Ukulinganisa iziphumo zemethylene blue kwiimitochondria: ioksijini, i-ATP, nomahluko wombane kwinwebu

Masithi ilabhoratri yongeza imethylene blue kwiimitochondria ezinokuphefumla okuphazamisekileyo ize ibone ukusetyenziswa kweoksijini kunyuka. Isivakalisi esinokubhalwa lula kukuba idayi ibuyisele ukusebenza kweemitochondria. Eso sivakalisi sisenokuba asichanekanga. Ukuhamba kweoksijini, ukwenziwa kwe-ATP, nomahluko wombane kwinwebu zizinto ezintathu ezahlukileyo. Zenzeka kwi-organelle enye ngendlela efanayo naleyo ugcino lwekopi, ungqamaniso, nokwabelana ezenzeka ngayo kwifolda enye esefini: ziyazalana, kodwa zilawulwa yimithetho eyahlukileyo. Ukuba ubhidanisa enye nenye, utoliko luyaphazama, nokuba umlinganiselo ngamnye uchanekile ngokobugcisa.

Imethylene blue yenza ukuba oku kubhideka kwenzeke lula kuba ithatha inxaxheba kwinkqubo eyilinganiselayo. Ijikeleza kwi-redox, yamkele ii-electron ekuqaleni kwendlela ize izinikele kamva; iyikationi ethanda amafutha eqokelelana kwiimitochondria ezinamandla; kwaye iyikhompawundi eluhlaza okwesibhakabhaka ngamandla efunxa ize ikhuphe ukukhanya kumabhanti afanayo nawezikhangeli ezisetyenziswa rhoqo. Umlinganiselo omnye awunakuthwala wonke loo mthwalo. Indlela ethembekileyo, eboniswe kwizifundo zeemitochondria zaseBudapest, kukulinganisa ukuhamba kweoksijini kunye nesantya sokwenziwa kwe-ATP kunye nomahluko wombane kwinwebu kunye nokukhutshwa kwehydrogen peroxide, kusetyenziswa amalinge olawulo ajolise kwidayi kwinyathelo ngalinye.

Ukulinganisa ukuphefumla kulinganisa ukuhamba kweoksijini, kungekhona imveliso yamandla

Ukulinganisa ukuphefumla ngokuchaneka okuphezulu, nokuba kusetyenziswa i-Oroboros Oxygraph okanye isihlalutyi sokuhamba ngaphandle kweeseli seSeahorse, kunika ingxelo yento enye: isantya ioksijini eyamkela ngaso ii-electron. Akuchazi ukuba zingaphi iiproton ezimpontshwe nge-electron nganye, ungakanani umahluko wamandla ogciniweyo, okanye ukuba i-ATP synthase iwusebenzisile na.

Isigama sibalulekile. Kwiimitochondria ezahluliweyo, isubstrate ngaphandle kwe-ADP inika ukuphefumla okunxulumene nokuvuza, isubstrate ene-ADP eyaneleyo ukufikelela ekugcwaleni inika umthamo we-oxidative phosphorylation, kwaye ukongeza ngokuthe ngcembe into esahlula ukuthuthwa kwee-electron ekwenziweni kwe-ATP kunika umthamo ophezulu wokuthuthwa kwee-electron. Kwiiseli ezingaphazanyiswanga, imilinganiselo ehambelanayo yeSeahorse kukuphefumla okusisiseko, ukuphefumla okunxulumene ne-ATP okuchatshazelwa yi-oligomycin, ukuvuza kweeproton, ukuphefumla okuphezulu okuchazwa yi-FCCP, kunye nokusetyenziswa kweoksijini okungekho kwiimitochondria okungathintelwayo yirotenone kunye ne-antimycin. Ukubiza iziphumo zeSeahorse ngokuba yi-State 3 ne-State 4 kuboleka ukuchaneka okungaxhaswanga lulungiselelo lwesampulu, njengoko isikhokelo se-Agilent Mito Stress Test sicacisa.

