Ka taea e te kikorangi metilīni te karo i te Matatini III o te pungao? He aha i rerekē ai ngā kitenga rangahau

E rua ngā taiwhanga i ui mēnā ka taea e te kikorangi metilīni te karo i te Matatini III o te pungao kua aukatia. I kī tētahi, kāore. I kī tētahi atu, āe. He raraunga ā rāua tahi.

I kitea e te rangahau tuatahi, ki ngā pungao roro o te kiore iti, kāore e taea e te tae. I kitea e te rangahau tuarua, ki ngā pungao roro o ngā momo kiore e toru, ka taea i tētahi wāhanga. He maha ngā wā ka kōrerotia tētahi rangahau tuatoru, ki te ate o te kiore iti, i te taha o ēnei, engari he mea kē tāna i whakamātau. Ki te noho taua rangahau ki tōna horopaki tika, ka mārama te haurua o te rangirua.

Kāore he tikanga mahi whānui e taea te whakapuaki. E toru kē ngā whakamātau, he rerekē ngā aukati, ngā rahinga, me ngā inenga. Pānuitia ngā āhuatanga whakamātau i te tuatahi. Ka whai mai ngā whakatau.

Te tikanga o te karo i konei

Ka uru ngā irahiko ki te mekameka mā te Matatini I (mai i NADH), mā te Matatini II rānei (mai i succinate), ka haere mā te Matatini III ki te haitokaroma c, kātahi mā te Matatini IV ki te hāora. Ko te rōnaki ka hangaia i taua haerenga te mea ka whakamahia e te pūtau hei hanga ATP.

E ai ki te kerēme karo, ka tango te kikorangi metilīni i ngā irahiko i mua i tētahi aukatinga, ka tuku ai i muri. Ka tango te tae kua whakahāoratia i ngā irahiko mai i NADH, mai i ngā wāhanga tōmua rānei o te mekameka, ka huri hei leucomethylene blue. Kātahi te āhua kua whakahekea ka tuku i aua irahiko ki te haitokaroma c, ki roto anō rānei i te mekameka. Ki te tau te tukunga ki muri i te aukatinga, ka ora ake tētahi wāhanga o te whakahā me te pūmanawa kiriuhi. Ki te tau ki te aukatinga tonu, ki mua rānei, kāore he whakaoranga.

He taipitopito kotahi o te Matatini III e whakatau ana i te nuinga o tēnei tautohe. Ka whakahaere te matatini i tētahi huringa Q, e rua ōna wāhi here aukati. Ka here te antimycin ki te wāhi Qi. Ka here te myxothiazol ki te wāhi Qo. Ko te rangahau e whakamahi ana i tētahi anake o ēnei, ko te haurua anake o te matatini tāna i whakamātau.

Rangahau tuatahi: kāore he karo i ngā pungao roro o te kiore iti

I whakamahia e Gureev rātou ko ōna hoa ngā pungao nō te kiri roro o ngā kiore iti C57BL/6, i wehea mā te digitonin, ā, i inea ki te kukū pūmua 0.1 mg per ml. E toru ngā huinga tïpako: pyruvate me malate, 5 mM mō ia mea; succinate, 10 mM, me glutamate, 2 mM; me L-proline, 10 mM. Ko ngā aukati ko rotenone, 1 micromolar (Matatini I), me antimycin, 1 microgram per ml (Matatini III). Ko ngā rahinga tae ko 0.5, 1, me 2 micromolar, ā, ko 1 micromolar te kukū matua. Kei te pūrongo FEBS Letters ngā tikanga me ngā kauwhata inenga.

I mahi te karo i te Matatini I. Nā te rotenone i whakaheke te whakahā i whakaohohia e ADP mai i te āhua 121 ki te āhua 2 nmol per minute per mg, ā, nā te tae 1 micromolar i whakahoki ki te āhua 21. I ora ake te pūmanawa kiriuhi i runga i te pyruvate me te malate, me te L-proline.

Kāore i mahi te karo i te Matatini III. Nā te antimycin i whakaheke te whakahā ki te āhua 16, ā, nā te tae i hiki paku noa ki te āhua 20. Kāore te pūmanawa kiriuhi i ora ake i runga i tētahi tïpako, ahakoa ngā rahinga tae tae atu ki te 5 micromolar. I tohu anō ngā raraunga peroxide ki taua whakatau: nā te tae anake i hiki te tukunga peroxide mai i te tere taketake tata ki te 76 pmol per minute per mg ki te āhua 1170, 1460, me 1945 i ngā rahinga e toru. Heoi, nā te antimycin i whakaheke taua tohu i ahu mai i te tae mā te āhua 25, 88, me 96 ōrau, mō ia rahinga. Mehemea ka tauhohe tika te tae kua whakahekea ki te hāora, kāore te aukati i te Matatini III e pēhi i taua tohu. I whakatau ngā kaituhi ka whakahāoratia te tae kua whakahekea ki te wāhi Qo o te Matatini III, i mua i te aukatinga antimycin. Hei kupu mō te ara poto: ngā kaituku ki te tae, te tae ki Qo, kātahi ko te ara noa ki mua. Ka karohia te rotenone. Kāore te antimycin e karohia.

