Glucosidase II has been purified from crude pig liver microsomes by a
convenient procedure involving DEAE-Sephacel, Con A-Sepharose and affi
nity chromatography on N-5-carboxypentyl-1-deoxynojirimycin-AH-Sepharo
se. Specific binding of glucosidase II to the affinity matrix required
its prior separation from glucosidase I, which was accomplished by fr
actional Con A-Sepharose chromatography. The three-step procedure yiel
ded, with approximate to 15% enzyme recovery, a > 190-fold enriched gl
ucosidase II, consisting of two proteins (107 kDa and 112 kDa). Both p
olypeptides are N-glycosylated with probably one glycan chain, in line
with their binding to Con A-Sepharose. Immunological cross-reactivity
and other experimental data indicate that the 107 kDa N-glycoprotein
is derived from the 112 kDa species by partial proteolysis. The occasi
onal presence of a 60 kDa peptide co-eluting with the catalytic activi
ty suggests that glucosidase II may be associated with other protein s
ubunit(s) in a heteromeric membrane complex. Glucosidase II hydrolyzes
the alpha 1,3-glucosidic linkages in Glc(2-1)-Man(9)-GlcNac(2), as we
ll as synthetic alpha-glucosides, efficiently but does not remove the
distal alpha 1,2-linked glucose in Glc(3)-Man(9)-GlcNAc(2). The enzyme
has a pH optimum close to 6.5 and is not metal ion-dependent. Catalyt
ic activity is strongly inhibited by basic sugar analogues including 1
-deoxynojirimycin (dNM; app. K-i approximate to 7.0 mu M)I N-5-carboxy
pentyl-dNM (app. K-i = 32 mu M) and castanospermine (app. K-i approxim
ate to 40 mu M) Substitution of the 3-OH or 6-OH group in dNM by a flu
oro group reduces the inhibitory potential drastically. We conclude fr
om these observations that the two hydroxy groups are essential for in
hibitor/substrate binding due to their ability to interfere as hydroge
n bond donors. A polyclonal antibody raised against the 107 kDa polype
ptide reacted specifically with two proteins from different cell types
on Western blots. Their molecular masses were identical with those fr
om pig liver microsomes, pointing to a highly conserved amino acid seq
uence of glucosidase II. This suggests that the variance in molecular
mass for glucosidase II reported for the enzyme from other tissues and
species may be due to partial proteolysis.