The purification, properties and characterization of three forms of α-L-fucosidase from monkey brain

Abstract

α-L-Fucosidase (α-L-fucoside fucohydrolase, EC 3.2.1.51) has been purified to apparent homogeneity (about 22 000-fold over the crude homogenate) from monkey brain. Values of kinetic constants for the purified enzyme were as follows: pH optimum, 5.0; k_m, 0.22 mM; V, 913 μmol/mg per h. α-L-Fucose was a competitive inhibitor (K_i, 0.275 mM) of the enzyme. Evidence for the involvement of sulphydryl group(s) and carboxyl group containing amino acid(s) in the catalytic process is presented. The purified enzyme was a tetramer of molecular weight of 285 000 of identical subunits of 73 500 held together by non-covalent forces. Gel filtration studies revealed the presence of three molecular forms of the activity in the purified preparation which appeared to be the tetramer, dimer and monomer. The existence of three types of activities was also supported by a triphasic heat inactivataion profile of the enzyme at 50 or 55°C and the distinctly different pH activity profiles of the differentially heat-inactivated enzymes. Immunodiffusion studies using antibody developed against purified monkey brain α-L-fucosidase showed that the monkey brain enzyme had only partial immunological identity with the enzymes from the non-neural tissues of monkey as well as the human and rat liver and the rat brain. However, the monkey brain and liver enzymes appeared to be similar to the human brain and liver enzymes, respectively.