Protein phosphorylation in human peripheral blood lymphocytes. Subcellular distribution and partial characterization of adenosine 3':5'-cyclic monophosphate-dependent protein kinase

Cytoplasmic and membrane fractions prepared from human peripheral-blood lymphocytes both contained cyclic AMP-dependent protein kinase activity and endogenous protein kinase substrates. Protein kinase activity in the particulate fractions was not eluted with 0.25 M-NaCl, suggesting that it was not d...

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Bibliographic Details
Published in:Biochemical journal 1979-08, Vol.182 (2), p.525-536
Main Authors: Chaplin, D D, Wedner, H J, Parker, C W
Format: Article
Language:English
Subjects:
Caseins - metabolism
Chromatography, DEAE-Cellulose
Cyclic AMP - pharmacology
Detergents
Histones - metabolism
Humans
In Vitro Techniques
Kinetics
Lymphocytes - drug effects
Lymphocytes - enzymology
Manganese - pharmacology
Phosphorylation
Protein Kinases - blood
Solubility
Subcellular Fractions - enzymology
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Summary:Cytoplasmic and membrane fractions prepared from human peripheral-blood lymphocytes both contained cyclic AMP-dependent protein kinase activity and endogenous protein kinase substrates. Protein kinase activity in the particulate fractions was not eluted with 0.25 M-NaCl, suggesting that it was not derived from non-specifically absorbed soluble cytoplasmic protein kinase. Nor was the particulate protein kinase activity eluted by treatment with cyclic AMP, suggesting that the catalytic subunit is membrane-bound and arguing against cyclic AMP-induced translocation of particulate activity. Cyclic AMP-dependent protein-phosphorylating activity in the cytoplasmic fraction was highly sensitive to inhibition by Mn2+, and was co-eluted from DEAE-cellulose primarily with type-I rabbit skeletal-muscle kinase. Cyclic AMP-dependent phosphorylating activity in the plasma-membrane fractions was stimulated at low [Mn2+] and inhibited only at high [Mn2+]. When solubilized with Nonidet P-40, plasma-membrane protein kinase was co-eluted from DEAE-cellulose with type-II rabbit muscle kinase. These differences, together with the strong association of the particulate kinases with the particulate fraction, suggest the possibility of compartmentalized protein phosphorylation in intact lymphocytes.
ISSN:0264-6021
0306-3283
1470-8728
DOI:10.1042/bj1820525