Shin methylene blue zai iya tsallake Complex III na mitochondria? Dalilin da ya sa bincike ya saɓa

Dakunan gwaje-gwaje biyu sun tambayi ko methylene blue zai iya tsallake Complex III na mitochondria da aka toshe. Ɗaya ya ce a'a. Ɗayan ya ce eh. Dukansu suna da bayanan gwaji.

Binciken farko, a mitochondria na kwakwalwar mice, ya gano cewa rinin ba zai iya ba. Na biyu, a mitochondria na kwakwalwa daga nau'ikan dabbobi masu haƙoran cizo uku, ya gano cewa zai iya zuwa wani mataki. Ana yawan ambaton bincike na uku, a hantar mice, tare da su, amma ya gwada wani abu dabam. Idan aka ajiye shi a inda ya dace, rabin ruɗanin zai gushe.

Babu wata hanyar aiki guda da za a bayyana cewa tana aiki a kowane yanayi. Akwai gwaje-gwaje uku masu sinadaran toshewa, adadin rini da ma'aunai daban-daban. A fara karanta yanayin gwaje-gwajen. Daga nan sai sakamakon.

Abin da tsallakewa ke nufi a nan

Electron suna shiga jerin jigilar ta Complex I (daga NADH) ko Complex II (daga succinate), su bi ta Complex III zuwa cytochrome c, sannan ta Complex IV zuwa oxygen. Bambancin da ake ginawa a kan hanyar shi ne abin da salula ke amfani da shi don samar da ATP.

Da'awar tsallakewa na nufin methylene blue yana ɗaukar electron kafin wurin toshewa, ya miƙa su bayan wurin. Rinin da aka yi wa oxidation yana karɓar electron daga NADH ko sassan farkon jerin jigilar, ya koma leucomethylene blue, sannan siffarsa da aka yi wa reduction ta miƙa su ga cytochrome c ko ta mayar da su cikin jerin jigilar. Idan an miƙa su bayan wurin toshewa, numfashi da ƙarfin lantarki na membrane suna murmurewa zuwa wani mataki. Idan an miƙa su a wurin toshewar ko kafin shi, babu abin da ke murmurewa.

Wani bayani guda game da Complex III yana da babban tasiri a wannan saɓanin. Wannan complex yana gudanar da zagayen Q mai wuraren ɗaure sinadaran hanawa biyu. Antimycin yana ɗaurewa a wurin Qi. Myxothiazol yana ɗaurewa a wurin Qo. Binciken da ya yi amfani da ɗaya kawai daga cikinsu ya gwada rabin complex ɗin ne kawai.

Bincike na ɗaya: babu tsallakewa a mitochondria na kwakwalwar mice

Gureev da abokan aikinsa sun yi amfani da mitochondria daga bawon kwakwalwar mice na C57BL/6, an ware su da digitonin, an gwada su a 0.1 mg na furotin per ml. Sinadaran farawa sun ƙunshi tsare-tsare uku: pyruvate da malate a 5 mM kowannensu, succinate a 10 mM tare da glutamate a 2 mM, da L-proline a 10 mM. Sinadaran toshewar su ne rotenone a 1 micromolar (Complex I) da antimycin a 1 microgram per ml (Complex III). Adadin rinin sun kasance 0.5, 1, da 2 micromolar, inda 1 micromolar ya kasance babban matakin taro. Hanyoyin gwaji da layukan bayanan ma'auni suna cikin rahoton FEBS Letters.

Tsallake Complex I ya yi aiki. Rotenone ya rage numfashin da ADP ya ƙarfafa daga kusan 121 zuwa kusan 2 nmol per minute per mg, sannan rini a 1 micromolar ya dawo da shi zuwa kusan 21. Ƙarfin lantarki na membrane ya murmure da pyruvate tare da malate, da kuma L-proline.

Tsallake Complex III bai yi aiki ba. Antimycin ya rage numfashi zuwa kusan 16, kuma rinin ya ɗaga shi zuwa kusan 20 kawai. Ƙarfin lantarki na membrane bai murmure da kowane sinadarin farawa ba, har da adadin rini da ya kai 5 micromolar. Bayanan peroxide sun nuna abu iri ɗaya: rini kaɗai ya ɗaga fitar peroxide daga matakin asali na kusan 76 pmol per minute per mg zuwa kusan 1170, 1460, da 1945 a adadin ukun, amma antimycin ya rage wannan siginar da rinin ya haifar da kusan 25, 88, da 96 percent, bi da bi. Idan rinin da aka yi wa reduction yana yin martani kai tsaye da oxygen, toshe Complex III bai kamata ya danne siginar ba. Marubutan sun yi hasashen cewa rinin da aka yi wa reduction yana samun oxidation a wurin Qo na Complex III, kafin wurin toshewar antimycin. A taƙaice, gajeriyar zagayawa ita ce: masu bayar da electron zuwa rini, rini zuwa Qo, sannan hanyar da aka saba zuwa gaba. Ana tsallake rotenone. Ba a tsallake antimycin.

