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J. Biochem, 1988, Vol. 104, No. 5 807-816
© 1988 Japanese Biochemical Society

research-article

Tyrosine Protein Kinases in Membrane Fractions from Rat Cerebral Cortex1

Haruhisa Tsukamoto, Kazuyoshi Azuma, Takahiro Miyauchi, Hirofumi Usui and Masao Takeda2

The Department of Biochemistry, Hiroshima University School of Medicine Minami-ku, Hiroshima, Hiroshima 734

2 To whom correspondence should be addressed

Specific activities of tyrosine tubulin kinase in the particulate fractions from rat cerebellum, medulla oblongata, hypothalamus, striatum, midbrain, and cerebral cortex ranged within 30% of each other and more than 3 times higher than those in the soluble fractions. 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 Kmvalues of II-1->5 for tubulin were nearly 10 times higher than those of I-1->5 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 I-3, I-4, II-3, and II-4 was immunoprecipitable with a saturating amount of monoclonal antibody against pp60c–src. Incubation of the immunoprecipitates with ATP resulted in the autophosphorylation of a 60 kDa protein in I-3 and I-4, and a 52 kDa protein in II-3 and II-4. Immunoblotting also indicated I-3 and I-4 as 60 kDa bands and II-3 and II-4 as 52 kDa bands on SDS-polyacrylamide gels. The relative specific activities of I-3, I-4, II-3, and II-4 were 1:1:6:3 with tubulin and 1:1:9:7 with tyrosine-glutamate copolymers. V8 peptide maps of 32P-labeled I-3, I-4, II-3, and II-4 revealed that I-3 and I-4 were pp60c-src and the neuronal variant, pp60c–src+ respectively, and suggested that II-3 and II-4 could be derived from pp60c–src and pp60c–src+, respectively, by removing the 8 kDa amino termini.

1 This work was supported in part by Grants-in-Aid for Cancer Research (1984–1986) and Scientific Research (1984–1987) from the Ministry of Education, Science and Culture of Japan.


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