Purification and characterization of glycoprotein processing enzyme. cap alpha. -glucosidase II from bovine mammary tissue

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

OSTI ID:5022325

Saxena, S K; Shailubhai, K; Pratta, M A; Vijay, I K

..cap alpha..-Glucosidase II removes the two inner (..cap alpha..1-3) linked glucose residues of glycoprotein precursor Glc/sub 3/Man/sub 9/(GlcNAc)/sub 2/...cap alpha..-Glucosidase II has been purified to homogeneity from bovine mammary tissue. Briefly, purification involved solubilization of glucosidase II with Triton X-100 from microsomes followed by 20-60% (NH/sub 4/)/sub 2/SO/sub 4/ fractionation, Con-A Sepharose 4B, hydroxylapatite and DEAE-Sephacel column chromatography. The purified glucosidase II showed single band on 10% non-denaturing PAGE and enzyme activity band on visualization with fluorogenic substrate 4-methylumbelliferyl (MUF) ..cap alpha..-D-glucopyranoside. The purified enzyme exhibited broad pH optima (6.0-7.5). The enzyme hydrolyzed MUF..cap alpha..-D-glucoside (K/sub m/ = 13 ..mu..M) but was inactive against the corresponding D-glucoside and the ..cap alpha..-D-mannoside. Cleavage of MUF ..cap alpha..-glucoside was enhanced by mannose and starch and was inhibited by maltose, turanose and glucose. The enzyme released glucose residues from (/sup 3/H)Glc/sub 2/Man/sub 9/(GlcNAc)/sub 2/ but did not release glucose from (/sup 3/H)Glc/sub 3/Man/sub 9/(GlcNAc)/sub 2/. Reductive SDS-PAGE revealed two closely associated major bands showing mol. wt. of 66 and 64 ksSa. These bands also showed PAS-positive staining. Gel filtration revealed a molecular size of 290 kDa for purified enzyme.

• Research Organization: Univ. of Maryland, College Park
• OSTI ID: 5022325
• Report Number(s): CONF-8606151-; TRN: 86-034847
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
GLUCOSIDASE
CHEMICAL COMPOSITION
PURIFICATION
BIOCHEMICAL REACTION KINETICS
CATTLE
CHROMATOGRAPHY
GLUCOPROTEINS
MAMMARY GLANDS
MICROSOMES
ANIMALS
BODY
CARBOHYDRATES
CELL CONSTITUENTS
DOMESTIC ANIMALS
ENZYMES
GLANDS
GLYCOSYL HYDROLASES
HYDROLASES
KINETICS
MAMMALS
O-GLYCOSYL HYDROLASES
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PROTEINS
REACTION KINETICS
RUMINANTS
SACCHARIDES
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques