Tyrosine protein kinases in membrane fractions from rat cerebral cortex.
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 Km values 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Tsukamoto
H
H
Department of Biochemistry, Hiroshima University School of Medicine.
Azuma
K
K
Miyauchi
T
T
Usui
H
H
Takeda
M
M
D000818:Animals; D015153:Blotting, Western; D002540:Cerebral Cortex / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000276:immunology / Q000302:isolation & purification; D016392:Proto-Oncogene Proteins pp60(c-src); D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)