Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 625-629 |
| Number of pages | 5 |
| Journal | Journal of Biological Chemistry |
| Volume | 264 |
| Issue number | 1 |
| State | Published - 1989 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane anchorage region. / Chakravarty, R.; Rice, R. H.
In: Journal of Biological Chemistry, Vol. 264, No. 1, 1989, p. 625-629.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane anchorage region
AU - Chakravarty, R.
AU - Rice, R. H.
PY - 1989
Y1 - 1989
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=0024570089&partnerID=8YFLogxK
M3 - Article
C2 - 2462562
AN - SCOPUS:0024570089
VL - 264
SP - 625
EP - 629
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 1
ER -