Metabolic Labeling with Fatty Acids

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Covalent attachment of radiolabeled fatty acids (e.g., [³ H]myristate or palmitate) is an alternative method for labeling proteins. This unit contains methods for biosynthetic labeling with fatty acids, analysis of the fatty acid linkage with protein, analysis of total protein?bound fatty acid level in cell extracts, and analysis of the identity of the bound fatty acid.

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Basic Protocol 1: Biosynthetic Labeling with Fatty Acids
Basic Protocol 2: Analysis of Fatty Acid Linkage to Protein
Basic Protocol 3: Analysis of Total Protein‐Bound Fatty Acid Label in Cell Extract
Basic Protocol 4: Analysis of Fatty Acid Label Identity
Reagents and Solutions
Commentary
Figures

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Materials

Basic Protocol 1: Biosynthetic Labeling with Fatty Acids

Materials

Cells for culture
Complete tissue culture medium appropriate for cells
Labeling medium: complete tissue culture medium containing the relevant dialyzed serum and 5 mM sodium pyruvate, 37°C
5 to 10 µCi/µl [9,10(n )‐³ H]fatty acid, e.g., [9,10(n ) ‐³ H]palmitic acid or [9,10(n )‐³ H]myristic acid (30 to 60 Ci/mmol; Amersham International, American Radiolabeled Chemicals, or NEN Research Products) in ethanol
PBS, pH 7.2 ( appendix 2A ), ice‐cold
1% (w/v) SDS or SDS sample buffer (for SDS‐PAGE, when using adherent or nonadherent cells respectively; unit 6.1 ) or RIPA lysis buffer (for immunoprecipitation; see recipe )
5× SDS sample buffer (see recipe )

Cell scrapers
Nitrogen gas

Additional reagents and equipment for culturing cells (unit 1.1 ), immunoprecipitation (unit 7.2 ), SDS‐PAGE (unit 6.1 ), treating a gel with sodium salicylate (unit 6.3 ), and fluorography (unit 6.3 )

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO₂ to maintain pH 7.4.

Basic Protocol 2: Analysis of Fatty Acid Linkage to Protein

Materials

Lysate or immunoprecipitate from [³ H]fatty acid–labeled cells (see protocol 1 , step )
0.2 M potassium hydroxide (KOH) in methanol
Methanol
1 M hydroxylamine⋅HCl, titrated to pH 7.5 with NaOH
1 M Tris⋅Cl, pH 7.5 ( appendix 2A )

Additional reagents and equipment for SDS‐PAGE (unit 6.1 ), treating a gel with sodium salicylate (unit 6.3 ), and fluorography (unit 7.2 )

Basic Protocol 3: Analysis of Total Protein‐Bound Fatty Acid Label in Cell Extract

Materials

0.1 M HCl/acetone, −20°C
Lysate from [³ H]fatty acid–labeled cells in 1% SDS (see protocol 1 , step or )
1% (w/v) SDS
2:1 (v/v) chloroform/methanol
Diethyl ether
6 M HCl (concentrated HCl diluted 1:1 with H₂ O)
Hexane
5 to 10 µCi/µl [9,10(n )‐³ H]fatty acid standards (30 to 60 Ci/mmol; Amersham International, American Radiolabeled Chemicals, or NEN Research Products) in ethanol
90:10 (v/v) acetonitrile/acetic acid
EN³ HANCE spray (NEN Research Products)

15‐ml polypropylene centrifuge tubes
Mistral 3000i benchtop centrifuge with swing‐out four‐bucket rotor or equivalent
Nitrogen gas
30‐ml thick‐walled Teflon container with an air‐tight screw top
110°C oven
Thin‐layer chromatography tank
RP18 thin‐layer chromatography plate (e.g., Merck)
Kodak XAR‐5 film, preflashed

Basic Protocol 4: Analysis of Fatty Acid Label Identity

Materials

SDS‐PAGE gel of lysate from [³ H]fatty acid–labeled cells
Additional reagents and equipment for analysis of protein‐bound label (see protocol 3 )

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Figures

Figure 7.4.1 Fluorogram of thin‐layer chromatography plate showing analysis of acylated nerve growth factor (NGF) receptor.Outside lanes, migration of 0.5 µCi [³ H]palmitate and [³ H]myristate standards. Lane 1, NGF receptor immunoprecipitated from cells labeled with [³ H]palmitic acid. Lane 2, NGF receptor immunoprecipitated from cells labeled with [³ H]myristic acid. Although the cells were labeled with different fatty acids, the protein was labeled with palmitic acid due to chain elongation of [³ H]myristic acid to [³ H]palmitic acid by the cells. Exposure for standards, 1 week; exposure for lanes 1 and 2, 1 month.

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Figure 7.4.2 Structures of myristic and palmitic acids.

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Videos

Literature Cited

Literature Cited
	Aitken, A., 1992. Structure determination of acylated proteins. In Lipid Modification of Proteins, A Practical Approach (N.M. Hooper and A.J. Turner, eds.) pp. 63‐88. Oxford University Press, Oxford.
	Camp, L.A. and Hoffmann, S.L. 1993. Purification and properties of a palmitoyl‐protein thioesterase that cleaves palmitate from H‐Ras. J. Biol. Chem. 268: 22566‐22574.
	DeGrella, R.F. and Light, R.J. 1980. Uptake and metabolism of fatty acids by dispersed adult rat heart myocytes. J. Biol. Chem. 255: 9739‐9745.
	Dunphy, J.T. and Linder, H.E. 1998. Signaling functions of protein palmitoylation. Biochim. Biophys. Acta 1436: 245‐261.
	French, S.A., Christakis, H., O'Neill, R.R., and Miller, S.P.F. 1994. An assay for myristoyl‐CoA:protein N‐myristoyltransferase activity based on ion‐exchange exclusion of [3H]myristoyl peptide . Anal. Biochem. 220: 115‐121.
	Glover, C.J., Goddard, C., and Felsted, R.L. 1988. N‐Myristoylation of p60src. Biochem. J. 250: 485‐491.
	Gordon, J.I., Duronio, R.J., Rudnick, D.A., Adams, S.P., and Gokel, G.W. 1991. Protein N‐myristoylation. J. Biol. Chem. 266: 8647‐8650.
	James, G. and Olson, E.N. 1989. Identification of a novel fatty acylated protein that partitions between the plasma membrane and cytosol and is deacylated in response to serum and growth factor stimulation. J. Biol. Chem. 264: 20988‐21006.
	Jochen, A., Hays, J., Lianos, E., and Hager, S. 1991. Insulin stimulates fatty acid acylation of adipocyte proteins. Biochem. Biophys. Res. Commun. 177: 797‐801.
	Kamps, M.P., Buss, J.E., and Sefton, B.M. 1986. Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation. Cell 45: 105‐112.
	King, M.J. and Sharma, R.K. 1991. N‐Myristoyl transferase assay using phosphocellulose paper binding. Anal. Biochem. 199: 149‐153.
	Magee, A.I., Wootton, J., and de Bony, J. (1995). Optimized methods for detecting radiolabeled lipid‐modified proteins in polyacrylamide gels. Methods in Enzymology 250: 330‐336.
	Moench, S.J., Terry, C.E., and Dewey, T.G. 1994a. Fluorescence labeling of the palmitoylation sites of rhodopsin. Biochemistry 33: 5783‐5790.
	Moench, S.J., Moreland, J., Stewart, D.H. and Dewey, T.G. 1994b. Fluorescence studies of the location and membrane accessibility of the palmitoylation sites of rhodopsin. Biochemistry 33: 5791‐5796.
	Muszbek, L. and Laposata, M. 1993. Myristoylation of proteins in platelets occurs predominantly through thioester linkages. J. Biol. Chem. 268: 8251‐8255.
	Newman, C.M.H. and Magee, A.I. 1993. Post‐translational processing of the ras superfamily of small GTP‐binding proteins. Biochim. Biophys. Acta 1155: 79‐96.
	Peseckis, S.M., Deichaite, I. and Resh, M.D., 1993. Iodinated fatty acids as probes for myristate processing and function. J. Biol. Chem. 268: 5107‐5114.
	Resh, M.D. 1994. Myristoylation and palmitoylation of Src family members: The fats of the matter. Cell 76: 411‐413.
	Rodgers, W., Crise, B., and Rose, J.K. 1994. Signals determining protein tyrosine kinase and glycosyl‐phosphatidylinositol‐anchored protein targeting to a glycolipid‐enriched membrane fraction. Mol. Cell. Biol. 14: 5384‐5391.
	Rudnick, D.A., McWherter, C.A., Rocque, W.J., Lennon, P.J., Getman, D.P., and Gordon, J.I. 1991. Kinetic and structural evidence for a sequential ordered bi bi mechanism of catalysis by Saccharomycescerevisiae myristoyl‐CoA:protein N‐myristoyltransferase. J. Biol. Chem. 266: 9732‐9739.
	Rudnick, D.A., Duronio, R.J., and Gordon, J.I. 1992. Methods for studying myristoyl‐CoA:protein N‐myristoyltransferase. In Lipid Modification of Proteins, A Practical Approach (N.M. Hooper and A.J. Turner, eds.) pp. 37‐61. Oxford University Press, Oxford.
	Tomoda, H., Igarashi, K., and Ømura, S. 1987. Inhibition of acyl‐CoA synthetase by triacsins. Biochim. Biophys. Acta 921: 595‐598.
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Key Reference
	Casey P.J. and Buss J.E. 1995. Lipid modification of proteins. Methods Enzymol. Vol. 250.
	A compilation of methods used in studying lipid modification of proteins.

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Metabolic Labeling with Fatty Acids

Abstract

Table of Contents

Materials

Basic Protocol 1: Biosynthetic Labeling with Fatty Acids

Basic Protocol 2: Analysis of Fatty Acid Linkage to Protein

Basic Protocol 3: Analysis of Total Protein‐Bound Fatty Acid Label in Cell Extract

Basic Protocol 4: Analysis of Fatty Acid Label Identity

Figures

Videos

Literature Cited