Articles
Mutsumi Shimada, Saori Yasui, Azusa Hotta, Sumio Ishijima and Masatake Ohnishi Graduate School of Agriculture, The Prefecture University of Kyoto (Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan) Received September 20, 2002, Accepted February 10, 2003 Glucocerebrosidase (EC 3.2.1.45) is a lysosomal β-glucosidase that catalyses the hydrolysis of glucocerebroside to glucose and ceramide.  A deficiency of glucocerebrosidase is the cause of Gaucher's disease.  Glucocerebrosidase was partially purified from bovine brain with cholate extraction followed by ammonium sulfate fractionation, acid precipitation at pH 5.35 and butanol extraction.   Molecular and kinetic properties of this enzyme were characterized with 4-methylumbeliferyl-β-D-glucopyranoside as substrate.    The enzyme activity was increased by addition of 0.05%—0.2% Triton X-100 and sodium taurocholate. Dextran sulfate noncompetitively inhibited the reaction.  The Ki value was estimated to be 1.5 mM.   Inhibition by Dextran sulfate was independent of the substrate concentration, but was pH dependent.  Maximal inhibition was observed at pH 4.1. Key words: Glucocerebrosidase; Dextran sulfate; Bovine brain Enzyme activities have been studied in various conditions, such as pH, temperature and ionic strength.  These are important environmental factors to regulate enzyme activities. The environment of membrane and intracellular organelles is much different from experimental conditions.   Analysis of protein function in these intracellular organelles is important to reveal We biological function of proteins. attracted attention to membrane-bound participating in biological proteins function.  We have examined reaction in     the mechanisms  of  enzymes hydrophobic circumstances.  We here selected an enzyme acting on sugar chain. Glucocerebrosidase (glucosyl- ceramide-β-glucosidase, EC 3.2.1.45) is a membrane-bound lysosomal hydrolase that participates in the degradation of glycosphingolipids glucosylceramide (glucocerebroside) to yield glucose and ceramide 1 .  The metabolic defect in patients with Gaucher's disease is a deficiency of this enzyme activity, which results in the accumulation of glucocerebroside in tissues of Gaucher's patients. Glucocerebrosidase       has been purified from a variety of mammalian tissues 2,3).  The enzyme is able to hydrolyze not only its natural lipid substrate, glucocerebroside, but also artificial  water-soluble  compounds  such 4-methylumbeliferyl-β-D-gluco- as pyranoside (MUG).  Delipidation and purification, that make the enzyme water-soluble, markedly impair the enzyme activity towards both lipid and water-soluble substrates; the activity can be restored by the addition of detergents or acidic lipids to assay mixture. Human 4,5) and mouse 6) cDNA clones encoding glucocerebrosidase       were isolated.    The likely precursor protein of human glucocerebrosidase is 536 amino acids long.  The N-terminal 39 amino acids constitute a leader sequence that is cleaved from the mature protein. In this paper, we describe the characterization of glucocerebrosidase from bovine brain
Materials and methods
Materials. Phenylmethylsulphonyl fluoride (PMSF), cholic acid sodium salt, taurocholic acid sodium salt, MUG, p-nitrophenyl-β-D-glucopyranoside  and cellobiose were obtained from Nacalai Tesque Inc.  Conduritol-B-epoxide and ceramide were purchased from Wako Pure Chemical Ind., Ltd.  Glucocere- broside and Dextran sulfate (average Mr 5000) were obtained from Sigma. Preparation of bovine brain enzyme. Bovine brains were obtained from Keiji slaughter house immediately after the sacrifice of the animals and kept frozen at −80°C. Brain (about 100 g) was homogenized in 3 volumes of 25 mM sodium phosphate buffer (pH 6.0) containing 1 mM 2-mercaptoethanol and 1 mM PMSF.  All procedures were performed at 4°C.   The homogenate was centrifuged at 40,000xg for 30 min. The pellet was rehomogenized in 2 8 volumes of 25 mM sodium phosphate buffer (pH 7.0) containing 1% sodium cholate, 5 mM EDTA and 1 mM 2-mercaptoethanol, and centrifuged at 100,000xg  for 1 h. Glucocerebrosidase was  partially purified and delipidated by ammonium sulfate fractionation, acid precipitation at pH 5.35 and butanol extraction as described by Reddy et al.³) with modifications. The      supernatant obtained above was adjusted to 30% saturation with ammonium sulfate.   The suspension was gently stirred for 30 min and centrifuged at 10,000xg for 1 h. This supernatant was adjusted to pH 5.35 by addition of 1 M citric acid, and the suspension was centrifuged at 10,000xg for 1 h.  The acid precipitate was suspended in 50 mM sodium phosphate buffer (pH 5.0) containing 25 mM citric acid, 5 mM EDTA and 1 mM 2-mercaptoethanol.   This suspension was brought to a concentration of 20% (v/v) butanol by slow addition of 1-butanol, stirred for 30 min, and centrifuged at 20,000g for 30 min.    The lower aqueous layer was removed and mM dialyzed overnight against 50 phosphate/citric acid buffer (pH 6.5) containing 0.02% Triton X-100. Enzyme assays  The standard assay mixture with artificial substrate contained 5 mM MUG in 0.1 M sodium phosphate buffer (pH 5.5), 0.15% Triton X-100 and 0.125% sodium taurocholate.  Time courses  of  the  enzyme-catalyzed hydrolysis of MUG were measured at 30°C.  The reaction was initiated by addition of 100 µl of the enzyme solution to 900 µl of the substrate solution.   The aliquots (200 μl) were removed at appropriate intervals and mixed with 50 mM NaOH (1 ml) to inactivate the enzyme.  The amount of produced 4-methylumbeliferon was determined by fluorescence intensity exited at 340 nm. The fluorescence intensity was measured Union  RS-401  fluorescence using spectrophotometer.
Protein determination.
Protein concentration was determined by the method of Bradford 7 using bovine serum albumin as standard. Molecular weight determination .   The purity of preparations and molecular weight were determined by SDS-10% polyacrylamide   gel    electrophoresis. Reference proteins used as molecular weight standards were phosphorylase b, 97,400;     serum     albumin,      66,200; ovalbumin, 45,000; carbonic anhydrase, 31,000 and trypsin inhibitor, 21,500. Kinetic studies .  Inhibitor constant, Ki, of Dextran sulfate was determined by standard assay using MUG as substrate in the presence of various concentrations of The Ki value was determined inhibitor. by Dixon plots.
Results
Purification of enzyme. Glucocere- brosidase was partially purified from bovine brain with cholate extraction followed ammonium sulfate by fractionation, acid precipitation at pH 5.35 and butanol extraction. Upon SDS-10% polyacrylamide gel electrophoresis, the final preparation of main gave  two bands enzyme corresponding to apparent molecular weights of 65,000 and 55,000 (Fig. 1). The final enzyme preparation had the specific activity, determined by using the standard assay, of 80 nmol/min/mg protein. Addition  of  21  μM conduritol-B-epoxide, a specific active site inhibitor of glucocerebrosidase 8 ), to the assay mixture resulted in 90% inhibition of the β-glucosidase activity. enzyme hydrolyzed a natural The substrate, glucocerebroside, but was inactive for cellobiose and p-nitrophenyl-β-D-glucopyranoside. These results indicated that the activity measured in this study was due to glucosylceramide-β-glucosidase, and not to some other β-glucosidases.   The final enzyme preparation was used for the subsequent characterization.

Effect of detergent on enzyme activity .
