PMID:8390354
| Citation |
Erneux, C, Moreau, C, Vandermeers, A and Takazawa, K (1993) Interaction of calmodulin with a putative calmodulin-binding domain of inositol 1,4,5-triphosphate 3-kinase. Effects of synthetic peptides and site-directed mutagenesis of Trp165. Eur. J. Biochem. 214:497-501 |
|---|---|
| Abstract |
Recombinant rat brain inositol 1,4,5-triphosphate [Ins(1,4,5)P3] 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion product. It could be adsorbed onto calmodulin-Sepharose and eluted in Ca(2+)-free medium as a 48-kDa protein. Purification could be achieved in a single step. Molecular evidence for a calmodulin-binding domain on Ins(1,4,5)P3 3-kinase can be shown by the following approaches. (a) Inhibition of Ca2+/calmodulin stimulation by a synthetic peptide based on a candidate calmodulin-binding domain. The inhibition was mimicked by a well-characterized peptide derived from the sequence of smooth muscle myosin light-chain kinase calmodulin-binding site. (b) The construction of two mutants by site-directed mutagenesis of Trp165 to Gly or Arg. Both mutants displayed kinase activity but were no longer Ca2+/calmodulin sensitive, supporting, therefore, the role of Trp165 in calmodulin binding. |
| Links | |
| Keywords |
Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Brain/enzymology; Calcium/pharmacology; Calmodulin/metabolism; Calmodulin/pharmacology; Escherichia coli; Kinetics; Mutagenesis, Site-Directed; Phosphotransferases/chemistry; Phosphotransferases/genetics; Phosphotransferases/metabolism; Phosphotransferases (Alcohol Group Acceptor); Rats; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/metabolism; Tryptophan/genetics; beta-Galactosidase |
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