RESUMO
Cyanogen bromide splitting of pike parvalbumin III was carried out and fragment 38-108 containing two calcium-binding domains was isolated. The obtained fragment binds calcium rather well (Kd = 1.6.10(-5) M), possesses a secondary structure, its CD spectrum changes after removal of calcium.
Assuntos
Cálcio/metabolismo , Proteínas Musculares , Parvalbuminas , Sequência de Aminoácidos , Animais , Sítios de Ligação , Fenômenos Químicos , Química , Dicroísmo Circular , Peixes , Cinética , Fragmentos de Peptídeos/metabolismo , Ligação ProteicaRESUMO
The possibility of creating new screening methods with a cell-free translation system has been demonstrated with a quantitative determination of diphtheria toxin and some antibiotics (puromycin, kanamycin and tetracycline) as examples. The approach proposed follows from the ability of various substances to inhibit protein synthesis. We used a wheat-germ cell-free translation system stabilized by freeze-drying in the presence of trehalose with the mRNA of the Ca(2+)-activated photoprotein obelin as a reporter template. This freeze-dried cell-free translation system allows prolonged storage of the detecting system before it is required, increases the reproducibility of the results and simplifies the application procedure. The obelin mRNA extends the sensitivity of the method owing to the high sensitivity of detection of the synthesized protein.