Open AccessPurification and Properties of α‐ d ‐Mannosidase from Medicago sativa L.
Author(s) -
PRIJCKER Jozef,
VERVOORT Antoine,
BRUYNE Clement
Publication year - 1974
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1974.tb03726.x
Subject(s) - chemistry , sephadex , mannosidase , moiety , hydrolysis , size exclusion chromatography , chromatography , reagent , ion chromatography , column chromatography , ammonium , enzyme , stereochemistry , biochemistry , organic chemistry
A purification method for α‐mannosidase from the seeds of Medicago sativa L. is described. It includes ammonium sulphate precipitation, phosphocellulose chromatography, DEAE‐Sephadex ion‐exchange chromatography, and Sephadex G‐200 gel filtration. The enzyme has a molecular weight of about 220000. At pH values below neutrality, α‐mannosidase undergoes inactivation. This process can be prevented by Zn 2+ , whereas EDTA accelerates inactivation. The influence of pH on K m and V have been determined with p ‐nitrophenyl α‐ d ‐mannoside as substrate. From the pH dependence it could be deduced that a dissociable group with p K ∼ 6 and functioning as a proton donor is necessary for catalytic activity. p ‐Chloromercuribenzoate and other sulfhydryl reagents were not inhibitory. Glycon specificity studies reveal strict requirements at C‐3, C‐4 and C‐5 of the mannopyranosyl moiety. The trans‐diaxial position of the C‐2 hydroxyl and the aglycon group seems necessary for hydrolysis, but not for binding. The strong inhibitory power of mannono‐(1 → 5)‐lactone suggests that the glycon part of the substrates is bound to the enzyme in a half‐chair like conformation.