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

Roger L. Berkow, Robert W. Dodson, Andrew S. Kraft

Research output: Contribution to journalArticle

53 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)292-301
Number of pages10
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume997
Issue number3
DOIs
StatePublished - Aug 31 1989
Externally publishedYes

Keywords

  • Inhibitor
  • Neutrophil
  • Protein purification
  • Tyrosine kinase

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'Human neutrophils contain distinct cytosolic and particulate tyrosine kinase activities: possible role in neutrophil activation'. Together they form a unique fingerprint.

  • Cite this