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Properties of three distinct phosphatidylinositol-specific phospholipase C enzymes from bovine brain

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:7171338
; ;
The soluble proteins precipitated at pH 5.0 and the membrane bound proteins extracted with 2M KCl are chromatographed on a HPLC DEAE column. The phospholipase C (PLC) activities specific to phosphatidylinositol (PI) are monitored at pH 5.5 and 7.2 using sonicated (/sup 3/H)inositol-PI vesicles as the substrate. Elution with a KCl gradient yields two well separated peaks, S1 and S2 for the soluble proteins, and M1 and M2 for the membrane derived proteins. The peak S2 is followed by a poorly resolved minor peak. The ratio of the PI-PLC activity at pH 5.5 to that at pH 7.2 is 1.4 for both S1 and M1 which come out at the same retention time, 11 for S2, and 30 for M2. The effects of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which are included in the PI vesicle in a ratio of 1:1 are studied for each fraction. PE does not have much effect at pH 7.2. PC inhibits the activities of all fractions, but by different degrees. The activity of each fraction is also measured using three different vesicles prepared from (/sup 32/P)-PI, (/sup 3/H-Inositol)-PI-4,5-P/sub 2/ and a 1:1 mixture of both, respectively. All three fractions hydrolyze both PI and PIP/sub 2/. Their activities are variable depending on the preparation of substrates, pH and the availability of Ca/sup + +/. Generally, the fraction M2 is more specific to PIP/sub 2/. In the absence of Ca/sup + +/, S1 (M1) and S2 hydrolyze only PIP/sub 2/. M2 also hydrolyzes PIP/sub 2/ preferentially, but this membrane derived enzyme shows significant activity toward PI when present with PIP/sub 2/.
Research Organization:
NIH, Bethesda, MD
OSTI ID:
7171338
Report Number(s):
CONF-8606151-
Conference Information:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
BIOLOGICAL EFFECTS
BODY
BRAIN
CALCIUM COMPOUNDS
CARBOHYDRATES
CARBOXYLESTERASES
CATIONS
CATTLE
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
CHARGED PARTICLES
CHEMICAL PROPERTIES
CHROMATOGRAPHY
DOMESTIC ANIMALS
ENZYME ACTIVITY
ENZYMES
ESTERASES
ESTERS
HYDROLASES
INOSITOL
INOSITOLS
IONS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIPASE
LIPIDS
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
MEMBRANES
MONOSACCHARIDES
NERVOUS SYSTEM
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
ORGANS
PHOSPHOLIPIDS
RUMINANTS
SACCHARIDES
SEPARATION PROCESSES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES