Ululu wa njoka uli ndi ma enzymes omwe amatsitsimutsa bond carboxyl ester.Magawo a hydrolysis ndi phospholipids, acetylcholine ndi onunkhira acetate.Ma enzymes awa ali ndi mitundu itatu: phospholipase, acetylcholinesterase ndi onunkhira esterase.Arginine esterase mu njoka ya njoka imathanso hydrolyze synthetic arginine kapena lysine, koma makamaka hydrolyze mapuloteni peptide zomangira m'chilengedwe, choncho ndi protease.Ma enzyme omwe akukambidwa pano amangogwira ntchito pagawo la ester ndipo sangathe kuchitapo kanthu pamtundu uliwonse wa peptide.Pakati pa ma enzymes, ntchito zachilengedwe za acetylcholinesterase ndi phospholipase ndizofunikira kwambiri ndipo zaphunziridwa mokwanira.Njoka zina za njoka zimakhala ndi fungo lamphamvu la esterase, lomwe limatha hydrolyze p-nitrophenyl ethyl ester, a - kapena P-naphthalene acetate ndi indole ethyl ester.Sizikudziwikabe ngati ntchitoyi imapangidwa ndi enzyme yodziyimira payokha kapena mbali yodziwika ya carboxylesterase, osasiyapo tanthauzo lake lachilengedwe.Pamene chiwopsezo cha Agkistrodon halys Japonicus chinachitidwa ndi p-nitrophenyl ethyl ester ndi indole ethyl ester, ma hydrolysates a p-nitrophenol ndi indole phenol sanapezeke;M'malo mwake, ngati ma esters achita ndi cobra Zhoushan subspecies njoka ya njoka ndi Bungarus multicinctus njoka ya njoka, iwo adzakhala mofulumira hydrolyzed.Zimadziwika kuti majeremusi awa ali ndi ntchito yolimba ya cholinesterase, yomwe imatha kuyambitsa hydrolysis ya magawo omwe ali pamwambapa.M'malo mwake, Mclean et al.(1971) inanena kuti majeremusi ambiri a njoka a m'banja la Cobra amatha hydrolyze indole ethyl ester, naphthalene ethyl ester ndi butyl naphthalene ester.Mitsempha ya njoka imeneyi imachokera ku: cobra, cobra, cobra yakuda, cobra yakuda, cobra yagolide, cobra yaku Egypt, king cobra, golden cobra mamba, black mamba ndi white lipped mamba (D. aw amadziwabe eastern rhombola rattlesnake).
Utsi wa njoka umatha hydrolyze methyl indole ethyl ester, yomwe ndi gawo lapansi lodziwira ntchito ya cholinesterase mu seramu, koma utsi wa njokawu suwonetsa ntchito ya cholinesterase.Izi zikuwonetsa kuti pali esterase yosadziwika mu cobra venom, yomwe ndi yosiyana ndi cholinesterase.Kuti mumvetsetse mtundu wa enzyme imeneyi, ntchito yolekanitsa ikufunika.
1, Phospholipase A2
(I) Mwachidule
Phospholipase ndi enzyme yomwe imatha hydrolyze glyceryl phosphate.Pali mitundu 5 ya phospholipase m'chilengedwe, yomwe ndi phospholipase A2 ndi phospholipase.
A. , phospholipase B, phospholipase C ndi phospholipase D. Njoka ya njoka makamaka imakhala ndi phospholipase A2 (PLA2), tizilombo tochepa ta njoka timakhala ndi phospholipase B, ndi ma phospholipases ena makamaka amapezeka mu minofu ya nyama ndi mabakiteriya.Chithunzi 3-11-4 chikuwonetsa malo ochitirapo ma phospholipases pa gawo lapansi la hydrolysis.
Pakati pa phospholipases, PLA2 yaphunziridwa kwambiri.Itha kukhala enzyme yophunziridwa kwambiri muululu wa njoka.Gawo lake laling'ono ndilo mgwirizano wa ester pa malo achiwiri a Sn-3-glycerophosphate.Enzyme imeneyi imapezeka kwambiri mu utsi wa njoka, utsi wa njuchi, utsi wa zinkhanira ndi minofu ya nyama, ndipo PLA2 imakhala yochuluka m'magulu anayi a njoka za m'banja.Chifukwa enzymeyi imaphwanya maselo ofiira a magazi ndikuyambitsa hemolysis, imatchedwanso "hemolysin".Anthu ena amatchanso PLA2 hemolytic lecithinase.
Ludeeke adapeza koyamba kuti utsi wa njoka ukhoza kupanga hemolytic compound pochita lecithin kudzera mu michere.Pambuyo pake, Delezenne et al.zinatsimikizira kuti pamene njoka ya cobra imagwira ntchito pa seramu ya kavalo kapena yolk, imapanga mankhwala a hemolytic.Tsopano zikudziwika kuti PLA2 ikhoza kuchitapo kanthu mwachindunji pa phospholipids ya erythrocyte nembanemba, kuwononga mapangidwe a erythrocyte nembanemba ndikuyambitsa hemolysis mwachindunji;Imathanso kuchitapo kanthu pa seramu kapena kuwonjezera lecithin kuti ipange hemolytic lecithin, yomwe imagwira ntchito pama cell ofiira amwazi kuti ipange hemolysis yosalunjika.Ngakhale kuti PLA2 ndi yochuluka m'mabanja anayi a njoka za njoka, zomwe zili m'matumbo amtundu wa njoka za njoka ndizosiyana pang'ono.Rattlesnake (C
Utsi wa njoka umangowonetsa zofooka za PLA2.Gulu 3-11-11 likuwonetsa kufananiza kwa ntchito ya PLA2 ya ma 10 akuluakulu a njoka zapoizoni ku China.
Table 3-11-11 Kuyerekeza kwa phospholipase VIII ntchito za 10 njoka za njoka ku China
Utsi wa njoka
Kutulutsa mafuta
Aliphatic acid,
Cjumol/mg)
Hemolytic ntchito CHU50/^ g * ml)
Utsi wa njoka
Kutulutsa mafuta acids
(^ raol/mg)
Ntchito ya Hemolytic "(HU50/ftg * 1111)
Najanaja atra
9. 62
khumi ndi chimodzi
Micracephal ophis
mfundo zisanu ziro imodzi
kalyspallas
8. 68
zikwi ziwiri mazana asanu ndi atatu
gracilis
V, akuto
7. 56
* * #
Ofiophagus Hana
atatu mfundo eyiti awiri
zana limodzi ndi makumi anayi
Bnugarus fasctus
7,56
mazana awiri mphambu makumi asanu ndi atatu
B. multicinctus
mfundo imodzi naini sikisi
mazana awiri mphambu makumi asanu ndi atatu
Viper ndi russelli
seven point ziro three
T, mucrosquamatus
mfundo imodzi eyiti zisanu
Siamensis
T. stejnegeri
0.97 pa
(2) Kupatukana ndi kuyeretsedwa
Zomwe zili mu PLA2 mu njoka ya njoka ndizochuluka, ndipo zimakhala zokhazikika kutentha, asidi, alkali ndi denaturant, kotero kuti ndizosavuta kuyeretsa ndikulekanitsa PLA2.Njira wamba ndikuyamba kusefera gel osakaniza pa utsi wakuda, ndiye kuchita ion kuwombola chromatography, ndi sitepe yotsatira akhoza kubwerezedwa.Tikumbukenso kuti amaundana-kuyanika kwa PLA2 pambuyo ion-kusinthanitsa chromatography sayenera aggregation, chifukwa amaundana-kuyanika ndondomeko nthawi zambiri kumawonjezera ma ionic mphamvu mu dongosolo, amene ndi chinthu chofunika kuchititsa aggregation wa PLA2.Kuphatikiza pa njira zomwe zili pamwambazi, njira zotsatirazi zagwiritsidwanso ntchito: ① Wells et al.② Analogue ya gawo lapansi ya PLA2 idagwiritsidwa ntchito ngati ligand kwa affinity chromatography.Ligand iyi imatha kumangirira ku PLA2 muululu wa njoka ndi Ca2+.EDTA imagwiritsidwa ntchito kwambiri ngati chidziwitso.Pambuyo pa Ca2 + kuchotsedwa, mgwirizano pakati pa PLA2 ndi ligand umachepa, ndipo ukhoza kulekanitsidwa ndi ligand.Ena amagwiritsa ntchito 30% organic solution kapena 6mol/L urea ngati sakudziwa.③ Hydrophobic chromatography idachitidwa ndi PheiiylSephar0SeCL-4B kuchotsa trace PLA2 mu cardiotoxin.④ Anti PLA2 antibody idagwiritsidwa ntchito ngati ligand kupanga affinity chromatography pa PLA2.
