Kodi methylene blue ingadutse pambali pa Complex III ya mitochondria? Chifukwa chake maphunziro sakugwirizana

Ma laboratories awiri anafufuza ngati methylene blue ingadutse pambali pa Complex III ya mitochondria yomwe yatsekedwa. Limodzi linayankha kuti ayi. Lina linayankha kuti inde. Onse anali ndi deta.

Phunziro loyamba, pogwiritsa ntchito mitochondria za ubongo wa mbewa, linapeza kuti utotowu sungatero. Lachiwiri, pogwiritsa ntchito mitochondria za ubongo wa mitundu itatu ya nyama zokuta, linapeza kuti ungatero pang'ono. Phunziro lachitatu, la chiwindi cha mbewa, nthawi zambiri limatchulidwa pamodzi nawo koma linayesa chinthu china. Mukalisiyanitsa moyenera, theka la chisokonezo limatha.

Palibe njira imodzi yogwirira ntchito yomwe tinganene kuti imagwira m'mikhalidwe yonse. Pali mayesero atatu okhala ndi zoletsa, milingo ndi miyeso yosiyana. Werengani mikhalidwe yake kaye. Kenako mudzamvetsa ziganizo zake.

Tanthauzo la kudutsa pambali pano

Maelekitironi amalowa mu unyolo kudzera mu Complex I (kuchokera ku NADH) kapena Complex II (kuchokera ku succinate), amadutsa mu Complex III kupita ku cytochrome c, kenako mu Complex IV kupita ku oxygen. Kusiyana kwa mphamvu komwe kumapangidwa m'njirayi ndi kumene selo limagwiritsa ntchito kupanga ATP.

Kunena kuti methylene blue imadutsa pambali kumatanthauza kuti imatenga maelekitironi asanafike pamalo otsekedwa ndi kuwapereka atadutsa malowo. Utoto womwe wataya maelekitironi umatenga maelekitironi kuchokera ku NADH kapena ku zigawo zoyambirira za unyolowo, n'kukhala leucomethylene blue. Kenako utoto womwe walandira maelekitironiwo umawapereka ku cytochrome c kapena kuwabwezera mu unyolowo. Ngati maelekitironi aperekedwa pambuyo pa malo otsekedwa, kupuma ndi mphamvu ya magetsi pa nembanemba zimabwezeretseka pang'ono. Ngati aperekedwa pamalo otsekedwa kapena asanafike pamenepo, palibe chimene chimabwezeretseka.

Mbali imodzi ya Complex III ndi yofunika kwambiri pa mkanganowu. Complex imeneyi imagwiritsa ntchito mkombero wa Q wokhala ndi malo awiri omangirapo zoletsa. Antimycin imamangirira pamalo a Qi. Myxothiazol imamangirira pamalo a Qo. Phunziro logwiritsa ntchito chimodzi chokha mwa ziwirizi limakhala litayesa theka lokha la complex imeneyi.

Phunziro loyamba: palibe kudutsa pambali mu mitochondria za ubongo wa mbewa

Gureev ndi anzake anagwiritsa ntchito mitochondria za kotekisi ya ubongo wa mbewa za C57BL/6, zolekanitsidwa ndi digitonin, ndipo anaziyesa pa 0.1 mg protein per ml. Ma substrate anagwiritsidwa ntchito m'magulu atatu: pyruvate ndi malate pa 5 mM iliyonse, succinate pa 10 mM limodzi ndi glutamate pa 2 mM, ndiponso L-proline pa 10 mM. Zoletsa zinali rotenone pa 1 micromolar (Complex I) ndi antimycin pa 1 microgram per ml (Complex III). Milingo ya utoto inali 0.5, 1, ndi 2 micromolar, ndipo 1 micromolar ndiyo inali ndende yaikulu yogwiritsidwa ntchito. Njira ndi mizere ya zotsatira zili mu lipoti la FEBS Letters.

Kudutsa pambali pa Complex I kunagwira ntchito. Rotenone inachepetsa kupuma kolimbikitsidwa ndi ADP kuchokera pa pafupifupi 121 kufika pa pafupifupi 2 nmol per minute per mg, ndipo utoto wa 1 micromolar unakubwezeretsa kufika pa pafupifupi 21. Mphamvu ya magetsi pa nembanemba inabwezeretseka pogwiritsa ntchito pyruvate ndi malate komanso L-proline.

Kudutsa pambali pa Complex III sikunagwire ntchito. Antimycin inachepetsa kupuma kufika pa pafupifupi 16, ndipo utoto unangokweza kufika pa pafupifupi 20. Mphamvu ya magetsi pa nembanemba sinabwezeretseke ndi substrate iliyonse, ngakhale pamlingo wa utoto wofika pa 5 micromolar. Deta ya peroxide inasonyezanso zomwezo: utoto wokha unakweza kutulutsidwa kwa peroxide kuchokera pamlingo woyambira wa pafupi ndi 76 pmol per minute per mg kufika pa pafupifupi 1170, 1460, ndi 1945 pa milingo itatuyo. Komabe, antimycin inachepetsa chizindikiro choyambitsidwa ndi utotocho ndi pafupifupi 25, 88, ndi 96 percent motsatana. Ngati utoto wolandira maelekitironi unkagwirizana mwachindunji ndi oxygen, kutseka Complex III sikunayenera kuchepetsa chizindikirocho. Olemba anaganiza kuti utoto wolandira maelekitironi umataya maelekitironiwo pamalo a Qo a Complex III, asanafike pamalo otsekedwa ndi antimycin. Mkombero waufupiwo tingaufotokoze motere: opereka maelekitironi kupita ku utoto, utoto kupita ku Qo, kenako kupitiriza m'njira yanthawi zonse. Rotenone imadutsidwa pambali. Antimycin sidutsidwa pambali.

Phunziro lachiwiri: kudutsa pambali pang'ono m'mitundu itatu, pa malo onse awiri a zoletsa

Svab ndi anzake anakulitsa kapangidwe ka mayeserowo. Anagwiritsa ntchito mitochondria za ubongo wa mbewa, makoswe ndi guinea pig, zolekanitsidwa ndi Percoll, n'kuziyesa pa mlingo womwewo wa 0.1 mg per ml, pogwiritsa ntchito pyruvate ndi malate kapena glutamate ndi malate pa 5 mM iliyonse. Anagwiritsa ntchito zoletsa ziwiri za Complex III: antimycin A pa 0.3 micromolar ndi myxothiazol pa 1 micromolar. Utoto unali pa 2 micromolar m'mayeso ambiri, ndi 0.5 m'mayeso a cytochrome c. Mitundu yonse itatuyi, yoyesedwa ndi njira zofanana, ili mu nkhani yopezeka kwa onse.

Malo onse awiri otsekedwa anadutsidwa pambali pang'ono, m'mitundu yonse itatu. Mu mitochondria za mbewa, antimycin inachepetsa kupuma ndipo utoto unakukweza ndi pafupifupi 60 percent, kuchokera pa pafupifupi 37 kufika pa 59 nmol per minute per mg. Myxothiazol inakuchepetsa ndi 88 percent, kufika pa pafupifupi 4.5, ndipo utoto unakukweza kasanu kufika pa pafupifupi 23. Mphamvu ya magetsi pa nembanemba inabwezeretseka pang'ono ndi choletsa chilichonse. Inayesedwa ndi utoto uwiri (safranin O ndi TMRM yoyezera chiŵerengero cha zizindikiro) kuti achotse kuthekera kwa zotsatira zabodza zobwera chifukwa cha kuwala.

Nkhaniyi inayesanso mwachindunji kupereka maelekitironi, zomwe phunziro loyamba silinachite. Cytochrome c yopangidwa ndi acetylation, yowonjezedwa kuchokera kunja pa 25 micromolar, sinalandire maelekitironi kuchokera ku mitochondria zokhala ndi mphamvu, kaya panali zoletsa kapena ayi. Koma kuwonjezera utoto kunachititsa kuti ilandire maelekitironi m'mikhalidwe yonse iwiriyi. Mu buffer yopanda maselo, NADH pamodzi ndi utoto zinapereka maelekitironi ku cytochrome c pamlingo wofanana ndi kuchuluka kwa NADH. Njira yodumphira kutali imeneyi tingaufotokoze motere: mphamvu zopereka maelekitironi kupita ku utoto, utoto mwachindunji kupita ku cytochrome c, Complex IV kupita ku oxygen. Njirayi imadumpha Complex III yonse, choncho zoletsa zonse ziwirizi zingadutsidwe pambali.

Miyeso ina iwiri imathandiza kutanthauzira kumeneku. Fluorescence ya NAD(P)H inatsika utoto utawonjezedwa ku mitochondria zomwe zinapatsidwa myxothiazol, kutanthauza kuti mphamvu zopereka maelekitironi zinkatuluka kudzera m'njira yatsopanoyo. Ndipo kuyamwa kwa kuwala ndi utoto pa 660 nm kunatsatira chinthu chilichonse chowonjezedwa motsatana. Madzi a pamwamba otengedwa pambuyo pozunguliza zitsanzo anasonyeza kuti utoto wotaya maelekitironi unkasonkhana mkati mwa mitochondria zokhala ndi mphamvu kupitirira kuchuluka kwa kunja kasanu ndi kawiri ka khumi, kenako n'kutuluka mphamvu ya magetsi itatha. Kuwerengera kwa ma proton kunagwirizananso ndi zimenezi: kudumpha masitepe opopera ma proton kumasiya pafupifupi ma proton 2 pa maelekitironi awiri m'malo mwa 10. Choncho kugwiritsa ntchito oxygen mofulumira koma mphamvu ya magetsi ikubwerera pang'ono kokha ndi mmene kudutsa pambali kwenikweni kumaonekera, osati umboni wotsutsa kuti kunachitika.

Zitsanzo zomwezo zinapereka zotsatira zimene zimafunika kuziwerenga mosamala. Panali choletsa ndi utoto, ADP inkachepetsa kupuma m'malo mokukweza, ndipo kutseka kunyamula zinthu kunkalimbikitsa kupuma m'malo mokuchepetsa. Komabe, mphamvu ya magetsi pa nembanemba inkasinthabe m'njira zanthawi zonse. Kugwiritsa ntchito oxygen kokha kungasocheretse m'dongosolo limeneli.

Phunziro lachitatu: phunziro la chiwindi lomwe linayesa Complex I ndi II, osati III

Lee ndi Boelsterli anagwiritsa ntchito mitochondria za chiwindi cha mbewa ndi maselo a chiwindi okulitsidwa m'malo oyesera, owonongeka ndi efavirenz (Complex I, IC50 pafupi ndi 30 micromolar) komanso hydrazine, chinthu chopangidwa pakusinthidwa kwa isoniazid m'thupi (Complex II, IC50 pafupi ndi 30 micromolar). Utoto wa pafupifupi 30 micromolar unabwezeretsa kutaya maelekitironi kwa NADH, mphamvu ya magetsi pa nembanemba ndi ATP, uku ukuchepetsa zinthu zotenga maelekitironi ndi kufa kwa maselo mwa necrosis. Nkhani ya Redox Biology imafotokoza zimenezi ngati utoto wonyamula maelekitironi kudutsa pambali pa Complex I ndi II zowonongekazo.

Zotsatira zimenezo ndi maziko ovomerezedwa okhudza zigawo zoyambirira za unyolo. Sizikunena chilichonse chokhudza Complex III, chifukwa palibe chimene chinatsekedwa pamenepo. Sizingatsimikizire kapena kutsutsa phunziro lililonse mwa awiri a ubongo aja.

Tchati cha mikhalidwe

Zifukwa zinayi zimene maphunziro onse awiri a Complex III angakhale olondola pa mikhalidwe yawo

1. Choletsa sichinali chinthu chofanana choyesedwa. Antimycin yokha pa 1 microgram per ml poyerekeza ndi antimycin pa 0.3 micromolar limodzi ndi choletsa china cha malo a Qo. Njira ya utoto yopereka maelekitironi pamalo a Qo imalephera pomwe njira yopereka mwachindunji ku cytochrome c imapambana. Malo amodzi sangaimire onse awiri.

