Purification and characterization of a novel protamine kinase in HL60 cells.
A protamine kinase from HL60 cells was purified to near homogeneity by DEAE-Sephacel, protamine-agarose, Hydroxylapatite, and S-200 chromatography. It was purified by 75.8-fold through four chromatographic steps, and 0.67% of total activity was recovered. The purified enzyme had an apparent molecular mass of 120 kDa and was activated by Mg(2+) or Mn(2+), but inhibited by Ca(2+). Neither phospholipid nor phorbol ester significantly affected the enzyme activity. Staurosporine was the most potent inhibitor of the enzyme among the protein kinase inhibitors tested, K(252a), H(7), heparin, and staurosporine. The purified protamine kinase exhibited a maximum velocity of 5,000 pmol/min/mg and K(m) of 1.3 mM for protamine sulfate as a substrate. Myelin basic protein and protamine sulfate served as the best substrates for the protamine kinase among those tested. The activity of the protamine kinase remained unchanged upon treatment with PMA, retinoic acid, dimethyl sulfoxide, or 1,25 dihydroxy vitamin D(3) for 15 min, while treatment with a differentiating agent, 1,25 dihydroxy vitamin D(3), for one week increased its activity. These results suggest that protamine kinase in HL60 cells is involved in the late stage of the macrophage-monocytic differentiation pathway and may play a role in maintenance of the differentiation after HL60 cells are committed.
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Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA. yuns@nms.kyunghee.ac.kr
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D002117:Calcitriol / Q000494:pharmacology; D002413:Cations, Divalent / Q000737:chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D017886:Durapatite; D004789:Enzyme Activation / Q000187:drug effects; D018922:HL-60 Cells / Q000201:enzymology*; D006801:Humans; D006656:Protamine Kinase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology
D002413:Cations, Divalent; D014212:Tretinoin; D017886:Durapatite; D006656:Protamine Kinase; D011493:Protein Kinase C; D002117:Calcitriol; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide