Interaction of S-acyl fatty acid synthase thioester hydrolase with fatty acid synthase. Direct measurement of binding by fluorescence anisotropy.
Treatment of S-acyl fatty acid synthase thioester hydrolase from the uropygial gland of Peking duck with pyrenebutylmethanephosphonofluoridate resulted in inactivation of the enzyme with covalent attachment of the pyrene derivative to the enzyme. One mole of the derivative was attached/mol of protein, most probably at the active serine. When avian fatty acid synthase was added to the modified thioesterase, the fluorescence anisotropy of the pyrene derivative increased dramatically. That this increase represented the functionally significant binding between the two proteins was suggested by the fact that increasing salt concentration resulted in concomitant loss in enzyme activity and fluorescence anisotropy. As the synthase concentration increased, anisotropy increased giving a saturation pattern. From a Scatchard plot analysis the association constant for the binding of the two proteins was calculated to be 10(6) M-1 and one-to-one stoichiometry was shown for this association. These results show that fluorescence anisotropy of the pyrene derivative attached to the thioesterase can be used to directly measure the binding of this enzyme to fatty acid synthase.
D000818:Animals; D004372:Ducks / Q000378:metabolism*; D064429:Fatty Acid Synthases / Q000378:metabolism*; D005260:Female; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005777:Geese / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000201:enzymology; D009943:Organophosphorus Compounds / Q000378:metabolism / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D012627:Sebaceous Glands / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005456:Fluorescent Dyes; D009943:Organophosphorus Compounds; C017692:4-(1-pyrenyl)butyl methylphosphonofluoridate; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C018748:oleoyl-(acyl-carrier-protein) hydrolase