Purification and characterization of rhodopsin kinase.

摘要: Rhodopsin kinase was purified to near homogeneity by affinity binding light-exposed rod cell outer segment membranes, followed DEAE-cellulose and hydroxyapatite chromatography. This resulted in a 1055-fold purification of highly active rhodopsin with an overall recovery 19%. is single polypeptide chain Mr = 67,000-70,000 as determined gel filtration SDS-PAGE. The kinetic parameters the enzyme for freshly bleached are Km 4 microM Vmax 700 nmol/min/mg whereas ATP 2 (which low value kinases generally, about 20 times lower than comparable measurements similar type, beta-adrenergic-receptor (Benovic, J.L., Mayor, F. Jr., Staniszewski, C., Lefkowitz, R.J., Caron, M.G. (1987) J. Biol. Chem. 262, 9026-9032). GTP, on other hand, very poor substrate (Km 1 mM, 10 nmol/min/mg). competitively inhibited adenosine its mono- diphosphate derivatives, but not most derivatives. Based upon 28 nucleotide ATP-binding site appears have more specific requirements that kinases. Compounds such cGMP, inositol trisphosphate, others change concentration during exposure cells light only minor inhibitory effects activity, exception monophosphate, which can activate 20% at 50-100 microM. has been difficult store retention be successfully stored frozen -20 degrees C adonitol.