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Characterization of a rat brain phospholipase D isozyme
1Department of Molecular Physiology and Biophysics, and Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
The Journal of Biological Chemistry
|April 18, 1998
Summary
Researchers cloned rat brain phospholipase D1 (rPLD1), finding it activated by specific phospholipids and G proteins. Protein kinase C (PKC) phosphorylation inhibited rPLD1 activity, suggesting a regulatory mechanism for this key enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Phospholipase D (PLD) enzymes play crucial roles in cellular signaling pathways.
- Understanding the specific isoforms, such as rat brain PLD1 (rPLD1), is essential for elucidating their functions.
- Previous research has identified various activators and regulators of PLD activity.
Purpose of the Study:
- To clone, express, and characterize a rat brain phospholipase D isoform, designated rPLD1.
- To investigate the substrate specificity, cofactor requirements, and regulatory mechanisms of rPLD1.
- To explore the potential roles of G proteins and protein kinase C (PKC) in modulating rPLD1 activity.
Main Methods:
- Cloning of rat brain cDNA encoding rPLD1.
- Expression of histidine-tagged rPLD1 in insect (Sf9) cells and purification via Ni2+-agarose affinity chromatography.
- Enzymatic assays to determine substrate specificity, pH optimum, and activation/inhibition by various molecules (phospholipids, divalent cations, G proteins, PKC).
Main Results:
- rPLD1 showed equipotent activation by phosphatidylinositol 4,5-P2 and phosphatidylinositol 3,4,5-P3, with specificity for phosphatidylcholine.
- Enzyme activity was dependent on Mg2+ and Ca2+, with optimal activity at pH 6.5-7.5.
- rPLD1 was activated by ARF3 and RhoA, and by PKC-alpha and -betaII (but not other PKC isoforms), with phosphorylation by PKC leading to inhibition of catalytic activity.
Conclusions:
- rPLD1 is a glycosylated protein regulated by phosphorylation by PKC.
- PKC-mediated phosphorylation negatively regulates rPLD1 catalytic activity.
- These findings provide insights into the biochemical properties and regulatory network of rPLD1 in rat brain.