E. Bause et M. Burbach, PURIFICATION AND ENZYMATIC-PROPERTIES OF ENDO-ALPHA-1,2-MANNOSIDASE FROM PIG-LIVER INVOLVED IN OLIGOSACCHARIDE PROCESSING, Biological chemistry, 377(10), 1996, pp. 639-646
An endo-alpha 1,2-mannosidase, which is involved in N-linked oligosacc
haride processing, has been purified to homogeneity from crude pig liv
er microsomes using conventional techniques. Two catalytically active
polypeptides, of 48 kDa and 50 kDa, have been isolated which degrade [
C-14]Glc(3-1)-Man(9)-GlcNAc(2) to [C-14]Glc(3-1)-Man and a specific Ma
n(8)-GlcNAc(2) isomer. They are not, however, active on synthetic alph
a-mannosides. [C-14]Glc(1)-Man(9)-GlcNAc(2) was found to be approximat
ely sevenfold more rapidly hydrolyzed than the [C-14]Glc(2)- and [C-14
]Glc(3)-homologues. The 48 kDa and 50 kDa proteins are not N-glycosyla
ted and ran on Superdex((TM)) 75 as monomers. Kinetic studies showed t
hat these proteins had similar catalytic properties: (i) the pH optima
were found to be close to 6.5; (ii) neither activity was metal ion de
pendent; (iii) hydrolysis of [C-14]Glc(3)-Man(9)-GlcNAc(2) was inhibit
ed strongly by Glc-alpha 1,3-Man (app. K-i approximate to 120 mu M), b
ut not by 1-deoxymannojirimycin or swainsonine, Other evidence, includ
ing immunological data, strongly suggests that the 48 kDa and 50 kDa p
olypeptides are proteolytic degradation products of a single endo-alph
a 1,2-mannosidase, rather than distinct subunits of an oligomeric comp
lex, Possible functions of the endo-alpha 1,2-mannosidase in N-linked
oligosaccharide processing are discussed.