Formation of a phorbol ester-binding fragment from protein kinase C by proteolytic digestion
When washed human platelets were disrupted by sonication in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, both the catalytic and (/sup 3/H)phorbol-12,13-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 microM Ca2+-ionophore A23187 for 5 min at 37 degrees C in the medium containing 3 mM CaCl/sub 2/ and then disrupted under the same conditions, the catalytic and (/sup 3/H)phorbol-12,13-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 (/sup 3/H)phorbol-12,13-dibutyrate-binding activity. The molecular weight of this fragment was estimated to be about 35,000 by sodium dodecyl sulfate-polyacrylamide 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.
- Research Organization:
- Kobe Univ. School of Medicine, Japan
- OSTI ID:
- 5563128
- Journal Information:
- Cancer Res.; (United States), Vol. 6
- Country of Publication:
- United States
- Language:
- English
Similar Records
Immunochemical characterization of rat brain protein kinase
Agonist-induced ADP-ribosylation of a cytosolic protein in human platelets
Related Subjects
PEPTIDE HYDROLASES
ENZYME ACTIVITY
PHORBOL ESTERS
BIOCHEMICAL REACTION KINETICS
BLOOD PLATELETS
CALCIUM
CHROMATOGRAPHY
FILTRATION
IN VITRO
MOLECULAR WEIGHT
PHOSPHOLIPIDS
PHOSPHOTRANSFERASES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ALKALINE EARTH METALS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARCINOGENS
ELEMENTS
ENZYMES
ESTERS
HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIPIDS
MATERIALS
METALS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
REACTION KINETICS
SEPARATION PROCESSES
TRANSFERASES
550201* - Biochemistry- Tracer Techniques