Purification and characterization of a protein-tyrosine kinase from lymphocytes

Thomas Francis Zioncheck, Purdue University

Abstract

The major NaCl-stimulated protein-tyrosine kinase activity has been isolated from a soluble extract of bovine thymus based on its ability to phosphorylate the tyrosine-containing peptide angiotensin I. The enzyme was purified by sequential column chromatography on DEAE-cellulose, heparin-agarose, casein-agarose, butyl-agarose, and Sephadex G-75. The purified enzyme (p40) is a monomer of 40,000 Daltons. The p40 kinase contains an ATP-binding site as shown by its specific labeling with photoaffinity analogs of ATP. Furthermore, the kinase catalyzes an intramolecular autophosphorylation reaction that leads to the modification of tyrosine residues. Polyclonal antisera raised against p40 cross-react with a 72 kDa protein-tyrosine kinase (p72) from spleen and thymus. Like p40 the 72 kDa protein is phosphorylated on tyrosine in vitro and phosphopeptide maps indicate that the proteins are also structurally related. The following observations suggest a precursor-product relationship between p72 and p40. The recovery of p40 from spleen homogenates is reduced, while the recovery of p72, is enhanced if high concentrations of leupeptin or soybean trypsin inhibitor are added to the homogenization buffer. The generation of p40 in spleen homogenates occurs with a concomitant increase in protein-tyrosine kinase activity. Activated catalytic fragments of 38-43 kDa can be generated by the treatment of partially purified p72 with exogenously added proteases. Western blotting studies reveal the highest levels of p72 are in spleen, thymus and lung, tissues that also have high protein-tyrosine kinase activity and generate high levels of p40 after homogenization. Consistent with this, we have observed high levels of p72 in certain B and T cell-derived cell lines. The presence of immunoreactive p72 in NIH3T3 cells suggests that the kinase may be expressed in cells other than those of hematopoietic origin. p40 is of higher specific activity and has been more stringently characterized than its partially purified precursor (p72), however, preliminary experiments reveal many similarities between the two enzymes. For example, both kinases strongly prefer Mn$\sp {+2}$ over Mg$\sp {+2}$ in in vitro autophosphorylation reactions, show an enhanced ability to phosphorylate angiotensin I in the presence of high concentrations of NaCl and exhibit the same preference for substrates, with the cytoplasmic domain of erythrocyte band 3 serving as the best substrate for both kinases.

Degree

Ph.D.

Advisors

Geahlen, Purdue University.

Subject Area

Biochemistry

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