Redukcija vanadata u micelijumu gljive Phycomyces blakesleeanus Burgeff: određivanje oksidacionih stanja u ćeliji

Abstract

Ova doktorska teza predstavlja jedan od prvih pokušaja korišćenja polarografije za in situ ispitivanja oksido - redukcionih procesa na celom organizmu. Rezultati dobijeni pomoću tehnika polarografije jednosmerne struje (DCP) i diferencijano pulsne polarografije (DPP) pokazuju da micelijum gljive P. blakesleeanus smanjuje koncentraciju V(V) monomera u spoljašnjem rastvoru za oko 27 % u celokupnom ispitivanom opsegu koncentracija (0.1 – 1 mM). Kako dosadašnji rezultati ukazuju da je tetramer V(V) podložan redukciji do V(IV) posredstvom enzima sa fericijanid reduktaznom aktivnošću (FeCNR), fericijanid je korišćen kao supstrat za koji FeCNR pokazuje veći afinitet nego za V(V), da bi se utvrdilo koji deo smanjenja koncentracije V(V) je posledica redukcije do V(IV). DCP merenja su ukazala da je oko 8 % V(V) redukovano do V(IV), dok je 18 % uklonjeno iz medijuma na neki drugi način. Potpuno odsustvo efekta Cd2+, potentnog blokatora FeCNR, ukazuje da redukcija nije katalizovana FeCNR. Značajnija redukcija monomera V(V) nije pokazana EPR metodom što otvara mogućnost da inhibitorni efekti fericijanida registrovani DCP metodom nisu vezani za redukciju monomera V(V) već za njegov transport i vezivanje za micelijum. Polarografski eksperimenti su dalje pokazali da aktivnost micelijuma P. blakesleaanus dovodi samo do snižavanja vrednosti Id talasa c5, bez uticaja na njegov polutalasni potencijal E1/2, što isključuje mogućnost da je stvaranje kompleksa V(V) sa eksudatima gljive uzrok smanjenja koncentracije V(V) monomera. DCP i 51V NMR eksperimenti sa izolovanim ćelijskim zidom P. blakesleeanus su pokazali da nema značajne adsorpcije za ćelijski zid. Nakon dodavanja 1 mM V(V) micelijumu signal monomera nestaje iz 51V NMR spektra, a u spektru se pojavljuje nov signal (– 532 ppm) sličnog intenziteta, kao rezultat vezivanja vanadata za neki od intracelularnih molekula. To ukazuje da je osnovni vid interakcije V(V) monomera sa micelijumom njegov transport u ćeliju...

This Thesis represents one of the first applications of polarographic method for in situ investigations of oxido - reduction processes in whole organism. The results obtained using DCP and DPP techniques have shown that in the presence of P. blakesleeanus mycelium concentration of added V(V) monomer decreased by 27 % in investigated concentration range (0.1 – 1 mM). To find out if enzyme with ferricyanide reductase activity (FeCNR) is capable for monomer reduction, as it is a case for tetramer, ferricyanide was used as competitor for V(V) reduction. DCP results have shown that about 8 % of V(V) was reduced, while 18 % was removed from the solution. The addition of Cd2+ has shown no effect on DCP results, suggesting no participation of FeCNR in reduction. The absence of V(IV) signal in EPR spectrum after addition of V(V) monomer indicated that the main reason for fading out V(V) is its transport (in)to mycelium. Polarographic experiments were also performed on isolated cell wall of P. blakesleeanus. It was concluded that there was no interaction of V(V) with cellular exudates, no adsorbtion / reduction on the cell wall was detected in 51V NMR spectrum. However, in 51V NMR spectra of mycelium treated with 1mM V(V) disappearance of monomer signal was recorded, with concomitant appearance of new signal at – 532 ppm, of similar intensity. This result confirms transport of V(V) monomer into the cell, and its binding to some of intracellular molecules. The interaction of V(IV), as the main product of V(V) reduction, was also investigated using polarographic techniques. The obtained result has shown that all added V(IV) was removed from the buffer solution, manifested as disappearance of polarographic waves. Therefore, the capacity of P. blakesleeanus mycelium to uptake was estimated for V(IV) (185 ± 12 μmol / gfw); V(V) (104 ± 2 μmol / gfw) i Cd2+ (330 μmol / gfw). This capacity puts P. blakesleeanus into the group of fungi known as good bioaccumulators...