Purification and properties of membrane and cytosolic phosphatidylinositol-specific phospholipases C from human spleen.
Two phosphatidylinositol-specific phospholipases C (PI-PLC) have been purified from human spleen. PI-PLCm represents the main activity detected in the membrane, while PI-PLCc is the main activity present in the cytoplasm. PI-PLCm can be resolved into two peaks of activity of high Mr (60,000-70,000) and low Mr (16,000-18,000). High salt concentration ((NH4)2SO4, 2M) dissociates the high Mr form yielding the low molecular form and increasing the specific activity. The same effect of dissociation and potentiation of the activity is observed when membranes solubilized by n-octyl glucoside are subjected to the high voltage conditions of an isoelectric focusing run. The purified Pi-PLCm has a Mr of about 18,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration and a basic pI (9.0-9.2). Purified PI-PLCc has a Mr of 57,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration) and a slightly acid pI (6.2). Other characteristics of both enzymes, such as cations dependence, substrate specificity, optimum pH, and kinetic parameters, are also discussed.
Roy
G
G
Servicio de Inmunologia, Hospital Ramon y Cajal, Madrid, Spain.
Villar
L M
LM
Lazaro
I
I
Gonzalez
M
M
Bootello
A
A
Gonzalez-Porque
P
P
D002853:Chromatography, Liquid; D004587:Electrophoresis, Agar Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008670:Metals / Q000737:chemistry; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D013154:Spleen / Q000201:enzymology*
D008565:Membrane Proteins; D008670:Metals; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase