lournal ofNeurochemiscry. 1977. Val. 28, pp. 423427. Pergarnan Press. Printed in Great Britain
PURIFICATION AND PROPERTIES OF HUMAN BRAIN a-L-FUCOSIDASE J. A. ALHADEFFand A . J. JANOWSKY' Depariments of Neurosciences and 'Biology, University of California, San Diego, La Jolla, CA 92093, U.S.A. (Received 16 June 1976. Accepted 4 August 1976) Abstract-Human brain a-L-fucosidase has been extracted and the soluble portion has been purified 9388-fold with 25% yield by a two-step affinity chromatographic procedure utilizing agarose-epsilonaminocaproyl-fucosamine. Isoelectric focusing revealed that all seven isoelectric forms of the enzyme were purified. Trace amounts of eight glycosidases, with hexosaminidase being the largest contaminant (1% by activity)were found in the purified a-L-fucosidase preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a single subunit of molecular weight 51,OOO f 2500. The purified enzyme has a pH optimum of 4.7 with a suggested second optimum of 6.6. The apparent Michaelis constant and maximal velocity of the purified enzyme with respect to the p-nitrophenyl substrate are 0.44 mM and 10.7 pmol/min/mg protein, respectively. Agz+ and Hg2+ completely inactivated the enzyme at concentrations of 0.14.3 mM. Antibodies made previously against purified human liver a-L-fucosidase cross-reacted with the purified brain a-L-fucosidase and gave a single precipitin line coincident with that from purified liver a-L-fucosidase. From all our studies it appears that at least the soluble portion of brain a-L-fucosidase is identical to human liver a-L-fucosidase.
FUCOSIDOSIS is a rare neurovisceral storage disease due to absence of activity of the lysosomal enzyme a-L-fucosidase (fucoside-fucohydrolase EC 3.2.1.51) (DURAND et al., 1969). The disease involves storage of fucose-containing molecules in visceral tissues and brain (VAN HOOF, 1973) and is characterized by
appears to be identical to the previously characterized liver enzyme (ALHADEFF et a/., 197%). To our knowledge this is the first report on the purification and characterization of human brain a-tfucosidase.
MATERIALS AND METHODS neurological involvement including progressive menGeneral. Protein was determined by the Lowry method tal and motor retardation, decorticate rigidity and progressive spasticity (DURAND et al., 1969). In a pre- (LOWRYet al., 1951) using human serum albumin (Sigma Chemical Co., St. Louis, MO) as standard. All procedures vious communication we studied the multiple forms were carried out at 04°C unless otherwise stated. Enzyme of human liver a-c-fucosidase with regard to its solutions were concentrated by ultrafiltration using greatly diminished activity in the liver of a fucosidosis Amicon concentrators with UM-10 Diaflo membranes at patient (ALHADEFFet al., 1974b). We have also puri- 5&70 psi, Agarose-epsilon-aminocaproyl-fucosamine(lot fied human liver a-tfucosidase to apparent homo- numbers AF-5, 7, 10 and 11) was purchased from Milesgeneity by an affinity chromatographic procedure and Yeda Ltd (Elkhart, IND.). a-L-Fucosidase activity was characterized the purified enzyme kinetically and assayed either by using pnitrophenyl-a-L-fucopyranoside (Kochimmunochemically (ALHADEFFet al., 1975a). In our (Sigma) or 4-methylumbelliferyl-a-~-fucopyranoside ongoing studies of fucosidosis and a-L-fucosidase, we Light, Ltd., Colnbrook, Buckinghamshire, England) as previously reported (ALHADEFF et al., 19746; ZIELKEet al,, have turned to a study of human brain a-L-fucosidase. 1972). Absorbances were read at 420nm on a Gilford Knowledge of the brain enzyme is not only important 24004 spectrophotometer and fluorescence was read on for a n understanding of the molecular defect in fuco- a Turner Fluorometer using an absorption wavelength of sidosis but also for a general understanding of the 356nm and an emission wavelength of 450nm. Enzyme catabolism of fucose-containing glycoproteins and assays were performed under conditions where activity was & MAR- linear with the amount of protein and time of incubation glycopeptides found in brain (MARGOLIS OOLIS,1972; G L A ~ etWal., 1972; DUTTON & BAR- except in the isoelectric focusing experiments. A unit of ONDES, 1970; ZATZ & BARONDES, 1970; BRUNN- activity is defined as the amount of enzyme that hydrolyzes 1 nmol of substrate per min at 37°C. GRABER, 1970). Few investigators have concerned Polycrylamide gels. The presence of subunits was deterthemselves with brain a-L-fucosidase and these studies mined in 10% polyacrylamide gels (0.6 x 6.0 cm) containhave been done in rat brain (QUARLES & BRADY, ing 0.1% sodium dodecyl sulfate according to the general 1970; BOSMANN& HEMSWORTH, 1971). In the present method of Laemmli (LAEMMLI, 1970). The a-L-fucosidase communication we describe the affinity chromato- (19.6 pg protein) was subjected to electrophoresis after graphic purification and characterization of the sol- treating with 1% sodium dodecylsulfate, 5% 8-mercapuble portion of human brain a-L-fucosidase. From the toethanol, and 8 M-Urea in 62.5 mM-Tris-HC1, pH 6.8, in properties studied, soluble brain a-L-fucosidase a boiling water bath for 5min. Electrophoresis was at 423
J. A. ALHADEFFand A. J. JANOWSKY
424
2mA/tube for 5 h at room temperature. The gels were stained with 0.2% Coomassie blue in 10% acetic acid (v/v) and 50% methanol (v/v). The gels were destained in a transverse destainer with 10% acetic acid. The following proteins were used for molecular weight determination: bovine serum albumin (67,000), ovalbumin (45,000), soy bean agglutinin (30,000) and cytochrome C (1 2,300). Isoelectric focusing. Isoelectric focusing was performed using an LKB 8101 (IlOml) apparatus as previously described (ALHADEFF er al., 197%). One percent ampholytes (pH 5-8) were used in a gradient of 0 to 67% (w/v) sucrose. The starting amperage was 2.5 mA and 350450 V. Electrofocusing was conducted for 6 M 6 h, after which 0.7 ml fractions were collected. The pH of each fraction was determined at 0-2°C with a Beckman digital pH meter. Ten microliter samples were assayed for 1 h with the 4-methylumbelliferyl substrate and 100 p1 samples were assayed for 72 min with the p-nitrophenyl substrate. Isoelectric focusing was performed on concentrated crude human brain supernatant fluid (125 units and 233 mg protein) and on the purified brain a-L-fucosidase (270 units and 39 pg protein). The purified fucosidase was focused in the presence of 3mg human serum albumin to stabilize the enzyme. Kinetic studies. An apparent Michaelis constant (K,) and maximal velocity (Vmex)was determined for the purified enzyme graphically by the Lineweaver-Burk method (LINEWEAVER & BURK,1934) using the p-nitrophenyl substrate. The purified a-L-fucosidase (0.2 pg protein) was incubated in substrates of varying concentrations containing human serum albumin (3.0 mg/ml) in 100 mM-Citratesodium citrate buffer (pH 5.0). All samples were incubated in duplicate for 20 min at 37°C. The pH optimum of the purified a-L-fucosidase was determined as previously described (ALHADEFF et al., 1975~)using 0.2 pg enzyme-protein per assay. All incubations were carried out in duplicate for 20min at 37°C. Various salts (Ag+, Hg2+,Ca2+,Mn2+)were incubated with 0.2pg of the purified fucosidase to determine their effect on enzymatic activity. Incubations were carried out in duplicate for 8 min at 37°C. Immunochemical studies. The IgG fraction of antiserum to purified human liver a-L-fucosidase (ALHADEFF et a/., 19751) was run in double immunodiffusion experiments on Hyland immunoplates against both pure liver a-L-fucosidase (2 pg protein) and the purified brain a-L-fucosidase (1 pg protein). The plates were spotted with samples, placed at 0-2°C and read after 48 h. Tissue extraction. In a preliminary experiment 20.5 g (wet weight) of frozen human cerebrum (white and gray matter) was cut into small pieces and homogenized for 1.5min at low speed in a Waring Blendor in 82ml of 1 0 m ~ pH , 5.5 NaH,PO, containing 0.2% (w/v) NaN,. The crude homogenate was assayed for a-L-fucosidase and centrifuged for 20min at 48,250g. The pellet was resusTABLE1. DATAON Brain number
pended in the extraction buffer and the resuspended pellet and supernatant were assayed for a-L-fucosidase. The preliminary experiment was performed on four different human brains to determine the distribution of a-L-fucosidase between the soluble and pellet fractions. Whole human brain (1039 g, wet weight) from an autopsied individual who appeared normal on gross pathological examination (brain 4, Table 1) was cut into small pieces and extracted in 4156 ml of 10 mM, pH 5.5 NaH,P04 containing 0.02% (w/v) NaN, in a Waring Blendor for five, 0.5 min intervals at low speed. The crude homogenate was centrifuged for 20 min at 23,500g.The supernatant fluid was used for purifying a-L-fucosidase. Purijication of a-L-fucosidnse. Brain a-L-fucosidase was purified by the same affinity chromatographic technique previously used for liver a-L-fucosidase (ALHADEFF et al., 1975~).Brain supernatant (3510 ml) containing 7301 units of a-L-fucosidase and 9.09 g protein was applied to a column (2.8 x 35 cm) of agarose-epsilon-aminocaproylfucosamine (affinity column I) at a rate of 60 ml/h at room temperature. The column was subsequently washed with 7.5 1 of 10 mM NaH,PO, (pH 5.5) containing 0.02% (w/v) NaN, until the absorbance at 280nm was 0.013. The enzyme was then eluted from the column with the wash buffer containing 100 mM-L-fucose. Those fractions which contained activity were combined, concentrated and assayed for a-L-fucosidase in the presence of human albumin (3 mg/ml) to stabilize the enzyme. The partially purified enzyme preparation was dialyzed for a total of 2.25 h against three changes (2 l/change) of 10 mM-NaH,PO, (pH 5.5) containing 0.02% (w/v) NaN, to remove the L-fucose. The dialyzed sample (7188 units and 1.28g protein) was put on a second column (1 x 27cm) of agarose-epsilonaminocaproyl-fucosamine (affinity column 11) and eluted as in the first column. The fractions containing fucosidase activity were combined, concentrated and assayed for a-Lfucosidase in the presence of human albumin (3mg/ml). This purified preparation of brain a-L-fucosidase (postaffinity column I1 sample) was assayed for contaminating glycosidases as previously described (ALHADEFFet al., 1975a)and used for the subunit, kinetic, isoelectric focusing and immunochemical studies. RESULTS
Table 1 summarizes the data on the four human brains used in this study. It can be seen that the range of specific activities is relatively large: 0.80-3.2 nmol/ min/mg protein. In the four brains studied the supernatant contained approximately 69% of the activity present in the homogenate. It was this soluble portion of brain cr-L-fucosidase which was purified by affinity chromatography.
HUMAN BRAINS STUDIED
Percentage activity in supernatant
Percent activity in pellet
Units* activity per ml supernatant
Supernatant specific activity (units/mg protein)
71 70 67 68
29 30 33 32
3.1 3.2 4.8 2.1
2.1 3.2 0.8
~~
I I1 111
1v
*A
unit of activity is the amount of enzyme that hydrolyzes 1 nmol substrate/min at 37°C.
1.5
Human brain a-L-fucosidase OF SOLUBLE TABLE2. PURIFICATION
Fraction
425
HUMAN BRAIN
a-L-fucosidase
Total protein (mg)
Total activity (units)
Specific activity (nmol/min/mg)
Purification factor
Over-all yield
909 1 1.28 0.24
7301 7188 1813
0.80 5610 7510
7012 9388
98% 25%
23,500 g supernatant which was put on affinity column I Postaffinity column I Postaffinity column I1
Table 2 summarizes the data on the affinity chromatographic purification of the soluble portion of human brain a-L-fucosidase. The two-step procedure results in 9388-fold purification with a 25% yield. Since the elution profiles from the two affinity columns are the same as for liver a-L-fucosidase (ALHADEFFet al., 1975a), they are not included in this paper. The purified brain fucosidase had only trace amounts of other glycosidase activity. Hexosaminidase is the major contaminant (1% by activity) in this preparation. Figure 1 depicts the results of isoelectric focusing on crude supernatant brain a-L-fucosidase using the 4-methylumbelliferyl substrate. This isoelectric profile shows seven forms of fucosidase. The most neutral form is a very small proportion of the total activity and the two most acidic forms are not well resolved. The purified fucosidase showed a similar isoelectric profile with all seven forms of the enzyme present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified a-L-fucosidase (postaffinity column I1 sample) indicated the presence of a single subunit. Standard proteins were subjected to electrophoresis on a sodium dodecylsulfate gel and the molecular weight of the subunit was graphically determined to be 51,000 f 2500 by a plot of log molecular weight versus protein migration distance in the gel. An apparent K , and V,, were determined on the purified brain a-L-fucosidase for the p-nitrophenyl substrate. The Lineweaver-Burk double reciprocal
(%)
plot yielded a straight line with an apparent K, of 0.44 mM and V,, of 10.7 pmol/min/mg protein. The pH optimum curve of the purified a-L-fucosidase using the p-nitrophenyl substrate is shown in Fig. 2. The optimum is centered around pH 4.7 with a second optimum suggested by a shoulder at pH 6.6. A similar pH curve was obtained with the 4-methylumbelliferyl substrate. Most salts had little or no effect on brain fucosidase activity. Hg2+ and Ag' (0.1-0.3 m ~completely ) inactivated enzymatic activity whereas the presence of 1.1 m-dithiothreitol protected against this inactivation. The IgG fraction of antiserum made against pure et al., 1975a) was used liver a-L-fucosidase (ALHADEFF in double immunodiffusion experiments against both the pure liver enzyme and the highly purified brain fucosidase. Figure 3 indicates that the antibody (IgG fraction) against liver a-L-fucosidase crossreacts with the purified brain a-L-fucosidase to give a single precipitin line coincident with that from liver. DISCUSSION
In this study we have utilized an affinity chromatographic method previously used to purify liver a-Lfucosidase (ALHADEFF et al., 197.5~)to purify the soluble portion (69%) of human brain a-L-fucosidase. The two-step method resulted in a 9388-fold purification with 25% yield. We feel that this yield is lower than that for human liver (66%) because of the low protein
m
i
f 0 PH
FIG.1. Isoelectric focusing (PH 5-8) of concentrated crude soluble human brain a-L-fucosidase. See Materials and Methods for details.
1
2
5
4
5
6
7
8
PH
FIG. 2. pH optimum curve for purified human brain a-Lfucosidase using p-nitrophenyl-a-L-fucopyranosideas substrate. See Materials and Methods for details.
J. A. ALHADEFFand A. J. JANOWSKY
426
concentration (0.2 mg/ml) in the postaffinity column line coincident with that from liver (Fig. 3). This indi11 preparation. We have previously noted that pure cates that the soluble brain fucosidase is antigenically liver fucosidase is labile at low protein concentrations similar, if not identical, to the liver fucosidase. From the results obtained in this investigation it (0.4mg/ml) (ALHADEWet al., 1975~).A larger scale preparation should result in an increased yield. Isoe- appears that the soluble brain a-L-fucosidase is identilectric focusing revealed the presence of seven isoen- cal to the liver enzyme. As yet we are not sure if zymes in both the crude and purified brain fucosidase. the 31% of the brain fucosidase found in the pellet Furthermore, .the pattern of isoenzymes (number of after centrifugation is the same as the soluble portion. forms, relative amounts and isoelectric points) in Studies are currently in progress to solubilize and brain is nearly identical to that for liver fucosidase characterize this pellet enzyme. et al., 1975a, b). A previous study demon(ALHADEFF strated only five a-L-fucosidase isoenzymes in human et al., 1976). As it was in the purifi- Acknowledgements-We gratefully acknowledge the excelbrain (DIMATTEO and we thank Dr. cation of placental and liver a-L-fucosidase (ALHADEFF lent technical assistance of G. CIMINO J. S. OBRIENfor providing laboratory space and equipet a!., 1974a, 1975a), hexosaminidase was the major ment for carrying out the work. This work was supported contaminating glycosidase in the purified brain a - ~ - in part by National Foundation-March of Dimes Grant fucosidase. 1-395. Treatment of the purified brain fucosidase with 1% sodium dodecyl sulfate, 5% p-mercaptoethanol and REFERENCES 8 M-urea followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of ALHADEFFJ. A., MILLER A. L. & OBRIENJ. S. (1974a) a single subunit with 51,000 molecular weight. This Analyt. Biochem. 60,424430. is identical to the result we obtained for the liver ALHADEFF J. A., MILLER A. L., WENGERD. A. & OBRIEN a-L-fucosidase subunit (ALHADEFFet al., 1975a) but J. S. (1974b) Clin. Chim. Acta 57, 307-313. different from the two, non-identical subunits (49,700 ALHADEFF J. A., MILLERA. L., WENAASH., VEDVICK T. and 53,700 molecular weights) found for rat epididy& OBRIEN J. S. (1975a) J. biol. Chem. 250, 7106-7113. ALHADEFFJ. A., TENNANT L. & OBRIENJ. S . (19756) Deul ma1 a-L-fucosidase (CARLSEN & PIERCE,1972). Biol. 47, 319-324. Kinetic studies also indicate that the soluble brain B. A. (1971) Biochim. biofucosidase is the same as the liver enzyme. Ag+ and BOSMANNH. B. & HEMSWORTH phys. Acta 242, 152-171. HgZ (0.14.3 mM) completely inactivates the brain BRUNNGRABER E. G. (1970) in Protein Metabolism of the et al., enzyme as it did the liver enzyme (ALHADEFF Nervous System (LAITHA A., ed), Chap. 19, pp. 383407. 1975a) and dithiothreitol at 1.1 mM protects both the Plenum Press, New York. brain and liver fucosidase against inactivation. It has CARLSEN R. B. & PIERCE J. G. (1972) J . bid. Chem. 247, also been found that HgZ inhibits rat brain a-L-fuco23-32. sidase (BOSMANN & HEMSWORTH, 1971). The apparent DIMATTEO G., ORFEO M. S . & ROMEOG. (1976) Biochim. biophys. Acta 429. 527-537. K , for the p-nitrophenyl substrate is the same for P., BORRONE C. & DELLACELLAG. (1969) J. the purified brain and liver fucosidases (0.44m~and DURAND Pediat. 75, 665-674. 0.43mM, respectively) but lower than the 1.33 mM G. R. & BARONDESS. H. (1970) J . Neurochem. found for rat brain fucosidase (BOSMANN& H ~ s - DUTTON 17. 913-920. WORTH, 1971). The V,,, of the liver enzyme is slightly GLASGOW M. S., QUARLES R. H. & GROLLMAN S. (1972) higher than that of the brain enzyme, 19.6 vs. Brain Res. 42, 129-137. 10.7 pmol/min/mg protein, respectively (ALHADEFFet LAEMMLI U.K. (1970) Nature, Lond. 227, 680-685. al., 19750). The pH optimum profile (Fig. 2) for the LINEWFAVER H. & BURKD. (1934) J . Am. Chem. SOC.56. brain fucosidase indicates a major optimum at 4.7 658-666. with a suggested second optimum of 6.6 and is nearly LOWRY 0.H., ROSEBROUGH N. J., FARRA. L. & RANDALL R. J. (1951) J. biol. Chem. 193, 265-275. identical to the pH curve for liver fucosidase: major R. K. & MARGOLIS R. U. (1972) J. Neurochem. optimum at pH 4.6 with suggested second optimum MARGOLIS 19, 1023-1030. at 6.5 (ALHADFFF et al., 19751). Rat brain a-L-fucosiR. H. & BRADYR. 0. (1970) J. Neurochem. 17. dase has been shown to have a pH optimum of 4.3 QUARLE~ 801-807. (BOSMANN & HEMSWORTH, 1971). VAN HOOFF. (1973) in Lysosomes and Storage Diseases The IgG fraction of antiserum made against pure (HERS H. G. & VAN HOOFF., eds) Chap. 10, pp. 277-290. liver a-L-fucosidase(ALHADEFFet al., 1975a) was used Academic Press, New York. to determine if the brain a-L-fucosidase is antigeni- ZATZ M. & BARONDESS. H. (1970) J . Neurochern. 17. cally similar to the liver enzyme. Antibody (IgG frac157-1 63. tion) against liver a-L-fucosidase cross-reacted with ZIELKE K., OKADA S. & O’BRIENJ. S. (1972) J. Lab. Clin. Med. 79. 164169 the purified brain enzyme and gave a single precipitin +
421
FIG. 3. Double immunodiffusion (Ouchtcrlony) plate of anti-liver-z-L-fucosidase antiserum (IgG fraction; 14Opg protein in center well) against purified human brain r-L-fucosidase (A, I p g protein) and against purified human liver fucosidase (B, 2 p g protein).
PURIFICATION AND PROPERTIES OF HUMAN BRAIN a-L-FUCOSIDASE J. A. ALHADEFFand A . J. JANOWSKY' Depariments of Neurosciences and 'Biology, University of California, San Diego, La Jolla, CA 92093, U.S.A. (Received 16 June 1976. Accepted 4 August 1976) Abstract-Human brain a-L-fucosidase has been extracted and the soluble portion has been purified 9388-fold with 25% yield by a two-step affinity chromatographic procedure utilizing agarose-epsilonaminocaproyl-fucosamine. Isoelectric focusing revealed that all seven isoelectric forms of the enzyme were purified. Trace amounts of eight glycosidases, with hexosaminidase being the largest contaminant (1% by activity)were found in the purified a-L-fucosidase preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a single subunit of molecular weight 51,OOO f 2500. The purified enzyme has a pH optimum of 4.7 with a suggested second optimum of 6.6. The apparent Michaelis constant and maximal velocity of the purified enzyme with respect to the p-nitrophenyl substrate are 0.44 mM and 10.7 pmol/min/mg protein, respectively. Agz+ and Hg2+ completely inactivated the enzyme at concentrations of 0.14.3 mM. Antibodies made previously against purified human liver a-L-fucosidase cross-reacted with the purified brain a-L-fucosidase and gave a single precipitin line coincident with that from purified liver a-L-fucosidase. From all our studies it appears that at least the soluble portion of brain a-L-fucosidase is identical to human liver a-L-fucosidase.
FUCOSIDOSIS is a rare neurovisceral storage disease due to absence of activity of the lysosomal enzyme a-L-fucosidase (fucoside-fucohydrolase EC 3.2.1.51) (DURAND et al., 1969). The disease involves storage of fucose-containing molecules in visceral tissues and brain (VAN HOOF, 1973) and is characterized by
appears to be identical to the previously characterized liver enzyme (ALHADEFF et a/., 197%). To our knowledge this is the first report on the purification and characterization of human brain a-tfucosidase.
MATERIALS AND METHODS neurological involvement including progressive menGeneral. Protein was determined by the Lowry method tal and motor retardation, decorticate rigidity and progressive spasticity (DURAND et al., 1969). In a pre- (LOWRYet al., 1951) using human serum albumin (Sigma Chemical Co., St. Louis, MO) as standard. All procedures vious communication we studied the multiple forms were carried out at 04°C unless otherwise stated. Enzyme of human liver a-c-fucosidase with regard to its solutions were concentrated by ultrafiltration using greatly diminished activity in the liver of a fucosidosis Amicon concentrators with UM-10 Diaflo membranes at patient (ALHADEFFet al., 1974b). We have also puri- 5&70 psi, Agarose-epsilon-aminocaproyl-fucosamine(lot fied human liver a-tfucosidase to apparent homo- numbers AF-5, 7, 10 and 11) was purchased from Milesgeneity by an affinity chromatographic procedure and Yeda Ltd (Elkhart, IND.). a-L-Fucosidase activity was characterized the purified enzyme kinetically and assayed either by using pnitrophenyl-a-L-fucopyranoside (Kochimmunochemically (ALHADEFFet al., 1975a). In our (Sigma) or 4-methylumbelliferyl-a-~-fucopyranoside ongoing studies of fucosidosis and a-L-fucosidase, we Light, Ltd., Colnbrook, Buckinghamshire, England) as previously reported (ALHADEFF et al., 19746; ZIELKEet al,, have turned to a study of human brain a-L-fucosidase. 1972). Absorbances were read at 420nm on a Gilford Knowledge of the brain enzyme is not only important 24004 spectrophotometer and fluorescence was read on for a n understanding of the molecular defect in fuco- a Turner Fluorometer using an absorption wavelength of sidosis but also for a general understanding of the 356nm and an emission wavelength of 450nm. Enzyme catabolism of fucose-containing glycoproteins and assays were performed under conditions where activity was & MAR- linear with the amount of protein and time of incubation glycopeptides found in brain (MARGOLIS OOLIS,1972; G L A ~ etWal., 1972; DUTTON & BAR- except in the isoelectric focusing experiments. A unit of ONDES, 1970; ZATZ & BARONDES, 1970; BRUNN- activity is defined as the amount of enzyme that hydrolyzes 1 nmol of substrate per min at 37°C. GRABER, 1970). Few investigators have concerned Polycrylamide gels. The presence of subunits was deterthemselves with brain a-L-fucosidase and these studies mined in 10% polyacrylamide gels (0.6 x 6.0 cm) containhave been done in rat brain (QUARLES & BRADY, ing 0.1% sodium dodecyl sulfate according to the general 1970; BOSMANN& HEMSWORTH, 1971). In the present method of Laemmli (LAEMMLI, 1970). The a-L-fucosidase communication we describe the affinity chromato- (19.6 pg protein) was subjected to electrophoresis after graphic purification and characterization of the sol- treating with 1% sodium dodecylsulfate, 5% 8-mercapuble portion of human brain a-L-fucosidase. From the toethanol, and 8 M-Urea in 62.5 mM-Tris-HC1, pH 6.8, in properties studied, soluble brain a-L-fucosidase a boiling water bath for 5min. Electrophoresis was at 423
J. A. ALHADEFFand A. J. JANOWSKY
424
