Na+/K+ ATPaza je membranski enzim koji koristi energiju oslobođenu iz molekula
adenozin trifosfata da bi regulisao odnos koncentracije jona kalijuma i natrijuma sa obe
strane ćelijske membrane većine živih bića. Pored transporterske uloge, on obavlja i
funkciju signalnog molekula, jer je uključen u regulaciju aktivnosti Src kinaze, koja je dalje
uključena u signalne puteve unutar ćelije i reguliše preživljavanje ćelije. Zbog ovoih
svojstava Na+/K+ ATPaza je prepoznata kao važna terapeutska meta u velikom broju
različitih patogenih stanja uključujući i tumore. Kompleksi plemenitih metala, a naročito
zlata predstavljaju novu veliku grupu jedinjenja koja pokazuje sposobnost inhibicije Na+/K+
ATPaze. Ovo ih čini potencijalnim terapeuticima, analognim kompleksima platine koji su u
sastavu modernih hemoterapija. Osim kompleksnih jedinjenja zlata, interesantna svojstva
pokazuju i nanočestice zlata. U ovoj studiji ispitivani su efekti 9 kompleksnih jedinjenja
zlata i 3 tipa koloidnih nanočesti...ca zlata na različite preparate enzima Na+/K+ ATPaze.
Dobijeni rezultati ukazuju na to da 6 od 9 ispitivanih kompleksa ima umereno jak
inhibitorni uticaj na Na+/K+ ATPazu. Pored toga, pokazano je da je reč o nekompetitivnoj
reverzibilnoj inhibiciji, koja se može otkloniti dodatkom donora –SH grupe kao što su
cistein i glutation. Osim toga, inhibicija se može i u potpunosti preduprediti istim ovim
supstancijama. Stoga je zaključeno da se vezivanje kompleksa za enzim odvija preko –SH
ostataka cisteina u enzimu. Pokazano je da neki od ispitivanih kompleksa imaju izuzetno
jak citostatski efekat, što ih čini dobrim kandidatima za dalje medicinske studije.
Upotrebom MALDI TOF masene spektrometrije pokazano je da postoji direktna reakcija
između kompleksa i donora –SH grupe, tj da ove dve vrste direktno reaguju kada su
prisutne u rastvoru.
Nanočestice zlata koje su ispitivane nisu dale zapažen inhibitorni efekat. Tip nanočestica
obeležen kao C3 koji sadrži nanočestice zlata prečnika 9,5 nm, pokazao je neobična
svojstva - dodatkom u preparat sinaptozomalnih plazma membrana, doveo je do porasta
aktivnosti Na+/K+ ATPaze u tom preparatu dvostruko, dok se aktivnost ostalih prisutnih
enzima nije menjala. Ispitivanjem ove interakcije pokazano je da je u pitanju fizička
promena u preparatu do koje dolazi usled dva tipa hemijske interakcije između koloida i
preparata enzima. Uočene su promene u UV-vis i FTIR spektrima koloida po dodatku
enzima, a upotrebom atomske mikroskopije uočeno je da dolazi do promena u
nanostrukturi preparata enzima koje izaziva povećanje aktivne površine ćelijske membrane,
te tako veći broj molekula enzima postaje dostupan za reakciju i javlja se porast aktivnosti
enzima u preparatu. Selektivnost enzima za njegove prirodne ligande ostaje neizmenjena, a
dobijeni preparat pojačane aktivnosti stabilan je najmanje sat vremena.
Zaključeno je da ispitivani kompleksi zlata mogu da budu od značaja za dalji razvoj
antitumorskih terapija, kao i da koloidne nanočestice zlata mogu poslužiti za razvoj
osetljivijih testova za merenje aktivnosti membranskih enzima.
Na+/K+ ATPase is a membrane enzyme that uses the enegry released from the molecule of
adenosine triphosphate to regulate the sodium and potasium ion concentration on either side
of the cell membrane in most living creatures. Appart from its transporter role, it also has a
role as a signal molecule, because it regulates the activity of Src kinase, an important
enzyme in cell signaling and survival. Beacuse of these properties, Na+/K+ ATPase is
recognized as an important molecular target in many disorders including tumors. Noble
metal complexes, and especially gold complexes have shown the tendency to inhibit
Na+/K+ ATPase. This makes them potential terapeutics, analogous to platinum based
complexes that are a part of modern chemotherapy. Colloid nanoparticles of gold have also
shown interesting properties in the past. This study presents the findings on the interaction
of several preparations of Na+/K+ ATPase with 9 different complexes of gold and 3
different types of gold colloid nanop...articles.
Results shown in this thesis indicate that 6 out of 9 investigated complexes have a
moderatley strong inhibitory effect upon the Na+/K+ ATPase. It is shown that this inhibition
is noncompetitive and reversible, and that it can be reversed by –SH group donors such as
cysteine and glutathione. Moreover, this inhibition can be fully prevented by adding these
substances prior to the addition of the complex. Therefore it is concluded that the binding
of 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 human
lymphocytes, which makes them good candidates for further clinical studies. MALDI TOF
mass spectroscopy has shown that there is a direct reaction between –SH group donors and
the gold complex.
Investigated nanoparticles yielded no significant inhibitory effect. One type of the
nanoparticles, however, has shown some very unusual properties. Upon the addition of this
type of colloid nanoparticles, labeled C3 and with an average diameter of 9.5 nm, to the
preparation of synaptosomal plasma membranes, the activity of Na+/K+ ATPase doubled,
while the activity of other present enzymes remained the same. Further investigation of this
interaction has shown that the cause of this is a physical change in the structure of the
membrane fragments. It is the result of two paralel chemical interactions that take place
when the nanoparticles are added to the enzyme preparation. Changes in UV/vis and FTIR
spectra of the nanoparticles were observed and using atomic force microscopy it was
possible to visualize the change in the nanostructure of the enzyme preparation upon the
addition of nanoparticles. This change resulted in an increase of the active membrane
surface, exposing more molecules of the enzyme and making them available for the
reaction. This caused an increase of the enzyme activity. Selectivity of the Na+/K+ ATPase
towards its natural ligands was preserved, and the effect of increased activity persisted for
at least an hour before diminishing.
In conclusion, investigated complexes and nanoparticles of gold have shown important
properties. The effects of gold complexes make them potential antitumor drugs, while the
nanoparticles of gold may be used to develop better assays for membrane enzymes.
