Methylene blue inogona kupfuura Komplekisi III yemitokondiriya here? Nei zvidzidzo zvisingawirirani

Marabhoritari maviri akabvunza kana methylene blue ichigona kupfuura Komplekisi III yemitokondiriya yakavharwa. Imwe yakapindura kuti kwete. Imwe yakapindura kuti hongu. Dzose dzaiva nemashoko akabva muzviyedzo.

Chidzidzo chekutanga, chakashandisa mitokondiriya yeuropi hwembeva, chakawana kuti dhayi iyi haigoni. Chechipiri, chakashandisa mitokondiriya yeuropi kubva kumhando nhatu dzemhuka dzinoruma-ruma, chakawana kuti inogona kuzviita zvishoma. Chidzidzo chechitatu, chakashandisa chiropa chembeva, chinowanzotaurwa pamwe nezvimwe izvi asi chakaedza chimwe chinhu. Kana chikaiswa panzvimbo yacho yakakodzera, hafu yenyonganiso inobva yapera.

Hapana nzira imwe yekushanda inoshanda mumamiriro ose ingaziviswa pano. Pane zviyedzo zvitatu zvakashandisa zvinovharisa, huwandu hwedhayi nezviyero zvakasiyana. Tanga waverenga mamiriro ezviyedzo. Mhedziso dzinobva ipapo.

Zvinorehwa nekupfuura pano

Maerekitironi anopinda muketani kuburikidza neKomplekisi I (achibva kuNADH) kana Komplekisi II (achibva kusuccinate), opfuura nemuKomplekisi III achienda kucytochrome c, ozopfuura nemuKomplekisi IV achienda kuokisijeni. Musiyano unovakwa pakufamba uku ndiwo unoshandiswa nesero kugadzira ATP.

Kutaura kuti pane nzira yokupfuura kunoreva kuti methylene blue inotora maerekitironi pamberi pechivhariso yoaendesa pashure pacho. Dhayi iri muchimiro cheokisidhesheni inotora maerekitironi kubva kuNADH kana kuzvikamu zvekutanga zveketani, ichiva leucomethylene blue, uye chimiro ichi chagamuchira maerekitironi chinoapa kucytochrome c kana kuadzorera muketani. Kana maerekitironi achiendeswa pashure pechivhariso, kufema nepotenshiyari yemembrane zvinodzorerwa zvishoma. Kana achiendeswa pachivhariso kana pamberi pacho, hapana chinodzorerwa.

Chimwe chinhu chine chokuita neKomplekisi III chinotsanangura zvakawanda pakupokana uku. Komplekisi iyi inoshandisa kutenderera kweQ kune nzvimbo mbiri dzinosungirirwa nezvinovharisa. Antimycin inosungirira panzvimbo yeQi. Myxothiazol inosungirira panzvimbo yeQo. Chidzidzo chinoshandisa chimwe chete chezviviri izvi chinenge chaedza hafu chete yekomplekisi.

Chidzidzo chekutanga: hapana nzira yokupfuura mumitokondiriya yeuropi hwembeva

Gureev nevamwe vake vakashandisa mitokondiriya kubva mucortex yeuropi hwembeva dzeC57BL/6, yakaparadzaniswa nedigitonin, vakaiongorora pahuwandu hweprotein hwe0.1 mg per ml. Masubstrate akashandiswa mumamiriro matatu: pyruvate pamwe nemalate pa5 mM chimwe nechimwe, succinate pa10 mM pamwe neglutamate pa2 mM, uye L-proline pa10 mM. Zvinovharisa zvaiva rotenone pa1 micromolar (Komplekisi I) neantimycin pa1 microgram per ml (Komplekisi III). Huwandu hwedhayi hwaiva 0.5, 1, ne2 micromolar, 1 micromolar iri iyo yakanyanya kushandiswa. Nzira dzakashandiswa nemitsetse yezviyero zviri muchinyorwa cheFEBS Letters.

