwa da tsarkakewa na PLA2 kowace shekara.A nan, mun mai da hankali kan rabuwa da tsarkakewar PLA daga malaman kasar Sin.
Chen Yuancong et al.(1981) ya raba nau'ikan PLA2 guda uku daga dafin Agkistrodon halys Pallas a cikin Zhejiang, wanda za'a iya raba su zuwa acidic, tsaka tsaki da alkaline PLA2 bisa ga abubuwan da suka dace.Dangane da gubarsa, tsaka tsaki PLA2 ya fi mai guba, wanda aka gano azaman presynaptic neurotoxin Agkistrodotoxin.Alkaline PLA2 ba shi da ɗanɗano mai guba, kuma PLA2 acidic kusan ba shi da guba.Wu Xiangfu et al.(1984) idan aka kwatanta da halayen PLA2 guda uku, ciki har da nauyin kwayoyin halitta, amino acid abun da ke ciki, N-terminal, isoelectric point, thermal kwanciyar hankali, aikin enzyme, mai guba da aikin hemolytic.Sakamakon ya nuna cewa suna da nauyin kwayoyin halitta iri ɗaya da kwanciyar hankali na thermal, amma suna da bambance-bambance masu mahimmanci a wasu bangarori.A cikin yanayin aikin enzyme, aikin enzyme acid ya fi girma fiye da aikin enzyme na alkaline;Tasirin hemolytic na enzyme alkaline akan ƙwayoyin jajayen jinin bera shine mafi ƙarfi, sannan enzyme tsaka tsaki ya biyo baya, kuma enzyme acid ɗin da kyar aka samu.Saboda haka, ana hasashen cewa tasirin hemolytic na PLAZ yana da alaƙa da cajin kwayoyin PLA2.Zhang Jingkang et al.(1981) sun sanya Agkistrodotoxin lu'ulu'u.Tu Guangliang et al.(1983) ya ba da rahoton cewa PLA mai guba tare da madaidaicin isoelectric na 7. 6 an keɓe kuma an tsarkake shi daga dafin Vipera rotundus daga Fujian, da abubuwan da ke cikin jiki da sinadarai, abun da ke tattare da amino acid da jerin ragowar amino acid 22 a N. - an ƙaddara ta ƙarshe.Li Yuesheng et al.(1985) keɓe kuma ya tsarkake wani PLA2 daga dafin Viper rotundus a Fujian.Subunit na PLA2 * shine 13 800, madaidaicin isoelectric shine 10.4, kuma takamaiman aikin shine 35/xnioI/miri mg. An yi wa LD5 allura ta hanyar jijiya a cikin beraye.0.5 ± 0.12mg/kg.Wannan enzyme yana da tasirin anticoagulant da tasirin hemolytic.Kwayoyin PLA2 mai guba ya ƙunshi ragowar 123 na nau'ikan amino acid 18.Kwayoyin kwayoyin suna da wadata a cikin cysteine (14), aspartic acid (14) da glycine (12), amma kawai ya ƙunshi methionine guda ɗaya, kuma N-terminal shine ragowar serine.Idan aka kwatanta da PLA2 wanda Tuguang ya keɓe, nauyin kwayoyin halitta da adadin ragowar amino acid na isoenzymes guda biyu suna da kama da juna, kuma abun da ke tattare da amino acid shima yayi kama da juna, amma adadin aspartic acid da ragowar proline ya ɗan bambanta.Guangxi sarkin macijin dafin maciji ya ƙunshi wadataccen PLA2.Shu Yuyan et al.