Bincike na biyu: tsallakewa zuwa wani mataki a nau'ikan dabbobi uku, a wuraren hanawa biyu

Svab da abokan aikinsa sun faɗaɗa tsarin gwajin. Sun yi amfani da mitochondria na kwakwalwar mice, rats, da guinea pigs, an ware su da Percoll, an gwada su a 0.1 mg per ml iri ɗaya, tare da pyruvate da malate ko glutamate da malate a 5 mM kowannensu. Sun yi amfani da sinadaran toshe Complex III biyu: antimycin A a 0.3 micromolar da myxothiazol a 1 micromolar. Rinin ya kasance 2 micromolar a yawancin gwaje-gwajen, 0.5 a gwajin cytochrome c. Duk nau'ikan dabbobi ukun da tsarin gwajinsu iri ɗaya suna cikin takardar binciken da kowa zai iya karantawa.

An tsallake toshewar biyu zuwa wani mataki, a duk nau'ikan dabbobi ukun. A mitochondria na mice, antimycin ya danne numfashi kuma rinin ya ɗaga shi da kusan 60 percent, daga kusan 37 zuwa 59 nmol per minute per mg. Myxothiazol ya danne shi da 88 percent, zuwa kusan 4.5, kuma rinin ya ɗaga shi sau biyar zuwa kusan 23. Ƙarfin lantarki na membrane ya sake kafuwa zuwa wani mataki ƙarƙashin kowane sinadarin hanawa, an auna shi da rini biyu (safranin O da TMRM mai auna rabon sigina) don kawar da kuskuren ma'aunin haske.

Wannan takardar ta kuma gwada miƙa electron kai tsaye, abin da binciken farko bai taɓa yi ba. Mitochondria masu samun kuzari ba su yi wa cytochrome c mai rukunin acetyl da aka ƙara daga waje a 25 micromolar reduction ba, ko da akwai sinadaran hanawa ko babu, amma ƙara rini ya haifar da reduction a duka yanayin biyun. A maganin buffer da babu salula, NADH tare da rini sun yi wa cytochrome c reduction a saurin da ya yi daidai da adadin NADH. A taƙaice, doguwar tsallakewar ita ce: abubuwan bayar da electron zuwa rini, rini kai tsaye zuwa cytochrome c, Complex IV zuwa oxygen. Wannan hanyar tana tsallake Complex III gaba ɗaya, don haka ana iya tsallake sinadaran toshewar biyu.

Wasu ƙarin ma'aunai biyu suna goyon bayan wannan fahimta. Hasken fluorescence na NAD(P)H ya ragu lokacin da aka ƙara rini ga mitochondria da aka yi wa maganin myxothiazol, yana nufin abubuwan bayar da electron sun fita ta sabuwar hanyar. Kuma shan haske na rinin a 660 nm ya nuna kowace ƙarin sinadari bi da bi, yayin da ruwan saman da aka samu bayan juyawa da sauri ya nuna cewa rinin da aka yi wa oxidation yana taruwa cikin mitochondria masu samun kuzari fiye da sau ɗari, sannan ya fita lokacin da ƙarfin lantarki ya rushe. Lissafin proton ma ya dace: tsallake matakan tura proton yana barin kusan 2 proton ga kowane ma'auratan electron maimakon 10, don haka saurin amfani da oxygen tare da sake kafuwar ƙarfin lantarki zuwa wani mataki kawai shi ne abin da ake gani a tsallakewa ta gaske, ba hujjar musunta ta ba.

Samfurin mitochondria ɗin nan ya kuma samar da martanin da ke iya ruɗar da mai karatu marar kulawa. Tare da sinadarin hanawa da rini, ADP ya rage numfashi maimakon ƙara shi, kuma toshe jigilar ya ƙarfafa shi maimakon rage saurinsa, yayin da ƙarfin lantarki na membrane har yanzu ya canza a hanyoyin da aka saba. Amfani da oxygen kaɗai yana ɓatar da fahimta a wannan tsarin.

Bincike na uku: binciken hanta da ya gwada Complex I da II, ba III ba

Lee da Boelsterli sun yi amfani da mitochondria na hantar mice da ƙwayoyin hanta da aka raina a dakin gwaje-gwaje, waɗanda efavirenz (Complex I, IC50 kusa da 30 micromolar) da hydrazine, samfurin sarrafa isoniazid (Complex II, IC50 kusa da 30 micromolar), suka lalata. Rini a kusan 30 micromolar ya dawo da oxidation na NADH, ƙarfin lantarki na membrane, da ATP, tare da rage sinadaran oxidation da mutuwar salula ta necrosis. Takardar Redox Biology ta bayyana wannan da tsarin da rini ke ɗaukar electron ya tsallake Complex I da II da suka lalace.

Wannan sakamako bayani ne da aka tabbatar game da abin da ke faruwa a farkon jerin jigilar, kuma bai ce komai game da Complex III ba saboda ba a toshe wani abu a can ba. Ba zai iya tabbatarwa ko musunta ɗaya daga cikin binciken kwakwalwar biyu ba.

Teburin yanayin gwaje-gwajen

Dalilai huɗu da binciken Complex III biyu za su iya zama daidai game da tsarin gwajinsu

1. Sinadarin toshewar ba tsoma baki iri ɗaya ba ne. Antimycin kaɗai a 1 microgram per ml idan aka kwatanta da antimycin a 0.3 micromolar da wani sinadarin toshe wurin Qo na dabam. Hanyar rini da ke miƙa electron ga wurin Qo tana gaza a daidai inda hanyar kai tsaye zuwa cytochrome c take nasara. Wuri ɗaya ba zai wakilci wuraren biyu ba.

2. Adadin rini da lokacin gwaji sun bambanta. Manyan matakan taro na 1 idan aka kwatanta da 2 micromolar, ga sinadarin da ke yin zagayen redox wanda shigarsa da yin reduction na cytochrome c kai tsaye suka dogara da matakin taro da lokaci. Ƙananan bambance-bambancen adadi tare da lokutan auna sakamako daban-daban na iya bayyana sassa daban-daban na tsarin hanyoyi guda.

3. Shirye-shiryen samfuran sun bambanta. Warewa da digitonin idan aka kwatanta da warewa ta bambancin yawa na Percoll yana zaɓar rukunin mitochondria masu kamanceceniya amma ba iri ɗaya ba. Matakin taron furotin ya kasance daidai a 0.1 mg per ml, don haka wannan abin ya fita daga lissafi, amma ƙananan rukunin mitochondria da ingancin tsarinsu ba su fita ba. Kuma an kawar da zargin bambancin nau'in dabba: ƙungiya ta biyu ta sake samun irin sakamakonta a mice ma.

4. Yanayin sinadarin farawa yana ƙayyade abin da rinin zai iya yi. Tsare-tsaren sinadaran farawa uku idan aka kwatanta da nau'i-nau'i masu alaƙa da NADH. Samar da NADH da ƙarfin lantarki na membrane tare suna ƙayyade saurin yin reduction na rinin da inda zai taru, kuma hanyar da ke buƙatar rini da aka yi wa reduction kusa da cytochrome c da aka yi wa oxidation tana dogara ga wannan zaɓin.

Yadda za a karanta da'awar tsallakewa ta gaba

A tambayi wane complex aka toshe, da wane sinadarin hanawa, a wane wuri da wane adadi. A tambayi matakin taron rinin da lokacin da aka ɗauki ma'aunin. A tambayi nau'in nama, nau'in dabba, hanyar warewa, matakin taron furotin, da sinadarin farawa. Kuma a tambayi ko ƙarfin lantarki, numfashi, yanayin redox, da gwajin miƙa electron kai tsaye sun yi daidai, domin amfani da oxygen kaɗai ya nuna hanyar da ba daidai ba fiye da sau ɗaya a nan.

Takaitaccen hukuncin mai iyaka yana nan. An yarda da tsallake Complex I ta rini. Game da Complex III, mitochondria na mice tare da antimycin a 1 microgram per ml da rini a 1 micromolar ba su nuna tsallakewa mai dawo da aiki ba, yayin da nau'ikan dabbobi uku tare da antimycin a 0.3 micromolar ko myxothiazol a 1 micromolar da rini a 2 micromolar suka nuna tsallakewa zuwa wani mataki tare da miƙa electron kai tsaye daga rini zuwa cytochrome c. Waɗannan bayanai biyu suna iya kasancewa tare ba tare da karo ba. Duk wani hukunci da ya fi wannan faɗi hasashe ne da ya wuce abin da gwaje-gwajen suka nuna.

Karatu mai alaƙa yana ajiye da'awar da ake saɓani a kanta cikin ilimin sinadarai da ba a saɓani a kansa: yadda methylene blue ke motsa electron don fahimtar miƙawar, gwaje-gwajen tsallake Complex I don lamarin farkon jerin jigilar da aka tabbatar, ma'aunan ƙarfin lantarki na membrane da kurakuransu don fahimtar dalilin buƙatar rini biyu, da yadda ake sarrafa sinadaran zagayen redox a dakin gwaje-gwaje don fahimtar tarkunan adadi da lokaci.