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Characterization of phosphatidylinositol-specific phospholipase C defects associated with thrombin-induced
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Insights
A Chinese hamster lung fibroblast mutant (D1-6b) shows diminished phospholipase C (PLC) activity due to loss of PLC-delta and compromised PLC-gamma, impacting inositol phosphate production and thrombin-induced mitogenesis.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Previous work identified a Chinese hamster lung fibroblast mutant (D1-6b) with defects in thrombin-induced mitogenesis.
- This mutant exhibits reduced inositol phosphate production and decreased phospholipase C (PLC) activity.
Purpose of the Study:
- To further characterize the phospholipase C (PLC) activities in wild-type CCL39 and D1-6b cytosolic extracts.
- To identify the specific PLC isoenzymes affected in the D1-6b mutant.
Main Methods:
- Anion exchange chromatography to separate PLC isoenzymes.
- Thermal inactivation studies to differentiate isoenzyme behavior.
- Immunoblotting using antibodies against bovine brain PLC isoenzymes (PLC-delta and PLC-gamma).
- Enzyme activity assays using mixed micelles and phospholipid vesicles.
Main Results:
- Wild-type CCL39 cytosol contains at least two phosphatidylinositol-specific PLC isoenzymes, identified as PLC-delta and PLC-gamma.
- Mutant D1-6b cytosol lacks detectable PLC-delta activity.
- Mutant PLC-gamma exhibits altered activity, being functional in mixed micelles but not phospholipid vesicles, unlike wild-type PLC-gamma.
- Mutant PLC-gamma shows similar elution and thermal inactivation kinetics to wild-type PLC-gamma.
Conclusions:
- The D1-6b mutant's diminished phosphatidylinositol-specific phospholipase C activity results from both the absence of PLC-delta and a functional defect in PLC-gamma.
- These PLC defects contribute to the previously observed reduction in inositol phosphate production and impaired thrombin-induced mitogenesis in the D1-6b mutant.
Abstract:
In a previous paper (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390), we reported a selection for the isolation of Chinese hamster lung fibroblasts (CCL39) defective in thrombin-induced mitogenesis. One mutant, D1-6b, had decreased production of inositol phosphates when challenged with activators of phosphatidylinositol turnover and extracts of this mutant showed a marked decrease in phospholipase C (PLC) activity toward phosphatidylinositol. In the current studies, the PLC activities of wild type CCL39 and D1-6b cytosolic extracts are further characterized. Wild type cytosol had at least two phosphatidylinositol-specific PLC isoenzymes, which could be separated by anion exchange chromatography and behaved differently in thermal inactivation studies. Since gel filtration of PLC activity in wild type extracts gave Mr values similar to that of previously characterized PLCs (140,000-200,000), immunoblots with antibodies to bovine brain isoenzymes were used to show that the PLC activities obtained by anion exchange chromatography were PLC-delta and PLC-gamma. Immunoblots with mutant D1-6b cytosol confirmed the presence of the PLC-gamma but showed no detectable PLC-delta. This activity in the mutant extracts eluted at the same conductivity on anion exchange columns and had the same kinetics of thermal inactivation as the PLC-gamma found in the wild type extracts. PLC-gamma from mutant extracts was active in assays containing phospholipid detergent mixed micelles but not in assays utilizing phospholipid vesicles, in sharp contrast to PLC-gamma from CCL39 extracts, which was active under either condition. Thus, the phosphatidylinositol-specific phospholipase C activity of mutant D1-6b is diminished both by the loss of PLC-delta and by the compromised behavior of PLC-gamma.