Altered patterns of protein phosphorylation and synthesis caused by methyl mercury in cerebellar granule cell culture.
In the preceding report we demonstrated a dose-dependent increase in 32P-phosphoprotein labeling after 24-h exposure of cultured cerebellar granule neurons to methyl mercury (MeHg), a response that was not observed in glial cultures. In the present study we have examined 32P-labeled phosphoproteins by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. At concentrations of 0.5 and 1 microM, which were not extensively cytotoxic, MeHg enhanced phosphorylation of numerous acidic proteins, particularly a cluster of proteins with Mr approximately 28,000 and pI approximately 5.7-5.9 (pp 28/5.7-5.9) and a protein with Mr approximately 58,000 and pI approximately 5.6. The pp28 cluster displayed considerable two-dimensional pattern variability from one experiment to the next, suggesting susceptibility to subtle structural modifications. Time course studies revealed that increased 32P phospholabeling of pp28/5.7-5.9 was detectable after 12-h exposure to 3 microM MeHg and reached values of 300-500% of control by 24 h. These studies also showed that among the 21 proteins analyzed by two-dimensional densitometry, 32P phospholabeling of four proteins increased by 20-50% and of two proteins decreased by 20-50% after 24-h treatment. However, exposure to 10 microM MeHg produced stimulation of pp28/5.7-5.9 32P phospholabeling within 2 h. Under these conditions a relatively high stimulation (sevenfold) of pp28/5.7 phospholabeling occurred, while pp28/5.9 32P phospholabeling was only moderately (5-20%) enhanced. 35S and 32P double-label analysis of cells treated with 0, 0.5, and 1 microM MeHg indicated specific stimulation of 32P phospholabeling of these proteins without increased polypeptide synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Sarafian
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Department of Pathology (Neuropathology), UCLA School of Medicine 90024-173216.
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D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology* / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D007700:Kinetics; D008767:Methylmercury Compounds / Q000494:pharmacology*; D008970:Molecular Weight; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D014176:Protein Biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains
D008767:Methylmercury Compounds; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes