Accelerated Enzymatic Galactosylation of N-Acetylglucosaminolipids in Lipid Microdomains

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• 作者：Gavin T. Noble ; Faye L. Craven ; Josef Voglmeir ; Robert 艩ardz铆k ; Sabine L. Flitsch ; Simon J. Webb
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：August 8, 2012
• 年：2012
• 卷：134
• 期：31
• 页码：13010-13017
• 全文大小：399K
• 年卷期：v.134,no.31(August 8, 2012)
• ISSN：1520-5126

文摘

A fluoro-tagged N-acetylglucosamine-capped glycolipid that can form lipid microdomains in fluid phospholipid bilayers has been shown to be enzymatically galactosylated by bovine 尾(1,4)-galactosyltransferase. MALDI MS, HPLC, and LC鈥揗S revealed that the rate of enzymatic transformation was significantly enhanced by lipid clustering; at a 1% mol/mol loading, clustered glycolipids were galactosylated 9-fold faster than glycolipids dispersed across the bilayer surface. The transformation of the GlcNAc 鈥済lycocalyx鈥?into a Gal(尾1鈥?)GlcNAc 鈥済lycocalyx鈥?relabeled these vesicles, making them susceptible to agglutination by Erythrina cristagalli lectin (ECL). The kinetic parameters for this transformation revealed a lower apparent K_m when the substrate lipids were clustered, which is attributed to multivalent binding to an extended substrate cleft around the active site. These observations may have important implications where soluble enzymes act on substrates embedded within cellular lipid rafts.