Solubilization and characterization of a novel tyrosine kinase from rat adipocytes

The authors report the efficient solubilization and characterization of a Triton X-100 insoluble tyrosine kinase from rat adipocytes. Plasma membranes were prepared from rat epididymal fat pads and were solubilized in 1% Triton X-100. Following centrifugation, the pellet was solubilized for 15 min at 40C using both ionic and non-ionic detergents. Tyrosine kinase activity was measured in the soluble and particulate fractions using the exogenous substrate poly(glu-tyr) in a TCA precipitation assay. Reactions were performed in 50mM Hepes, 10mM MgCl2 and 100μM gamma[32P]-ATP (10Ci/mmol) at 40C with or without 1mg/ml of the polyaminoacid. Incorporation rates of 100 to 1000 pmol/min/mg were obtained, while endogenous [32P] incorporation was typically less than 10% of that in the presence of poly(glu-tyr). More than 75% of the tyrosine kinase activity was recovered in the soluble supernatant using this assay methodology. The solubilized tyrosine kinase was found to require Mg2+ or Mn2+ but preferred Mg2+ and was inhibited by high levels of Mn2+. Kinase activity was strongly inhibited by Ca2+ (>50% at 1mM), NaCl (>50% at 250mM) and NH4SO4 (>50% at 50mM) but was activated by 10μM heparin and 5mM dithiothreitol. These properties distinguish the solubilized tyrosine kinase from other cellular tyrosine kinases