Purification and properties of cAMP dependent and independent histone kinases from human leukocytes |
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Authors: | Henning Juhl Viggo Esmann |
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Institution: | (1) Department of Medicine, Marselisborg Hospital, DK-8000 Århusc., Denmark |
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Abstract: | Summary Histone kinase activity was purified from human polymorphonuclear leukocytes by ammonium sulphate precipitation of a 180 000 × g supernatant, followed by DEAF-cellulose chromatography and gelfiltration. On DEAE-cellulose cAMP dependent kinase activity eluted in two peaks, I and III, at 1.2 mmho and 6.5 mmho, respectively. Catalytic subunit (C) from both peaks had Mr 33 000, 3.0S. Regulatory subunit (R) from peak I and III both had Mr 33 000 upon gelfiltration, but sedimented at 2.8–3.0S and 3.0–3.2S, respectively. R2 and R4 subunits were identified. The R-C dimer from peak I and III sedimented at 4.8S and (4.8)–5.1S, respectively. The holoenzyme from peak I had Mr 165 000, 6.7S, which suggest a R2C2 structure, while that of peak III sedimented at 6.7S, but eluted at Mr 330 000 (2R2C2) by gelfiltration.The K
m
app
for peak I and III enzymes were, respectively: histone IIA 0.5 mg/ml (both forms), ATP 18 m and 23 m, and cAMP 5 × 10–8
m and 6.3 × 10–8
m. Both enzymes had pH optimum 6.7–6.9 and were equally sensitive to Ca2+ temperature and protein kinase inhibitor. The substrate specificity was histone VS histone IIA = histone VIS casein > phosvitin. Peak I enzyme, but not peak III enzyme, was dissociated by histone and high ionic strength and reassociation of R and C subunits were facilitated by ATP-Mg. It is concluded that peak I and III enzymes represent type I and II cAMP dependent protein kinases, respectively. Type I comprises 20–30% of cAMP dependent protein kinase activity and is absent from the 180 000 × g supernatant of gently disrupted cells.Purified catalytic subunit had K
m
app
(ATP) 20 m with rabbit muscle glycogen synthase I as substrates. Synthase I from rabbit muscle and human leukocytes were phosphorylated by catalytic subunit to synthase D (ratio of independence less than 0.07).cAMP independent histone kinase activity eluted in one peak (Peak II) at 3 mmho. The enzymatic activity sedimented at 3.4S and eluted from gelfiltration with Mr 78 000. K
m
app
for ATP was 78 m and for histone IIA 0.5 mg/ml. The enzyme was sensitive to temperature, but less sensitive than cAMP dependent protein kinase to Ca2+, and insensitive to protein kinase inhibitor. The substrate specificity was histone IIA > histone VS = histone VIS, while casein and phosvitin were poor substrates. Glycogen synthase I was not phosphorylated. The cAMP independent histone kinase activity comprised 15% of the total histone kinase activity in a crude homogenate of leukocytes. Its physiological substrate is unknown.Abbreviations AR
activity ratio for cAMP dependent protein kinase
- cAMP
adenosine cyclic 3:5-monophosphate
- cIMP
inosine cyclic 3:5-monophosphate
- cGMP
guanosine cyclic 3:5-monophosphate
- Glucose-6-P
glucose-6-phosphate
- DDT
dithiothreitol
- EGTA
ethylene glycol-bis-(-aminoethylether)-N, N-tetraacetic acid
- PMSF
phenylmethylsulfonylfluoride
- PKI
protein kinase inhibitor
- RI
ratio of independence for glycogen synthase
- SDS
sodium dodecyl sulphate |
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