2mA/tube for 5 h at room temperature. The gels were stained with 0.2% Coomassie blue in 10% acetic acid (v/v) and 50% methanol (v/v). The gels were destained in a transverse destainer with 10% acetic acid. The following proteins were used for molecular weight determination: bovine serum albumin (67,000), ovalbumin (45,000), soy bean agglutinin (30,000) and cytochrome C (1 2,300). Isoelectric focusing. Isoelectric focusing was performed using an LKB 8101 (IlOml) apparatus as previously described (ALHADEFF er al., 197%). One percent ampholytes (pH 5-8) were used in a gradient of 0 to 67% (w/v) sucrose. The starting amperage was 2.5 mA and 350450 V. Electrofocusing was conducted for 6 M 6 h, after which 0.7 ml fractions were collected. The pH of each fraction was determined at 0-2°C with a Beckman digital pH meter. Ten microliter samples were assayed for 1 h with the 4-methylumbelliferyl substrate and 100 p1 samples were assayed for 72 min with the p-nitrophenyl substrate. Isoelectric focusing was performed on concentrated crude human brain supernatant fluid (125 units and 233 mg protein) and on the purified brain a-L-fucosidase (270 units and 39 pg protein). The purified fucosidase was focused in the presence of 3mg human serum albumin to stabilize the enzyme. Kinetic studies. An apparent Michaelis constant (K,) and maximal velocity (Vmex)was determined for the purified enzyme graphically by the Lineweaver-Burk method (LINEWEAVER & BURK,1934) using the p-nitrophenyl substrate. The purified a-L-fucosidase (0.2 pg protein) was incubated in substrates of varying concentrations containing human serum albumin (3.0 mg/ml) in 100 mM-Citratesodium citrate buffer (pH 5.0). All samples were incubated in duplicate for 20 min at 37°C. The pH optimum of the purified a-L-fucosidase was determined as previously described (ALHADEFF et al., 1975~)using 0.2 pg enzyme-protein per assay. All incubations were carried out in duplicate for 20min at 37°C. Various salts (Ag+, Hg2+,Ca2+,Mn2+)were incubated with 0.2pg of the purified fucosidase to determine their effect on enzymatic activity. Incubations were carried out in duplicate for 8 min at 37°C. Immunochemical studies. The IgG fraction of antiserum to purified human liver a-L-fucosidase (ALHADEFF et a/., 19751) was run in double immunodiffusion experiments on Hyland immunoplates against both pure liver a-L-fucosidase (2 pg protein) and the purified brain a-L-fucosidase (1 pg protein). The plates were spotted with samples, placed at 0-2°C and read after 48 h. Tissue extraction. In a preliminary experiment 20.5 g (wet weight) of frozen human cerebrum (white and gray matter) was cut into small pieces and homogenized for 1.5min at low speed in a Waring Blendor in 82ml of 1 0 m ~ pH , 5.5 NaH,PO, containing 0.2% (w/v) NaN,. The crude homogenate was assayed for a-L-fucosidase and centrifuged for 20min at 48,250g. The pellet was resusTABLE1. DATAON Brain number
pended in the extraction buffer and the resuspended pellet and supernatant were assayed for a-L-fucosidase. The preliminary experiment was performed on four different human brains to determine the distribution of a-L-fucosidase between the soluble and pellet fractions. Whole human brain (1039 g, wet weight) from an autopsied individual who appeared normal on gross pathological examination (brain 4, Table 1) was cut into small pieces and extracted in 4156 ml of 10 mM, pH 5.5 NaH,P04 containing 0.02% (w/v) NaN, in a Waring Blendor for five, 0.5 min intervals at low speed. The crude homogenate was centrifuged for 20 min at 23,500g.The supernatant fluid was used for purifying a-L-fucosidase. Purijication of a-L-fucosidnse. Brain a-L-fucosidase was purified by the same affinity chromatographic technique previously used for liver a-L-fucosidase (ALHADEFF et al., 1975~).Brain supernatant (3510 ml) containing 7301 units of a-L-fucosidase and 9.09 g protein was applied to a column (2.8 x 35 cm) of agarose-epsilon-aminocaproylfucosamine (affinity column I) at a rate of 60 ml/h at room temperature. The column was subsequently washed with 7.5 1 of 10 mM NaH,PO, (pH 5.5) containing 0.02% (w/v) NaN, until the absorbance at 280nm was 0.013. The enzyme was then eluted from the column with the wash buffer containing 100 mM-L-fucose. Those fractions which contained activity were combined, concentrated and assayed for a-L-fucosidase in the presence of human albumin (3 mg/ml) to stabilize the enzyme. The partially purified enzyme preparation was dialyzed for a total of 2.25 h against three changes (2 l/change) of 10 mM-NaH,PO, (pH 5.5) containing 0.02% (w/v) NaN, to remove the L-fucose. The dialyzed sample (7188 units and 1.28g protein) was put on a second column (1 x 27cm) of agarose-epsilonaminocaproyl-fucosamine (affinity column 11) and eluted as in the first column. The fractions containing fucosidase activity were combined, concentrated and assayed for a-Lfucosidase in the presence of human albumin (3mg/ml). This purified preparation of brain a-L-fucosidase (postaffinity column I1 sample) was assayed for contaminating glycosidases as previously described (ALHADEFFet al., 1975a)and used for the subunit, kinetic, isoelectric focusing and immunochemical studies. RESULTS
Table 1 summarizes the data on the four human brains used in this study. It can be seen that the range of specific activities is relatively large: 0.80-3.2 nmol/ min/mg protein. In the four brains studied the supernatant contained approximately 69% of the activity present in the homogenate. It was this soluble portion of brain cr-L-fucosidase which was purified by affinity chromatography.
