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 (/sup 3/H) acetic, (/sup 3/H)myristic, or (/sup 3/H)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 NH/sub 2/ or COOH terminus.
- Research Organization:
- Harvard School of Public Health, Boston, MA (USA)
- OSTI ID:
- 6569533
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 264:1
- Country of Publication:
- United States
- Language:
- English
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AMIDASES
ACYLATION
CELL MEMBRANES
BIOCHEMISTRY
ANIMAL CELLS
EPIDERMIS
HEXADECANOIC ACID
KERATIN
LIQUID COLUMN CHROMATOGRAPHY
MAN
TETRADECANOIC ACID
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ANIMAL TISSUES
ANIMALS
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHEMISTRY
CHROMATOGRAPHY
ENZYMES
EPITHELIUM
HYDROLASES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
MEMBRANES
MONOCARBOXYLIC ACIDS
NON-PEPTIDE C-N HYDROLASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PRIMATES
PROTEINS
SCLEROPROTEINS
SEPARATION PROCESSES
SKIN
TISSUES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques