An extracellular inositol phospholipid-specific phospholipase C is released by cultured Swiss 3T3 cells.
A phosphatidylinositol-cleaving phospholipase C (PI-PLC) activity is released into the extracellular environment by intact Swiss 3T3 cell cultures. The activity is found in both serum-containing and serum-free defined culture medium. The cells remain attached and intact by Trypan Blue exclusion and lactate dehydrogenase assays. The activity is specific for phosphoinositides as no cleavage of phosphatidylcholine is observed. The activity is a phospholipase C rather than D since the water soluble products formed from cleavage of [3H]phosphatidylinositol were inositol phosphates and not inositol. Analysis of the inositol phosphate products showed a variation in composition with the pH of the assay, the ratio of noncyclic:cyclic forms being 60:40 at pH 7.5 and 40:60 at pH 5.5. This external phospholipase C resembles the well-characterized intracellular isozymes in that it is calcium-dependent and has a pH optimum between 5 and 6. From membrane filter assays the molecular weight of the native enzyme is estimated to be between 50 and 100 kDa.
Birrell
G B
GB
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.
Hedberg
K K
KK
Griffith
O H
OH
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D005110:Extracellular Space; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*
D003470:Culture Media; D007295:Inositol Phosphates; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium