Ecto-protein kinase activity of fibroblasts

Abstract

Isolated, intact dermal fibroblasts can transfer the terminal phosphate of adenosine triphosphate, [γ- 32P]ATP, to an exogenously added macromolecule (histone). The incorporation of labeled phosphate to histone is attributed to an extracellularly directed protein kinase activity (ecto-kinase) which cannot be accounted for by soluble cytoplasmic protein kinase that might have been released and become bound to cell membranes during the cell preparation. The addition of soluble cytoplasmic enzyme preparations to the cell suspension was fully recoverable in the supernatant and the first wash. The activity of ectokinase was abolished by incubation of intact cells with trypsin for 5 min, whereas the activity of cytoplasmic enzyme was unaffected by the trypsin treatment. These data suggest that dermal fibroblasts contain protein kinase on the outer surface of plasma membrane which can phosphorylate exogenously added macromolecules. The ecto-protein kinase activity is dependent on cell number, time of incubation, and the concentration of Mg 2+ in the reaction mixture. Lineweaver-Burk plot analyses yielded K m values for ATP and histone of 7 × 10 −5 and 3 × 10 −6 m, respectively. The ecto-protein kinase activity of normal fibroblasts and fibrosarcoma cells were also compared. The enzyme activity of normal cells was higher than that of the malignant cells and was not significantly affected by cyclic nucleotides, whereas the activity of the malignant cells were stimulated by the addition of micromolar concentrations of the cyclic nucleotides.