Purification and properties of a glycoprotein processing alpha-mannosidase from mung bean seedlings

The microsomal fraction of mung bean seedlings contains mannosidase activities capable of hydrolyzing [3H]mannose from the $[{}^{3}\text{H}]\text{Man}_{9}\text{GlcNAc}$ as well as for releasing mannose from p-nitrophenyl-α-D-mannopyranoside. The glycoprotein processing mannosidase was solubilized from the microsomes with 1.5% Triton X-100 and was purified 130-fold by conventional methods and also by affinity chromatography on mannan-Sepharose and mannosamine-Sepharose. The final enzyme preparation contained a trace of aryl-mannosidase, but this activity was inhibited by swainsonine whereas the processing enzyme was not. The pH optimum for the processing enzyme was 5.5 to 6.0, and activity was optimum in the presence of 0.1% Triton X-100. The enzyme was inhibited by ethylenediaminetetraacetate while Ca2+ was the most effective cation for reversing this inhibition. Mn2+ was considerably less effective than Ca2+ and Mg2+ was without effect. The processing mannosidase was inhibited by α1,2- and α1,3-linked mannose oligosaccharides (50% inhibition at 3 millimolar), whereas free mannose and α1,6-linked mannose oligosaccharides were ineffective. Mannosamine was also an inhibitor of this enzyme. The aryl-mannosidase and the processing mannosidase could also be distinguished by their susceptibility to various processing inhibitors. The aryl-mannosidase was inhibited by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol but not by deoxymannojirimycin or other inhibitors, while the processing mannosidase was only inhibited by deoxymannojirimycin. The processing mannosidase was incubated for long periods with $[{}^{3}\text{H}]\text{Man}_{9}\text{GlcNAc}$ and the products were identified by gel filtration. Even after a 24 hour incubation, the only two radioactive products were Man5GlcNAc and free mannose. Thus, this enzyme appears to be similar to the animal processing enzyme, mannosidase I, and is apparently a specific α1,2-mannosidase.