I-methylene blue neemitochondria: ukuthuthwa kwee-elektroni nobungqina

Yiba nomfanekiso-ngqondweni weesampuli ezimbini ezibekwe ecaleni kwenye. Zombini ziqulethe iimitochondria zobuchopho ezifakwe ityhefu yerotenone, ikhemikhali evala i-Complex I yekhonkco lokuphefumla. Komnye, umphandi ufaka ngepipette idosi ye-methylene blue ekumlinganiselo we-micromolar. Ukusetyenziswa kweoksijini kubuyela ngokuyinxenye. Umahluko wombane kwinwebu ubuyela ngokuyinxenye. Kuqalisa kwakhona ukuveliswa kwe-ATP okuncinane. Kwi-cuvette esecaleni kwayo, isivamvo seperoxide sibalisa elinye icala: ukuveliswa kwehydrogen peroxide kuphindaphindeke izihlandlo eziliqela, kuyo yonke idosi evavanyiweyo, nakuyo yonke isubstrate ezanyiweyo.

Zombini ezi ziphumo zichanile. I-methylene blue ngumthuthi wee-elektroni onokugcina umsinga uhamba udlula indawo eyonakeleyo yekhonkco lokuphefumla, kodwa lo mthuthi ukwanexabiso lokunyuka kweperoxide ngoxa esenza oko. Xa uzigcina zombini ezi nyaniso engqondweni, uncwadi ngomsebenzi we-methylene blue kwiimitochondria luyacaca. Xa ugcina enye kuphela, lubonakala njengommangaliso okanye isoyikiso, kuxhomekeke kwiphepha lophando oqale walifunda.

Umthuthi wee-elektroni, uchazwe ngokucacileyo

Ikhonkco lokuphefumla lifana nomgca wokudlulisela. Ii-elektroni zingena kwi-Complex I (zisuka kwi-NADH) okanye kwi-Complex II nakwii-enzyme zeflavin ezinxulumene nayo (zisuka kwis succinate nakwabanye abanikeli abafana nayo), zidlule kwi-Complex III ziye kumthwali oshukumayo, icytochrome c, zize zidlule kwi-Complex IV ziye kwioksijini. Ukudluliselwa ngakunye kumpompa iiproton zinqumle inwebu yangaphakathi, yaye umahluko woxinaniso ovelayo uqhuba ukuveliswa kwe-ATP. Xa nasiphi na isikhululo sivalekile, inxalenye yomgca engaphambi kwaso iyema.

I-methylene blue izifaka njengomthuthi osebenzisa enye indlela ehambelana nalo mgca. Uhlobo lwayo olukwimo ye-oxidation luthatha ii-elektroni kubathwali abangaphambili, lube luhlobo olungenambala olukwimo ye-reduction, ileucomethylene blue, oluthi emva koko ludlulisele ezo elektroni phambili luze lubuyele kumbala oluhlaza okwesibhakabhaka. Ezi ntlobo zimbini zichazwe kwingcaciso yesibini se-redox, yaye indlela ebanzi yokudluliselwa ichazwe kwingcaciso ngokubanzi yendlela esebenza ngayo. Umbuzo osavulekileyo kukuba umthuthi ubuyela phi kulo mgca: kwipokotho ebopha iquinol kwi-Complex III, eyaziwa njengendawo ye-Qo, okanye ngqo kwicytochrome c. Indlela ephambili yokuphuma ixhomekeke kwiimeko, njengoko uhlalutyo lokudlula i-Complex III luchaza ngokweenkcukacha.

Oko kwalinganiswa ngokwenene zizifundo ezintathu zeempuku

Zontathu izifundo zasebenzisa iimitochondria zobuchopho ezahluliweyo, oko kukuthi ii-organelle ezikhutshwe kwiseli zaza zabekwa kwisisombululo sebuffer esichazwe ngokucacileyo. Kwakungekho seli zipheleleyo, kungekho zilwanyana zinikwe idosi, kungekho bantu bathatha inxaxheba. Uxinaniso lubhekisa kwisisombululo esijikeleze ii-organelle, hayi kwidosi ethathwa ngumntu ngomlomo. Gcina loo mda engqondweni, yaye amanani aya kuthetha ngokucacileyo.

Isifundo sokuqala, esasisebenzisa iimitochondria zobuchopho zehagu yeguinea ezahluliweyo kuxinaniso olususela kwi-100 nanomolar ukuya kwi-2 micromolar, sabonisa inzuzo nexabiso layo kwisampuli enye. Kwiimitochondria eziphilileyo idayi yandisa ukusetyenziswa kweoksijini kwimeko yokuphumla, kodwa ayizange itshintshe ukuphefumla okuvuselelwe yi-ADP, ngoko ukuveliswa kwe-ATP okunxulunyaniswa nako akuzange kuphucuke. Xa i-Complex I yayivalekile, i-2 micromolar yabuyisela ukuveliswa kwe-ATP ukusuka kwi-46 ukuya kwi-93 nanomoles ngomzuzu nge-milligram, xa kuthelekiswa nomlinganiselo oqhelekileyo omalunga ne-1109. Xa i-Complex III yayivalekile, kwaloo dosi yabuyisela i-ATP ukusuka malunga ne-46 ukuya kwi-63, xa kuthelekiswa nomlinganiselo oqhelekileyo omalunga ne-910. Umahluko wombane nokungeniswa kwecalcium kwabuyela ngokuyinxenye. Ukuvalwa kwe-Complex IV yicyanide kwakungenakudlulwa, nto leyo ebeka indawo eluncedo yokuphuma kwee-elektroni ngaphambi kwe-Complex IV esebenzayo. Konke ukubuyiselwa komsebenzi kwakubalulekile ngokwezibalo kodwa kukuncinane: iipesenti ezinamanani anedijithi enye zemveliso ye-ATP eqhelekileyo.

Isifundo sesibini, esasisebenzisa iimitochondria zecortex yempuku kuxinaniso olumalunga ne-1 micromolar, saqinisekisa ukudlulwa kwe-Complex I saza sabeka umda kwi-Complex III. Irotenone yehlisa ukuphefumla ukusuka malunga ne-121 ukuya ngaphantsi kwe-2 nanomoles zeoksijini ngomzuzu nge-milligram, yaza idayi yakunyusa kwaba malunga ne-21, phantse isinye kwisithandathu somlinganiselo oqhelekileyo, ngoxa umahluko wombane kwinwebu wabuyela phantse ngokupheleleyo. Kodwa emva kokuvalwa kwe-Complex III, ukuphefumla nomahluko wombane akuzange kubuyele kuxinaniso olususela kwi-1 ukuya kwi-5 micromolar, yaye ukuvalwa kwacinezela umqondiso weperoxide obangelwa yidayi. Ababhali bacebisa ukuba idayi ekwimo ye-reduction idlulisela ii-elektroni zayo kwindawo ye-Qo ye-Complex III: xa loo ndawo ithathiwe, umthuthi akanandawo yokuzisa umthwalo wakhe.

Isifundo sesithathu, esasisebenzisa iimitochondria zobuchopho zeempuku ezincinane, iimpuku ezinkulu neehagu zeguinea, ubukhulu becala kuxinaniso lwe-2 micromolar, savavanya oku kungavisisani ngokuthe ngqo saza safumanisa ukuba ukuvalwa kwe-Complex III kunokudlulwa. Xa imy xothiazol, isithinteli sendawo ye-Qo, yehlisa ukuphefumla okusisiseko nge-88 percent, idayi yabuyisela ukusetyenziswa kweoksijini malunga ne-62 percent yomlinganiselo ongavalwanga. Kuvavanyo oluthe ngqo, isiqingatha se-micromolar sabangela i-reduction yecytochrome c eyongezwe ngaphandle kwanaxa i-Complex III yayivalekile, nto leyo exhasa indlela esuka kwi-NADH idlule kwidayi iye kwicytochrome c ize iqhubeke iye kwi-Complex IV. Kodwa ukuphefumla okubuyiselweyo kwaziphatha ngendlela engaqhelekanga: i-ADP eyongeziweyo yehlisa ukusetyenziswa kweoksijini endaweni yokukunyusa, yaye ukuthintela ukuveliswa kwe-ATP kwavuselela ukuphefumla. Umahluko wombane wabuyela kuphela ngokuyinxenye. Ukusetyenziswa okuphezulu kweoksijini okunonxulumano olujikileyo nokuveliswa kwe-ATP akuyomveliso yamandla eqhelekileyo.

