Location of long chain fatty acid-binding sites of bovine serum albumin by affinity labeling.
1986; Elsevier BV; Volume: 261; Issue: 33 Linguagem: Inglês
10.1016/s0021-9258(18)66760-2
ISSN1083-351X
Autores Tópico(s)Muscle metabolism and nutrition
ResumoAffinity labeling with palmitic acid was used to identify long chain fatty acid-binding sites of bovine serum albumin.[l-'4C]Palmitic acid was activated by esterification with N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's Reagent K).The product was purified by chromatography and shown to compete with unesterified fatty acids for binding sites on bovine serum albumin.Activated ["C]palmitic acid coupled covalently to albumin producing 114C]palmitoyl-albumins containing from 0.12 to a maximum of 6.9 mol of attached label per mol of albumin.The presence of the covalently attached affinity label depressed binding of other long chain fatty acids to albumin.Albumin carrying 1 eq. of [ *4C]palmitate was cleaved using cyanogen bromide, pepsin, and trypsin.Radioactive peptides were isolated by high pressure liquid chromatography.Three peptides accounted for greater than 90% of the label.Residues labeled with [14C]palmitate were identified as Lys-116, Lys-349 and Lys-473, and the relative distribution of label was 10,45, and 45% respectively, consistent with the presence of two strong binding sites in the COOH-terminal half of albumin and a somewhat weaker site in the NH2-terminal half.Serum albumin is the major vehicle for transport of nonesterified fatty acids in the circulation (1-4).It accepts long chain fatty acids generated by the action of lipase on triglycerides and delivers long chain fatty acids to various tissue for storage or metabolism.Albumin's ability to modulate the availability of free fatty acids, which are essential nutrients for growth but which may be toxic at high concentrations, makes it an important component of media for the culturing of microorganisms, tissue, and isolated cells (2,4).Circulating albumin typically carries 0.5-2.0mol of fatty acid/mol of albumin although, in vitro, albumin has been demonstrated to be capable of binding as many as 6 mol of stearate, 7 mol of palmitate, or 9 mol of oleate ( 5 ) .The binding has been characterized as heterogeneous-involving numerous sites of varying affinity.Stepwise association constants, ranging from 10' to lo5 "I, are so similar in magnitude that no single site can be said to dominate.Even at mol ratios less than 1, fatty acid molecules are distributed over several sites producing a heterogeneous population of albumin-fatty acid complexes (4).The major fatty acid binding region of albumin appears to
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