Pakalipano, chiwerengero chachikulu cha njoka za PLAZ zayeretsedwa.Ndi et al.(1977) adalemba PLA2 yoyeretsedwa ku njoka ya njoka isanafike 1975. M'zaka zaposachedwapa, nkhani zambiri zokhudzana ndi kulekana ndi kuyeretsedwa kwa PLA2 zakhala zikudziwika chaka chilichonse.Apa, timayang'ana kwambiri kulekanitsa ndi kuyeretsedwa kwa PLA ndi akatswiri aku China.
Chen Yuancong et al.(1981) adalekanitsa mitundu itatu ya PLA2 kuchokera ku utsi wa Agkistrodon halys Pallas ku Zhejiang, womwe ungathe kugawidwa kukhala acidic, neutral ndi alkaline PLA2 malinga ndi mfundo zawo za isoelectric.Malinga ndi kawopsedwe ake, ndale PLA2 ndi poizoni kwambiri, yemwe amadziwika kuti presynaptic neurotoxin Agkistrodotoxin.Alkaline PLA2 imakhala ndi poizoni pang'ono, ndipo acidic PLA2 ilibe poizoni.Wu Xiangfu et al.(1984) anayerekezera makhalidwe a PLA2 atatu, kuphatikizapo kulemera kwa maselo, amino acid, N-terminal, isoelectric point, kutentha kwa kutentha, ntchito ya enzyme, poizoni ndi ntchito ya hemolytic.Zotsatirazo zinasonyeza kuti anali ndi kulemera kwa maselo ofanana ndi kukhazikika kwa kutentha, koma anali ndi kusiyana kwakukulu pazinthu zina.Mu mbali ya enzyme ntchito, asidi enzyme ntchito anali apamwamba kuposa zamchere enzyme ntchito;Mphamvu ya hemolytic ya enzyme ya alkaline pama cell ofiira a makoswe inali yamphamvu kwambiri, kutsatiridwa ndi enzyme yolowererapo, ndipo enzyme ya asidi idalephera kutulutsa magazi.Chifukwa chake, zimaganiziridwa kuti zotsatira za hemolytic za PLAZ zimagwirizana ndi mtengo wa molekyulu ya PLA2.Zhang Jingkang et al.(1981) apanga makhiristo a Agkistrodotoxin.Tu Guangliang et al.(1983) inanena kuti PLA yapoizoni yokhala ndi isoelectric point ya 7. 6 idapatulidwa ndikuyeretsedwa ku chiwopsezo cha Vipera rotundus kuchokera ku Fujian, ndi zinthu zake zakuthupi ndi zamankhwala, kapangidwe kake ka amino acid ndi kutsatizana kwa zotsalira za 22 amino acid ku N. -terminal zidatsimikiziridwa.Li Yuesheng et al.(1985) adadzipatula ndikuyeretsa PLA2 ina ku ululu wa Viper rotundus ku Fujian.Subunit ya PLA2 * ndi 13 800, isoelectric point ndi 10.4, ndipo ntchito yeniyeni ndi 35/xnioI/miri mg LD5 anabayidwa mtsempha wa mbewa.Ndi 0.5 ± 0.12mg/kg.Enzyme iyi imakhala ndi anticoagulant komanso hemolytic zotsatira.Molekyu yapoizoni ya PLA2 imakhala ndi zotsalira 123 za mitundu 18 ya ma amino acid.Molekyu imakhala ndi cysteine (14), aspartic acid (14) ndi glycine (12), koma imakhala ndi methionine imodzi yokha, ndipo N-terminal yake ndi zotsalira za serine.Poyerekeza ndi PLA2 yomwe imasiyanitsidwa ndi Tuguang, kulemera kwa maselo ndi chiwerengero cha amino acid zotsalira za isoenzymes ziwiri ndizofanana kwambiri, ndipo mawonekedwe a amino acid ndi ofanana kwambiri, koma chiwerengero cha aspartic acid ndi proline zotsalira ndizosiyana.Ululu wa njoka ya Guangxi king cobra uli ndi PLA2 yolemera.Shu Yuyan et al.