Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. II. Proenzyme and its activation by calcium-dependent protease from rat brain

Inoue, M.; Kishimoto, A.; Takai, Y.; Nishizuka, Y.

Journal of Biological Chemistry 252(21): 7610-7616

1977


ISSN/ISBN: 0021-9258
PMID: 199594
Accession: 068523786

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Abstract
A proenzyme of the protein kinase was found in the soluble fraction of rat brain. Upon limited proteolysis by Cu-dependent protease occurring in the same tissue, the proenzyme was converted to an active protein kinase which could phosphorylate 5 species of histone fractions. Trypsin also catalyzed the conversion. The proenzyme and protease were separated by DEAE-cellulose (DE52) column chromatography. The proenzyme was resolved into 2 components; each was purified further by gel filtration followed by isoelectrofocusing electrophoresis. Both components showed a sedimentation coefficient of about 5.1 with a MW of 7.7 .times. 104 and a Stokes radius of about 42 .ANG. Upon isoelectrofocusing electrophoresis the proenzyme exhibited heterogeneity with isoelectric points of 4.0-5.6. The proenzyme showed no glycogen phosphorylase kinase activity but was always associated with activity to phosphorylate protamine. Ca2+-dependent protease was also purified further by gel filtration followed by DE52 column chromatography. The protease showed a sedimentation coefficient of about 5.8 with a MW of 9.3 .times. 104, a Stokes radius of about 47 .ANG., and an isoelectric point of 4.8. The protease required a divalent cation almost absolutely; Ca2+ was most active and the maximum activity was obtained at 3 mM. Sr2+ and Mn2+ were 24% and 11% as active as Ca2+ respectively, but other cations including Mg2+, Ba2+, Zn2+ and Cu2+ were inactive. The optimum pH of the protease was 7.5-8.5. The active protein kinase thus produced from the proenzyme in vitro showed a sedimentation coefficient of about 3.9 with a MW of 5.1 .times. 104 and a Stokes radius of about 38 .ANG. Although these values were slightly different from those of the protein kinase which was obtained from bovine cerebellum stored frozen, the active protein kinases were indistinguishable from each other in their kinetic and catalytic properties. A preliminary survey revealed that such proenzyme and protease were distributed in many other tissues including lung, liver, kidney, cerebellum, heart, skeletal muscle and adipose tissue.