The phosphoinositide pathway of lymphoid cells: labeling after permeabilization by alveolysin, a bacterial sulfhydryl-activated cytolysin.
An improved method allowing incorporation of [3H]myo-inositol into the phosphoinositide pool of human lymphoid cells is described. The procedure devised involves cell permeabilization with a thiol-activated membranolytic toxin, alveolysin, and optimization of the phosphoinositide labeling and extraction. In these conditions 4 to 10% of the added [3H]myo-inositol is found intracellularly and half of this amount (2-5%) is incorporated into the phosphoinositide pool in only 1 h as compared to the classical 0.2 to 0.3% incorporation obtained after 10 to 20 h. The integrity of coupling between receptors and phospholipase C was assessed by the inositol phosphate production after cell stimulation by various agonists.
Berthou
L
L
Formation de Recherche Associée Claude Bernard, Neurochimie des Communications Cellulaires et Service de Biochimie, Hôpital Saint-Louis, Paris/France.
Corvaia
N
N
Geoffroy
C
C
Mutel
V
V
Launay
J M
JM
Alouf
J E
JE
D002051:Burkitt Lymphoma; D006460:Hemolysin Proteins / Q000494:pharmacology*; D006801:Humans; D007294:Inositol / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism*; D009930:Organic Chemicals; D015398:Signal Transduction; D014407:Tumor Cells, Cultured / Q000187:drug effects
D006460:Hemolysin Proteins; D007295:Inositol Phosphates; D009930:Organic Chemicals; C002647:inositol 1-phosphate; C044259:inositol 1,4-bis(phosphate); D007294:Inositol; C014963:alveolysin; D015544:Inositol 1,4,5-Trisphosphate