(1989) analekanitsa PLA2 ku chiwopsezo, amene ali ndi ntchito yeniyeni nthawi 3.6 kuposa chiwembu choyambirira, molekyulu kulemera 13000, zikuchokera 122 amino asidi zotsalira, ndi isoelectric mfundo 8.9, ndi wabwino matenthedwe bata.Kuchokera pa maikulosikopu ya elekitironi ya momwe PLA2 imakhudzira maselo ofiira a magazi, zitha kuwoneka kuti imakhudza kwambiri maselo ofiira a magazi a munthu, koma ilibe mphamvu zowonekera pama cell ofiira a mbuzi.PLA2 iyi ili ndi kuchedwetsa koonekeratu pa liwiro la electrophoretic la maselo ofiira a magazi mwa anthu, mbuzi, akalulu ndi nkhumba.Chen et al.Enzyme iyi imatha kuletsa kuphatikizika kwa mapulateleti opangidwa ndi ADP, collagen ndi sodium arachidonic acid.Pamene ndende ya PLA2 ndi 10/xg/ml~lOOjug/ml, kuphatikizika kwa mapulateleti kumaletsedwa kotheratu.Ngati mapulateleti otsukidwa adagwiritsidwa ntchito ngati zida, PLA2 sakanatha kuletsa kuphatikizika pamlingo wa 20Mg/ml.Aspirin ndi inhibitor ya cyclooxygenase, yomwe imatha kuletsa mphamvu ya PLA2 pamapulateleti.PLA2 imatha kuletsa kuphatikizika kwa mapulateleti ndi hydrolyzing arachidonic acid kupanga thromboxane A2.Mayankho a PLA2 opangidwa ndi Agkistrodon halys Pallas venom m'chigawo cha Zhejiang adawunikidwa pogwiritsa ntchito ma circular dichroism, fluorescence ndi mayamwidwe a UV.Zotsatira zoyesera zidawonetsa kuti kuphatikizika kwakukulu kwa enzyme iyi kunali kofanana ndi mtundu womwewo wa enzyme kuchokera ku mitundu ina ndi ma genera, ma skeleton conformation anali ndi kukana kwabwino kwa kutentha, ndipo kusintha kwachilengedwe kwa asidi kunasinthika.Kuphatikiza kwa activator Ca2 + ndi enzyme sikukhudza chilengedwe cha zotsalira za tryptophan, pomwe inhibitor Zn2 + imachita mosiyana.Momwe pH phindu la yankho limakhudzira ntchito ya enzyme ndi yosiyana ndi ma reagents omwe ali pamwambapa.
M'kati mwa PLA2 kuyeretsa utsi wa njoka, chodziwika bwino ndi chakuti ululu wa njoka uli ndi nsonga ziwiri kapena zingapo za PLA2.Izi zitha kufotokozedwa motere: ① chifukwa cha kukhalapo kwa isozymes;② Mtundu umodzi wa PLA2 umapangidwa polima mumitundu yosiyanasiyana ya PLA2 yokhala ndi masikelo osiyanasiyana a mamolekyulu, ambiri omwe ali mu 9 000 ~ 40 000;③ Kuphatikiza kwa PLA2 ndi zigawo zina zaululu wa njoka zimasokoneza PLA2;④ Chifukwa chomangira cha amide mu PLA2 ndi hydrolyzed, mtengowo umasintha.① Ndipo ② ndizofala, kupatulapo zochepa, monga PLA2 mu CrWa/w utsi wa njoka
Pali zinthu ziwiri: ① ndi ②.Mkhalidwe wachitatu wapezeka mu PLA2 mu ululu wa njoka zotsatirazi: Oxyranus scutellatus, Parademansia microlepidota, Bothrops a ^>er, Palestinian viper, sand viper, ndi terrible rattlesnake km.