Imethylene blue iqhawula unxulumano olubonakala lulula phakathi kweoksijini eninzi namandla amaninzi. Kwiimitochondria ezahluliweyo zobuchopho behagu yaseGuinea, idayi esuka ku-100 nM ukuya ku-1 uM yandise ukusetyenziswa kweoksijini ngexesha lokuphumla xa kusetyenziswa iisubstrate zeglutamate kunye nemalate, isuccinate, kunye ne-alpha-glycerophosphate, ngokunyuka okumalunga nama-64 ekhulwini kwi-alpha-glycerophosphate. Kodwa ukuphefumla okukhuthazwa yi-ADP kwiimitochondria ezisempilweni akuzange kutshintshe ngokubonakalayo, ngokutsho kwesifundo seemitochondria zobuchopho ezahluliweyo. Ngenxa yokuba ukuvuza kwenyuka ngoxa ukuhamba okukhuthazwa yi-ADP kwahlala kungatshintshanga, umlinganiselo obaliweyo wolawulo lokuphefumla uyehla. Oko kwehla akuthethi ukuba idayi yonakalise iimitochondria. Kuthetha ukuba idayi yongeze umjikelo we-redox osebenzisa ioksijini phezu kokuphefumla okuqhelekileyo. Uvavanyo lokusetyenziswa kweoksijini lwemethylene blue oluchaza kuphela ukunyuka lulinganise ikhemistri yokwenene kodwa aluthethanga nto ngokugcinwa kwamandla.

Amalinge okubuyisela ukusebenza abonisa umsantsa ofanayo ngomlinganiselo omkhulu ngakumbi. Xa i-Complex I ithintelwe, ukuhamba kweoksijini kwabuyela malunga ne-3 phinda, ngoxa ukwenziwa kwe-ATP kwabuyela kuphela malunga ne-1.5 phinda. Ioksijini eninzi yafikelela kwi-electrode. Inxalenye encinane yayo yancedisa ekwenzeni i-ATP. Naliphi na ibango lokuphucuka kwe-oxidative phosphorylation lifuna icala le-ATP kwingxelo, ngokufanelekileyo lichazwe njengomlinganiselo we-ATP ngeoksijini nganye, kuchazwe nendlela yokubala idinomineyitha.

Iimvavanyo ze-ATP zilinganisa isantya sokwenziwa kwayo, esingenakuthatyathelwa indawo sisixa esikhoyo

Uvavanyo lwe-ATP luphendula umbuzo owahlukileyo: ingakanani i-ATP eyenziwa ngeyunithi yexesha nganye. Umlinganiselo olandela utshintsho ngokuhamba kwexesha, nokuba kukukhanya kwe-luciferase yenkanyezi elandelwa kangangemizuzu okanye luvavanyo oludibanisa i-hexokinase kunye neglucose-6-phosphate dehydrogenase olufundwa ku-340 nm, unika isantya. Umlinganiselo omnye we-ATP iyonke ekupheleni kovavanyo unika isixa esikhoyo, esibonisa ukwenziwa kuthatyathwe ukusetyenziswa, kwaye sisodwa sixela okuncinane nge-oxidative phosphorylation.

Iqela laseBudapest lasebenzisa indlela yee-enzyme ezidityanisiweyo ngoxa i-adenylate kinase icinezelwe, laza lafumanisa ukuba imethylene blue eku-1 ukuya ku-2 uM yandise imveliso ye-ATP kancinane kodwa ngendlela ebalulekileyo kwiimitochondria ezinyangwe ngerotenone nezo zinyangwe nge-antimycin. Oko kukubuyiselwa kokusebenza kokwenene, kwaye kuyinxalenye kuphela. Isifundo sobutyhefu be-TEGDMA safumana uhlobo olufanayo lwempendulo kwimodeli eyahlukileyo: i-5 mM yemonoma yamazinyo i-TEGDMA yehlisa ukuphefumla okunxulumene ne-Complex I ukusuka malunga ne-286 ukuya ku-28 yeeyunithi zokuhamba, yaza idayi eku-2 uM yakubuyisela malunga ne-86.

Amalinge amabini olawulo ayimfuneko xa kusetyenziswa ikhompawundi enemibala esebenza kwi-redox. Okokuqala, sebenzisa i-oligomycin ukumisela ukuba ingakanani i-ATP elinganisiweyo eyenziwa ngokuxhomekeka kwi-ATP synthase. Okwesibini, yenza uvavanyo lokubuyiswa kwesixa esaziwayo esongeziweyo ngaphandle kweeseli, usebenzisa i-ATP eyaziwayo kunye nomxube opheleleyo wezivuseleli kunye nemilinganiselo yoxinaniso lwedayi kanye esetyenzisiweyo, ubuncinane i-0, 100 nM, 300 nM, 1 uM, kunye ne-2 uM. Idayi inokufunxa ukukhanya okukhutshwayo, ithintele i-enzyme enika umqondiso, okanye itshintshe ikhemistri ye-redox yezivuseleli. Isikhokelo esiqhelekileyo seemvavanyo ze-luciferase sicebisa iimvavanyo ezongezelelweyo zebhayokhemistri zokuchonga kanye olu hlobo lokuphazamiseka. Musa ukucinga ukuba i-1 uM ayinaziphazamiso zomlinganiselo ngenxa yokuba ukuncitshiswa kokukhanya kudla ngokuba kubi ngakumbi xa uxinaniso luphezulu.

Izikhangeli zomahluko wombane kwinwebu zilinganisa ukusasazeka kwedayi, kungekhona ukuhamba

I-Safranine O, i-TMRM ne-TMRE, irhodamine 123, kunye ne-JC-1 zonke ziikationi ezinokudlula kwinwebu. Ziqokelelana kwimatriksi enentlawulo engalunganga ngokolungelelwano lwe-electrochemical, ngoko ukukhanya kwefluorescence kwesampulu iyonke kubonisa umahluko wombane ngokungangqalanga, ngokwendawo esikuyo isikhangeli. Akukho nanye kuzo elinganisa i-ATP okanye ukuhamba kwee-electron.

Isikhangeli ngasinye sinolungiselelo lwesampulu olusifaneleyo. I-Safranine O ifanele iimitochondria ezahluliweyo neeseli ezenziwe zavumela izinto ukuba zingene, ngokuqhelekileyo kufutshane ne-2 uM, ivuselelwa kukukhanya okufutshane ne-495 nm ize ikhuphe ukukhanya kufutshane ne-585 nm. Isebenza kwimeko yokuncitshiswa kokukhanya apho ukufunxwa kwayo kutshintsha ukukhanya kwefluorescence kwesampulu iyonke. I-TMRM ne-TMRE zifanele iiseli ezingaphazanyiswanga xa zifakwa ngoxinaniso oluphantsi lwe-nanomolar kwimeko engenakho ukuncitshiswa kokukhanya, apho ukuphela komahluko wombane kukhupha idayi kuze kwehle ukukhanya kwefluorescence kweemitochondria; xa kufakwe kakhulu, indlela otshintsha ngayo umqondiso inokujika ngenxa yokuncitshiswa kokukhanya yidayi ngokwayo. I-JC-1 inika umlinganiselo oluncedo wokukhanya okuluhlaza ukuya kokubomvu kodwa ixhomekeke kwisixa esifakiweyo nakubuninzi beemitochondria, nto leyo eyenza ingabi namandla kangako kumsebenzi wokulinganisa amanani xa ithelekiswa ne-TMRM elawulwa ngononophelo. Ezi nzuzo nemiqobo zishwankathelwe kuphononongo olusetyenziswa ngokubanzi lwezikhangeli zomahluko wombane kwinwebu yeemitochondria.

Imethylene blue iphazamisa ukukhanya kanye kwimilinganiselo yoxinaniso esetyenziswayo. Amabhanti ayo okufunxa ukukhanya kufutshane ne-600 ukuya ku-665 nm ayadibana nokukhanya okubomvu okukhutshwa zizikhangeli, ngoko iziphumo zokuhluza ukukhanya ngaphakathi kwesampulu, ukuncitshiswa kokukhanya, nokufunxwa kokukhanya okuvuselelayo zonke zinokwenzeka. Idayi ikwayikationi yesibini ethanda amafutha, enokusasazeka okuguquka ngokomahluko wombane olinganiswayo. Ukuzikhusela koko kufuna imilinganiselo engaphezu kwenye kunye neesampulu zolawulo: isikhangeli sodwa, idayi yodwa, isikhangeli kunye nomlinganiselo ngamnye woxinaniso lwedayi, ukupheliswa ngokupheleleyo komahluko wombane nge-FCCP njengomlinganiselo onguziro, kwaye ngokufanelekileyo isikhangeli sesibini esineempawu zokukhanya ezahlukileyo. Isifundo sokudlula kwi-Complex III ngenye indlela sabeka loo mzekelo ngokuphinda uvavanyo lokubuyiselwa komahluko wombane kwinwebu kusetyenziswa zombini i-safranine O ne-TMRM emva kokuthintelwa nge-antimycin ne-myxothiazol, saza safumana ukubuyiselwa okufanayo ngokohlobo. Isixhobo esinye esinika umqondiso wokukhanya sisalathiso. Izixhobo ezimbini ezizimeleyo zibubungqina.

