Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane Anchorage region.
The membrane-bound form of keratinocyte transglutaminase was found to be labeled by addition of [3H] acetic, [3H]myristic, or [3H]palmitic acids to the culture medium of human epidermal cells. Acid methanolysis and high performance liquid chromatography analysis of palmitate-labeled transglutaminase yielded only methyl palmitate. In contrast, analysis of the myristate-labeled protein yielded approximately 40% methyl myristate and 60% methyl palmitate. Incorporation of neither label was significantly affected by cycloheximide inhibition of protein synthesis. The importance of the fatty acid moiety for membrane anchorage was demonstrated in three ways. First, the enzyme was solubilized from the particulate fraction of cell extracts by treatment with neutral 1 M hydroxylamine, which was sufficient to release the fatty acid label. Second, solubilization of active enzyme from the particulate fraction upon mild trypsin treatment resulted in a reduction in size by approximately 10 kDa and removal of the fatty acid radiolabels. Third, the small fraction of soluble transglutaminase in cell extracts was found almost completely to lack fatty acid labeling. Keratinocyte transglutaminase translated from poly(A+) RNA in a reticulocyte cell-free system was indistinguishable in size from the native enzyme, suggesting anchorage requires only minor post-translational processing. Thus, the data are highly compatible with membrane anchorage by means of fatty acid acylation within 10 kDa of the NH2 or COOH terminus.
Chakravarty
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Charles A. Dana Laboratory of Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115.
Rice
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RH
D000085:Acetates / Q000378:metabolism; D019342:Acetic Acid; D000215:Acylation; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D002478:Cells, Cultured; D004817:Epidermis / Q000201:enzymology*; D006801:Humans; D007633:Keratins; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011503:Transglutaminases / Q000235:genetics* / Q000378:metabolism; D014316:Tritium
D000085:Acetates; D009227:Myristic Acids; D010169:Palmitic Acids; D019814:Myristic Acid; D014316:Tritium; D019308:Palmitic Acid; D007633:Keratins; D011503:Transglutaminases; D019342:Acetic Acid