Rat brain inositol 1,4,5-trisphosphate 3-kinase. Ca2(+)-sensitivity, purification and antibody production.
Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase was purified from rat brain by Blue-Sepharose, phosphocellulose and calmodulin (CaM)-Sepharose affinity chromatography. The purified enzyme was stimulated by Ca2+/CaM by 3-6-fold as compared with the activity measured in the presence of EGTA. Rat brain InsP3 3-kinase activity was associated with two silver-stained bands of about equal activity which migrated with an apparent Mr of 50,000 on SDS/polyacrylamide gels. InsP3 3-kinase activity from rat brain could be immunoprecipitated by an antiserum against the SDS/PAGE-purified 50,000-Mr protein doublet. InsP3 kinase activity from bovine brain and the InsP3 5-phosphatase activity from rat brain were not immunoprecipitated. On Western blot, the human brain crude InsP3 3-kinase reacted specifically, but less strongly than the rat brain enzyme, with the antiserum.
Institute of Interdisciplinary Research, School of Medicine, Free University of Brussels, Campus Erasme, Belgium.
K
K
Takazawa
M
M
Lemos
A
A
Delvaux
C
C
Lejeune
J E
JE
Dumont
C
C
Erneux
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007163:Immunosorbent Techniques; D007700:Kinetics; D008970:Molecular Weight; D010770:Phosphotransferases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats
D002147:Calmodulin; D000939:Epitopes; D007106:Immune Sera; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D002118:Calcium