Rangahau tuarua: he karo wāhanga i ngā momo e toru, ki ngā wāhi aukati e rua

I whakawhānui a Svab rātou ko ōna hoa i te hoahoa whakamātau. I whakamahia ngā pungao roro o ngā kiore iti, ngā kiore nui, me ngā poaka kini, i wehea mā te Percoll, ā, i inea ki taua kukū tonu, 0.1 mg per ml, i runga i te pyruvate me te malate, i te glutamate me te malate rānei, 5 mM mō ia mea. I whakamahia e rātou e rua ngā aukati Matatini III: antimycin A, 0.3 micromolar, me myxothiazol, 1 micromolar. Ko te kukū tae he 2 micromolar i te nuinga o ngā whakamātau, he 0.5 i ngā whakamātau haitokaroma c. Kei te tuhinga uru tuwhera ngā momo e toru katoa, me ngā kawa whakamātau ōrite.

I karohia tētahi wāhanga o ngā aukatinga e rua, i ngā momo e toru katoa. I ngā pungao o te kiore iti, nā te antimycin i pēhi te whakahā, ā, nā te tae i hiki mā te āhua 60 ōrau, mai i te āhua 37 ki te 59 nmol per minute per mg. Nā te myxothiazol i pēhi mā te 88 ōrau, ki te āhua 4.5, ā, nā te tae i hiki kia rima whakareatanga, ki te āhua 23. I hoki mai tētahi wāhanga o te pūmanawa kiriuhi i raro i ia aukati. I inea tēnei mā ngā tae e rua (safranin O me te TMRM ōwehenga-inenga), hei whakakore i ngā hapa whatu.

I whakamātau tika hoki tēnei rangahau i te tukunga irahiko, kāore nei i mahia e te rangahau tuatahi. Kāore te haitokaroma c kua tāpirihia mai i waho, kua acetylated hoki, i te kukū 25 micromolar, i whakahekea e ngā pungao whai pūngao, ahakoa he aukati, kāore rānei. Engari, nā te tāpiri tae i whakakaha te whakahekenga i ngā āhuatanga e rua. I roto i te wairewa pūreirei kore pūtau, nā NADH me te tae i whakaheke te haitokaroma c, i tētahi tere e hāngai ana ki te nui o NADH. Hei kupu mō te peke roa ake: ngā taurite whakaheke ki te tae, te tae ki te haitokaroma c tonu, te Matatini IV ki te hāora. Ka hipa katoa taua ara i te Matatini III, nō reira ka taea te karo i ngā aukati e rua.

E rua anō ngā inenga e tautoko ana i taua whakamāramatanga. I heke te kōrekoreko NAD(P)H i te tāpiritanga o te tae ki ngā pungao i tukuna ki te myxothiazol, e tohu ana kua rere atu ngā taurite whakaheke mā te ara hou. Waihoki, i whai te ngongo mārama a te tae ki te 660 nm i ia tāpiritanga. I whakaatu ngā wai o runga i muri i te hurihanga whakatau he neke atu i te kotahi rau whakareatanga te kukū o te tae kua whakahāoratia i roto i ngā pungao whai pūngao, kātahi ka puta atu i te hinganga o te pūmanawa. Ka hāngai hoki te kaute iraoho: mā te hipa i ngā takahanga papu, ka toe mai te āhua 2 iraoho mō ia takirua irahiko, kaua ko te 10. Nō reira, ko te tere ake o te whakamahi hāora, me te hokinga wāhanga noa o te pūmanawa kiriuhi, te āhua o tētahi karo tūturu, ehara i te taunakitanga whakahē.

I hua mai hoki i taua takanga tonu ētahi urupare e whakaatu ana i te mōrearea o te pānui pāpaku. I te aroaro o te aukati me te tae, nā ADP i whakaheke te whakahā, kaua i hiki, ā, nā te aukatinga kawe i whakaoho, kaua i whakaroa. Heoi, i neke tonu te pūmanawa kiriuhi ki ngā aronga noa. Ka whakapohehe te kohinga hāora anake i tēnei pūnaha.

Rangahau tuatoru: te rangahau ate i whakamātau i ngā Matatini I me II, kaua i te III

I whakamahia e Lee rāua ko Boelsterli ngā pungao ate o te kiore iti me ngā pūtau ate i whakatipuria, i tūkinohia e efavirenz (Matatini I, IC50 tata ki te 30 micromolar) me hydrazine, te hua pākia o isoniazid (Matatini II, IC50 tata ki te 30 micromolar). Nā te tae, i te āhua 30 micromolar, i whakahoki te whakahāoratanga NADH, te pūmanawa kiriuhi, me te ATP, me te whakaheke i ngā matū whakahāora me te matenga necrotic. Ka whakatauira te tuhinga Redox Biology i tēnei hei kawe irahiko a te tae mā tētahi ara huri i ngā Matatini I me II kua tūkinohia.

