Formation of a Phorbol Ester-Binding Fragment from Protein Kinase C by Proteolytic Digestion

Masahiko Hoshijima, Akira Kikuchi, Tetsuji Tanimoto, Kozo Kaibuchi, Yoshimi Takai

研究成果: Article査読

40 被引用数 (Scopus)

抄録

When washed human platelets were disrupted by sonication in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N‘,N’-tetra-acedc acid, both the catalytic and [3H]phorbol-l 2,13-dibutyrate (PDBu)-binding activities of protein kinase C were recovered in the soluble fraction and were not separable from each other upon several column chromatographies. Platelet protein kinase C required diacylglycerol, Ca2+, and phospholipid for its activation and showed a molecular weight of about 87,000 as estimated by gel filtration analysis. However, when platelets were first incubated with 2 μM Ca2+-ionophore A23187 for 5 min at 37°C in the medium containing 3 mM CaCl2 and then disrupted under the same conditions, the catalytic and [3H]phorbol-l 2,13-dibutyrate-binding activities were separately recovered in the soluble and particulate fractions, respectively; moreover, the catalytic activity recovered in the soluble fraction became independent of diacylglycerol, Ca2+, and phospholipid, and showed a molecular weight of about 50,000 as estimated by gel filtration analysis. The kinetic properties of this Mr 50,000 enzyme were similar to those of the catalytic fragment of rat brain protein kinase C described previously. In a cell-free system, digestion with trypsin of protein kinase C highly purified from rat brain caused the generation of a fragment which had no catalytic activity but showed full [3H]phorbol-12,13-dibutyrate-binding activity. The molecular weight of this fragment was estimated to be about 35,000 by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. These results indicate that protein kinase C consists of at least two functionally different domains, a hydrophobic phorbol ester- or diacylglycerol-binding and hydrophilic catalytic domains.

本文言語English
ページ(範囲)3000-3004
ページ数5
ジャーナルCancer Research
46
6
出版ステータスPublished - 01-06-1986
外部発表はい

All Science Journal Classification (ASJC) codes

  • 腫瘍学
  • 癌研究

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