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Title: Monoclonal antibodies against type II rat brain protein kinase

Abstract

Three monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C (PKC) were used to carry out the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II PKC in a dose-dependent manner but did neither to type I nor type III isozyme. Purified type II PKC has a molecular weight of 82,000 and consists of heterogeneous isoelectric point species, all of which are cross reactive with these antibodies. Immunoblot analysis of the tryptic fragments from PKC revealed that all three antibodies recognized the 33-38-KDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-KDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained the kinase activity which was dependent on Ca/sup 2 +/ and phosphatidylserine (PS) and further activated by diacylglycerol. With antibody 8/1, the apparent Km values of the kinase for Ca/sup 2 +/ and PS were not influenced. The initial rate and final extent of autophosphorylation were reduced. The concentration of PS required for half-maximal (/sup 3/H)phorbol 12,13-dibutyrate (PDBu) binding was increased and the total PDBu binding was reduced. In the presence of optimum concentrations of Ca/sup 2 +/ and PS, the Kd of PDBu was unaffectedmore » by the antibody but the total binding was reduced. These results demonstrate that the PS/PDBu-binding domain contains the major epitope for the antibodies and the antibody mainly influences the PS/PDBu binding to the kinase.« less

Authors:
;
Publication Date:
Research Org.:
National Institutes of Health, Bethesda, MD
OSTI Identifier:
6366411
Report Number(s):
CONF-870644-
Journal ID: CODEN: FEPRA; TRN: 87-034174
Resource Type:
Conference
Journal Name:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
Additional Journal Information:
Journal Volume: 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; 59 BASIC BIOLOGICAL SCIENCES; MONOCLONAL ANTIBODIES; IMMUNOASSAY; PHORBOL ESTERS; BIOLOGICAL EFFECTS; PHOSPHOTRANSFERASES; ENZYME ACTIVITY; BRAIN; CALCIUM COMPOUNDS; DOSE-RESPONSE RELATIONSHIPS; GLYCEROL; PHOSPHOLIPIDS; RATS; ALCOHOLS; ALKALINE EARTH METAL COMPOUNDS; ANIMALS; ANTIBODIES; BODY; CARCINOGENS; CENTRAL NERVOUS SYSTEM; ENZYMES; ESTERS; HYDROXY COMPOUNDS; LIPIDS; MAMMALS; NERVOUS SYSTEM; ORGANIC COMPOUNDS; ORGANIC PHOSPHORUS COMPOUNDS; ORGANS; PHOSPHORUS-GROUP TRANSFERASES; RODENTS; TRANSFERASES; VERTEBRATES; 560300* - Chemicals Metabolism & Toxicology; 550201 - Biochemistry- Tracer Techniques

Citation Formats

Nakabayashi, C H, and Huang, K P. Monoclonal antibodies against type II rat brain protein kinase. United States: N. p., 1987. Web.
Nakabayashi, C H, & Huang, K P. Monoclonal antibodies against type II rat brain protein kinase. United States.
Nakabayashi, C H, and Huang, K P. 1987. "Monoclonal antibodies against type II rat brain protein kinase". United States.
@article{osti_6366411,
title = {Monoclonal antibodies against type II rat brain protein kinase},
author = {Nakabayashi, C H and Huang, K P},
abstractNote = {Three monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C (PKC) were used to carry out the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II PKC in a dose-dependent manner but did neither to type I nor type III isozyme. Purified type II PKC has a molecular weight of 82,000 and consists of heterogeneous isoelectric point species, all of which are cross reactive with these antibodies. Immunoblot analysis of the tryptic fragments from PKC revealed that all three antibodies recognized the 33-38-KDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-KDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained the kinase activity which was dependent on Ca/sup 2 +/ and phosphatidylserine (PS) and further activated by diacylglycerol. With antibody 8/1, the apparent Km values of the kinase for Ca/sup 2 +/ and PS were not influenced. The initial rate and final extent of autophosphorylation were reduced. The concentration of PS required for half-maximal (/sup 3/H)phorbol 12,13-dibutyrate (PDBu) binding was increased and the total PDBu binding was reduced. In the presence of optimum concentrations of Ca/sup 2 +/ and PS, the Kd of PDBu was unaffected by the antibody but the total binding was reduced. These results demonstrate that the PS/PDBu-binding domain contains the major epitope for the antibodies and the antibody mainly influences the PS/PDBu binding to the kinase.},
doi = {},
url = {https://www.osti.gov/biblio/6366411}, journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 46:6,
place = {United States},
year = {Fri May 01 00:00:00 EDT 1987},
month = {Fri May 01 00:00:00 EDT 1987}
}

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