TY - JOUR
T1 - Human neutrophils contain distinct cytosolic and particulate tyrosine kinase activities
T2 - possible role in neutrophil activation
AU - Berkow, Roger L.
AU - Dodson, Robert W.
AU - Kraft, Andrew S.
N1 - Funding Information:
This work was supporteidn part by a grantf romthe American Cancer Society BC522, and the NIH CA42533D. r. Berkowi s supporteidn part by a Clinical OncologyC areerD evelopmenAtw ardfrom the American CancerS ociety.
PY - 1989/8/31
Y1 - 1989/8/31
N2 - Tyrosine protein kinase activities were partially purified from circulatinghuman 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 × g cytosolic fraction which is distinct from the activity in a detergent extractable 150 000 × 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 μM, and an apparrent Km for the particulate extract activity of 42.7±6.0 μM. By gel filtration chromatography, the cytosolic and particulate tyrose 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 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.
AB - Tyrosine protein kinase activities were partially purified from circulatinghuman 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 × g cytosolic fraction which is distinct from the activity in a detergent extractable 150 000 × 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 μM, and an apparrent Km for the particulate extract activity of 42.7±6.0 μM. By gel filtration chromatography, the cytosolic and particulate tyrose 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 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.
KW - Inhibitor
KW - Neutrophil
KW - Protein purification
KW - Tyrosine kinase
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U2 - 10.1016/0167-4838(89)90200-8
DO - 10.1016/0167-4838(89)90200-8
M3 - Article
C2 - 2765566
AN - SCOPUS:0024446512
SN - 0167-4838
VL - 997
SP - 292
EP - 301
JO - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
JF - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
IS - 3
ER -