Deletion and site-directed mutagenesis of EF-hand domain of phospholipase C-delta 1: effects on its activity.
In order to elucidate a role of a putative EF-hand motif (144-172) in phospholipase C-delta 1 (PLC-delta 1), deletion and point mutation of the enzyme were performed and the mutated cDNAs were expressed in CHO cells and E. coli AD202 strain. Deletion of amino acid residues of 141-236 or 173-236 resulted in abolition of PLC activity. However, the decreased PLC activity to 15-20% by deletion of the EF-hand motif (144-172) was still Ca(2+)-dependent. Furthermore, mutants, in which conserved Asp153, Asp157, Glu164 or all these acidic amino acids in the EF-hand motif were replaced with alanine residues, showed nearly the same PLC activity and Ca(2+)-dependency as those of wild-type. These results suggest that the region containing the EF-hand motif may not play a role in regulation of Ca(2+)-sensitivity of PLC-delta 1, but is important for its activity.
Nakashima
S
S
Department of Biochemistry, Gifu University School of Medicine, Japan.
Banno
Y
Y
Watanabe
T
T
Nakamura
Y
Y
Mizutani
T
T
Sakai
H
H
Zhao
Y
Y
Sugimoto
Y
Y
Nozawa
Y
Y
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli; D018698:Glutamic Acid; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D000409:Alanine