Isizathu sokungavisisani kweelebhu, noko kusombulula umba

Ingcaciso esekelwe kwiintlobo zezilwanyana yeyokuqala engaxhaswayo. Iqela lesithathu lawuphinda umsebenzi kwiintlobo ezintathu zeempuku laza lafumana indlela efanayo yokudlula kuzo zonke, kuquka nodidi lwempuku apho kwakuxelwe ukuba akukho ndlela yokudlula. Endaweni yoko, umahluko ukuxinaniso (i-1 xa ithelekiswa ne-2 micromolar), kwiidosi zezithinteli, kwizisombululo zebuffer, kwiindlela zokwahlula, yaye ngaphezu kwako konke kwimeko yamandla ethelekiswayo: indlela yokudlula icace kakhulu ngaphambi kokongezwa kwe-ADP, kanye apho kubonakala khona impendulo ephikisanayo kwi-ADP. Ngoko umzobo ochanekileyo ubonisa iindlela ezimbini zokuphuma ezixhomekeke kwiimeko, enye kwindawo ye-Qo nenye kwicytochrome c. Umthuthi uhambisa umthwalo apho imichiza yaloo ndawo imvumela khona.

Ixabiso leperoxide

Icala lexabiso liyahambelana njengelokubuyiselwa komsebenzi. Kumsebenzi wehagu yeguinea, idayi yandisa ukukhutshwa kweperoxide phantsi kwazo zonke iimeko ezavavanywayo, xa iimitochondria ziphumle, zivelisa i-ATP okanye ziphazanyiswe yimichiza, kuzo zontathu iisubstrate. Ngexesha lokuveliswa kwe-ATP, imveliso yenyuka ukusuka malunga ne-148 ukuya ngaphezu kwe-1500 picomoles ngomzuzu nge-milligram kuxinaniso lwe-1 micromolar, yaye nkqu ne-100 nanomolar yawandisa umqondiso ngaphezu kwezihlandlo ezine. Idayi yakwacothisa ukususwa kweperoxide malunga nezihlandlo eziyi-2.6, ngoko umqondiso ubonisa ukuveliswa okukhawulezileyo kunye nokususwa okucothayo. Indlela yemichiza ecetywayo yi-reduction ethe ngqo yeoksijini yidayi ekwimo ye-reduction, ngaphandle kwesuperoxide enokufunyanwa, nangona oku kuseyingcaciso kunokuba kube linyathelo elingqiniweyo.

Umsebenzi weempuku ezincinane wenza umfanekiso ube nzima ngakumbi ngendlela eluncedo. Iperoxide yenyuka kakhulu ngokunyuka kwedosi, ukusuka kumlinganiselo wokuqala omalunga ne-76 ukuya phantse kwi-2000 picomoles ngomzuzu nge-milligram kuxinaniso lwe-2 micromolar, kodwa ukuvalwa kwe-Complex III kwacinezela inxalenye ebangelwa yidayi. Ukuba iperoxide yayivela kuphela kwidayi ekwimo ye-reduction esabela kwioksijini, ukuvala ikhonkco kwakungafanele kuchaphazele kangako. Kuyachaphazela, ngoko ikhonkco lithatha inxaxheba. Uphononongo lweempawu ze-antioxidant ne-pro-oxidant lubeka la macala mabini kumxholo obanzi, yaye uhlalutyo lweoksijini xa ithelekiswa ne-ATP luchaza ukuba kutheni amanani eoksijini ewodwa engakwazi ukugqiba umba webango lamandla.