In studies with membrane-bound enzyme, lipid or detergent is usually added for reconstitution of enzyme activity.   Table 1 shows β-glucosidase activity assayed in the presence and absence of detergents (0.15% Triton X-100 and 0.125% sodium taurocholate) for enzyme preparation samples obtained after each purification The enzyme activity was step. increased by addition of Triton X-100 Figure   2 and sodium taurocholate. shows the change of glucocerebrosidase activity of the final preparation by addition of Triton X-100 and sodium taurocholate (1:1).  High activity was obtained when 0.05%—0.2% detergent was added, but if the concentration in the assay exceeded 0.5%, the enzyme activity The enzyme was decreased. was of assayed below in the presence The enzyme activity was measured by assay  as  described standard under Materials and methods, but with the concentration of detergents (Triton X-100 and sodium taurocholate The maximal activity was taken 0.125% sodium taurocholate, under which the highest enzyme activity was obtained as shown in Fig. 2.  The final enzyme preparation had the specific activity of 80 nmol/min/mg protein in the presence of detergents (Table 1). Inhibition of enzyme by Dextran sulfate. Shafit-Zagardo and Turner 91 reported that the enzyme from human placenta was inhibited by sulfated macromolecules such as Dextran sulfate, and that Dextran sulfate was an effective inhibitor of the enzyme assayed with either natural or artificial substrates. Effects of Dextran sulfate on the bovine brain glucocerebrosidase-catalyzed hydrolysis of MUG were examined. Dextran sulfate noncompetitively inhibited the reaction.    The Ki value was estimated to be 1.5 mM.  Effects of substrate concentration, [MUG], in the inhibition of enzyme activity by Dextran sulfate were examined.  The assay mixture contained 5 mg/ml Dextran sulfate and various concentrations of MUG.    As shown in Fig. 3, inhibition by Dextran sulfate was independent of substrate concentration. Effects of pH on the inhibition by in Dextran sulfate were examined phosphate/citrate    buffer. The assay mixture contained 1 mM MUG, and 5 mg/ml Dextran sulfate.  As shown in Fig. 4, inhibition by Dextran sulfate was pH dependent.  Maximal inhibition was observed at pH 4.1. indicated concentrations of MUG in the absence (open bars) and presence (closed bars) of 5 mg/ml Dextran sulfate.
Discussion
Glucocerebrosidase is localized in the lysosome and is a membrane-bound Human skin fibroblast enzyme. glucocerebrosidase requires detergents for its solubilization 10 .  Thus, cholate was used for extraction in the initial step of purification of this enzyme.  The enzyme activity was increased by addition of Triton X-100 and sodium taurocholate (Table 1 and Fig. 2).  The enzyme was assayed here in the presence of 0.15% Triton X-100 and 0.125% sodium taurocholate. The final preparation of enzyme gave two main bands corresponding to apparent molecular weights of 65,000 and 55,000 upon SDS-polyacrylamide gel electrophoresis (Fig. 1).  These two bands  may  be  the  bands  of glucocerebrosidase, and the observed heterogeneity in apparent molecular weight may be due to the presence of complex carbohydrate chains in the enzyme. From the deduced amino acid sequence, the molecular weight of the mature human glucocerebrosidase is be 55,384 without calculated  to or glycosylation carboxyl-terminal processing 4,5) . Placental  glucocere- brosidase contains approximately 7% carbohydrate.  The enzyme contains high mannose-type oligosaccharides as well as triantennary and biantennary complex- type oligosaccharides¹¹).  The potential glycosylation sites (Asn-X-Ser/Thr) are identified in the Of nucleic acid sequence of cDNA. these five possible sites for N-linked are glycosylation, the four sites the glycosylated expression experiments of the cDNAs in insect (Sf9) and COS-1 cells 12 ) Glucocerebrosidase from human placenta is inhibited by Dextran sulfate9). Dextran sulfate also inhibited the bovine brain         enzyme         noncompetitively. Inhibition was independent of substrate concentration, but was pH dependent, with a maximal inhibition at pH 4.1. The inhibition by sulphated macro- molecule could be attributed to the interaction of this compound with the enzyme or with the activators which are essential for activity of the solubilized enzyme 13 ).  However, the binding of human  β-glucosidase to immobilized Dextran sulphate and its specific elution with sodium taurocholate 9 , argues for a direct interaction between the inhibitor and the enzyme itself.