The effects of mastoparan on the carrot cell plasma membrane polyphosphoinositide phospholipase C.
When [3H]inositol-labeled carrot (Daucus carota L.) cells were treated with 10 or 25 microM wasp venom peptide mastoparan or the active analog Mas-7 there was a rapid loss of more than 70% of [3H]phosphatidylinositol-4-monophosphate (PIP) and [3H]phosphatidylinositol-4,5-bisphosphate (PIP2) and a 3- and 4-fold increase in [3H]inositol-1,4-P2 and [3H]inositol-1,4,5-P3, respectively. The identity of [3H]inositol-1,4,5-P3 was confirmed by phosphorylation with inositol-1,4,5-P3 3-kinase and co-migration with inositol-1,3,4,5-P4. The changes in phosphoinositides were evident within 1 min. The loss of [3H]PIP was evident only when cells were treated with the higher concentrations (10 and 25 microM) of mastoparan or Mas-7. At 1 microM Mas-7, [3H]PIP increased. The inactive mastoparan analog Mas-17 had little or no effect on [3H]PIP or [3H]PIP2 hydrolysis in vivo. Neomycin (100 microM) inhibited the uptake of Mas-7 and thereby inhibited the Mas-7-stimulated hydrolysis of [3H]PIP and [3H]PIP2. Plasma membranes isolated from mastoparan-treated cells had increased PIP-phospholipase C (PLC) activity. However, when Mas-7 was added to isolated plasma membranes from control cells, it had no effect on PIP-PLC activity at low concentrations and inhibited PIP-PLC at concentrations greater than 10 microM. In addition, guanosine-5'-O-(3-thiotriphosphate) had no effect on the PIP-PLC activity when added to plasma membranes isolated from either the Mas-7-treated or control cells. The fact that Mas-7 did not stimulate PIP-PLC activity in vitro indicated that the Mas-7-induced increase in PIP-PLC in vivo required a factor that was lost from the membrane during isolation.
Cho
M H
MH
Department of Botany, North Carolina State University, Raleigh 27695-7612.
Tan
Z
Z
Erneux
C
C
Shears
S B
SB
Boss
W F
WF
D000595:Amino Acid Sequence; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D018552:Daucus carota / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D007811:Lanthanum / Q000494:pharmacology; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D010455:Peptides; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000187:drug effects*; D014862:Wasp Venoms / Q000494:pharmacology*
D002121:Calcium Channel Blockers; D007295:Inositol Phosphates; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007811:Lanthanum; C037500:mastoparan; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D009355:Neomycin