Ispitivanje in vitro interakcija jedinjenja zlata sa Na+/K+ ATPazom

Abstract

Na+/K+ ATPaza je membranski enzim koji koristi energiju oslobođenu iz molekulaadenozin trifosfata da bi regulisao odnos koncentracije jona kalijuma i natrijuma sa obestrane ćelijske membrane većine živih bića. Pored transporterske uloge, on obavlja ifunkciju signalnog molekula, jer je uključen u regulaciju aktivnosti Src kinaze, koja je daljeuključena u signalne puteve unutar ćelije i reguliše preživljavanje ćelije. Zbog ovoihsvojstava Na+/K+ ATPaza je prepoznata kao važna terapeutska meta u velikom brojurazličitih patogenih stanja uključujući i tumore. Kompleksi plemenitih metala, a naročitozlata predstavljaju novu veliku grupu jedinjenja koja pokazuje sposobnost inhibicije Na+/K+ATPaze. Ovo ih čini potencijalnim terapeuticima, analognim kompleksima platine koji su usastavu modernih hemoterapija. Osim kompleksnih jedinjenja zlata, interesantna svojstvapokazuju i nanočestice zlata. U ovoj studiji ispitivani su efekti 9 kompleksnih jedinjenjazlata i 3 tipa koloidnih nanočestica zlata na različite preparate enzima Na+/K+ ATPaze.Dobijeni rezultati ukazuju na to da 6 od 9 ispitivanih kompleksa ima umereno jakinhibitorni uticaj na Na+/K+ ATPazu. Pored toga, pokazano je da je reč o nekompetitivnojreverzibilnoj inhibiciji, koja se može otkloniti dodatkom donora –SH grupe kao što sucistein i glutation. Osim toga, inhibicija se može i u potpunosti preduprediti istim ovimsupstancijama. Stoga je zaključeno da se vezivanje kompleksa za enzim odvija preko –SHostataka cisteina u enzimu. Pokazano je da neki od ispitivanih kompleksa imaju izuzetnojak citostatski efekat, što ih čini dobrim kandidatima za dalje medicinske studije.Upotrebom MALDI TOF masene spektrometrije pokazano je da postoji direktna reakcijaizmeđu kompleksa i donora –SH grupe, tj da ove dve vrste direktno reaguju kada suprisutne u rastvoru.Nanočestice zlata koje su ispitivane nisu dale zapažen inhibitorni efekat. Tip nanočesticaobeležen kao C3 koji sadrži nanočestice zlata prečnika 9,5 nm, pokazao je neobičnasvojstva - dodatkom u preparat sinaptozomalnih plazma membrana, doveo je do porastaaktivnosti Na+/K+ ATPaze u tom preparatu dvostruko, dok se aktivnost ostalih prisutnihenzima nije menjala. Ispitivanjem ove interakcije pokazano je da je u pitanju fizičkapromena u preparatu do koje dolazi usled dva tipa hemijske interakcije između koloida ipreparata enzima. Uočene su promene u UV-vis i FTIR spektrima koloida po dodatkuenzima, a upotrebom atomske mikroskopije uočeno je da dolazi do promena unanostrukturi preparata enzima koje izaziva povećanje aktivne površine ćelijske membrane,te tako veći broj molekula enzima postaje dostupan za reakciju i javlja se porast aktivnostienzima u preparatu. Selektivnost enzima za njegove prirodne ligande ostaje neizmenjena, adobijeni preparat pojačane aktivnosti stabilan je najmanje sat vremena.Zaključeno je da ispitivani kompleksi zlata mogu da budu od značaja za dalji razvojantitumorskih terapija, kao i da koloidne nanočestice zlata mogu poslužiti za razvojosetljivijih testova za merenje aktivnosti membranskih enzima.

Na+/K+ ATPase is a membrane enzyme that uses the enegry released from the molecule ofadenosine triphosphate to regulate the sodium and potasium ion concentration on either sideof the cell membrane in most living creatures. Appart from its transporter role, it also has arole as a signal molecule, because it regulates the activity of Src kinase, an importantenzyme in cell signaling and survival. Beacuse of these properties, Na+/K+ ATPase isrecognized as an important molecular target in many disorders including tumors. Noblemetal complexes, and especially gold complexes have shown the tendency to inhibitNa+/K+ ATPase. This makes them potential terapeutics, analogous to platinum basedcomplexes that are a part of modern chemotherapy. Colloid nanoparticles of gold have alsoshown interesting properties in the past. This study presents the findings on the interactionof several preparations of Na+/K+ ATPase with 9 different complexes of gold and 3different types of gold colloid nanoparticles.Results shown in this thesis indicate that 6 out of 9 investigated complexes have amoderatley strong inhibitory effect upon the Na+/K+ ATPase. It is shown that this inhibitionis noncompetitive and reversible, and that it can be reversed by –SH group donors such ascysteine and glutathione. Moreover, this inhibition can be fully prevented by adding thesesubstances prior to the addition of the complex. Therefore it is concluded that the bindingof these complexes takes place through –SH groups of cysteine residues in the enzyme.Some of the investigated complexes have shown a strong cytotoxic effect upon humanlymphocytes, which makes them good candidates for further clinical studies. MALDI TOFmass spectroscopy has shown that there is a direct reaction between –SH group donors andthe gold complex.Investigated nanoparticles yielded no significant inhibitory effect. One type of thenanoparticles, however, has shown some very unusual properties. Upon the addition of thistype of colloid nanoparticles, labeled C3 and with an average diameter of 9.5 nm, to thepreparation of synaptosomal plasma membranes, the activity of Na+/K+ ATPase doubled,while the activity of other present enzymes remained the same. Further investigation of thisinteraction has shown that the cause of this is a physical change in the structure of themembrane fragments. It is the result of two paralel chemical interactions that take placewhen the nanoparticles are added to the enzyme preparation. Changes in UV/vis and FTIRspectra of the nanoparticles were observed and using atomic force microscopy it waspossible to visualize the change in the nanostructure of the enzyme preparation upon theaddition of nanoparticles. This change resulted in an increase of the active membranesurface, exposing more molecules of the enzyme and making them available for thereaction. This caused an increase of the enzyme activity. Selectivity of the Na+/K+ ATPasetowards its natural ligands was preserved, and the effect of increased activity persisted forat least an hour before diminishing.In conclusion, investigated complexes and nanoparticles of gold have shown importantproperties. The effects of gold complexes make them potential antitumor drugs, while thenanoparticles of gold may be used to develop better assays for membrane enzymes.