Antibodies against the PH domain of phospholipase C-delta1 inhibit Ins(1,4,5)P3-mediated Ca2+ release from the endoplasmic reticulum.
The pleckstrin homology domain (PH domain) is now well known as a structural module for the binding of inositol compounds. In the present study, polyclonal antibodies against the peptide KVKSSSWRRERFYK, derived from the N-terminal of the PH domain of phospholipase C-delta1 (PLC-delta1), were raised in rabbits. These were then tested for their ability to inhibit the binding of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to the binding proteins including the receptor molecule. The Fab fragment of the antibodies but not the whole molecule inhibited the binding of Ins(1,4,5)P3 not only to PLC-delta1 but also to the Ins(1,4,5)P3 receptor, indicating that the antibodies raised recognized the binding site for Ins(1,4, 5)P3 in the receptor. Rat basophilic leukemic cells were permeabilized with saponin and assayed for Ins(1,4,5)P3-mediated Ca2+ release. Pretreatment of permeabilized RBL cells with the Fab fragment of the antibodies diminished the release of Ca2+ caused by Ins(1,4,5)P3, and further absorption experiments using a variety of synthetic peptides suggested that the tripeptide KVK is the epitope of the antibodies. Structural information about KVK will help in screening for Ins(1,4,5)P3 antagonists.
Matsuki
N
N
Department of Biochemistry, Faculty of Dentistry, Fukuoka, 812-8582, Japan.
Tateishi
K
K
Takeuchi
H
H
Yagisawa
H
H
Kanematsu
T
T
Oishi
M
M
Hirata
M
M
D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D000915:Antibody Affinity; D002118:Calcium / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D011817:Rabbits; D051381:Rats; D013997:Time Factors; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000276:immunology*
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D002118:Calcium