Ingabe i-methylene blue ingayidlula iNkimbinkimbi III ye-mitochondria? Kungani izifundo zingavumelani

Amalabhorethri amabili abuza ukuthi i-methylene blue ingayidlula yini iNkimbinkimbi III ye-mitochondria evinjiwe. Elinye lathi cha. Elinye lathi yebo. Womabili ayenemininingwane yokuhlola.

Isifundo sokuqala, sama-mitochondria obuchopho bamagundane amancane, sathola ukuthi udayi awukwazi. Esesibili, sama-mitochondria obuchopho bezinhlobo ezintathu zamagundane, sathola ukuthi ungakwenza ngokwengxenye. Isifundo sesithathu, sesibindi samagundane amancane, sivame ukucashunwa kanye nalezi kodwa sahlola okunye. Uma sigcinwa endaweni yaso efanele, ingxenye yokudideka iyaphela.

Ayikho indlela yokusebenza esebenza kuzo zonke izimo engamenyezelwa. Kukhona ukuhlolwa okuthathu okusebenzisa izivimbeli, imithamo nezilinganiso ezihlukene. Funda izimo kuqala. Iziphetho ziyalandela.

Kusho ukuthini ukudlula lapha

Ama-electron angena ochungechungeni ngeNkimbinkimbi I (evela ku-NADH) noma ngeNkimbinkimbi II (evela ku-succinate), adlule kuNkimbinkimbi III aye ku-cytochrome c, bese edlula kuNkimbinkimbi IV aye ku-oxygen. Umehluko wokugxila owakhiwa endleleni yiwo iseli eliwusebenzisa ukwenza i-ATP.

Isimangalo sokudlula sithi i-methylene blue ithatha ama-electron ngaphambi kwendawo evinjiwe iwahambise ngale kwayo. Udayi osulahlekelwe ama-electron uthatha ama-electron ku-NADH noma ezingxenyeni zokuqala zochungechunge, ube yi-leucomethylene blue, bese isimo sawo esamukele ama-electron siwanikeza i-cytochrome c noma siwabuyisele ochungechungeni. Uma ukudluliselwa kwenzeka ngale kwendawo evinjiwe, ukuphefumula nomehluko wamandla kagesi olwelwesini kuyabuyela ngokwengxenye. Uma kwenzeka endaweni evinjiwe noma ngaphambi kwayo, akukho okubuyayo.

Umniningwane owodwa weNkimbinkimbi III unquma okuningi kule mpikiswano. Le nkimbinkimbi isebenzisa umjikelezo we-Q onezindawo ezimbili okubopha kuzo izivimbeli. I-antimycin ibopha endaweni ye-Qi. I-myxothiazol ibopha endaweni ye-Qo. Isifundo esisebenzisa esisodwa kuphela salezi sivivinye ingxenye eyodwa kuphela yale nkimbinkimbi.

Isifundo sokuqala: akukho ukudlula kuma-mitochondria obuchopho bamagundane amancane

UGureev nozakwabo basebenzisa ama-mitochondria oqweqwe lobuchopho bamagundane amancane ohlobo lwe-C57BL/6, ahlukaniswa nge-digitonin, ahlolwa ku-0.1 mg wamaprotheni nge-ml. Ama-substrate asetshenziswa ngezinhlelo ezintathu: i-pyruvate kanye ne-malate ku-5 mM ngakunye, i-succinate ku-10 mM kanye ne-glutamate ku-2 mM, ne-L-proline ku-10 mM. Izivimbeli kwakuyi-rotenone ku-1 micromolar (iNkimbinkimbi I) ne-antimycin ku-1 microgram nge-ml (iNkimbinkimbi III). Imithamo kadayi yayingu-0.5, 1, no-2 micromolar, u-1 micromolar uyizinga eliyinhloko lokugxila. Izindlela nemidwebo yezilinganiso kusembikweni we-FEBS Letters.

Ukudlula iNkimbinkimbi I kwasebenza. I-rotenone yehlisa ukuphefumula okukhuthazwa yi-ADP kusuka cishe ku-121 kuya cishe ku-2 nmol ngomzuzu nge-mg, kwathi udayi ongu-1 micromolar wakubuyisela cishe ku-21. Umehluko wamandla kagesi olwelwesini wabuyela lapho kusetshenziswa i-pyruvate kanye ne-malate, nalapho kusetshenziswa i-L-proline.

Ukudlula iNkimbinkimbi III akuzange kusebenze. I-antimycin yehlisa ukuphefumula kuya cishe ku-16, udayi wakunyusa kuphela kuya cishe ku-20. Umehluko wamandla kagesi olwelwesini awuzange ubuyele kunoma iyiphi i-substrate, ngisho nasemithameni kadayi efika ku-5 micromolar. Imininingwane ye-peroxide yakhomba okufanayo: udayi uwodwa wanyusa ukukhishwa kwe-peroxide kusuka ezingeni eliyisisekelo eliseduze no-76 pmol ngomzuzu nge-mg kuya cishe ku-1170, 1460, no-1945 kule mithamo emithathu, kodwa i-antimycin yehlisa leso silinganiso esidalwa udayi cishe ngamaphesenti angu-25, 88, no-96 ngokulandelana. Uma udayi osamukele ama-electron ubusabela ngokuqondile ne-oxygen, ukuvimba iNkimbinkimbi III bekungafanele kucindezele lesi silinganiso. Ababhali baphetha ngokuthi udayi osamukele ama-electron ulahlekelwa yiwo endaweni ye-Qo yeNkimbinkimbi III, ngaphambi kwendawo evinjwe yi-antimycin. Umjikelezo omfushane, ngamazwi: abanikeli bama-electron baya kudayi, udayi uye ku-Qo, bese indlela evamile iqhubekela phambili. I-rotenone iyadlulwa. I-antimycin ayidlulwa.

Isifundo sesibili: ukudlula ngokwengxenye ezinhlotsheni ezintathu, kuzo zombili izindawo zezivimbeli

USvab nozakwabo banweba uhlelo lokuhlola. Basebenzisa ama-mitochondria obuchopho bamagundane amancane, amagundane amakhulu nama-guinea pig, ahlukaniswa nge-Percoll, ahlolwa ezingeni elifanayo elingu-0.1 mg nge-ml, kusetshenziswa i-pyruvate kanye ne-malate noma i-glutamate kanye ne-malate ku-5 mM ngakunye. Basebenzisa izivimbeli ezimbili zeNkimbinkimbi III: i-antimycin A ku-0.3 micromolar ne-myxothiazol ku-1 micromolar. Udayi wawungu-2 micromolar ezivivinyweni eziningi, no-0.5 emsebenzini we-cytochrome c. Zontathu izinhlobo, ezahlolwa ngezinqubo ezifanayo, zikhona ephepheni elifinyeleleka mahhala.

Zombili izivimbelo zadlulwa ngokwengxenye, kuzo zontathu izinhlobo. Kuma-mitochondria amagundane amancane, i-antimycin yacindezela ukuphefumula, udayi wakunyusa cishe ngamaphesenti angu-60, kusuka cishe ku-37 kuya ku-59 nmol ngomzuzu nge-mg. I-myxothiazol yakucindezela ngamaphesenti angu-88, kuya cishe ku-4.5, udayi wakunyusa ngokuphindwe kahlanu kuya cishe ku-23. Umehluko wamandla kagesi olwelwesini wabuyela ngokwengxenye ngaphansi kwesivimbeli ngasinye, walinganiswa ngodayi ababili (i-safranin O ne-TMRM esebenzisa isilinganiso samasignali) ukuze kukhishwe amathuba amaphutha okubangelwa ukukhanya.

Leli phepha laphinde lahlola ukudluliselwa kwama-electron ngokuqondile, okuyinto isifundo sokuqala esingazange siyenze. I-cytochrome c ene-acetyl eyengezwe ivela ngaphandle ku-25 micromolar ayizange yamukele ama-electron kuma-mitochondria anamandla, kungakhathaliseki ukuthi izivimbeli zazikhona noma cha, kodwa ukwengeza udayi kwabangela ukwamukelwa kwama-electron kuzo zombili izimo. Esixazululweni se-buffer esingenawo amaseli, i-NADH kanye nodayi kwenza i-cytochrome c yamukele ama-electron ngesivinini esilingana nezinga le-NADH. Ukugxuma okude, ngamazwi: ama-electron atholakalayo aya kudayi, udayi uwadlulisele ngokuqondile ku-cytochrome c, iNkimbinkimbi IV iwadlulisele ku-oxygen. Leyo ndlela yeqa iNkimbinkimbi III ngokuphelele, ngakho zombili izivimbeli zingadlulwa.

Izilinganiso ezimbili ezengeziwe ziyakusekela lokho kuchazwa. Ukukhanya kwe-fluorescence kwe-NAD(P)H kwehla lapho kwengezwa udayi kuma-mitochondria aphathwe nge-myxothiazol, okusho ukuthi ama-electron atholakalayo ayedlula ngale ndlela entsha. Futhi ukumunca ukukhanya kukadayi ku-660 nm kwalandela ukwengezwa ngakunye ngokulandelana, kuyilapho uketshezi olungaphezulu olwahlukaniswa ngokuphothwa lwabonisa ukuthi udayi osulahlekelwe ama-electron wawunqwabelana ngaphakathi kwama-mitochondria anamandla ngokuphindwe ngaphezu kwekhulu, bese uphuma lapho umehluko wamandla kagesi uwohloka. Ibhalansi yama-proton nayo iyahambisana: ukweqa izinyathelo zokumpompa kushiya cishe ama-proton angu-2 ngepheya lama-electron esikhundleni sangu-10, ngakho ukusetshenziswa kwe-oxygen okusheshayo kanye nokubuyela ngokwengxenye kuphela komehluko wamandla kagesi kuyilokho okulindelekile ekudluleni kwangempela, hhayi ubufakazi obuphikisana nakho.

Lawo ma-mitochondria alungiselelwe ukuhlolwa abonisa nokusabela okungenza ukufunda ngokunganaki kudukise. Lapho kukhona isivimbeli kanye nodayi, i-ADP yehlisa ukuphefumula esikhundleni sokukunyusa, futhi ukuvinjwa kokuthutha kwakukhuthaza esikhundleni sokukwehlisa, kuyilapho umehluko wamandla kagesi olwelwesini usashintsha ngendlela evamile. Ukusetshenziswa kwe-oxygen kukodwa kuyadukisa kulolu hlelo.

Isifundo sesithathu: isifundo sesibindi esahlola iNkimbinkimbi I no-II, hhayi u-III

ULee noBoelsterli basebenzisa ama-mitochondria esibindi samagundane amancane namaseli esibindi akhuliswe elabhorethri, alinyazwa yi-efavirenz (iNkimbinkimbi I, i-IC50 eseduze no-30 micromolar) ne-hydrazine, umkhiqizo wokucutshungulwa kwe-isoniazid emzimbeni (iNkimbinkimbi II, i-IC50 eseduze no-30 micromolar). Udayi ocishe ube ngu-30 micromolar wabuyisela ukulahlekelwa kwama-electron kwe-NADH, umehluko wamandla kagesi olwelwesini ne-ATP, ngesikhathi wehlisa izinto ezidala i-oxidation nokufa kwamaseli nge-necrosis. Iphepha le-Redox Biology likuchaza ngemodeli lapho udayi uthwala ama-electron udlule iNkimbinkimbi I no-II ezilimele.

Lowo mphumela uwumongo ovunyelwanayo wezingxenye ezingaphambili, futhi awusho lutho ngeNkimbinkimbi III ngoba akukho okwakuvinjwe kuyo. Awukwazi ukuqinisekisa noma ukuphikisa noma yisiphi salezi zifundo zobuchopho.

Ithebula lezimo

Izizathu ezine ezenza zombili izifundo zeNkimbinkimbi III zibe neqiniso ngezimo zazo

1. Isivimbeli sasingesona isenzo esifanayo. I-antimycin iyodwa ku-1 microgram nge-ml uma iqhathaniswa ne-antimycin ku-0.3 micromolar kanye nesivimbeli esihlukile sendawo ye-Qo. Indlela kadayi efaka ama-electron endaweni ye-Qo ihluleka khona lapho indlela eqonde ngqo ku-cytochrome c iphumelela khona. Indawo eyodwa ayikwazi ukumela zombili.

