Študija vpliva izbranih ionov težkih kovin na aktivnost katepsinov B, L in S

magistrsko delo

Primož Bembič (Avtor), Marko Novinec (Mentor), Gregor Gunčar (Član komisije za zagovor), Marina Klemenčič (Član komisije za zagovor), Iztok Turel (Komentor)

Povzetek

Encimi predstavljajo pomembno skupino makromolekul, ki v telesu sodelujejo pri veliki večini procesov. Zaradi povezave s fiziološkimi in patološkimi procesi je regulacija njihovega delovanja izjemno pomembna. Pri patoloških stanjih so encimi pogosto prekomerno aktivni zato predstavljajo inhibitorji encimske aktivnosti pomembno terapevtsko sredstvo za nadzorovanje in zdravljenje bolezni. Kot regulatorji encimske aktivnosti lahko delujejo proteini, peptidi, organske molekule ali pa kovine oz. kovinski kompleksi. Med ene izmed bolj raziskanih encimov spadajo katepsini, lizosomske, cisteinske proteaze, ki se izražajo v vseh tipih celic, kjer nespecifično razgrajujejo različne proteinske komponente celic. Njihova patološka vloga je običajno povezana z zunajcelično lokalizacijo ter razgradnjo komponent zunajceličnega matriksa. Povezani so s kardiovaskularnimi, avtoimunskimi ter nevrološkimi obolenji, osteoporozo, aterosklerozo ter nenazadnje z napredovanjem in metastaziranjem malignih tumorjev. V predhodnih študijah so preučevali vpliv izbranih ionov težkih kovin (Cd(II), Ce(III), Ce(IV), Ga(III), La(III), Pb(II) ter Zn(II)) na delovanje katepsina K, ki ima pomembno vlogo pri razgradnji in obnavljanju kostnine. Kovinski ioni predstavljajo dobro izhodišče za pripravo kovinskih kompleksov, ki lahko selektivno delujejo samo na izbran encim ter delno ali v celoti inhibirajo njegovo delovanje. V okviru te študije smo preučili vpliv izbranih kovinskih ionov na aktivnost treh katepsinov (B, L ter S). Vpliv kovinskih ionov smo spremljali s pomočjo kinetičnih meritev encimske aktivnosti, s pomočjo katerih smo nato določili vrednosti Ki oz. EC50 ter mehanizme delovanja za posamezen kovinski kation. Pri uporabi sintetičnih substratov, se je pri vseh encimih izkazalo, da Cd(II), Pb(II) ter Zn(II) ioni delujejo kot linearni kompetitivni inhibitorji, kot najbolj učinkovit inhibitor so delovali Zn(II), kot najšibkejši pa Pb(II) ioni. Preostali kationi (Ce(III), Ce(IV), Ga(III) ter La(III)) so pri različnih katepsinih delovali na različne načine. Pri katepsinu B so vsi kationi razen Ce(III) ionov delovali kot delni inhibitorji, v primeru Ce(III) ionov pa je do inhibicije prišlo šele pri visokih koncentracijah kationa. Vsi štirje kationi so prav tako delovali kot delni inhibitorji katepsina S, pri katepsinu L pa so delovali po mehanizmu linearne kompetitivne inhibicije. V vseh primerih so se kot boljši inhibitorji izkazali Ga(III) ter Ce(IV) ioni, kar je najverjetneje povezano z manjšim ionskim polmerom teh kationov. Pri razgradnji makromolekulskih substratov pa so bili rezultati nekoliko drugačni, inhibicija je bila v vseh primerih nižja. Pri razgradnji elastina so kovinski ioni nekoliko bolje inhibirali delovanje katepsina S kakor katepsina L, pri razgradnji azokazeina pa so se kot učinkovit inhibitor izkazali samo Zn(II) ioni. Ostali kovinski ioni bodisi niso vplivali na aktivnost encima, ali pa je bil njihov vpliv zanemarljivo majhen.

Ključne besede

encimi;encimska aktivnost;inhibicija encimske aktivnosti;mehanizem inhibicije;kationi težkih kovin;magistrska dela;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2022
Tipologija:	2.09 - Magistrsko delo
Organizacija:	UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik:	[P. Bembič]
UDK:	577.15:546.3(043.2)
COBISS:	111257603
Št. ogledov:	92
Št. prenosov:	18
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	Effect of selected heavy metal ions on activity of cathepsins B, L and S
Sekundarni povzetek:	Enzymes, an important group of macromolecules, are involved in most processes in the human body. Because of their involvement in both physiological and pathological processes, regulation of enzyme activity is of the utmost importance. Enzymes are often overly active in various pathological conditions. Inhibitors of enzyme activity therefore represent an important therapeutical implement for both managing and treating the disease. Different types of molecules, such as proteins, peptides, organic molecules, metals, or complex compounds can regulate enzyme activity. The lysosomal, cysteine proteases called cathepsins, which are expressed in all cell types, where they non-specifically degrade various protein components of cells are among the most often studied enzymes. Their pathological role is usually associated with their extracellular localization and degradation of the components of extracellular matrix. They are involved in different diseases, such as cardiovascular, autoimmune, and neurological diseases as well as osteoporosis, atherosclerosis and last but not least, in the progression and metastasis of malignant tumours. Previous studies have already examined the effect of selected heavy metal ions (Cd(II), Ce(III), Ce(IV), Ga(III), La(III), Pb(II) and Zn(II)) on the activity of cathepsin K, an important enzyme in bone degradation and repair. Metal ions offer a good basis for the development of complex compounds that can selectively act on a specific enzyme and partially or fully inhibit its activity. The aim of this study was to examine the effect of these heavy metal ions on activity of other cathepsins, namely cathepsins B, L and S. We tested the effects of selected metal ions by means of kinetic measurement of enzyme activity, which in turn enabled us to determine the Ki or EC50 values and mechanism of action for each metal cation. When using a synthetic substrate Cd(II), Zn(II) and Pb(II) ions act as linear competitive inhibitors of all three enzymes, of these the Zn(II) ions were the most effective inhibitor, while Pb(II) ions were the least effective inhibitor. The remaining cations (Ce(III), Ce(IV), Ga(III) ter La(III)) acted differently on different cathepsins. All cations except Ce(III) ions acted as partial inhibitors of cathepsin B, whilst low inhibition occurred only at very high concentrations of Ce(III) ions. All 4 cations acted as partial inhibitors of cathepsin S activity and as full inhibitors of cathepsin L activity. In all cases Ga(III) and Ce(IV) ions proved to be better inhibitors, which is most likely due to their smaller ionic radii. The results of macromolecular degradation assay the results were slightly different, with inhibition being lower in all cases. In the elastin degradation assay, metal ions proved to inhibit the activity of cathepsin S slightly better than that of cathepsin L. On the other hand, in the azocasein assay, only Zn(II) ions proved to be an effective inhibitor of both cathepsin L and S, while the effect of other metal ions was either non-existent or negligible.
Sekundarne ključne besede:	enzyme inhibition;cathepsins;heavy metal cations;Katepsini;Težke kovine;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS):	Magistrsko delo/naloga
Študijski program:	1000377
Konec prepovedi (OpenAIRE):	1970-01-01
Komentar na gradivo:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Strani:	91 str.
ID:	15563682