Isolation and characterization of thelck protein-tyrosine kinase from bovine thymus
Abstract
Soluble extracts prepared from bovine thymus contain an NaCl-stimulated protein-tyrosine kinase activity when measured by the phosphorylation of angiotensin I. This activity is found at much higher levels in extracts from bovine thymus as compared to bovine spleen. The enzyme responsible for the major thymic NaCl-activated tyrosine kinase activity has been partially purified by sequential chromatography on DEAE-cellulose, heparin-agarose, butyl-agarose, protamine-agarose, and Sephacryl S200. The isolated enzyme was identified as p56$\sp{lck}$, a major T lymphocyte protein-tyrosine kinase, on the basis of covalent modification with 5$\sp\prime$-fluorosulfonylbenzoyladenosine (FSBA) and reactivity with anti-FSBA antibodies, reactivity with polyclonal anti-peptide antibodies against p56$\sp{lck}$, and peptide mapping. The phosphorylation of angiotensin I by the partially purified thymus p56$\sp{lck}$ is weakly stimulated by polyionic compounds such as heparin and polylysine, but is strongly activated by high concentrations of NaCl. The dilute enzyme present in fractions eluting from chromatographic columns fails to catalyze an autophosphorylation reaction. Autophosphorylation can be detected in more concentrated enzyme samples and is readily observed in immune-complex assays. The partially purified enzyme exhibits a very restricted substrate specificity in vitro. It is unable to phosphorylate the cytoplasmic domain of human erythrocyte band 3, casein or histones, but is able to efficiently phosphorylate acid-treated enolase and bovine myelin basic protein (MBP). The phosphorylation of MBP by bovine thymus p56$\sp{lck}$ has an apparent Km of 75 $\mu$M. Phosphoamino acid analysis indicates that thymus p56$\sp{lck}$ specifically phosphorylates MBP on tyrosine. When phosphorylated MBP is digested with proteases including trypsin and endoproteinase Lys-C, only one major phosphopeptide is identified. Results obtained from both peptide mapping and fast atom bombardment mass spectrometry indicate that tyrosine 67, in the sequence -Thr-Thr-His-Tyr$\sp{67}$-Gly-Ser-Leu-Pro-Gln-Lys- in bovine MBP, is the specific phosphorylation site for bovine thymus p56$\sp{lck}$.
Degree
Ph.D.
Advisors
Geahlen, Purdue University.
Subject Area
Biochemistry|Immunology
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