2. Mlingo wa utoto ndi nthawi zinasiyana. Ndende zazikulu zinali 1 poyerekeza ndi 2 micromolar, pa chinthu chomwe chimachita mkombero wa redox ndipo kulowa kwake ndi kupereka kwake maelekitironi mwachindunji ku cytochrome c zimadalira ndende ndi nthawi. Kusiyana pang'ono kwa milingo komanso nthawi yosiyanasiyana yoyezera kungavumbule mbali zosiyanasiyana za dongosolo limodzi.

3. Njira zokonzera zitsanzo zinasiyana. Kulekanitsa ndi digitonin poyerekeza ndi kulekanitsa pogwiritsa ntchito kusiyana kwa ndende ya Percoll kumasankha magulu a mitochondria omwe amafanana pang'ono koma osati ndendende. Ndende ya mapuloteni inali yofanana pa 0.1 mg per ml, choncho si chifukwa cha kusiyanako. Koma magulu ang'onoang'ono a mitochondria ndi mmene anakhalira osawonongeka zidakali zofunika. Ndipo kukayikira kuti mtundu wa nyama ndiwo unachititsa kusiyana kunachotsedwa: gulu lachiwiri linapezanso zotsatira zake mu mbewa.

4. Mkhalidwe wa substrate umakhazikitsa zimene utoto ungachite. Magulu atatu a ma substrate poyerekeza ndi ma substrate awiriawiri ogwirizana ndi NADH. Kupezeka kwa NADH ndi mphamvu ya magetsi pa nembanemba pamodzi zimakhazikitsa liwiro limene utoto umalandirira maelekitironi komanso malo amene umasonkhanako. Njira yomwe imafuna utoto wolandira maelekitironi pafupi ndi cytochrome c yotaya maelekitironi imadalira kwambiri kusankha kumeneku.

Mmene mungawerengere mfundo yotsatira yonena za kudutsa pambali

Funsani kuti ndi complex iti yomwe inatsekedwa, ndi choletsa chiti, pamalo ati komanso pamlingo uti. Funsani ndende ya utoto ndi nthawi imene muyeso unatengedwa. Funsani za minofu, mtundu wa nyama, njira yolekanitsa, ndende ya mapuloteni ndi substrate. Ndiponso funsani ngati mphamvu ya magetsi, kupuma, mkhalidwe wa redox ndi mayeso a kupereka maelekitironi mwachindunji zikugwirizana, chifukwa kugwiritsa ntchito oxygen kokha kunapereka chithunzi cholakwika kangapo pano.

Chidule chokhudza mikhalidwe imeneyi yokha chikadali cholondola. Kudutsa kwa utoto pambali pa Complex I kwavomerezedwa. Pa Complex III, mitochondria za mbewa zokhala ndi antimycin pa 1 microgram per ml ndi utoto pa 1 micromolar sizisonyeza kudutsa pambali komwe kumabwezeretsa ntchito. Koma mitundu itatu yokhala ndi antimycin pa 0.3 micromolar kapena myxothiazol pa 1 micromolar ndi utoto pa 2 micromolar imasonyeza kudutsa pambali pang'ono, ndi kupereka maelekitironi mwachindunji kuchokera ku utoto kupita ku cytochrome c. Mfundo zimenezi zingakhale zolondola pamodzi. Chilichonse chopitirira pamenepa ndi kutambasula zotsatira kupitirira zomwe zinayesedwa.

Zowerenga zina zogwirizana zimayika mfundo yotsutsidwayi mkati mwa chemistry yosatsutsidwa: mmene methylene blue imasunthira maelekitironi pa nkhani yopatsirana maelekitironi, mayesero a kudutsa pambali pa Complex I pa nkhani yovomerezedwa ya zigawo zoyambirira za unyolo, miyeso ya mphamvu ya magetsi pa nembanemba ndi zotsatira zake zabodza pa chifukwa chimene panafunikira utoto uwiri, komanso mmene zinthu zochita mkombero wa redox zimagwiritsidwira ntchito mu laboratory pa zolakwika zokhudza milingo ndi nthawi.