HUMAN BRAINS STUDIED
Percentage activity in supernatant
Percent activity in pellet
Units* activity per ml supernatant
Supernatant specific activity (units/mg protein)
71 70 67 68
29 30 33 32
3.1 3.2 4.8 2.1
2.1 3.2 0.8
~~
I I1 111
1v
*A
unit of activity is the amount of enzyme that hydrolyzes 1 nmol substrate/min at 37°C.
1.5
Human brain a-L-fucosidase OF SOLUBLE TABLE2. PURIFICATION
Fraction
425
HUMAN BRAIN
a-L-fucosidase
Total protein (mg)
Total activity (units)
Specific activity (nmol/min/mg)
Purification factor
Over-all yield
909 1 1.28 0.24
7301 7188 1813
0.80 5610 7510
7012 9388
98% 25%
23,500 g supernatant which was put on affinity column I Postaffinity column I Postaffinity column I1
Table 2 summarizes the data on the affinity chromatographic purification of the soluble portion of human brain a-L-fucosidase. The two-step procedure results in 9388-fold purification with a 25% yield. Since the elution profiles from the two affinity columns are the same as for liver a-L-fucosidase (ALHADEFFet al., 1975a), they are not included in this paper. The purified brain fucosidase had only trace amounts of other glycosidase activity. Hexosaminidase is the major contaminant (1% by activity) in this preparation. Figure 1 depicts the results of isoelectric focusing on crude supernatant brain a-L-fucosidase using the 4-methylumbelliferyl substrate. This isoelectric profile shows seven forms of fucosidase. The most neutral form is a very small proportion of the total activity and the two most acidic forms are not well resolved. The purified fucosidase showed a similar isoelectric profile with all seven forms of the enzyme present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified a-L-fucosidase (postaffinity column I1 sample) indicated the presence of a single subunit. Standard proteins were subjected to electrophoresis on a sodium dodecylsulfate gel and the molecular weight of the subunit was graphically determined to be 51,000 f 2500 by a plot of log molecular weight versus protein migration distance in the gel. An apparent K , and V,, were determined on the purified brain a-L-fucosidase for the p-nitrophenyl substrate. The Lineweaver-Burk double reciprocal
(%)
plot yielded a straight line with an apparent K, of 0.44 mM and V,, of 10.7 pmol/min/mg protein. The pH optimum curve of the purified a-L-fucosidase using the p-nitrophenyl substrate is shown in Fig. 2. The optimum is centered around pH 4.7 with a second optimum suggested by a shoulder at pH 6.6. A similar pH curve was obtained with the 4-methylumbelliferyl substrate. Most salts had little or no effect on brain fucosidase activity. Hg2+ and Ag' (0.1-0.3 m ~completely ) inactivated enzymatic activity whereas the presence of 1.1 m-dithiothreitol protected against this inactivation. The IgG fraction of antiserum made against pure et al., 1975a) was used liver a-L-fucosidase (ALHADEFF in double immunodiffusion experiments against both the pure liver enzyme and the highly purified brain fucosidase. Figure 3 indicates that the antibody (IgG fraction) against liver a-L-fucosidase crossreacts with the purified brain a-L-fucosidase to give a single precipitin line coincident with that from liver. DISCUSSION
In this study we have utilized an affinity chromatographic method previously used to purify liver a-Lfucosidase (ALHADEFF et al., 197.5~)to purify the soluble portion (69%) of human brain a-L-fucosidase. The two-step method resulted in a 9388-fold purification with 25% yield. We feel that this yield is lower than that for human liver (66%) because of the low protein
m
i
f 0 PH
FIG.1. Isoelectric focusing (PH 5-8) of concentrated crude soluble human brain a-L-fucosidase. See Materials and Methods for details.
1
2
5
4
5
6
7
8
PH
FIG. 2. pH optimum curve for purified human brain a-Lfucosidase using p-nitrophenyl-a-L-fucopyranosideas substrate. See Materials and Methods for details.