Kupfuura Komplekisi I kwakashanda. Rotenone yakaderedza kufema kunokurudzirwa neADP kubva panenge 121 kusvika panenge 2 nmol per minute per mg, uye dhayi pa1 micromolar yakakudzorera kusvika panenge 21. Potenshiyari yemembrane yakadzorerwa pakushandisa pyruvate pamwe nemalate uye pakushandisa L-proline.

Kupfuura Komplekisi III hakuna kushanda. Antimycin yakaderedza kufema kusvika panenge 16, uye dhayi yakangokukwidza kusvika panenge 20. Potenshiyari yemembrane haina kudzorerwa pasubstrate chero ipi zvayo, kunyangwe dhayi yakaiswa kusvika pa5 micromolar. Mashoko eperoxide airatidzawo zvakafanana: dhayi yoga yakakwidza kubudiswa kweperoxide kubva pachiyero chepakutanga chinenge 76 pmol per minute per mg kusvika panenge 1170, 1460, ne1945 pahuwandu hutatu hwedhayi, asi antimycin yakaderedza kubudiswa uku kwaikonzerwa nedhayi neinenge 25, 88, ne96 percent, zvichitevedzana. Kudai dhayi yakagamuchira maerekitironi yaipindirana zvakananga neokisijeni, kuvharisa Komplekisi III hakwaifanira kuderedza kubudiswa uku. Vanyori vakafungidzira kuti dhayi yakagamuchira maerekitironi inobva yaarasikirwa nawo panzvimbo yeQo yeKomplekisi III, iri pamberi pechivhariso cheantimycin. Kutenderera kupfupi uku, mumashoko: vanopa maerekitironi kuenda kudhayi, dhayi kuenda kuQo, yozotevera nzira yakajairika. Rotenone inopfuudzwa. Antimycin haipfuudzwi.

Chidzidzo chechipiri: kupfuura zvishoma mumhando nhatu dzemhuka, panzvimbo dzose mbiri dzezvinovharisa

Svab nevamwe vake vakawedzera zvakabatanidzwa muchiyedzo. Vakashandisa mitokondiriya yeuropi hwembeva, makonzo nenguruve dzeGuinea, yakaparadzaniswa nePercoll, vakaiongorora pa0.1 mg per ml imwe chete, vachishandisa pyruvate pamwe nemalate kana glutamate pamwe nemalate pa5 mM chimwe nechimwe. Vakashandisa zvinovharisa Komplekisi III zviviri: antimycin A pa0.3 micromolar nemyxothiazol pa1 micromolar. Dhayi yaiva pa2 micromolar muzviyero zvakawanda, uye pa0.5 mubasa recytochrome c. Mhando dzose nhatu dzemhuka, dzakaongororwa nenzira dzakafanana, dziri muchinyorwa chinowanikwa nemunhu wese.

Zvivhariso zvose zviri zviviri zvakapfuudzwa zvishoma, mumhando dzose nhatu dzemhuka. Mumitokondiriya yembeva, antimycin yakaderedza kufema uye dhayi yakakukwidza neinenge 60 percent, kubva panenge 37 kusvika pa59 nmol per minute per mg. Myxothiazol yakakuderedza ne88 percent, kusvika panenge 4.5, uye dhayi yakakukwidza kashanu kusvika panenge 23. Potenshiyari yemembrane yakadzorerwa zvishoma pachinovharisa chimwe nechimwe, ichiyerwa nedhayi mbiri (safranin O neratiometric TMRM) kuitira kubvisa mukana wekukanganisa kunokonzerwa nezvinhu zvine chokuita nechiedza.

Chinyorwa ichi chakaedzawo kuendeswa kwemaerekitironi zvakananga, izvo zvisina kuitwa nechidzidzo chekutanga. Cytochrome c yakaiswa mapoka eacetyl, yakawedzerwa kubva kunze pa25 micromolar, haina kugamuchira maerekitironi kubva kumitokondiriya ine simba, ingava ine zvinovharisa kana isina, asi kuwedzera dhayi kwakaita kuti igamuchire maerekitironi mumamiriro ose ari maviri. Mumushonga webuffer usina masero, NADH pamwe nedhayi zvakapa maerekitironi kucytochrome c pachiyero chaienderana nehuwandu hweNADH. Kusvetuka kwakareba uku, mumashoko: simba rokupa maerekitironi kuenda kudhayi, dhayi zvakananga kucytochrome c, Komplekisi IV kuenda kuokisijeni. Nzira iyi inosvetuka Komplekisi III yose, saka zvinovharisa zvose zviri zviviri zvinogona kupfuudzwa.

Zvimwe zviyero zviviri zvinotsigira kunzwisisa uku. Fluorescence yeNAD(P)H yakaderera pakawedzerwa dhayi kumitokondiriya yakaiswa myxothiazol, zvichireva kuti simba rokupa maerekitironi rakanga richibuda nenzira itsva. Uye kunyura kwechiedza nedhayi pa660 nm kwairatidza kuwedzerwa kwechinhu chimwe nechimwe, ukuwo mvura yepamusoro yakawanikwa mushure mekutenderedza musanganiswa muchina ichiratidza kuti dhayi iri muchimiro cheokisidhesheni yakanga yakaungana mukati memitokondiriya ine simba, nehuwandu hunopfuura hwekunze kanopfuura zana, yozobuda pakaparara potenshiyari. Kuverengwa kwemapurotoni kunoenderanawo nazvo: kusvetuka nhanho dzekupomba kunosiya anenge 2 mapurotoni papeya yemaerekitironi pane 10, saka kushandiswa kweokisijeni nekukurumidza uku potenshiyari ichidzorerwa zvishoma chete ndizvo zvinotarisirwa panzira chaiyo yokupfuura, kwete uchapupu hunoiramba.

Mitokondiriya yakagadzirirwa nenzira imwe chete iyi yakaratidza mhinduro dzinogona kukanganisa muverengi asinganyatsotarisisi. Paiva nechinovharisa pamwe nedhayi, ADP yakaderedza kufema pane kukukwidza, uye kuvhariswa kwekutakurwa kwezvinhu kwakakukurudzira pane kukuderedza, ukuwo potenshiyari yemembrane ichiri kuchinja nenzira dzakajairika. Kushandiswa kweokisijeni chete kunotsausa mugadziriro iyi.

Chidzidzo chechitatu: chidzidzo chechiropa chakaedza Komplekisi I neII, kwete III

Lee naBoelsterli vakashandisa mitokondiriya yechiropa chembeva nemasero echiropa akakuriswa murabhoritari, zvakanga zvakuvadzwa neefavirenz (Komplekisi I, IC50 pedyo ne30 micromolar) nehydrazine, chinhu chinobva pakushandurwa kweisoniazid (Komplekisi II, IC50 pedyo ne30 micromolar). Dhayi paanenge 30 micromolar yakadzorera okisidhesheni yeNADH, potenshiyari yemembrane neATP, uku ichideredza zvinhu zvinokonzera okisidhesheni nekufa kwemasero kuburikidza nenecrosis. Chinyorwa cheRedox Biology chinotsanangura izvi semaerekitironi anotakurwa nedhayi achipfuura Komplekisi I neII dzakakuvadzwa.

Mhedzisiro iyi inopa ruzivo rwunobvumiranwa pamusoro pezvinoitika pamberi peKomplekisi III, uye haitauri chinhu pamusoro peKomplekisi III nokuti hapana chakanga chavharwa ipapo. Haigoni kusimbisa kana kuramba chimwe chezvidzidzo zviviri zveuropi.

Tafura yemamiriro ezviyedzo

Zvikonzero zvina nei zvidzidzo zvose zviri zviviri zveKomplekisi III zvingava zvakarurama pamusoro pemamiriro azvo

1. Zvinovharisa hazvina kushandiswa nenzira imwe chete. Antimycin yoga pa1 microgram per ml ichienzaniswa neantimycin pa0.3 micromolar pamwe nechimwe chinovharisa nzvimbo yeQo chakashandiswa chakazvimirira. Nzira yedhayi inoendesa maerekitironi panzvimbo yeQo inokundikana chaipo panobudirira nzira inoenda zvakananga kucytochrome c. Nzvimbo imwe haigoni kumirira dzose dziri mbiri.

2. Huwandu hwedhayi nenguva zvakasiyana. Huwandu hwakanyanya kushandiswa hwaiva 1 huchienzaniswa ne2 micromolar, muchinhu chinotenderera pakati pemamiriro eredox, chine kupinda mukati uye kupa maerekitironi zvakananga kucytochrome c kunoenderana nehuwandu nenguva. Misiyano midiki yehuwandu pamwe nenguva dzakasiyana dzekuyera inogona kuratidza zvikamu zvakasiyana zvemambure mamwe chete enzira.

3. Kugadzirirwa kwemitokondiriya kwakasiyana. Kuparadzanisa nedigitonin kuchienzaniswa nekuparadzanisa nemusiyano wekuwanda kwePercoll kunosarudza mapoka emitokondiriya ane zvimwe zvakafanana asi asina kunyatsofanana. Huwandu hweprotein hwaifanana pa0.1 mg per ml, saka icho hachichatsananguri musiyano, asi mapoka madiki emitokondiriya nemamiriro ayo ekusakuvadzwa zvichiri kusiyana. Uye pfungwa yokuti mhando yemhuka ndiyo yaikonzera musiyano yakabviswa: boka rechipiri rakawana maitiro mamwe chete mumbeva zvakare.

4. Mamiriro emasubstrate ndiwo anoisa muganhu wezvingaitwa nedhayi. Mamiriro matatu emasubstrate achienzaniswa nemapeya akabatana neNADH. Kuwanikwa kweNADH nepotenshiyari yemembrane pamwe chete ndizvo zvinodzora kuti dhayi inokurumidza sei kugamuchira maerekitironi uye kuti inoungana kupi, uye nzira inoda dhayi yakagamuchira maerekitironi pedyo necytochrome c iri muchimiro cheokisidhesheni inosungirwa nemasarudziro iwayo.

Kuverenga chirevo chinotevera chenzira yokupfuura

Bvunza kuti komplekisi ipi yakavharwa, nechinovharisa chipi, panzvimbo ipi uye pahuwandu hupi. Bvunza huwandu hwedhayi uye kuti chiyero chakatorwa riini. Bvunza tishu, mhando yemhuka, nzira yekuparadzanisa, huwandu hweprotein nesubstrate. Uye bvunza kana potenshiyari, kufema, mamiriro eredox uye chiyero chekuendeswa kwemaerekitironi zvakananga zvichiwirirana, nokuti pano kushandiswa kweokisijeni chete kwakaratidza nzira isiri iyo kanopfuura kamwe.

Pfupiso ine muganhu uyu ichiri kushanda. Kupfuura Komplekisi I nedhayi kunobvumiranwa. Panyaya yeKomplekisi III, mitokondiriya yembeva ine antimycin pa1 microgram per ml nedhayi pa1 micromolar hairatidzi nzira yokupfuura inodzoreredza kushanda, nepo mhando nhatu dzemhuka dzine antimycin pa0.3 micromolar kana myxothiazol pa1 micromolar nedhayi pa2 micromolar dzichiratidza kupfuura zvishoma, nemaerekitironi achibva kudhayi achienda zvakananga kucytochrome c. Zvirevo izvi zviviri zvinogona kuva zvechokwadi pamwe chete. Chinhu chipi nechipi chinopfuura muganhu uyu kufungidzira kupfuura zvakayedzwa.

Zvinyorwa zvinoenderana nenyaya iyi zvinochengeta chirevo chinopikiswa chiri mukati mekemisitiri isingapikiswi: mafambisirwo emaerekitironi nemethylene blue kuti unzwisise kupfuudzana kwawo, zviyedzo zvekupfuura Komplekisi I pamusoro penyaya inobvumiranwa yezvinoitika pakutanga kweketani, zviyero zvepotenshiyari yemembrane nekukanganisa kwazvo kuti unzwisise nei dhayi mbiri dzaidiwa, uye mabatirwo emarabhoritari ezvinhu zvinotenderera pakati pemamiriro eredox kuti unzwisise misungo yehuwandu nenguva.