Structure of 18S and 14S Acetylcholinesterase. Identification of Collagen-Like Subunits That Are Linked by Disulfide Bonds to Catalytic Subunits

Terrone L. Rosenberry, Jeanne M. Richardson

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Abstract

The 14S and 18S acetylcholinesterase (EC 3.1.1.7) forms present in 1.0 M ionic strength extracts of fresh electric organ of the eel Electrophorus electricus were purified by affinity chromatography. The purification procedure involved a highly efficient acridinium resin whose synthesis and use are introduced. Previous electron microscopy studies have determined that the 14S and 18S acetylcholinesterase species as well as an 8S species not observed in this study contain a 40-nm tail structure associated with discrete numbers of presumably catalytic subunits, and other reports have established that either trypsin or collagenase will convert the 8S, 14S, and 18S forms to 11S catalytic subunit tetramers which appear devoid of the tail structure. A comparative analysis of polypeptides present in 18S plus 14S preparations and 11S preparations by polyacrylamide gel electrophoresis in sodium dodecyl sulfate is presented in this report. Prior to disulfide reduction the predominant 11S component in sodium dodecyl sulfate was an apparent catalytic subunit dimer possessing an intersubunit disulfide bond; in contrast, only half the 18S plus 14S components corresponded to this dimer while the remainder were found primarily as two oligomers, A and B, of high molecular weight (>300 000). Exposure of either 11S or 18S plus 14S preparations to disulfide reduction in the absence of a denaturant selectively reduced interpolypeptide bonds and generated primarily 75 000 molecular weight catalytic subunit monomers which appeared identical in the two preparations. In addition, this selective reduction of the 18S plus 14S enzyme produced a faint indication of polypeptides E with apparent molecular weights, relative to noncollagen standards, of 40 000 and 44 000. These polypeptides, which could not be obtained from the 11S preparation, were more clearly seen after complete disulfide reduction in a denaturant. Polypeptides E are thus prime candidates for the tail subunits. Fractionation of the nonreduced 18S plus 14S preparation by gel exclusion chromatography in sodium dodecyl sulfate revealed that all fractions contained catalytic subunits while polypeptides E derived only from oligomers A and B and not from catalytic subunit dimers. Difference amino acid compositions indicated that polypeptides E are largely collagen-like, containing about 30% glycine, 15% proline, 7% hydroxyproline, and 7% hydroxylysine. A proposed model of 18S acetylcholinesterase considers this molecule to consist of one oligomer A unit, composed of three pairs of catalytic subunits disulfide bonded to the tail structure, and three catalytic subunit dimers. It follows that the catalytic subunits in both 18S and 11S acetylcholinesterase are arranged asymmetrically. Both the indicated amino acid composition of polypeptides E and the presence of intersubunit disulfide bonds linking noncollagenous glycoprotein catalytic subunits to the apparently collagen-like filamentous tail structure are characteristic of basement membrane components.

Original languageEnglish (US)
Pages (from-to)3550-3558
Number of pages9
JournalBiochemistry
Volume16
Issue number16
DOIs
StatePublished - Aug 1 1977

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ASJC Scopus subject areas

  • Biochemistry

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