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Protein N-myristoylation

Thesis/Dissertation ·
OSTI ID:5674280
The covalent attachment of fatty acids to eukaryotic cellular proteins occurs via one of three linkages - amide linkage to amino-terminal glycine; ester linkage to cysteine, serine, or threonine; and carboxy terminal addition of glycophospholipid. This work investigates the fatty acid specificity of protein acylation and the enzymology of one type of acylation, protein N-myristoylation. Metabolic labelling studies of culture mammalian cells with {sup 3}H fatty acid or {sup 3}H-acetate indicated that myristate and palmitate become attached to different subsets of cellular proteins. Myrstate is bound via an amide bond, while palmitate is primarily esterified to protein. In one cell line, at least 70% of the myristate bound to protein in amide linkage was accounted for as N-myristoylglycine. Specific yeast cellular proteins were also shown to contain N-myristoyglycine. An assay was developed for detecting myristoyl CoA:protein N-myristoyl transferase (NMT) activity. This assay measures the enzymatic transfer of {sup 3}H-myristate from myristoyl CoA to the aminoterminal glycine of a synthetic octapeptide whose sequence was derived from the known N-myristoylprotein, bovine cAMP-dependent protein kinase. The radioactive myristoylpeptide was identified and quantified by reverse phase HPLC and liquid scintillation counting. Using the assay, NMT has been purified to apparent homogeneity from yeast. The enzyme is a 55 kDa monomer which exhibits high specificity for myristoyl CoA as the acyl donor. The primary structural requirements for substrate recognition by NMT have been examined by testing as substrates or inhibitors a series of synthetic peptides whose sequences have been systematically altered from those present at the amino-termini of known N-myristoyl proteins.
Research Organization:
Washington Univ., Seattle, WA (USA)
OSTI ID:
5674280
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETATES
ACYLATION
AMIDES
ANIMAL CELLS
ANIMALS
BIOLOGICAL PATHWAYS
CARBOXYLIC ACID SALTS
CARBOXYLIC ACIDS
CHEMICAL BONDS
CHEMICAL REACTIONS
CHROMATOGRAPHY
COUNTING TECHNIQUES
ENZYME ACTIVITY
ENZYMES
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PROTEINS
RADIOASSAY
SCINTILLATION COUNTING
SEPARATION PROCESSES
SPECIFICITY
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
VERTEBRATES