HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Joshi, A. K. Articles by Smith, S. Search for Related Content PubMed PubMed Citation Articles by Joshi, A. K. Articles by Smith, S. Social Bookmarking                      What's this?

J Biol Chem, Vol. 273, Issue 9, 4937-4943, February 27, 1998

Differential Affinity Labeling of the Two Subunits of the Homodimeric Animal Fatty Acid Synthase Allows Isolation of Heterodimers Consisting of Subunits That Have Been Independently Modified

From the Children's Hospital Oakland Research Institute, Oakland, California 94609

To explore the domain interactions that are required for catalytic activity of the multifunctional, homodimeric fatty acid synthase (FAS), we 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 amino terminus, within an internal noncatalytic domain, or at the carboxyl terminus. The presence of the tags had no effect on the activity of the 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. The doubly tagged heterodimers could be purified to homogeneity by chromatography on an anti-FLAG immunoaffinity column followed by a metal ion chelating column. This procedure for isolation of FAS heterodimers was utilized to determine whether the two centers for fatty acid synthesis in the FAS dimer can function independently of each other. Doubly tagged heterodimers, consisting of one wild-type subunit and one subunit in which the thioesterase activity had been eliminated, either by mutation or by treatment with phenylmethanesulfonyl fluoride, have 50% of the wild-type thioesterase activity and, in the presence of substrates, accumulate a long chain fatty acyl moiety on the modified subunit, thus blocking further substrate turnover at this center. Nevertheless, the ability of the heterodimer to synthesize fatty acids is also 50% of the wild-type FAS, demonstrating that an individual center for fatty acid synthesis has the same activity when paired with either a functional or nonfunctional catalytic center.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. Witkowski, A. K. Joshi, V. S. Rangan, A. M. Falick, H. E. Witkowska, and S. Smith Dibromopropanone Cross-linking of the Phosphopantetheine and Active-site Cysteine Thiols of the Animal Fatty Acid Synthase Can Occur Both Inter- and Intrasubunit. REEVALUATION OF THE SIDE-BY-SIDE, ANTIPARALLEL SUBUNIT MODEL J. Biol. Chem., April 23, 1999; 274(17): 11557 - 11563. [Abstract] [Full Text] [PDF]

				V. S. Rangan, A. K. Joshi, and S. Smith Fatty Acid Synthase Dimers Containing Catalytically Active beta -Ketoacyl Synthase or Malonyl/Acetyltransferase Domains in Only One Subunit Can Support Fatty Acid Synthesis at the Acyl Carrier Protein Domains of Both Subunits J. Biol. Chem., December 25, 1998; 273(52): 34949 - 34953. [Abstract] [Full Text] [PDF]