He horopaki tēnei hua kua whakaaetia mō ngā wāhanga tōmua o te mekameka. Kāore he kōrero mō te Matatini III, nō te mea kāore tētahi mea i aukatia ki reira. Kāore e taea e tēnei te whakaū, te whakahē rānei, i tētahi o ngā rangahau roro.

Te ripanga āhuatanga whakamātau

E whā ngā take ka tika ai ngā rangahau Matatini III e rua mō ō rāua ake āhuatanga

1. Kāore i ōrite te mahinga aukati. Ko te antimycin anake i te 1 microgram per ml, ki te antimycin i te 0.3 micromolar me tētahi aukati motuhake mō te wāhi Qo. Ka kore te ara tae e tuku ana ki te wāhi Qo e mahi ki taua wāhi tonu e angitu ai tētahi ara tika ki te haitokaroma c. Kāore e taea e te wāhi kotahi te whakakapi i ngā wāhi e rua.

2. He rerekē te rahinga tae me te wā. Ko ngā kukū matua he 1 me te 2 micromolar, i tētahi matū huringa whakahāora-whakaheke e whakawhirinaki ana tōna urunga me tōna whakahekenga tika i te haitokaroma c ki te kukū me te wā. Mā ngā rerekētanga rahinga iti, me ngā wā inenga rerekē, e kitea ai ngā peka rerekē o te whatunga kotahi.

3. He rerekē ngā takanga. Ka kōwhiri te wehenga digitonin me te wehenga rōnaki Percoll i ngā huinga pungao e īnaki ana, engari kāore i ōrite katoa. I ōrite te kukū pūmua, 0.1 mg per ml, nō reira kāore taua taurangi e whakamārama i te rerekētanga. Engari, kei te whai take tonu ngā huinga iti me te pai o te mau o ngā pungao. Ā, kua whakakorea te whakapae ko te momo kararehe te take: i whakaputaina anō e te rōpū tuarua tōna āhua urupare ki te kiore iti hoki.

4. Mā te āhua o ngā tïpako e whakatau ngā mahi ka taea e te tae. E toru ngā huinga tïpako, ki ngā takirua e hono ana ki NADH. Mā te wātea o NADH me te pūmanawa kiriuhi e whakatau ngātahi te tere o te whakahekenga o te tae, me te wāhi e tohatoha ai. Ko tētahi ara e hiahia ana ki te tae kua whakahekea i te taha o te haitokaroma c kua whakahāoratia ka whakawhirinaki ki taua kōwhiringa.

Me pēhea te pānui i te kerēme karo e whai ake nei

Ui ko tēhea matatini i aukatia, mā tēhea aukati, ki tēhea wāhi, me tēhea rahinga. Ui mō te kukū tae me te wā i mahia ai te inenga. Ui mō te kiko, te momo kararehe, te tikanga wehe, te kukū pūmua, me te tïpako. Ui hoki mēnā e hāngai ana ngā inenga pūmanawa, whakahā, āhua whakahāora-whakaheke, me tētahi whakamātau tukunga irahiko tika, nō te mea neke atu i te kotahi te wā i tohu ai te kohinga hāora anake ki te aronga hē i konei.

Ka mau tonu te whakarāpopototanga whāiti. E whakaaetia ana te karo a te tae i te Matatini I. Mō te Matatini III, kāore ngā pungao o te kiore iti, me te antimycin i te 1 microgram per ml me te tae i te 1 micromolar, e whakaatu ana i tētahi karo e mahi ana. Engari, e whakaatu ana ngā momo e toru, me te antimycin i te 0.3 micromolar, te myxothiazol rānei i te 1 micromolar, me te tae i te 2 micromolar, i te karo wāhanga me te whakawhiti tika mai i te tae ki te haitokaroma c. Ka taea e aua kōrero te noho tahi. Ko ngā whakatau whānui ake he whakawhānuitanga i tua atu i ngā raraunga.

Mā ngā pānuitanga e pā ana e pupuri te kerēme e tautohetia ana i roto i te matū e whakaaetia ana: te āhua e neke ai te kikorangi metilīni i ngā irahiko mō te tuku haere; ngā whakamātau karo i te Matatini I mō te take kua whakaaetia i te wāhanga tōmua o te mekameka; ngā inenga pūmanawa kiriuhi me ō rātou hapa mō te take i hiahiatia ai ngā tae e rua; me te whakahaere taiwhanga i ngā matū huringa whakahāora-whakaheke mō ngā raruraru o te rahinga me te wā.