Abstract
Rat liver alpha-mannosidase II, a hydrolase involved in the processing of asparagine-linked oligosaccharides, is an integral membrane glycoprotein facing the lumen of Golgi membranes. We have previously shown (Moremen, K. W., and Touster, O. (1986) J. Biol. Chem. 261, 10945-10951) that mild chymotrypsin digestion of permeabilized or solubilized Golgi membranes will result in the cleavage of the intact 124,000-dalton alpha-mannosidase II subunit, releasing a 110,000-dalton hydrophilic polypeptide which contains the catalytic site. Consistent with the removal of a membrane binding domain, the chymotrypsin-generated 110,000-dalton peptide was found exclusively in the aqueous phase in Triton X-114 phase separation studies, whereas the intact enzyme was found in the detergent phase. Taking advantage of this conversion in phase partitioning behavior, a purification procedure was developed to isolate the 110,000-dalton proteolytic digestion product as a homogeneous polypeptide for further characterization and protein sequencing at a yield of greater than 65% from a rat liver Golgi-enriched membrane fraction. An improved purification procedure for the intact enzyme was also developed. The two forms of the enzyme were compared yielding the following results. (a) The catalytic activity of the intact and cleaved forms of alpha-mannosidase II were indistinguishable in Km, Vmax, inhibition by the alkaloid, swainsonine, and in their activity toward the natural substrate GlcNAc-Man5GlcNAc. (b) Both the intact and cleaved forms of the enzyme appear to be disulfide-linked dimers. (c) The two forms of the enzyme contain different NH2-terminal sequences suggesting that the cleaved NH2 terminus contains the membrane-spanning domain. (d) Additional peptide sequences were obtained from proteolytic fragments and cyanogen bromide digestion products in order to create a partial protein sequence map of the enzyme. These results are consistent with a model common among Golgi processing enzymes of a hydrophilic catalytic domain anchored to the lumenal face of Golgi membranes through an NH2-terminal hydrophobic membrane-anchoring domain.
Highlights
From the $Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and the VDepartment of Molecular Biology, Vanderbilt Uniuersity, Nashuille, Tennessee 37235
Man I1 is the final processing hydrolase in the pathway catalyzing the removal of the final two mannosyl residues intact enzyme was found in the the detergent phase. (a1,3and a1,6 linkages) to generatethe GlcNAcManaGlcNAcz
Since many transmembrane enzymes have catalytic centers that are composed of hydro
Summary
Tion product as a homogeneous polypeptide for further Man I1 is one of the most well studied enzymes of the Golgi characterization and protein sequencingat a yield of complex It has been purified [2] and characterized [3], and. Purification of both the intact and Additional peptide sequenceswere obtained from proteolytic fragmentsand cyanogenbromidedigestion products in order to createa partial protein sequence map of the enzyme These resultsare consistent with a model common among Golgi processing enzymes of a hydrophilic catalytic domain anchored tothe lumenal proteolytically solubilized forms of these enzymes has not yet been accomplished, so a direct comparison of the two forms has not been possible.
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