Resolution of the phosphoinositide-specific phospholipase C isolated from porcine lymphocytes into multiple species. Partial purification of two isoenzymes.
Phospholipase C isolated from porcine mesenteric lymph node lymphocytes was distributed between the soluble and particulate fractions. Enzyme activity was found predominantly in the soluble fraction with optimal activity at pH 5.5. Gel filtration chromatography of the soluble phospholipase C revealed that it was composed of multiple species of enzyme activity. The activity associated with the particulate fraction had optimal activity at pH 7.0, as also did one of the species of soluble phospholipase C. Cellulose phosphate chromatography resolved the major soluble form into two species designated PLC-A and PLC-B. Both phenyl-Sepharose chromatography and hydroxyapatite chromatography purified these species still further. PLC-A and PLC-B demonstrated similar activities against phosphatidylinositol with a pH optimum near 5.5. The phospholipase C activities were abolished against this substrate by the addition of 1 mM-EDTA. When assayed in the presence of Ca2+-EDTA buffers providing a range of Ca2+ free concentrations, both enzymes exhibited optimal activity near 10(-3) M free Ca2+, but PLC-B was inhibited above this concentration more than PLC-A. PLC-B exhibited markedly lower activity against phosphatidylinositol 4,5-bisphosphate, suspended as liposomes of the pure phospholipid, than did PLC-A.
Carter
H R
HR
Department of Chemical Pathology, Middlesex Hospital Medical School, London, U.K.
Smith
A D
AD
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008214:Lymphocytes / Q000201:enzymology*; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism; D013552:Swine; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D004492:Edetic Acid; D010738:Type C Phospholipases