(1989) keɓe PLA2 daga dafin, wanda ke da takamaiman aiki sau 3.6 sama da dafin asali, nauyin kwayoyin halitta na 13000, abun da ke tattare da ragowar amino acid 122, madaidaicin isoelectric na 8.9, da kwanciyar hankali mai kyau.Daga na’urar duban na’ura mai kwakwalwa ta lantarki na tasirin PLA2 na asali akan jajayen kwayoyin halittar jini, ana iya ganin cewa yana da tasirin gaske a jikin jajayen kwayar halittar dan adam, amma ba shi da wani tasiri a fili kan kwayoyin jajayen jinin akuya.Wannan PLA2 yana da tabbataccen tasiri na jinkiri akan saurin electrophoretic na jajayen ƙwayoyin jini a cikin mutane, awaki, zomaye da aladun Guinea.Chen et al.Wannan enzyme zai iya hana tarin platelet wanda ADP, collagen da sodium arachidonic acid suka haifar.Lokacin da PLA2 maida hankali ne 10/xg/ml~lOOjug/ml, an hana tara platelet gaba ɗaya.Idan an yi amfani da platelets ɗin da aka wanke azaman kayan aiki, PLA2 ba zai iya hana haɗuwa ba a yawan adadin 20Mg/ml.Aspirin shine mai hana cyclooxygenase, wanda zai iya hana tasirin PLA2 akan platelets.PLA2 na iya hana haɗuwar platelet ta hydrolyzing arachidonic acid don haɗa thromboxane A2.An yi nazarin tsarin maganin PLA2 da Agkistrodon halys Pallas dafin dafin ya yi a lardin Zhejiang ta hanyar dichroism na madauwari, haske da kuma sha UV.Sakamakon gwaje-gwajen ya nuna cewa babban sarkar da ke tattare da wannan enzyme ya yi kama da irin nau'in nau'in enzyme daga wasu nau'o'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i) nau'i-nau'.Haɗin mai kunnawa Ca2 + da enzyme baya shafar yanayin ragowar tryptophan, yayin da mai hana Zn2 + yayi akasin haka.Hanyar da darajar pH na bayani ke shafar aikin enzyme ya bambanta da na sama reagents.
A cikin aiwatar da tsarkakewar PLA2 na dafin maciji, wani abu a fili shine cewa dafin maciji ya ƙunshi kololuwa biyu ko fiye da PLA2.Ana iya bayyana wannan lamari kamar haka: ① saboda kasancewar isozymes;② Ɗaya daga cikin nau'in PLA2 an yi shi ne a cikin nau'i-nau'i na PLA2 tare da nau'i-nau'i daban-daban, yawancin su suna cikin kewayon 9 000 ~ 40 000;③ Haɗin PLA2 da sauran abubuwan dafin maciji yana dagula PLA2;④ Saboda haɗin amide a cikin PLA2 yana da ruwa, cajin yana canzawa.① Kuma ② sun zama ruwan dare, tare da ƴan kaɗan kawai, kamar PLA2 a cikin dafin maciji na CrWa/w
Akwai yanayi guda biyu: ① da ②.An gano yanayi na uku a cikin PLA2 a cikin dafin macizai masu zuwa: Oxyranus scutellatus, Parademansia microlepidota, Bothrops a ^>er, Viper Palestine, Viper sand, da mummunan rattlesnake km.
Sakamakon yanayin ④ yana sa saurin ƙaura na PLA2 ya canza yayin electrophoresis, amma abun da ke tattare da amino acid baya canzawa.Ana iya karya peptide ta hanyar hydrolysis, amma gabaɗaya har yanzu ana ɗaure su tare ta hanyar haɗin disulfide.Dafin ramin ramin gabas ya ƙunshi nau'i biyu na PLA2, wanda ake kira type a da type p PLA2 bi da bi.Bambanci tsakanin waɗannan nau'ikan PLA2 guda biyu shine amino acid guda ɗaya, wato, glutamine a cikin kwayar PLA2 guda ɗaya ana maye gurbinsu da glutamic acid a cikin sauran kwayoyin PLA2.Kodayake ainihin dalilin wannan bambance-bambance ba a bayyana ba, an yi imani da cewa yana da alaƙa da deamination na PLA2.Idan PLA2 a cikin dafin viper na Falasdinu an kiyaye shi da dumi tare da dafin dafin, ƙungiyoyin ƙarshe a cikin ƙwayoyin enzyme ɗin sa zasu zama fiye da da.Daga C PLA2 ware daga dafin maciji yana da nau'in N-terminal guda biyu daban-daban, kuma nauyin kwayoyinsa shine 30000. Wannan al'amari na iya faruwa ta hanyar asymmetric dimer na PLA2, wanda yayi kama da simmetric dimer da PLA2 ya samar a cikin dafin gabashin diamondback rattlesnake. da yammacin diamondback rattlesnake.Asian wasan COBRA sun hada da yawancin sassa, waɗanda ba su da tabbatacciyar hanyar rarrabuwa.Misali, abin da a da ake kira Cobra Outer Caspian subspecies an gane yanzu
Ya kamata a dangana shi zuwa ga Cobra Sea Caspian Outer.Kamar yadda akwai wasu sassa da yawa kuma suna hade tare, tsarin da maciji ya bambanta sosai saboda kafofin daban-daban, da kuma abubuwan da ake ciki na isozymes ma sun yi yawa.Misali, dafin cobra
Akalla nau'ikan PLA2 isozymes 9 na nau'in r ll an samo su a ciki, kuma an sami nau'ikan isozymes 7 na PLA2 a cikin dafin nau'in cobra na Caspian.Durkin et al.(1981) yayi nazarin abubuwan da ke cikin PLA2 da adadin isozymes a cikin dafin maciji daban-daban, gami da dafin macizai guda 18, dafin mamba 3, dafin viper 5, dafin maciji 16 da dafin maciji na teku guda 3.Gabaɗaya, aikin PLA2 na dafin cobra yana da girma, tare da isozymes masu yawa.Ayyukan PLA2 da isozymes na dafin viper matsakaici ne.Ayyukan PLA2 na dafin mamba da dafin maciji ba su da yawa ko babu aikin PLA2.Ayyukan PLA2 na dafin maciji na teku kuma ba su da yawa.
A cikin 'yan shekarun nan, ba a ba da rahoton cewa PLA2 a cikin dafin maciji ya kasance a cikin nau'i na dimer mai aiki, irin su rhombophora rattlesnake na gabas (C. dafin maciji ya ƙunshi nau'in a da nau'in PLA2, dukansu sun ƙunshi nau'i biyu masu kama da juna. , kuma kawai dimerase yana da
Ayyuka.Shen et al.Hakanan ya ba da shawarar cewa kawai dimer na PLA2 na dafin maciji shine nau'in aikin enzyme.Nazarin tsarin sararin samaniya kuma ya tabbatar da cewa PLA2 na yammacin lu'u-lu'u rattlesnake yana wanzuwa ta hanyar dimer.Filayen Piscivorous
Akwai nau'ikan PLA ^ Ei da E2 daban-daban na dafin maciji, wanda 仏 ya kasance a cikin nau'in dimer, dimer yana aiki, kuma monomer ɗin da aka raba ba ya aiki.Lu Yinghua et al.(1980) ya kara nazarin abubuwan da ke cikin jiki da sinadarai da halayen halayen E. Jayanthi et al.(1989) keɓe ainihin PLA2 (VRVPL-V) daga dafin viper.Nauyin kwayoyin halitta na monomer PLA2 shine 10000, wanda ke da kisa, anticoagulant da tasirin edema.Enzyme na iya yin polymerize polymers tare da ma'auni daban-daban na kwayoyin halitta a ƙarƙashin yanayin PH 4.8, kuma digiri na polymerization da nauyin kwayoyin halitta na polymers yana ƙaruwa tare da karuwar zafin jiki.Nauyin kwayoyin halitta na polymer da aka samar a 96 ° C shine 53 100, kuma aikin PLA2 na wannan polymer yana ƙaruwa da biyu.
Lokacin aikawa: Nuwamba 18-2022