J. A. ALHADEFFand A. J. JANOWSKY
426
concentration (0.2 mg/ml) in the postaffinity column line coincident with that from liver (Fig. 3). This indi11 preparation. We have previously noted that pure cates that the soluble brain fucosidase is antigenically liver fucosidase is labile at low protein concentrations similar, if not identical, to the liver fucosidase. From the results obtained in this investigation it (0.4mg/ml) (ALHADEWet al., 1975~).A larger scale preparation should result in an increased yield. Isoe- appears that the soluble brain a-L-fucosidase is identilectric focusing revealed the presence of seven isoen- cal to the liver enzyme. As yet we are not sure if zymes in both the crude and purified brain fucosidase. the 31% of the brain fucosidase found in the pellet Furthermore, .the pattern of isoenzymes (number of after centrifugation is the same as the soluble portion. forms, relative amounts and isoelectric points) in Studies are currently in progress to solubilize and brain is nearly identical to that for liver fucosidase characterize this pellet enzyme. et al., 1975a, b). A previous study demon(ALHADEFF strated only five a-L-fucosidase isoenzymes in human et al., 1976). As it was in the purifi- Acknowledgements-We gratefully acknowledge the excelbrain (DIMATTEO and we thank Dr. cation of placental and liver a-L-fucosidase (ALHADEFF lent technical assistance of G. CIMINO J. S. OBRIENfor providing laboratory space and equipet a!., 1974a, 1975a), hexosaminidase was the major ment for carrying out the work. This work was supported contaminating glycosidase in the purified brain a - ~ - in part by National Foundation-March of Dimes Grant fucosidase. 1-395. Treatment of the purified brain fucosidase with 1% sodium dodecyl sulfate, 5% p-mercaptoethanol and REFERENCES 8 M-urea followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of ALHADEFFJ. A., MILLER A. L. & OBRIENJ. S. (1974a) a single subunit with 51,000 molecular weight. This Analyt. Biochem. 60,424430. is identical to the result we obtained for the liver ALHADEFF J. A., MILLER A. L., WENGERD. A. & OBRIEN a-L-fucosidase subunit (ALHADEFFet al., 1975a) but J. S. (1974b) Clin. Chim. Acta 57, 307-313. different from the two, non-identical subunits (49,700 ALHADEFF J. A., MILLERA. L., WENAASH., VEDVICK T. and 53,700 molecular weights) found for rat epididy& OBRIEN J. S. (1975a) J. biol. Chem. 250, 7106-7113. ALHADEFFJ. A., TENNANT L. & OBRIENJ. S . (19756) Deul ma1 a-L-fucosidase (CARLSEN & PIERCE,1972). Biol. 47, 319-324. Kinetic studies also indicate that the soluble brain B. A. (1971) Biochim. biofucosidase is the same as the liver enzyme. Ag+ and BOSMANNH. B. & HEMSWORTH phys. Acta 242, 152-171. HgZ (0.14.3 mM) completely inactivates the brain BRUNNGRABER E. G. (1970) in Protein Metabolism of the et al., enzyme as it did the liver enzyme (ALHADEFF Nervous System (LAITHA A., ed), Chap. 19, pp. 383407. 1975a) and dithiothreitol at 1.1 mM protects both the Plenum Press, New York. brain and liver fucosidase against inactivation. It has CARLSEN R. B. & PIERCE J. G. (1972) J . bid. Chem. 247, also been found that HgZ inhibits rat brain a-L-fuco23-32. sidase (BOSMANN & HEMSWORTH, 1971). The apparent DIMATTEO G., ORFEO M. S . & ROMEOG. (1976) Biochim. biophys. Acta 429. 527-537. K , for the p-nitrophenyl substrate is the same for P., BORRONE C. & DELLACELLAG. (1969) J. the purified brain and liver fucosidases (0.44m~and DURAND Pediat. 75, 665-674. 0.43mM, respectively) but lower than the 1.33 mM G. R. & BARONDESS. H. (1970) J . Neurochem. found for rat brain fucosidase (BOSMANN& H ~ s - DUTTON 17. 913-920. WORTH, 1971). The V,,, of the liver enzyme is slightly GLASGOW M. S., QUARLES R. H. & GROLLMAN S. (1972) higher than that of the brain enzyme, 19.6 vs. Brain Res. 42, 129-137. 10.7 pmol/min/mg protein, respectively (ALHADEFFet LAEMMLI U.K. (1970) Nature, Lond. 227, 680-685. al., 19750). The pH optimum profile (Fig. 2) for the LINEWFAVER H. & BURKD. (1934) J . Am. Chem. SOC.56. brain fucosidase indicates a major optimum at 4.7 658-666. with a suggested second optimum of 6.6 and is nearly LOWRY 0.H., ROSEBROUGH N. J., FARRA. L. & RANDALL R. J. (1951) J. biol. Chem. 193, 265-275. identical to the pH curve for liver fucosidase: major R. K. & MARGOLIS R. U. (1972) J. Neurochem. optimum at pH 4.6 with suggested second optimum MARGOLIS 19, 1023-1030. at 6.5 (ALHADFFF et al., 19751). Rat brain a-L-fucosiR. H. & BRADYR. 0. (1970) J. Neurochem. 17. dase has been shown to have a pH optimum of 4.3 QUARLE~ 801-807. (BOSMANN & HEMSWORTH, 1971). VAN HOOFF. (1973) in Lysosomes and Storage Diseases The IgG fraction of antiserum made against pure (HERS H. G. & VAN HOOFF., eds) Chap. 10, pp. 277-290. liver a-L-fucosidase(ALHADEFFet al., 1975a) was used Academic Press, New York. to determine if the brain a-L-fucosidase is antigeni- ZATZ M. & BARONDESS. H. (1970) J . Neurochern. 17. cally similar to the liver enzyme. Antibody (IgG frac157-1 63. tion) against liver a-L-fucosidase cross-reacted with ZIELKE K., OKADA S. & O’BRIENJ. S. (1972) J. Lab. Clin. Med. 79. 164169 the purified brain enzyme and gave a single precipitin +
421
FIG. 3. Double immunodiffusion (Ouchtcrlony) plate of anti-liver-z-L-fucosidase antiserum (IgG fraction; 14Opg protein in center well) against purified human brain r-L-fucosidase (A, I p g protein) and against purified human liver fucosidase (B, 2 p g protein).