2. Umthamo kadayi nesikhathi kwakuhlukile. Amazinga ayinhloko okugxila ayengu-1 uma eqhathaniswa no-2 micromolar, entweni ejikeleza phakathi kwezimo ze-redox, okumuncwa kwayo nokudlulisela kwayo ama-electron ngokuqondile ku-cytochrome c kuncike ezingeni lokugxila nesikhathi. Umehluko omncane wemithamo kanye nezikhathi ezihlukene zokulinganisa kungaveza amagatsha ahlukene enethiwekhi eyodwa.

3. Izindlela zokulungiselela zazihlukile. Ukuhlukanisa nge-digitonin uma kuqhathaniswa nokuhlukanisa nge-gradient ye-Percoll kukhetha amaqoqo ama-mitochondria agqagqene kodwa angafani ncamashi. Izinga lokugxila kwamaprotheni lalifana ku-0.1 mg nge-ml, ngakho lokho akusachazi umehluko, kodwa amaqoqo amancane nesimo sokungalimali kwawo kusengawuchaza. Futhi ukusola umehluko wezinhlobo kwasuswa: iqembu lesibili laphinda lathola indlela efanayo yokusabela nasemagundaneni amancane.

4. Isimo se-substrate sinquma ukuthi udayi ungenzani. Izinhlelo ezintathu zama-substrate uma ziqhathaniswa namapheya axhumene ne-NADH. Ukutholakala kwe-NADH nomehluko wamandla kagesi olwelwesini kunquma ngokuhlanganyela ukuthi udayi uwamukela ngokushesha kangakanani ama-electron nokuthi ugxila kuphi, futhi indlela edinga udayi osamukele ama-electron eduze kwe-cytochrome c esilahlekelwe ama-electron incike kakhulu kulokho kukhetha.

Indlela yokufunda isimangalo esilandelayo sokudlula

Buza ukuthi iyiphi inkimbinkimbi evinjiwe, ngasiphi isivimbeli, kuyiphi indawo nangamuphi umthamo. Buza izinga lokugxila kukadayi nokuthi isilinganiso sathathwa nini. Buza ngesicubu, uhlobo, indlela yokuhlukanisa, izinga lokugxila kwamaprotheni ne-substrate. Futhi buza ukuthi umehluko wamandla kagesi, ukuphefumula, isimo se-redox nokuhlolwa kokudluliselwa kwama-electron ngokuqondile kuyavumelana yini, ngoba ukusetshenziswa kwe-oxygen kukodwa kwakhomba ohlangothini olungafanele izikhathi ezingaphezu kwesisodwa lapha.

Isifinyezo esilinganiselwe sisamile. Kuyavunyelwana ngokudlula iNkimbinkimbi I ngodayi. NgokweNkimbinkimbi III, ama-mitochondria amagundane amancane ane-antimycin ku-1 microgram nge-ml kanye nodayi ku-1 micromolar awabonisi ukudlula okubuyisela ukusebenza, kuyilapho izinhlobo ezintathu ezine-antimycin ku-0.3 micromolar noma i-myxothiazol ku-1 micromolar kanye nodayi ku-2 micromolar zibonisa ukudlula ngokwengxenye ngokudluliselwa kwama-electron ngokuqondile kusuka kudayi kuya ku-cytochrome c. Le misho ingaba yiqiniso yomibili. Noma yini ebanzi kunalokho iyisiphetho esidlulela ngalé kokuhloliwe.

Ukufunda okuhlobene kugcina isimangalo esiphikiswanayo ngaphakathi kwekhemistri engaphikiswana ngayo: indlela i-methylene blue ehambisa ngayo ama-electron mayelana nokudlulisela, ukuhlolwa kokudlula iNkimbinkimbi I mayelana nesimo sezingxenye ezingaphambili okuvunyelwana ngaso, izilinganiso zomehluko wamandla kagesi olwelwesini namaphutha azo mayelana nokuthi kungani kwakudingeka odayi ababili, kanye nokuphathwa kwezinto ezijikeleza phakathi kwezimo ze-redox elabhorethri mayelana nezingibe zomthamo nesikhathi.