Protein kinase NII from calf thymus chromatin. Isolation, characterization and some functional properties.
1. A protein kinase type II was purified from calf thymus chromatin using ammonium sulphate fractionation, ion exchange chromatography on DEAE and phosphocellulose and affinity chromatography on phosvitin- and casein-sepharose columns. 2. The enzyme moves as a single band in non-denaturing gel electrophoresis at pH 8.3, which coincides with the enzyme activity assayed on gel slices. 3. Sodium dodecyl sulphate gel electrophoresis shows three separate polypeptide chains having M(r) of 40,000, 38,000 and 25,000, respectively. The native M(r) was about 130,000, as measured by HPLC on Superose 12 column, suggesting a subunit structure of alpha, alpha', beta 2 type. The enzyme incubated with [gamma 32P]ATP or [gamma 32P]GTP as phosphoryl donors undergoes autophosphorylation in the M(r) = 25,000 subunit. 4. The enzyme phosphorylates casein (Km = 7 microM) and phosvitin (Km = 5 microM) but not histones and was strongly deactivated by Zn2+ ions (I50 = 0.05 mM) and heparin (I50 = 0.1 micrograms/ml). 5. The enzyme seems to be the major phosphorylating system present in the 0.35 M NaCl chromatin extract of calf thymus. The RNA polymerase II from calf thymus and RNA polymerase from E. coli are both phosphorylated by protein kinase NII. The effect of phosphorylation, which causes a remarkable increase of DNA transcription rate, was studied in vitro and extensively discussed.
Angiolillo
A
A
Istituto di Biologia Cellulare, Università di Perugia, Italy.
Panara
F
F
Desgro
A
A
Petrelli
C
C
Gianfranceschi
G L
GL
D000818:Animals; D002417:Cattle; D002843:Chromatin / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D013696:Temperature; D013950:Thymus Gland / Q000201:enzymology*
D002843:Chromatin; D011494:Protein Kinases; C026813:protein kinase NII; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases