N-Acetylglucosaminyltransferase III, IV and V activities in Novikoff ascites tumour cells, mouse lymphoma cells and hen oviduct

Application of a sensitive and specific assay by use of high-performance liquid chromatography

Authors


Correspondence to D. H. van den Eijnden, Vakgroep Medische Chemie, Vrije Universiteit. P.O. Box 7161, NL-1007 MC Amsterdam, The Netherlands

Abstract

A specific and fast method for the determination of N-acetylglucosaminyltransferase III, IV and V activity in one assay is described. The method is based on the separation by HPLC of the three tranferase products formed from the common acceptor oligosaccharide substrate GlcNAcβ1→2Manα1→3(GlcNAcβ1→2Manαl→6)Manβ1→4GlcNAc. Assays are not interfered with by substances that result from enzymatic or chemical breadkdown of the donor substrate UDP-[14C]GlcNAc. Using this assay system N-acetylglucosaminyltransferase III, IV and V activities were estimated in Novikoff ascites tumour cells, mouse lymphoma BW 5147 cells and hen oviduct.

Enzymes
 

UDP-GlcNAc:β-mannoside β1→4-N-acetylglucosaminyltransferase, GlcNAc transferase III (EC 2.4.1.144)

 

UDP-GlcNAc: α-mannoside β1→-N-acetylglucosaminyltransferase, GlcNAc transferase IV (EC 2.4.1.145)

 

UDP-GlcNAc: α-mannoside β1→6-N-acetylglucosaminyltransferase, GlcNAc transferase V (EC 2.4.1.155)

 

UDP-GlcNAc: β-galactoside β1→3-N-acetylglucosaminyltransfease, β3-GlcNAc transferase (EC 2.4.1.149)

 

UDP-GlcNAc: β-galactoside β1→6-N-acetylglucosaminyltransferase A, β6-GlcNAc transferase A (EC 2.4.1.150)

 

UDP-GlcNAc: β-galactoside β1→6-N-acetylglucosaminyltransferase B, β6-GlcNAc transferase B (EC 2.4.1.102 and 2.4.1.148)

Ancillary