Purification and properties of the glycoprotein processing N-acetylglucosaminyltransferase II from plants

Journal Article · Tue Aug 25 04:00:00 EDT 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00391a043· OSTI ID:5520660

Szumilo, T; Kaushal, G P; Elbein, A D

The presence of an N-acetylglucosaminyltransferase (GlcNAc-transferase) capable of adding a GlcNAc residue to GlcNAcMan3GlcNAc was demonstrated in mung bean seedlings. This enzyme was purified about 3400-fold by using (diethylaminoethyl)cellulose and phosphocellulose chromatographies and chromatography on Concanavalin A-Sepharose. The transferase was assayed by following the change in the migration of the (/sup 3/H)mannose-labeled GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc on Bio-Gel P-4, or by incorporation of (/sup 3/H)GlcNAc from UDP-(3H)GlcNAc into a neutral product, (GlcNAc)2Man3GlcNAc. Thus, the purified enzyme catalyzed the addition of a GlcNAc to that mannose linked in alpha 1,6 linkage to the beta-linked mannose. GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc was an excellent acceptor while Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, Man alpha 1,6(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, and Man alpha 1,6(Man apha 1,3)Man alpha 1,6(GlcNAcMan alpha 1,3)Man beta 1,4GlcNAc were not acceptors. Methylation analysis and enzymatic digestions showed that both terminal GlcNAc residues on (GlcNAc)2Man3GlcNAc were attached to the mannoses in beta 1,2 linkages. The GlcNAc transferase had an almost absolute requirement for divalent cation, with Mn2+ being best at 2-3 mM. Mn2+ could not be replaced by Mg2+ or Ca2+, but Cd2+ showed some activity. The enzyme was also markedly stimulated by the presence of detergent and showed optimum activity at 0.15% Triton X-100. The Km for UDP-GlcNAc was found to be 18 microM and that for GlcNAcMan3GlcNAc about 16 microM.

• Research Organization: Univ. of Texas Health Science Center, San Antonio
• OSTI ID: 5520660
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:17; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALDEHYDES
BACTERIA
CARBOHYDRATES
CHEMICAL PROPERTIES
CHEMICAL REACTIONS
CHROMATOGRAPHY
ENZYME ACTIVITY
ENZYMES
FOOD
FRACTIONATION
HEXOSES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LEGUMINOSAE
MANNOSE
METHYLATION
MICROORGANISMS
MONOSACCHARIDES
MUNGBEANS
ORGANIC COMPOUNDS
PLANTS
RHIZOBIUM
SACCHARIDES
SEEDS
SEPARATION PROCESSES
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
VEGETABLES