Ngaba imethylene blue ingayidlula iKompleksi III yezimitokondriya? Kutheni izifundo zingavumelani

Iilabhoratri ezimbini zabuza ukuba imethylene blue ingayidlula na iKompleksi III yezimitokondriya evaliweyo. Enye yaphendula yathi hayi. Enye yathi ewe. Zombini zazinengcombolo yovavanyo.

Isifundo sokuqala, esenziwa kwiimitokondriya zobuchopho beempuku, safumanisa ukuba le dayi ayikwazi ukuyidlula. Esesibini, esenziwa kwiimitokondriya zobuchopho beentlobo ezintathu zezilwanyana eziziimpuku, safumanisa ukuba inokuyidlula ngokuyinxenye. Isifundo sesithathu, esenziwa kwisibindi seempuku, sisoloko sicatshulwa kunye nezi kodwa savavanya enye into. Xa usibeka kwindawo yaso efanelekileyo, isiqingatha sokubhideka siyaphela.

Akukho ndlela inye yokusebenza esebenza kuzo zonke iimeko enokubhengezwa. Kukho iimvavanyo ezintathu ezinezithinteli, ubungakanani bedayi nezilinganiso ezahlukileyo. Funda iimeko kuqala. Izigqibo zilandela apho.

Kuthetha ntoni ukudlula apha

Ii-elektroni zingena kwikhonkco ngeKompleksi I (zisuka kwi-NADH) okanye ngeKompleksi II (zisuka kwi-succinate), zidlule kwiKompleksi III ziye kwi-cytochrome c, zize zidlule kwiKompleksi IV ziye kwioksijini. Umahluko oxineneyo owakhiwa kule ndlela ngowona usetyenziswa yiseli ukwenza i-ATP.

Ibango lokudlula lithi imethylene blue ithatha ii-elektroni ngaphambi kwendawo evaliweyo ize izikhuphe emva kwayo. Idayi ekwimo eneoksideshini ithatha ii-elektroni kwi-NADH okanye kumacandelo okuqala ekhonkco, ibe yi-leucomethylene blue, ize imo encitshisiweyo izidlulisele kwi-cytochrome c okanye izibuyisele kwikhonkco. Ukuba olo dluliselo lufika emva kwendawo evaliweyo, ukuphefumla nomahluko wombane kwinwebu kubuyela ngokuyinxenye. Ukuba lufika kwindawo evaliweyo okanye ngaphambi kwayo, akukho nto ibuyelayo.

Inkalo enye yeKompleksi III igqiba okuninzi kule mpikiswano. Le kompleksi isebenzisa umjikelo we-Q oneendawo ezimbini zokubophelela izithinteli. I-antimycin ibophelela kwindawo ye-Qi. I-myxothiazol ibophelela kwindawo ye-Qo. Isifundo esisebenzisa esinye kuphela kwezi sivavanye isiqingatha kuphela sale kompleksi.

Isifundo sokuqala: akukho kudlula kwiimitokondriya zobuchopho beempuku

UGureev noogxa bakhe basebenzisa iimitokondriya zecortex yeempuku ze-C57BL/6, ezahlulwe nge-digitonin, baza bazivavanya kwi-0.1 mg yeprotheyini nge-ml. Iisubstrate zazibandakanya iimeko ezintathu: i-pyruvate kunye ne-malate kwi-5 mM nganye, i-succinate kwi-10 mM kunye ne-glutamate kwi-2 mM, ne-L-proline kwi-10 mM. Izithinteli zaziyi-rotenone kwi-1 micromolar (iKompleksi I) ne-antimycin kwi-1 microgram per ml (iKompleksi III). Ubungakanani bedayi babuyi-0.5, 1, no-2 micromolar, nge-1 micromolar njengoxinaniso oluphambili. Iindlela nemizila yezilinganiso zikolu papasho lwe-FEBS Letters.

Ukudlula kwiKompleksi I kwasebenza. I-rotenone yanciphisa ukuphefumla okuvuselelwa yi-ADP ukusuka malunga ne-121 ukuya malunga ne-2 nmol per minute per mg, yaza idayi ye-1 micromolar yakubuyisela malunga ne-21. Umahluko wombane kwinwebu wabuyela xa kusetyenziswa i-pyruvate kunye ne-malate, nakwi-L-proline.

Ukudlula kwiKompleksi III akuzange kusebenze. I-antimycin yanciphisa ukuphefumla ukuya malunga ne-16, yaza idayi yakunyusa kuphela ukuya malunga ne-20. Umahluko wombane kwinwebu awuzange ubuyele nakweyiphi isubstrate, nakubungakanani bedayi obufikelela kwi-5 micromolar. Ingcombolo ye-peroxide nayo yalatha kwicala elinye: idayi yodwa yanyusa ukukhutshwa kwe-peroxide ukusuka kwisantya esisisiseko esimalunga ne-76 pmol per minute per mg ukuya malunga ne-1170, 1460, ne-1945 kubungakanani bedayi obuthathu, kodwa i-antimycin yanciphisa loo mqondiso uveliswa yidayi malunga ne-25, 88, ne-96 pesenti ngokulandelelana. Ukuba idayi encitshisiweyo yayisabela ngqo kwioksijini, ukuvala iKompleksi III kwakungafanele kunciphise loo mqondiso. Ababhali bagqiba kwelokuba idayi encitshisiweyo ifakwa kwioksideshini kwindawo ye-Qo yeKompleksi III, ngaphambi kwendawo evalwe yi-antimycin. Umjikelo omfutshane, ngamazwi: abanikezeli bee-elektroni ukuya kwidayi, idayi ukuya kwi-Qo, kuze kuqhubeke indlela eqhelekileyo. I-rotenone iyadlulwa. I-antimycin ayidlulwa.

Isifundo sesibini: ukudlula ngokuyinxenye kwiintlobo ezintathu, kuzo zombini iindawo zezithinteli

USvab noogxa bakhe balwandisa uyilo lovavanyo. Basebenzisa iimitokondriya zobuchopho beempuku, iimpuku ezinkulu neehagu zaseGuinea, ezahlulwe nge-Percoll, zavavanywa kwakwi-0.1 mg per ml, kusetyenziswa i-pyruvate kunye ne-malate okanye i-glutamate kunye ne-malate kwi-5 mM nganye. Basebenzisa izithinteli ezimbini zeKompleksi III: i-antimycin A kwi-0.3 micromolar ne-myxothiazol kwi-1 micromolar. Idayi yayikwi-2 micromolar kuninzi lweemvavanyo, nakwi-0.5 kumsebenzi we-cytochrome c. Zontathu iintlobo, ezineenkqubo zovavanyo ezifanayo, zikolu papasho lufumaneka ngokukhululekileyo.

Zombini iindawo ezivaliweyo zadlulwa ngokuyinxenye, kuzo zontathu iintlobo. Kwiimitokondriya zeempuku, i-antimycin yathintela ukuphefumla yaza idayi yakunyusa malunga ne-60 pesenti, ukusuka malunga ne-37 ukuya kwi-59 nmol per minute per mg. I-myxothiazol yakunciphisa nge-88 pesenti, ukuya malunga ne-4.5, yaza idayi yakunyusa ngokuphindwe kahlanu ukuya malunga ne-23. Umahluko wombane kwinwebu wabuyela ngokuyinxenye phantsi kwesithinteli ngasinye, walinganiswa ngeedayi ezimbini (i-safranin O ne-TMRM ethelekisa umlinganiselo wemiqondiso) ukuze kukhutshwe iziphene zokulinganisa okubangelwa kukukhanya.

Olu papasho lwavavanya nokudluliselwa kwee-elektroni ngokuthe ngqo, into engazange yenziwe sisifundo sokuqala. I-cytochrome c ene-acetyl eyongezwe ngaphandle kwi-25 micromolar ayizange incitshiswe ziimitokondriya ezinikwe amandla, nokuba izithinteli zazikho okanye zingekho, kodwa ukongeza idayi kwabangela ukuncitshiswa kuzo zombini iimeko. Kwisisombululo se-buffer esingenaziseli, i-NADH kunye nedayi zanciphisa i-cytochrome c ngesantya esihambelana nobungakanani be-NADH. Ukutsiba okude ngakumbi, ngamazwi: amandla okunciphisa ukuya kwidayi, idayi ngqo kwi-cytochrome c, iKompleksi IV ukuya kwioksijini. Loo ndlela itsiba iKompleksi III ngokupheleleyo, ngoko zombini izithinteli zinokudlulwa.

Izilinganiso ezibini ezongezelelekileyo zixhasa loo ngcaciso. Ukukhanya kwe-fluorescence ye-NAD(P)H kwehla xa idayi yongezwa kwiimitokondriya ezinyangwe nge-myxothiazol, nto leyo ethetha ukuba amandla okunciphisa aphuma ngendlela entsha. Kwakhona ukufunxa ukukhanya kwidayi kwi-660 nm kwalandela ukongezwa ngakunye, ngoxa ulwelo olungaphezulu olwahlulwe nge-centrifuge lwabonisa ukuba idayi ekwimo eneoksideshini yaqokelelana ngaphakathi kwiimitokondriya ezinikwe amandla ngokuphindwe ngaphezu kwekhulu, yaza yaphuma xa umahluko wombane waphela. Ukubalwa kweeprotoni nako kuyahambelana: ukutsiba amanyathelo okumpompa kushiya malunga neeprotoni ezi-2 kwisibini see-elektroni endaweni ye-10, ngoko ukusetyenziswa ngokukhawuleza kweoksijini kunye nokubuya ngokuyinxenye komahluko wombane yindlela ukudlula kokwenene okubonakala ngayo, kungekhona ubungqina obuphikisa oko.

Iimitokondriya ezilungiswe ngendlela enye zavelisa iimpendulo ezenza ufundo olungacokisekanga lukhokelele kwimpazamo. Xa kwakukho isithinteli kunye nedayi, i-ADP yanciphisa ukuphefumla endaweni yokukunyusa, kwaza ukuvalwa kothutho kwakuvuselela endaweni yokukucothisa, ngoxa umahluko wombane kwinwebu wawusatshintsha ngendlela eqhelekileyo. Ukusetyenziswa kweoksijini kuphela kuyalahlekisa kule nkqubo.

Isifundo sesithathu: isifundo sesibindi esavavanya iiKompleksi I no-II, hayi III

ULee noBoelsterli basebenzisa iimitokondriya zesibindi seempuku kunye neeseli zesibindi ezikhuliswe elabhoratri ezonakaliswe yi-efavirenz (iKompleksi I, i-IC50 emalunga ne-30 micromolar) ne-hydrazine, imveliso yokuguqulwa kwe-isoniazid emzimbeni (iKompleksi II, i-IC50 emalunga ne-30 micromolar). Idayi emalunga ne-30 micromolar yabuyisela ioksideshini ye-NADH, umahluko wombane kwinwebu ne-ATP, ngoxa yayinciphisa izinto ezibangela ioksideshini nokufa kweeseli nge-necrosis. Upapasho lwe-Redox Biology luyichaza njengedayi ethwala ii-elektroni ijikeleza iiKompleksi I no-II ezonakeleyo.

Eso siphumo ngumxholo oqinisekisiweyo onxulumene namanyathelo angaphambili, kwaye asithethi nto ngeKompleksi III kuba akukho nto kuyo eyayivaliwe apho. Asinakuqinisekisa okanye siphikise nasiphi na kwezi zifundo zobuchopho.

Itheyibhile yeemeko

Izizathu ezine zokuba zombini izifundo zeKompleksi III zinokuchaneka ngeemeko zazo

1. Izithinteli zazingangeneleli ngendlela efanayo. I-antimycin yodwa kwi-1 microgram per ml ithelekiswa ne-antimycin kwi-0.3 micromolar kunye nesithinteli esahlukileyo sendawo ye-Qo. Indlela yedayi engenisa ii-elektroni kwindawo ye-Qo iyasilela kanye apho indlela eya ngqo kwi-cytochrome c iphumelela khona. Indawo enye ayinakumela zombini.

2. Ubungakanani bedayi namaxesha ayengafani. Uxinaniso oluphambili lwe-1 luthelekiswa ne-2 micromolar, kwinto ejikeleza phakathi kweemeko ze-redox, ekufunxwa kwayo nokunciphisa kwayo i-cytochrome c ngokuthe ngqo kuxhomekeke kuxinaniso nakwixesha. Umahluko omncinane kubungakanani bedayi kunye namaxesha ahlukileyo okulinganisa unokubonisa amacandelo ahlukileyo othungelwano olunye.

3. Iindlela zokulungiselela zazahlukile. Ukwahlula nge-digitonin xa kuthelekiswa nokwahlula ngomahluko woxinaniso lwe-Percoll kukhetha amaqela eemitokondriya afanayo ngokuyinxenye kodwa angafani ngokupheleleyo. Uxinaniso lweprotheyini lwalufana kwi-0.1 mg per ml, ngoko loo nto ayichazi umahluko, kodwa amaqela amancinci eemitokondriya nokungonakali kwazo kusenokuwuchaza. Kwaye ukurhanelwa kohlobo lwesilwanyana kwasuswa: iqela lesibini laphinda lafumana impendulo yalo nakwimpuku.

4. Imeko yeesubstrate igqiba oko idayi inokukwenza. Iimeko ezintathu zeesubstrate zithelekiswa nezibini ezixhomekeke kwi-NADH. Ukufumaneka kwe-NADH nomahluko wombane kwinwebu kunye kugqiba isantya sokuncitshiswa kwedayi nendawo esasazeka kuyo, kwaye indlela efuna idayi encitshisiweyo ecaleni kwe-cytochrome c ekwimo eneoksideshini ixhomekeke kolo khetho.

Indlela yokufunda ibango elilandelayo lokudlula

Buza ukuba yeyiphi ikompleksi evaliweyo, ngesiphi isithinteli, kweyiphi indawo nangobuphi ubungakanani. Buza ngoxinaniso lwedayi nexesha ekwenziwe ngalo isilinganiso. Buza ngethishu, uhlobo lwesilwanyana, indlela yokwahlula, uxinaniso lweprotheyini nesubstrate. Kwaye buza ukuba umahluko wombane, ukuphefumla, imeko ye-redox novavanyo lokudluliselwa ngokuthe ngqo kwee-elektroni ziyavumelana na, kuba ukusetyenziswa kweoksijini kuphela kwalatha kwicala elingalunganga ngaphezu kwesihlandlo esinye apha.

Isishwankathelo esinomda ocacileyo sisamile. Ukuvumelana kukhona ngokudlula kwedayi kwiKompleksi I. KwiKompleksi III, iimitokondriya zeempuku ezine-antimycin kwi-1 microgram per ml nedayi kwi-1 micromolar azibonisi kudlula okusebenzayo, ngoxa iintlobo ezintathu ezine-antimycin kwi-0.3 micromolar okanye i-myxothiazol kwi-1 micromolar nedayi kwi-2 micromolar zibonisa ukudlula ngokuyinxenye, ngokudluliselwa kwee-elektroni ngqo ukusuka kwidayi ukuya kwi-cytochrome c. Ezo nkcazelo zinokuba yinyaniso ngaxeshanye. Nantoni na ebanzi kunoko kukwandisa isigqibo ngaphaya kweemeko ezivavanyiweyo.

Ufundo olunxulumeneyo lugcina ibango eliphikiswayo ngaphakathi kwekhemistri engaphikiswayo: indlela imethylene blue ehambisa ngayo ii-elektroni ukucacisa udluliselo, iimvavanyo zokudlula kwiKompleksi I ngemeko eqinisekisiweyo yamanyathelo angaphambili, izilinganiso zomahluko wombane kwinwebu neziphene zazo zokulinganisa ukucacisa ukuba kutheni kwakufuneka iidayi ezimbini, nokuphathwa elabhoratri kwezinto ezijikeleza phakathi kweemeko ze-redox malunga nemigibe yobungakanani bedayi nexesha.