Brain histone kinase: the structure, the substrate specificity and the mechanism of action.

摘要: Cyclic adenosine 3′,5′-monophosphate-dependent histone kinase (ATP protein phosphotransferase, EC 2.7.1.37) was obtained from pig brain. The enzyme purified 1140 times. It homogeneous on polyacrylamide gel with electrophoresis and filtration. established molecular weight of the is 120,000. dissociates into catalytic regulatory subunits (molecular 40,000 90,000, respectively). With chromatography DEAE-cellulose in presence excess cyclic AMP subunit obtained. Using polylysylsepharose, which contains immobilized 8-(γ-carboxypropylthio)-cyclic affinity isolated. methods permit us to obtain also individual partially preparations enzyme. Subunits were SDS. The had a high specificity action according lysine-rich histones F1, F2a2 F2b. Arginine-rich other known substrates AMP-dependent kinases (casein, E. coli, RNA polymerase, etc.) not phosphorylated by this kinetics phosphorylation F2a2, F2b investigated corresponding sites determined primary structures these histones: Ser-38 Ser-19 Ser-14 Ser-36 Phosphorylated serine residues localized all cases sequence X-Y-Ser, where X always lysine or arginine residue, Y — an acidic neutral amino acid. Using various original ATP analogs containing reactive groups, functional topography active investigated. Specific regions binding centre imidazole ring histidine suggested as group participating phosphotransferase reaction. The existence at least three parts negatively charged hydroxyl cyclophosphate system, nucleophilic locus for 2′-hydroxyl ribose proton-donor group, may take part interaction exo-amino adenine base occurs. On basis, contribution separate groups molecule nucleotide activation studied. The investigation structure different subfractions F1 histone, chemical modification reaction PMR spectra native molecules complex DNA permitted suggest that during role two cationic loci (15–36) (107–212) realization typical electrostatic contacts. ionic one loci, probably part, destroyed incorporation phosphate molecule. leads disorganization secondary central segment its more free movement polyanion template. PMR data demonstrated residue led weakening histone-DNA influenced protein-protein interactions nucleoproteid complex. Using cytochemical approach influence physico-chemical properties chromatin investigated. The hepatocyte treatment increase acridine orange binding. DNP same both 2.5 hr after operation hepatocytes without operation: i.e., cell effect partial hepatectomy. Thus, exogenous appearance activated genome stimulation transcription process.