Ese methylene blue ishobora kunyura iruhande rwa Complex III yo muri mitokondiriya? Impamvu ubushakashatsi butavuga rumwe

Laboratwari ebyiri zabajije niba methylene blue ishobora kunyura iruhande rwa Complex III yabujijwe gukora muri mitokondiriya. Imwe yasubije ko bidashoboka. Indi isubiza ko bishoboka. Zombi zari zifite amakuru abigaragaza.

Ubushakashatsi bwa mbere, bwakoresheje mitokondiriya zo mu bwonko bw'imbeba nto, bwasanze iryo rangi ridashobora kubikora. Ubwa kabiri, bwakoresheje mitokondiriya zo mu bwonko bw'amoko atatu y'inyamaswa zihekenya, bwasanze rishobora kubikora igice. Ubushakashatsi bwa gatatu, bwakoresheje umwijima w'imbeba nto, bukunze kuvugwa hamwe n'ubwo, ariko bwo bwagerageje ikindi kintu. Kubutandukanya n'ibyo butagerageje bikuraho kimwe cya kabiri cy'urujijo.

Nta buryo bumwe bwo gukora bukoreshwa hose twatangaza. Hari amagerageza atatu yakoresheje ibibuza imikorere, ingano y'irangi n'ibipimo by'ibisubizo bitandukanye. Banza usome uko amagerageza yakozwe. Imyanzuro irakurikiraho.

Icyo kunyura iruhande bisobanura hano

Electron zinjira mu ruhererekane zinyuze muri Complex I (ziturutse kuri NADH) cyangwa Complex II (ziturutse kuri succinate), zikanyura muri Complex III zigera kuri cytochrome c, hanyuma zikanyura muri Complex IV zigera kuri ogisijeni. Itandukaniro ryubakwa muri iyo nzira ni ryo akaremangingo gakoresha mu gukora ATP.

Kuvuga ko methylene blue inyura iruhande rw'ahabujijwe gukora bisobanura ko yakira electron mbere y'aho hantu, ikazitanga nyuma yaho. Irangi ryataye electron ryakira electron zivuye kuri NADH cyangwa ku bice bibanza by'uruhererekane, rigahinduka leucomethylene blue; irangi ryakiriye electron rikazihereza cytochrome c cyangwa rikazisubiza mu ruhererekane. Iyo electron zitanzwe nyuma y'ahabujijwe gukora, ihumeka n'itandukaniro rya potansiyeli y'amashanyarazi ku gihu bigaruka igice. Iyo zitanzwe aho imikorere yabujijwe cyangwa mbere yaho, nta kigaruka.

Ikintu kimwe cyihariye cya Complex III kigira uruhare rukomeye muri uku kutavuga rumwe. Iyo complex ikoresha uruziga rwa Q rufite ahantu habiri ibibuza imikorere bifatira. Antimycin ifatira ku mwanya wa Qi. Myxothiazol ifatira ku mwanya wa Qo. Ubushakashatsi bukoresha kimwe gusa muri ibyo bintu buba bwagerageje igice kimwe gusa cy'iyo complex.

Ubushakashatsi bwa mbere: nta kunyura iruhande muri mitokondiriya zo mu bwonko bw'imbeba nto

Gureev na bagenzi be bakoresheje mitokondiriya zo mu gice cy'inyuma cy'ubwonko bw'imbeba nto za C57BL/6, bazitandukanya bakoresheje digitonin, bapima imikorere yazo kuri 0.1 mg ya poroteyine kuri ml. Ibitanga electron byakoreshejwe mu buryo butatu: pyruvate hamwe na malate kuri 5 mM buri kimwe, succinate kuri 10 mM hamwe na glutamate kuri 2 mM, na L-proline kuri 10 mM. Ibibuza imikorere byari rotenone kuri 1 micromolar (Complex I) na antimycin kuri 1 microgram kuri ml (Complex III). Ingano z'irangi zari 0.5, 1 na 2 micromolar, aho 1 micromolar ari yo yakoreshejwe cyane. Uburyo bwakoreshejwe n'imirongo y'ibipimo biri mu nyandiko ya FEBS Letters.

Kunyura iruhande rwa Complex I byarakoze. Rotenone yagabanyije ihumeka ryatewe imbaraga na ADP kuva hafi 121 kugera hafi 2 nmol ku munota kuri mg, maze irangi kuri 1 micromolar ririgarura kugeza hafi 21. Itandukaniro rya potansiyeli y'amashanyarazi ku gihu ryaragarutse hakoreshejwe pyruvate hamwe na malate, ndetse na L-proline.

Kunyura iruhande rwa Complex III ntibyakunze. Antimycin yagabanyije ihumeka kugeza hafi 16, irangi ririzamura gusa kugeza hafi 20. Itandukaniro rya potansiyeli y'amashanyarazi ku gihu ntiryagarutse ku gitanga electron na kimwe, nubwo ingano y'irangi yageze kuri 5 micromolar. Amakuru ya perokiside na yo yerekanye kimwe: irangi ryonyine ryazamuye irekurwa rya perokiside kuva ku kigero fatizo cya hafi 76 pmol ku munota kuri mg kugeza hafi 1170, 1460 na 1945 kuri za ngano eshatu, ariko antimycin yagabanyije icyo kimenyetso cyatewe n'irangi hafi 25, 88 na 96 ku ijana, uko zikurikirana. Iyo irangi ryakiriye electron riza gukorana na ogisijeni ako kanya, kubuza Complex III gukora ntibyari kugabanya icyo kimenyetso. Abanditsi banzuye ko irangi ryakiriye electron rizitakaza ku mwanya wa Qo wa Complex III, mbere y'aho antimycin ibuza imikorere. Urwo ruziga rugufi, mu magambo: ibitanga electron biziha irangi, irangi rikaziha Qo, hanyuma zigakomeza mu nzira isanzwe. Rotenone inyurwaho iruhande. Antimycin yo ntinyurwaho iruhande.

Ubushakashatsi bwa kabiri: kunyura iruhande igice mu moko atatu, ku myanya yombi y'ibibuza imikorere

Svab na bagenzi be baguye uburyo bw'igerageza. Bakoresheje mitokondiriya zo mu bwonko bw'imbeba nto, imbeba nini n'imbeba zo muri Gineya, bazitandukanya bakoresheje Percoll, bapima imikorere yazo kuri 0.1 mg kuri ml nk'uko byari byakozwe mbere, bakoresheje pyruvate hamwe na malate cyangwa glutamate hamwe na malate kuri 5 mM buri kimwe. Bakoresheje ibibuza Complex III gukora bibiri: antimycin A kuri 0.3 micromolar na myxothiazol kuri 1 micromolar. Irangi ryari kuri 2 micromolar mu bipimo byinshi, na 0.5 mu kazi karebanaga na cytochrome c. Amoko yose uko ari atatu, yageragejwe hakoreshejwe uburyo buhuye, ari mu nyandiko iboneka ku buntu.

Ahantu hombi habujijwe gukora hanyurwagaho iruhande igice, mu moko yose uko ari atatu. Muri mitokondiriya z'imbeba nto, antimycin yagabanyije ihumeka, maze irangi ririzamura hafi 60 ku ijana, kuva hafi 37 kugera kuri 59 nmol ku munota kuri mg. Myxothiazol yarigabanyije 88 ku ijana, rigera hafi 4.5, maze irangi ririzamura inshuro eshanu kugeza hafi 23. Itandukaniro rya potansiyeli y'amashanyarazi ku gihu ryaragarutse igice hakoreshejwe kimwe cyangwa ikindi muri ibyo bibuza imikorere. Ryapimwe hakoreshejwe amarangi abiri (safranin O na TMRM ipimwa hakurikijwe igipimo cy'uburinganire) kugira ngo hirindwe ibisubizo biyobya bituruka ku buryo bwo gupima hakoreshejwe urumuri.

Iyi nyandiko yanagerageje ihererekanya rya electron mu buryo butaziguye, ibyo ubushakashatsi bwa mbere butigeze bukora. Cytochrome c yashyizweho amatsinda ya acetyl, yongewemo ivuye hanze kuri 25 micromolar, ntiyakiriye electron zivuye kuri mitokondiriya zifite ingufu, haba hari ibibuza imikorere cyangwa bidahari; ariko kongeramo irangi byatumye yakira electron muri ibyo bihe byombi. Mu gisubizo ngengakigero kitagira uturemangingo, NADH hamwe n'irangi byatumye cytochrome c yakira electron ku muvuduko wajyanaga n'ingano ya NADH. Iyo nzira isimbuka intera ndende, mu magambo: ubushobozi bwo gutanga electron buhererezwa irangi, irangi rigatanga electron ako kanya kuri cytochrome c, Complex IV ikazitanga kuri ogisijeni. Iyo nzira isimbuka Complex III yose, bityo ibibuza imikorere byombi bikaba bishobora kunyurwaho iruhande.

Ibindi bipimo bibiri bishyigikira ubwo busobanuro. Urumuri rwa fluorescence rwa NAD(P)H rwaragabanutse igihe irangi ryongerwaga muri mitokondiriya zakoreshejwemo myxothiazol, bisobanura ko ubushobozi bwo gutanga electron bwagabanukaga binyuze muri iyo nzira nshya. Nanone, uko irangi ryinjizaga urumuri kuri 660 nm byakurikiye buri kintu cyongerwagamo, naho ibipimo by'amazi yo hejuru yabonetse nyuma yo kuzungurutswa byerekanye ko irangi ryataye electron ryirundanyaga imbere muri mitokondiriya zifite ingufu kugeza ku gipimo kirenze inshuro ijana, hanyuma rikavamo igihe itandukaniro rya potansiyeli ryasenyukaga. Imibare ya proton na yo irahuye: gusimbuka intambwe zo kuvoma proton bituma hasigara hafi proton 2 kuri buri electron ebyiri aho kuba 10. Bityo, gukoresha ogisijeni vuba kandi itandukaniro rya potansiyeli rigaruka igice gusa ni ko kunyura iruhande nyakuri kugaragara, si ikimenyetso kibihakana.

Mitokondiriya zateguwe muri ubwo buryo zanatanze ibisubizo bishobora kuyobya usoma atitonze. Iyo hari ikibuza imikorere hamwe n'irangi, ADP yagabanyaga ihumeka aho kurizamura, naho kubuza ubwikorezi byariryongeraga aho kurigabanya, mu gihe itandukaniro rya potansiyeli y'amashanyarazi ku gihu ryakomezaga guhinduka mu cyerekezo gisanzwe. Muri ubu buryo, kureba ikoreshwa rya ogisijeni ryonyine birayobya.

Ubushakashatsi bwa gatatu: ubushakashatsi ku mwijima bwagerageje Complex I na II, atari III

Lee na Boelsterli bakoresheje mitokondiriya zo mu mwijima w'imbeba nto n'uturemangingo tw'umwijima twakuze muri laboratwari, byangijwe na efavirenz (Complex I, IC50 hafi 30 micromolar) na hydrazine, igikomoka ku itunganywa rya isoniazid mu mubiri (Complex II, IC50 hafi 30 micromolar). Irangi kuri hafi 30 micromolar ryagaruye itakazwa rya electron za NADH, itandukaniro rya potansiyeli y'amashanyarazi ku gihu na ATP, ari na ko rigabanya ibitera itakazwa rya electron n'urupfu rw'uturemangingo ruturuka kuri nekoroze. Inyandiko ya Redox Biology ibisobanura nk'aho irangi ritwara electron rikanyura iruhande rwa Complex I na II zangiritse.

Icyo gisubizo ni amakuru yemejwe ku bice bibanza by'uruhererekane, kandi ntacyo kivuga kuri Complex III kuko nta kintu cyabujijwe gukora aho muri ubwo bushakashatsi. Ntigishobora kwemeza cyangwa guhakana bumwe muri bwa bushakashatsi ku bwonko.

Imbonerahamwe y'imiterere y'amagerageza

Impamvu enye zituma ubushakashatsi bwombi kuri Complex III bushobora kuba buri mu kuri ku miterere yabwo y'igerageza

1. Ikibuza imikorere nticyakoreshejwe mu buryo bumwe. Antimycin yonyine kuri 1 microgram kuri ml, ugereranyije na antimycin kuri 0.3 micromolar hamwe n'ikindi kibuza imikorere ku mwanya wa Qo. Inzira y'irangi itanga electron ku mwanya wa Qo irananirwa neza neza aho inzira izitanga ako kanya kuri cytochrome c ishobora gukora. Umwanya umwe ntushobora guhagararira yombi.

2. Ingano y'irangi n'igihe byari bitandukanye. Ingano zakoreshejwe cyane zari 1 ugereranyije na 2 micromolar, ku kintu gisubiramo kwakira no gutakaza electron, kandi kwinjira kwacyo muri mitokondiriya no gutanga electron ako kanya kuri cytochrome c biterwa n'ingano n'igihe. Itandukaniro rito ry'ingano, hamwe n'ibihe bitandukanye byo gufata ibipimo, bishobora kwerekana amashami atandukanye y'urusobe rumwe.

3. Uburyo bwo gutegura mitokondiriya bwari butandukanye. Kuzitandukanya hakoreshejwe digitonin, ugereranyije no kuzitandukanya hakoreshejwe ibice bya Percoll bifite ubucucike bugenda butandukana, bitoranya amatsinda ya mitokondiriya afite ibyo ahuriyeho ariko atari amwe neza. Ingano ya poroteyine yari imwe, kuri 0.1 mg kuri ml, bityo icyo gipimo ntigisobanura itandukaniro, ariko amatsinda mato ya mitokondiriya n'uko yari ameze byo biracyashobora kurigiramo uruhare. Kandi gukeka ko itandukaniro ryaturutse ku bwoko bw'inyamaswa byarakuweho: itsinda rya kabiri ryabonye ya myitwarire no mu mbeba nto.

4. Imiterere y'ibitanga electron igena icyo irangi rishobora gukora. Uburyo butatu bwo gukoresha ibitanga electron, ugereranyije n'ibintu bibiri bikoreshwa hamwe kandi bifitanye isano na NADH. Ingano ya NADH iboneka n'itandukaniro rya potansiyeli y'amashanyarazi ku gihu bifatanyiriza hamwe kugena umuvuduko irangi ryakiraho electron n'aho ryirundanyiriza. Inzira ikenera irangi ryakiriye electron hafi ya cytochrome c yataye electron iba ishingiye kuri iryo hitamo.

Uko wasuzuma ikindi kivugwa ku kunyura iruhande

Baza complex yabujijwe gukora, ikibuza imikorere cyakoreshejwe, umwanya cyakoreyeho n'ingano yacyo. Baza ingano y'irangi n'igihe igipimo cyafatiwe. Baza igice cy'umubiri, ubwoko bw'inyamaswa, uburyo bwo gutandukanya mitokondiriya, ingano ya poroteyine n'igitanga electron. Kandi ubaze niba ibipimo bya potansiyeli, ihumeka, uko electron zakiriwe cyangwa zatakajwe, n'igerageza ry'iherekanywa rya electron mu buryo butaziguye bihuriza ku gisubizo kimwe, kuko muri aya magerageza ikoreshwa rya ogisijeni ryonyine ryayobeje inshuro zirenze imwe.

Incamake yibanda ku byageragejwe gusa iracyafite ishingiro. Kunyura iruhande rwa Complex I kw'irangi biremeranywaho. Kuri Complex III, mitokondiriya z'imbeba nto zakoreshejwemo antimycin kuri 1 microgram kuri ml n'irangi kuri 1 micromolar ntizerekana kunyura iruhande gusubiza imikorere; naho amoko atatu yakoreshejwemo antimycin kuri 0.3 micromolar cyangwa myxothiazol kuri 1 micromolar n'irangi kuri 2 micromolar agaragaza kunyura iruhande igice, hamwe n'iherekanywa rya electron riva ku irangi rijya kuri cytochrome c mu buryo butaziguye. Izo nteruro zombi zishobora kuba ukuri icyarimwe. Ikindi cyose kirenze ibyo ni ugufata ibisubizo ukabigeza ku bitageragejwe.

Ibindi byo gusoma bifasha gushyira iki kivugwa kitavugwaho rumwe mu rwego rwa shimi yemejwe: uko methylene blue yimura electron ku bijyanye n'ihererekanya, amagerageza yo kunyura iruhande rwa Complex I ku byemejwe mu bice bibanza by'uruhererekane, ibipimo by'itandukaniro rya potansiyeli y'amashanyarazi ku gihu n'ibisubizo biyobya bishobora kubonekamo ku mpamvu hakenewe amarangi abiri, n'uburyo bwo gukoresha muri laboratwari ibintu bisubiramo kwakira no gutakaza electron, ku makosa ashobora guturuka ku ngano n'igihe.