Abstract
Two different forms of inositol phospholipid-specific phospholipase C (PLC) have been purified 2810- and 4010-fold, respectively, from a crude extract of rat brain. The purification procedures consisted of chromatography of both enzymes on Affi-Gel blue and cellulose phosphate, followed by three sequential high performance liquid chromatography steps, which were different for the two enzymes. The resultant preparations each contained homogeneous enzyme with a Mr of 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these enzymes (PLC-II) was found to hydrolyze phosphatidyl-inositol 4,5-bisphosphate (PIP2) at a rate of 15.3 mumol/min/mg of protein and also phosphatidylinositol 4-monophosphate and phosphatidylinositol (PI) at slower rates. For hydrolysis of PI, this enzyme was activated by an acidic pH and a high concentration of Ca2+ and showed a Vmax value of 19.2 mumol/min/mg of protein. The other enzyme (PLC-III) catalyzed hydrolysis of PIP2 preferentially at a Vmax rate of 12.9 mumol/min/mg of protein and catalyzed that of phosphatidylinositol 4-monophosphate slightly. The rate of PIP2 hydrolysis by this enzyme exceeded that of PI under all conditions tested. Neither of these enzymes had any activity on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid. These two enzymes showed not only biochemical but also structural differences. Western blotting showed that antibodies directed against PLC-II did not react with PLC-III. Furthermore, the two enzymes gave different peptide maps after digestion with alpha-chymotrypsin or Staphylococcus aureus V8 protease. These results suggest that these two forms of PLC belong to different families of PLC.
Highlights
These findings may support the idea that phatidylserine, or phosphatidicacid.Thesetwoeneach phospholipase C (PLC) enzyme within a cell is stimulated in a different zymes showed biochemical but alsstoructural manner and mediates a different physiological pathway for differences
Purification of the Two Forms of PLC-Over 70% of the total PLC activitywas extracted in thesoluble fraction of rat brain, and treatmenwt ith detergent caused marked reduction in the activity
When all chromatography fractions were assayedfor PIPz hydrolysis, at least three peaksof PLC were separated during thepurification procedure
Summary
Step 4 for PLC-ZIZ: Hydroxylapatite (HCA) HPLC-Pooled fractions of PLC-I11 from cellulose phosphate were dialyzed against the column buffer consisting of10 mM sodium phosphate (pH 7.2), 0.1 mM PMSF, 0.1 mM DFP, and 10% (v/v) glycerol. PLC activity in each fraction was determined using 500 p~ PIP, as a substrate as described under “Experimental Procedures.”
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