Ukubuyiselwa komsebenzi asikokulungiswa

Ibinzana ekufuneka liyekwe lithi “ngaba i-methylene blue iyazilungisa iimitochondria.” Ukulungiswa kungathetha ulungiso oluhlala ixesha elide: iicomplex ezakhiwe ngokutsha, unxulumano lokuphefumla nokuveliswa kwe-ATP oluqhubekayo emva kokuba ichiza limkile, ukubuyiselwa komsebenzi kwiiseli ezipheleleyo, kuze kulandele umsebenzi ophucukileyo kwinto ephilayo. Akukho nalinye kula maphepha mathathu elibonisa oko. Abonisa ukutshintshwa kwendlela ngokukhawuleza kwii-organelle ezisandul’ ukwahlulwa ngexesha lokuvalwa okwenziweyo ngemichiza, apho imveliso ye-ATP iyinxalenye encinane yesiqhelo yaye ngamanye amaxesha unxulumano lokuphefumla nayo lungaqhelekanga ngokucacileyo. Irotenone neantimycin zizixhobo zaselebhu, hayi iimodeli zokwaluphala okanye zesifo seemitochondria ebantwini, yaye akukho nto apha ethetha ukuba umntu ophilileyo ufumana amandla angakumbi; loo nto ngumbuzo wobungqina owahlukileyo ononcwadi lwawo lwezifundo zabantu.

Imida emithathu igcina nasiphi na isigqibo sebiohacking sinyanisekile. Okokuqala, uxinaniso lwesisombululo aluyodosi: i-0.1 ukuya kwi-2 micromolar ejikeleze ii-organelle ezahluliweyo ayithethi nto ngesixa esithathwa ngomlomo esivelisa olo xinaniso lungabotshelelwanga kwimitochondrion yobuchopho bomntu, ingakumbi kuba idayi iqokelelana okanye ikhutshwe ngokuxhomekeka kumahluko wombane kwinwebu ngokwawo. Okwesibini, ioksijini ayingomandla: idayi inokwandisa ukusetyenziswa kweoksijini ngokujikeleza nokuvuza nangokufosforileyisha okuvelisa i-ATP, ngoko ukulinganiswa kokuphefumla ngaphandle kweziphumo ze-ATP nomahluko wombane akunakuxhasa ibango lamandla. Okwesithathu, inzuzo nomthwalo kufuneka zibe kwisivakalisi esinye: nayiphi na ingxelo yokubuyiselwa komahluko wombane okanye kwe-ATP eshiya ngaphandle ukunyuka kweperoxide okuhamba nako ichaza uvavanyo ngokungachanekanga. Ezinye izinto ezinxulumeneyo ezifanele zithathelwe ingqalelo ziquka ibhalansi ye-redox ye-NAD, ukumila kwempendulo kwidosi kunye nesikhokelo sovavanyo.

Inqaku elinye elisebenzisekayo: ukuthutha ii-elektroni luphawu lwaloo molekyuli kanye. Iimolekyuli ezinxulumene nayo ezisuswe amaqela emethyl, njenge-Azure B, nazo ziyasebenza kwi-redox, kodwa ziziphatha ngokwahlukileyo, ngoko isisombululo esonakele ngokuyinxenye siluvavanyo olwahlukileyo kwidayi engonakalanga. Eso sesinye isizathu sokunyanzelisa iimfuno ezipheleleyo zepharmacopoeia noxwebhu lwebhetshi olupapashiweyo, endaweni yovavanyo olujonga izinyithi kuphela. Imichiza eqhutywa kukukhanya yongeza isilumkiso esifanayo kusetyenziso olwenziwa phantsi kokukhanya, esichazwe phantsi kokudityaniswa nokukhanya okubomvu.

Ibango elinomda lisamile emva kwako konke oku yaye lifanele ligcinwe: i-methylene blue inokusebenza njengomthuthi wee-elektroni oxhomekeke kwiimeko kwiimitochondria ezahluliweyo ezonakeleyo, igcine ukuhamba ngokuyinxenye kodwa ngexabiso leperoxide. Yonke into engaphaya koko, amandla, ubomi obude, ukulungiswa, ilindele ubungqina ezi zifundo ezazingazange ziyilelwe ukubunika.