Zotsatira za mlandu ④ zimapangitsa kuti liwiro la kusamuka kwa PLA2 lisinthe panthawi ya electrophoresis, koma mawonekedwe a amino acid sasintha.Ma peptides amatha kuthyoledwa ndi hydrolysis, koma nthawi zambiri amakhala omangidwa palimodzi ndi zomangira za disulfide.Utsi wa njoka yakum'mawa uli ndi mitundu iwiri ya PLA2, yotchedwa mtundu a ndi mtundu wa p PLA2 motsatana.Kusiyana pakati pa mitundu iwiriyi ya PLA2 ndi amino acid imodzi yokha, ndiye kuti, glutamine mu molekyulu imodzi ya PLA2 imasinthidwa ndi glutamic acid mu molekyulu ya PLA2.Ngakhale kuti chifukwa chenicheni cha kusiyana kumeneku sikudziwika bwino, amakhulupirira kuti chikugwirizana ndi deamination ya PLA2.Ngati PLA2 mu utsi wa njoka ya Palestine itenthedwa ndi utsi wosakhwima, magulu omalizira mu mamolekyu ake a enzyme adzakhala ambiri kuposa kale.Kuchokera ku C PLA2 yotalikirana ndi njoka ya njoka imakhala ndi N-terminal ziwiri zosiyana, ndipo kulemera kwake kwa maselo ndi 30000. Chodabwitsa ichi chikhoza kuyambitsidwa ndi asymmetric dimer ya PLA2, yomwe ili yofanana ndi dimer symmetric yopangidwa ndi PLA2 mu ululu wa eastern diamondback rattlesnake. ndi Western diamondback rattlesnake.Nsomba za ku Asia zimapangidwa ndi timagulu tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tambiri.Mwachitsanzo, zomwe kale zinkatchedwa Cobra Outer Caspian subspecies tsopano zadziwika
Izi ziyenera kunenedwa ndi Outer Caspian Sea Cobra.Popeza pali mitundu yambiri yamagulu ndipo imasakanizidwa pamodzi, mapangidwe a njoka ya njoka amasiyana kwambiri chifukwa cha magwero osiyanasiyana, komanso zomwe zili mu PLA2 isozymes ndizokwera kwambiri.Mwachitsanzo, poizoni wa cobra
Pafupifupi mitundu 9 ya ma isozymes a PLA2 a r ^ll anapezeka, ndipo mitundu 7 ya ma isozymes a PLA2 inapezeka mu utsi wa cobra subspecies Caspian.Durkin et al.(1981) adaphunzira zomwe zili mu PLA2 ndi kuchuluka kwa ma isozymes muzowopsa za njoka, kuphatikiza ma 18 a cobra, 3 mamba, 5 utsi wa njoka, 16 utsi wa njoka zam'nyanja ndi 3 utsi wa njoka zam'nyanja.Nthawi zambiri, ntchito ya PLA2 ya utsi wa cobra ndiyokwera, yokhala ndi ma isozyme ambiri.Ntchito ya PLA2 ndi ma isozymes a viper venom ndi apakatikati.Ntchito ya PLA2 ya utsi wa mamba ndi utsi wa rattlesnake ndiyotsika kwambiri kapena palibe ntchito ya PLA2.Ntchito ya PLA2 ya utsi wa njoka ya m'nyanja nayonso ndiyotsika.
M'zaka zaposachedwa, sikunanene kuti PLA2 mu njoka ya njoka ilipo mu mawonekedwe a dimer yogwira, monga eastern rhombophora rattlesnake (C. njoka ya njoka ili ndi mtundu A ndi mtundu wa P PLA2, onsewa amapangidwa ndi zigawo ziwiri zofanana. , ndipo ndi dimerase yokha
Zochita.Shen et al.Anatinso dimer ya PLA2 ya utsi wa njoka ndiyo njira yogwira ntchito ya enzyme.Kafukufuku wamapangidwe a malo amatsimikiziranso kuti PLA2 ya Western diamondback rattlesnake ilipo mu mawonekedwe a dimer.Piscivorous compound
Pali mitundu iwiri yosiyana ya PLA ^ Ei ndi E2 ya utsi wa njoka, momwe 仏 ilipo mu mawonekedwe a dimer, dimer imagwira ntchito, ndipo monomer yake yosiyana sikugwira ntchito.Lu Yinghua et al.(1980) anaphunziranso za thupi ndi mankhwala katundu ndi zochita kinetics wa E. Jayanthi et al.(1989) adapatula PLA2 yoyambira (VRVPL-V) ku utsi wa njoka.Kulemera kwa molekyulu ya monomer PLA2 ndi 10000, yomwe imakhala ndi zotsatira zakupha, anticoagulant ndi edema.Enzyme akhoza polymerize ma polima ndi osiyana maselo zolemera pansi pa chikhalidwe cha PH 4.8, ndi mlingo wa polymerization ndi maselo kulemera kwa ma polima kuwonjezeka ndi kuwonjezeka kutentha.Kulemera kwa maselo a polima opangidwa pa 96 ° C ndi 53 100, ndipo ntchito ya PLA2 ya polima iyi imawonjezeka ndi ziwiri.
Nthawi yotumiza: Nov-18-2022