članak: 1 od 1  
Jugoslovenska medicinska biohemija
2002, vol. 21, br. 3, str. 255-260
jezik rada: engleski
izvorni naučni članak
doi:10.2298/JMH0203255D
Interakcija intracelularnih petlji dopaminskog d1 receptora sa podjedinicama g proteina
Đuranović Sergeja, Predić Jelenab, Backović Marijac, Šoškić Vukića
aUniverzitet u Beogradu, Hemijski fakultet
bInstitut za hemiju, tehnologiju i metalurgiju - IHTM, Beograd
cOdeljenje za biohemiju i molekularnu biologiju, Univerzitet Ilinois, Čikago

Sažetak

Razvijena je jednostavna i brza metoda za kvalitativano i kvantitativno određivanje interakcija podjedinica Ga proteina sa drugom i trećom intracelularnom petljom, kao i sa terminalnim krajem D1 dopaminskog receptora čoveka. U tu svrhu su D1-ICL2 i D1-ICL3 klonirani u vektor pGEX-2T i eksprimirani u E. coli BL21 kao fuzioni proteini sa glutation-S-transferazom (D1-ICL2-GST i D1-ICL3-GST). C-terminalni deo je rastavljen u dva fragmenta koji su klonirani u pGEX-2T i eksprimirani in E. coli BL21 DE3 kao fuzioni proteini sa glutation-S-transferazom (D1-CTSF-GST i D1-CTLF-GST). Dobijeni solubilni konstrukti su prečišćavani afinitetnom hromatografijom na glutation-sefarozi. Podjedinice Ga su eksprimirane i prečišćene kao His-obeleženi proteini (Gao i Gai1 u E. coli BL21 DE3, a Gas u E. coli JM 109). Za kvantitativno određivanje su različite koncentracije prečišćenih His-obeleženih podjedinica Ga imobilisane na smoli His-Bind i titrovane fuzionim proteinima, a interakcije su određivane kolorimetrijskim postupkom za određivanje aktivnosti GST. Za kvalitativno dokazivanje interakcija je primenjen sličan pristup. U tu svrhu su poznate koncentracije fuzionih proteina imobilisane na glutation-sefarozi i tretirane poznatim koncentracijama prečišćenih His-obeleženih podjedinica Ga. Tako tretirani kompleksi su eluirani sa glutation-sefaroze i analizirani SDS elektroforezom na poliakrilamidnim gelovima (SDSPAGE). Pokazano je da D1-CTSF specifično interaguje sa podjedinicom Gas, a D1-CTLF sa podjedinicom Gao. Druge interakcije nisu zapažene. Kd vrednosti izračunate na osnovu eksperimenata vezivanja liganda do zasićenja bile su u nanomolarnom opsegu koncentracija, što ukazuje na najviši afinitet vezivanja His-Gas za D1-CTSF-GST i His-Gao za D1-CTLF-GST.

Ključne reči

D1 dopaminski receptor čoveka; intracelularne petlje; G proteini; podjedinice Ga; interakcije

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