Purification and biochemical characterization of two soluble alpha-mannosidases from Candida albicans.
Two soluble alpha-mannosidases, E-I and E-II, were purified from C. albicans yeast cells by a three-step procedure consisting of size exclusion and ion exchange chromatographies in Sepharose CL6B and Mono Q columns, respectively, and preparative nondenaturing electrophoresis. E-I and E-II migrated as monomeric polypeptides of 54.3 and 93.3 kDa in SDS-PAGE, respectively. Some biochemical properties of purified enzymes were investigated by using 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside as substrates. Hydrolysis of both substrates by either enzyme was optimum at pH 6.0 with 50 mM Mes-Tris buffer and at 42 degrees C. Apparent Kmvalues for hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside by E-I were 0.83 microM and 2. 4 mM, respectively. Corresponding values for E-II were 0.25 microM and 1.86 mM. Swansonine and deoxymannojirimicin strongly inhibited the hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside by both enzymes. On the contrary, hydrolysis of p-nitrophenyl-alpha-D-mannopyranoside by E-I and E-II was slightly stimulated or not affected, respectively, by both inhibitors. E-I and E-II did not depend on metal ions although activity of the latter was slightly stimulated by Mn2+and Ca2+in the range of 0.5-2 mM. At the same concentrations, Mg2+was slightly inhibitory of both enzymes. Substrate specificity experiments revealed that both E-I and E-II preferentially cleaved alpha-1,6 and alpha-1,3 linkages, respectively.
Vázquez-Reyna
A B
AB
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad Autónoma de Guanajuato, Apartado Postal 187, Guanajuato, Gto. CP 36000, México.
Ponce-Noyola
P
P
Calvo-Méndez
C
C
López-Romero
E
E
Flores-Carreón
A
A
D002176:Candida albicans / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008361:Mannosidases / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D043323:alpha-Mannosidase
D007527:Isoenzymes; D008361:Mannosidases; D043323:alpha-Mannosidase