1988 Volume 104 Issue 5 Pages 807-816
Specific activities of tyrosine tubulin kinase in the particulate fractions from ratcerebel-lum, medulla oblongata, hypothalamus, striatum, midbrain, and cerebralcortex ranged within 30% of each other and more than 3 times higher than those in the solublefractions. In the cerebral cortex, tyrosine protein kinase activity toward tubulin and tyrosine-glutamate (1 : 4) copolymers was mainly distributed in the plasma membrane and the microsome fractions. The kinase activity in cerebral cortex particulate fractions was quantitatively solubilized and separated into two peaks, kinase I and kinase II, by Sephacryl S-300 gel filtration in the presence of 0.2% Nonidet P-40 and 0.2 M NaCl. Kinases I and II were each resolved into 5 active peaks (I-1→5 and II-1→5) by casein-Sepharose column chromatography. The molecular weights of these kinases were estimated from the s20.w values to be 59, 000-65, 000. The Km values of II-1→5. for tubulin were nearly 10 times higher than those of 1-1 However, the Km values of the two groups of kinases for tyrosine-glutamate copolymers were not so significantly different. About 60% of the copolymers kinase activity in 1-3, 1-4, 11-3, and 11-4 was immunoprecipitable with a saturating amount of monoclonal antibody against pp60c-src. Incubation of the immuno-precipitates with ATP resulted in the autophosphorylation of a 60kDa protein in 1-3 and 1-4, and a 52 kDa protein in 11-3 and 11-4. Immunoblotting also indicated 1-3 and 1-4 as 60 kDa bands and 11-3 and 11-4 as 52 kDa bands on SDS-polyacrylamide gels. The relative specific activities of 1-3, 1-4, 11-3, and 11-4 were 1 : 1 : 6 : 3 with tubulin and 1 : 1 : 9 : 7 with tyrosine-glutamate copolymers. V8 peptide maps of 32P-labeled 1-3, 1-4, 11-3, and 11-4 revealed that 1-3 and 1-4 were pp60c-src and the neuronal variant, pp60s-src, respectively, and suggested that 11-3 and 11-4 could be derived from pp60s-src and pp60s-src, respectively, hv removing the 8 kDa amino termini.
