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.
Department of Pediatrics (Hematology-Oncology), University of Alabama, Birmingham 35233.
R L
RL
Berkow
R W
RW
Dodson
A S
AS
Kraft
D002413:Cations, Divalent; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002934:Cinnamates / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000097:blood* / Q000302:isolation & purification; D013440:Sulfides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002413:Cations, Divalent; D002934:Cinnamates; D007527:Isoenzymes; D013440:Sulfides; C060747:ST 638; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011505:Protein-Tyrosine Kinases; D013755:Tetradecanoylphorbol Acetate