Human neutrophils contain distinct cytosolic and particulate tyrosine kinase activities: possible role in neutrophil activation.

Tyrosine protein kinase activities were partially purified from circulating human neutrophils. Purification steps involved sequential chromatography on DEAE-Sephacel, gel filtration and affinity chromatography on a column composed of a glutamine:tyrosine copolymer linked to AH-Sepharose. The results indicate that human neutrophils contain a tyrosine kinase activity in the 150,000 x g cytosolic fraction which is distinct from the activity in a detergent extractable 150,000 x g particulate fraction. These enzyme activities are dependent on the divalent cations Mn2+ and Mg2+. Kinetics for the phosphorylation of a glutamine:tyrosine copolymer substrate demonstrated an apparent Km for the cytosolic tyrosine kinase activity of 22.3 +/- 0.3 microM, and an apparent Km for the particulate extract activity of 42.7 +/- 6.0 microM. By gel filtration chromatography, the cytosolic and particulate tyrosine kinase activities have approximate molecular masses of 80-90 and 50-60 kDa, respectively. The particulate but not the cytosolic neutrophil tyrosine kinase activity was inhibited by a novel tyrosine kinase inhibitor ST638. ST638 inhibited superoxide production in intact neutrophils stimulated with the chemotactic peptide F-Met-Leu-Phe, opsonized zymosan particles, and sodium fluoride. ST638 did not, however, inhibit superoxide production in neutrophils stimulated with phorbol myristate acetate or the calcium ionophore, A23187.