Improved purification and characterization of membraneous and cytosolic inositol phospholipid-specific phospholipases C from porcine brain cortex.
A phospholipase C was solubilized and purified from membranes of porcine brain cortex. Simultaneously, a phospholipase C was purified from a cytosolic fraction of porcine brain cortex. The enrichment of phospholipase C from either fraction was about 1000-fold as determined by hydrolysis of phosphatidylinositol 4,5-bisphosphate. Phospholipases C purified from membranes or from cytosol were indistinguishable with regard to the following properties: The enzyme activities copurified with a protein of 145 kDa. The standard sedimentation coefficients (s20,w values) of the purified enzymes were 6.2 S in the absence or presence of 0.3% (w/v) sodium cholate; Stokes' radii, estimated by gel filtration on a Superose 6 HR 10/30 column in the presence of 0.3% sodium cholate, were 4.5 nm; calculated molecular masses were about 120 kDa; no significant hydrolysis of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine by preparations of purified phospholipase C was observed; adenine and guanine nucleotides affected the activity of purified enzymes in a complex manner. Thus, the enzymes purified from membraneous and from cytosolic fractions exhibited properties of the phospholipase C-beta form. The enzymes purified from either fraction required Ca2+ at a low concentration (100 nM to 10 microM) for maximal activity. The advantage of the present purification procedure is that the purified enzymes were free of phosphatidylinositol 4,5-bisphosphate 5-phosphatase, inositol 1,4,5-trisphosphate 5-phosphatase and guanine nucleotide-binding proteins after three chromatographic steps. The purified enzymes may, therefore, prove useful for studying the hormonal regulation of phospholipase C in reconstituted systems and for the preparation of [5-32P]inositol 1,4,5-trisphosphate from [5-32P]phosphatidylinositol 4,5-bisphosphate.
Hoffmann
B
B
Institut für Pharmakologie, Freie Universität Berlin.
Seib
C
C
Höer
A
A
Höer
D
D
Oberdisse
E
E
Rosenthal
W
W
Schultz
G
G
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002540:Cerebral Cortex / Q000201:enzymology*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000032:analysis; D015151:Immunoblotting; D009711:Nucleotides / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D010738:Type C Phospholipases / Q000302:isolation & purification* / Q000378:metabolism
D009711:Nucleotides; D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins