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Tesi etd-02182010-104711


Thesis type
Tesi di dottorato di ricerca
Author
CALVANI, ENRICA
URN
etd-02182010-104711
Title
ON THE ROLE OF CYSTATHIONINE-γ-LYASE IN MODULATING TRANSSULFURATION PATHWAY IN THE LENS
Settore scientifico disciplinare
BIO/10
Corso di studi
BIOTECNOLOGIE MOLECOLARI
Commissione
tutor Prof. Mura, Umberto
Parole chiave
  • CGL
  • Transsulfuration pathway
Data inizio appello
05/03/2010;
Consultabilità
parziale
Data di rilascio
05/03/2050
Riassunto analitico
In Mammals transsulfuration pathway enables the conversion of homocysteine (Hcy), deriving from methionine (Met) transmetilation, into cysteine (Cys) through two steps, the first catalyzed by cystathionine β-synthase (CBS) and the second one by cystathionine-γ-lyase (CGL). The latter enzyme is considered to catalyze the rate limiting step of the whole process and one of its products, Cys, is the limiting amino acid for glutathione (GSH) synthesis. GSH, is a tripeptide that has the prominent function to balance the cell redox state. This role is particularly important in the lens where the oxidative stress is at the base of senile cataract onset. Experimental evidences obtained in our research unit point out the attention on the possible role of cysteinylglycine (CysGly) (an intermediate of GSH catabolism), in modulating the transsulfuration pathway in cultured bovine lens. Blocking γ-glutamyl transpeptidase (γ-GT), upon action of serine borate, appears to induce an extrasynthesis of GSH, which is released into medium without any change in intralenticular levels; while the supplementation of culture medium with CysGly, impaired the observed GSH extrasynthesis, as the use of PPG that inhibits CGL. Aim of this work was the accomplishment of CGL purification from calf lenses in order to assess the possible regulatory role of CysGly on CGL activity. The purification of CGL from bovine lens resulted a very hard task, since the extremely low activity detectable in the lens extracts. The assembled purification protocol did not allowed the availability of sufficient enzymatic sample to proceed to a characterization. Thus cultured bovine lens epithelial cells (BLEc), for which a CGL specific activity two order of magnitude higher than that measured in the lens extract, were selected as a better source to isolate CGL.<br>The purification of the enzyme from BLEc, even though did not allow to obtain an electrophoretically homogeneous sample provided information on the quaternary structure of the enzyme. A modest, even though of debateable statistical significance, inhibitory effect of CysGly on partially purified CGL was observed.<br>
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