Amalungiselelo amathathu, imibuzo emithathu eyahlukileyo

Iimitochondria ezahluliweyo, iiseli ezenziwe zavumela izinto ukuba zingene, neeseli ezingaphazanyiswanga azizozicwangciso zobuntununtunu ezinokutshintshana kuvavanyo olunye. Zizinto ezahlukileyo ezinemigaqo yokusebenza eyahlukileyo.

Iimitochondria ezahluliweyo zigcina ikhonkco lokuthuthwa kwee-electron, inwebu yangaphakathi, neenkqubo zematriksi ngoxa kususwa inwebu yeseli, icytosol, kunye nolwakhiwo lothungelwano. Oko kususwa yeyona njongo: iisubstrate, i-ADP, izithinteli, nemilinganiselo yoxinaniso lwedayi zichazwa ngokuchanekileyo, ukuze kuchongwe indlela ebandakanyekayo. Umqobo kukulahleka konikezelo lwendalo lweesubstrate, ukudluliselwa kwemiqondiso, nemfuno yamandla.

Iiseli ezenziwe zavumela izinto ukuba zingene zigcina iimitochondria ngaphakathi kwenxalenye enkulu yolwakhiwo lwazo lweseli, ngoxa zivumela iisubstrate zangaphandle ne-ADP ukuba zidlule kwinwebu yeseli evuliweyo. Zisisixhobo esifanelekileyo sokuchonga isiphene kwindlela yesubstrate ngaphakathi kumxholo weseli. Ukwenza inwebu ivuleke ngokugqithiseleyo konakalisa inwebu yangaphandle, kwaye ulawulo lwecytosol luyahlanjwa luphume.

Iiseli ezingaphazanyiswanga zigcina yonke into: umqobo wenwebu yeseli, izinto ezinikezela ngee-electron kwi-cytosol, ukuthuthwa kweesubstrate zangaphakathi, uthungelwano lweemitochondria, nemfuno ye-ATP. Zisondele kakhulu kwindlela umzimba osebenza ngayo kwaye zikude kakhulu ekucaciseni indlela esebenza ngayo inkqubo, kuba ukufunxwa kwedayi, ukwamkela kwayo ii-electron, nokubanjwa kwayo kwiindawo ezithile zonke ziphembelela ukuba iimitochondria zisesichengeni sedayi kangakanani.

Isifundo se-TEGDMA sasebenzisa omathathu amanqanaba ngabom: iimitochondria zobuchopho ezahluliweyo ukuvavanya ukubuyiselwa kwe-oxidation nomahluko wombane yidayi, iiseli ezisiqu zentlama yamazinyo ezenziwe zavumela izinto ukuba zingene ikakhulu ukuchonga ukuba isiphene sikwi-oxidation yeesubstrate enxulumene ne-Complex I, kunye neeseli ezisiqu ezingaphazanyiswanga ukuvavanya isiphumo esipheleleyo ngokokusebenza kweeseli sedayi eku-2 uM ekusetyenzisweni kweoksijini okucinezelweyo. Kubafundi abathelekisa imilinganiselo yeemitochondria kumalungiselelo ahlukeneyo, inqaku elihambelana neli elichaza indlela imethylene blue ehambisa ngayo ii-electron kwikhonkco lokuphefumla linika imvelaphi yendlela, kwaye isikhokelo sokufunda izifundo zokuphefumla ezisebenzisa imethylene blue sichaza ingqiqo yesixa nezithinteli.

Amalinge olawulo afunekayo kwidayi esebenza kwi-redox

Into ejikeleza kwi-redox ifuna uluhlu olude lwamalinge olawulo kunesithinteli esiqhelekileyo, kuba ikhemistri yedayi yodwa inokulinganisa iinkqubo zebhayoloji.

Xa ulinganisa ukuphefumla, sebenzisa igumbi elinesisombululo sokugcina i-pH kuphela ukujonga ukutyibilika komlinganiselo, igumbi elineso sisombululo kunye nedayi ngaphandle kwezinto zebhayoloji, idayi kunye nayo nayiphi na into enikezela ngee-electron ngaphandle kweeseli ekhoyo kuvavanyo, kunye nerotenone ne-antimycin ekupheleni ukuchaza ukusetyenziswa kweoksijini okungekho kwiimitochondria kwiiseli ezingaphazanyiswanga. Qaphela ukuba idayi eyamkele ii-electron iphinda yenze i-oxidation ngokudlulisela ii-electron kwioksijini ukwenza ihydrogen peroxide. Ngoko isampulu yolawulo enedayi yodwa ngaphandle komnikeli wee-electron iwujongela phantsi umthombo wokusetyenziswa kweoksijini ongenaziseli ovela xa i-NADH okanye i-NADPH ifumaneka.

Kwihydrogen peroxide, khetha i-Amplex UltraRed elungelelaniswe nomgangatho kunye nehorseradish peroxidase kuneedayi ze-DCFDA ezingakhethiyo, qinisekisa ngecatalase, uze uvavanye ukuxhomekeka ekukhanyisweni, kuba idayi ivuselela iimpendulo zekhemistri ngokukhanya. Isifundo seemitochondria zobuchopho safumanisa ukuba umthwalo weperoxide wanyuka ngaphezu kwe-4 phinda ngedayi eku-100 nM kuphela ngoxa ukususwa kwayo kwacotha, kwaye iperoxide yenziwa ngokuthe ngqo yidayi eyamkele ii-electron ngaphandle kwesigaba esiphakathi se-superoxide. Iinkcukacha zokukhetha isikhangeli namalinge olawulo okukhanya zikweli nqaku lihambelanayo lokulinganisa iintlobo zeoksijini ezisebenza kakhulu kumalinge edayi, kwaye umbuzo obanzi wokuba iperoxide isebenza nini njengomqondiso okanye njengomthwalo uxutyushwa kunye nolungelelwano lwe-redox lweeseli namabango ngezinto ezichasene ne-oxidation.

Kumabango ngeendlela zokusebenza, vavanya zombini iindawo zezithinteli ze-Complex III: i-antimycin A kwindawo ye-Qi ne-myxothiazol kwindawo ye-Qo, kunye nesiphelo se-Complex IV usebenzisa icyanide. Isifundo sangaphambili sobuchopho bempuku sachaza ukubuyiselwa kokuphefumla yidayi emva kokuthintelwa ngerotenone kodwa kungekhona emva kokuthintelwa nge-antimycin. Isifundo samva esisebenzise iintlobo ezintathu zezilwanyana safumana ukubuyiselwa okuyinxalenye ngezithinteli zombini nobungqina bokwamkeliswa kwee-electron ngokuthe ngqo kwi-cytochrome c. Phatha indlela yokudlula njengento ehambelana nedatha entsha kunokuba ibe yinto esele iqinisekisiwe, kuba uxinaniso lwedayi, indawo yesithinteli, imeko yesubstrate, nokusasazeka kwedayi zonke ziguqula indlela ebonwayo.

Itheyibhile yokukhetha uvavanyo

Sebenzisa le theyibhile ukukhetha umlinganiselo ngokombuzo, kungekhona ukukhetha umbuzo ngokomlinganiselo.

Ubuncinane bebango eliqinisekisayo bufuna ukuvavanya imilinganiselo efanayo yoxinaniso lwedayi, i-100 nM, 300 nM, 1 uM, kunye ne-2 uM, ekuhambeni kweoksijini phantsi kweemeko zokuvuza, ze-ADP, ze-oligomycin, nezento esahlula ukuthuthwa kwee-electron ekwenziweni kwe-ATP; kwisantya sokwenziwa kwe-ATP; kumahluko wombane kwinwebu kusetyenziswa ngokukhethekileyo izikhangeli ezimbini; nasekukhutshweni kweperoxide okulungelelaniswe nomgangatho, kusetyenziswa icatalase namalinge olawulo angenaziseli. Chaza iseti edityanisiweyo: ukuhamba kweoksijini kunye nesantya se-ATP kunye ne-ATP ngeoksijini nganye kunye nomahluko wombane kunye neperoxide. Naliphi na kula manani lilodwa lingumlinganiselo osafuna intsingiselo.