DIFFERENTIAL AFFINITY LABELING OF THE 2 SUBUNITS OF THE HOMODIMERIC ANIMAL FATTY-ACID SYNTHASE ALLOWS ISOLATION OF HETERODIMERS CONSISTING OF SUBUNITS THAT HAVE BEEN INDEPENDENTLY MODIFIED

Citation
Ak. Joshi et al., DIFFERENTIAL AFFINITY LABELING OF THE 2 SUBUNITS OF THE HOMODIMERIC ANIMAL FATTY-ACID SYNTHASE ALLOWS ISOLATION OF HETERODIMERS CONSISTING OF SUBUNITS THAT HAVE BEEN INDEPENDENTLY MODIFIED, The Journal of biological chemistry, 273(9), 1998, pp. 4937-4943
Citations number
36
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0021-9258
Volume
273
Issue
9
Year of publication
1998
Pages
4937 - 4943
Database
ISI
SICI code
0021-9258(1998)273:9<4937:DALOT2>2.0.ZU;2-J
Abstract
To explore the domain interactions that are required for catalytic act ivity of the multifunctional, homodimeric fatty acid synthase (FAS), w e have formulated a strategy that allows isolation of modified dimers containing independently mutated subunits, Either a hexahistidine or a FLAG octapeptide tag was incorporated into the FAS at either the amin o terminus, within an internal noncatalytic domain, or at the carboxyl terminus, The presence of the tags had no effect on the activity of t he wild-type FAS. His-tagged dimers were mixed with FLAG-tagged dimers , and the subunits were randomized to produce a mixture of His-tagged homodimers, FLAG-tagged homodimers, and doubly tagged heterodimers. Th e doubly tagged heterodimers could be purified to homogeneity by chrom atography on an anti-FLAG immunoaffinity column followed by a metal io n chelating column, This procedure for isolation of FAS heterodimers w as utilized to determine whether the two centers for fatty acid synthe sis in the FAS dimer can function independently of each other, Doubly tagged heterodimers, consisting of one wild-type subunit and one subun it in which the thioesterase activity had been eliminated, either by m utation or by treatment with phenylmethanesulfonyl fluoride, have 50% of the wild-type thioesterase activity and, in the presence of substra tes, accumulate a long chain fatty acyl moiety on the modified subunit , thus blocking further substrate turnover at this center, Nevertheles s, the ability of the heterodimer to synthesize fatty acids is also 50 % of the wild-type FAS, demonstrating that an individual center for fa tty acid synthesis has the same activity when paired with either a fun ctional or nonfunctional catalytic center.