Purification of. beta. -mannosyl transferase that synthesizes Man-. beta. -GlcNAc-GlcNAc-PP-dolichol

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5126755

Kaushal, G P; Elbein, A D

The ..beta..-mannosyl transferase that catalyzes the transfer of mannose from GDP-mannose to GlcNAc-GlcNAc-PP-dolichol (GlcNAc/sub 2/-lipid) to form Man-..beta..-GlcNAc/sub 2/-lipid was solubilized from the microsomal fraction of pig aorta by treatment with 0.5% NP-40. The enzyme was purified about 115-fold using DEAE-cellulose, hydroxylapatite, and epoxy-activated Sepharose. The purified enzyme was free of ..cap alpha.. 1.3 and ..cap alpha..1,6-mannosyl transferases since the only product seen when enzyme was incubated with GDP-(/sup 14/C)-mannose and GlcNAc/sub 2/-lipid was Man-..beta..-GlcNAc/sub 2/-lipid. The oligosaccharide portion of this lipid was released by mild acid hydrolysis and characterized as Man-..beta..-GlcNAc-GlcNAc by gel filtration, as well as susceptibility to ..beta..-mannosidase and resistance to a ..cap alpha..-mannosidase. This partially purified enzyme was stabilized by adding 20% glycerol and 0.5 mM dithiothreitol to the storage buffer. Thus, the transferase was stable for 5 or 6 days at 0/sup 0/ and could be kept for a month at -20/sup 0/. The activity was greatly stimulated by detergent with optimum activity being seen at 0.1% NP-40. However, phospholipids had no effect. The transferase had a pH optimum of 7.0, and showed an almost absolute requirement for Mg/sup + +/, with maximum activity at 5 mM. The K/sub m/ for GDP-mannose was about 5 x 10/sup -7/ M, and for GlcNAc/sub 2/-lipid about 1 x 10/sup -6/ M. The transferase was competitively inhibited by a variety of guanosine nucleotides.

• Research Organization: Univ. of Texas Health Science Center, San Antonio
• OSTI ID: 5126755
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALDEHYDES
ANIMALS
AORTA
ARTERIES
BLOOD VESSELS
BODY
CARBOHYDRATES
CARBON 14 COMPOUNDS
CARDIOVASCULAR SYSTEM
CELL CONSTITUENTS
CHEMICAL REACTIONS
DECOMPOSITION
DOMESTIC ANIMALS
ENZYME ACTIVITY
ENZYMES
FILTRATION
HEXOSES
HYDROLYSIS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIPIDS
LYSIS
MAMMALS
MANNOSE
MICROSOMES
MONOSACCHARIDES
NUCLEOTIDES
OLIGOSACCHARIDES
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PURIFICATION
SACCHARIDES
SEPARATION PROCESSES
SOLVOLYSIS
SWINE
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES