Solubilization and characterization of pellet-associated human brain α-L-fucosidase activity

Abstract

The pellet-associated portion of human brain alpha-L-fucosidase (which represents approx. 20% of the homogenate activity) was solubilized with 0.5% (w/v) Triton X-100, characterized with regard to several properties and compared with the corresponding properties of the soluble supernatant-fluid enzyme in an attempt to find a second alpha-L-fucosidase in human brain. The solubilized and soluble alpha-L-fucosidase activities exhibited complete stability after storage at 2-4 degrees C for up to 29 days, comparable thermostability after preincubation at 50 degrees C, comparable apparent Km values (0.07-0.08 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside, comparable hydrophobicity, comparable isoelectric-focusing profiles (six major forms, with pI values between 4.5 and 5.8) and comparable immunoprecipitation curves (with the IgG fraction of antisera prepared against human liver alpha-L-fucosidase). Differences in three properties were found between solubilized and soluble alpha-L-fucosidase activities: the solubilized activity was less stable to storage at -20 degrees C, had a 0.5-pH-unit neutral shift in its pH optimum (6.0) and had smaller Mr forms after gel filtration on Sephadex G-200. The overall results indicate that the pellet-associated and soluble portions of human brain alpha-L-fucosidase are quite similar in most of their properties. Thus there is still no compelling evidence for the existence of a second mammalian alpha-L-fucosidase.

Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-l-fucosidase from rat testis and epididymal spermatozoa

“…Our findings are very unusual since almost all previous studies have found that mammalian fucosidases are associated with lysosomes and have relatively acidic pH optima between 4 and 6 [1]. The pellet-associated human brain fucosidase has a pH optimum of 5.5-6.0 [21] and purified rat liver [23] and cerebral cortex [24] α--fucosidases have pH optima of 5.7-5.9 and 4.3, respectively. In addition and more specifically, previous studies on rat epididymal fucosidase [25][26][27] have found pH optima (5.5-6.5) lower than the pH 7.0 value which we have observed.…”

“…This is a very novel finding for this enzyme since the great majority (90-100 %) of α--fucosidase activity in almost all mammalian tissues investigated is in the soluble fraction [1]. Human brain is an exception in that it contains a pellet-associated α--fucosidase but it only represents approximately 30 % of the recovered activity from brain homogenates [21]. Approximately 90 % of the epididymal sperm pellet-associated α--fucosidase activity could be extracted with phosphate buffer containing 0.5 M NaCl compared with only 10 % of the activity with phosphate buffer containing 0.5 % Triton X-100.…”

Hydrolysis of fucosyl-GM1 ganglioside by purified pellet-associated human brain and human liver α-l-fucosidases without activator proteins or detergents

“…Resuspension was followed by recentrifugation at 25 500 g for 45 min. Solubilization of human brain pellet-associated a-L-fucosidase was carried out as previously described (Hopfer & Alhadeff, 1985). The twice-washed pellet was resuspended in phosphate buffer containing 0.5% (w/v) Triton X-100, homogenized by use of the Potter-Elvehjem tissue grinder as described above and centrifuged at 25000 g for 45 min.…”

“…Isoelectric focusing of purified pellet-associated brain a-L-fucosidase was accomplished as previously described for crude preparations of pellet-associated human brain a-L-fucosidase (Hopfer & Alhadeff, 1985). ac-L-Fucosidase activity (6.4 units) was loaded on an LKB 8100 (110 ml) Ampholine column with Ampholytes (pH 5-8;…”

“…The presence of these lipophilic compounds suggests that brain may contain a membrane-bound a-L-fucosidase involved in their degradation. In a previous investigation (Hopfer & Alhadeff, 1985), a pellet-associated a-L-fucosidase from human brain was solubilized, characterized with the use of the synthetic substrate 4-methylumbelliferyl a-L-fucopyranoside and found to be quite similar to soluble human brain a-L-fucosidase (Alhadeff & Janow-sky, 1977). Since only crude enzyme preparations and synthetic substrate were employed in our previous study on pellet-associated brain a-L-fucosidase (Hopfer & Alhadeff, 1985), an ac-L-fucosidase active only on natural substrates could have been overlooked.…”