Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Characterization of phosphatidylinositol-specific phospholipase C defects associated with thrombin-induced mitogenesis. 230 41

A mutant fibroblast, 2A4b, was isolated from the Chinese hamster lung cell line CCL39 by a previously described selection (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390) for cells deficient in thrombin-induced signaling. Although the antiporter activation by thrombin in 2A4b is only approximately 60% that in CCL39, the stimulation by serum is not significantly impaired, indicating that the defect in 2A4b lies upstream of the antiporter in the signaling pathway. The addition of thrombin to serum-starved 2A4b cells causes blunted responses both in production of inositol phosphates and in the cytosolic [Ca2+] transient, particularly when no Ca2+ is added to the external medium. The in vitro inositol phospholipid-specific phospholipase C (PLC) activity of 2A4b cytosol plus membrane extracts exceeds that in CCL39. However, immunoblots with antibodies to PLC isozymes show that although the levels of PLC-delta 1, PLC-gamma 1, and PLC-beta 3 are at least as great as those in CCL39, the amount of PLC-beta 1 in 2A4b is markedly deficient (< or = 10%). PLC-beta 1 is found primarily in the nucleus and in non-nuclear membranes of CCL39 and is proportionately low in these subcellular locations of 2A4b. Thrombin activation of phospholipases D and A2 is impaired in 2A4b. We postulate that the deficiency in PLC-beta 1 causes defective targeting of protein kinase C-alpha to specific membrane sites, which may be required for activation of these downstream phospholipases.
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PMID:A Chinese hamster fibroblast mutant defective in thrombin-induced signaling has a low level of phospholipase C-beta 1. 806 14