- Email: [email protected]
Isozymes of human α-l -fucosidase detectable by starch gel electrophoresis
29
Clinica Chimica
@ Elsevier
Acta,
Scientific
57 (1974) 29-35 Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6629
ISOZYMES OF HUMAN (Y-L-FUCOSIDASE GEL ELECTROPHORESIS
B.M. TURNER,
N.G. BERATIS,
Division of Medical the City University
(Received
VIRGINIA
DETECTABLE
S. TURNER
BY STARCH
and K. HIRSCHHORN
Genetics, Department of Pediatrics, Mount Sinai School of New York, New York, N.Y. 10029 (U.S.A.)
of Medicine
of
May 14, 1974)
Summary Methods are described for the electrophoresis in starch-gel of human O-Lfucosidase and for the detection of the enzyme using the fluorogenic substrate 4-methylumbelliferyl-a-l,-fucopyranoside. The electrophoretic pattern of the enzyme was examined in leucocytes, serum, cultured fibroblasts and long-term lymphoid cell lines. The enzyme from cultured lymphoid cell lines was found to consist of up to 6 clearly resolved electrophoretic isozymes plus a diffuse, more anodal region of lower staining intensity. The enzyme from leucocytes and cultured skin fibroblasts was less clearly resolved, but these cell types appeared to have components corresponding in mobility to the isozymes of lymphoid lines. In contrast, the enzyme from serum (or plasma) showed only a diffuse region of activity, with an anodal mobility slightly greater than that of the most anodal lymphoid line isozymes. Evidence is presented which indicates that the electrophoretic heterogeneity of a-L-fucosidase is due in part to the binding of sialic acid to the primary gene product. None of the isozymes was detectable in lymphoid cell lines, serum or cultured fibroblasts from a patient with fucosidosis, an inborn error of metabolism.
Introduction The enzyme oc-L-fucosidase has been detected in a wide range of human tissues [l-3] by assay methods employing the artificial substrate p-nitrophenyl fucoside. The properties of the human enzyme have not yet been extensively studied, but evidence has been presented that human liver contains at least two forms of a-L-fucosidase which differ in charge, molecular weight and thermostability [2]. Interest in the enzyme has been stimulated by the description of an inborn error of metabolism, fucosidosis, involving the excessive accumulation of fucose-containing glycolipids and glycoproteins [ 4-61. This accumulation is
30
accompanied by severe’ progressive> cerebral degeneration. loss 01’ muscles strength and increasing respiratory difficulties, leading to death in early <*hiltlhood [5]. A complete absence or marked deficiency of a-L-fucosidasc> activity has been reported in a number of tissues [1,5--Y 1, urine and k:ucoc:ytt~s [ 8 1 and cultured fihroblasts [9] from patients. The recent availability of the fluorogenic substrate 4-n~t~thylumt~ellifery1cr-L-fucopyranoside has made possible tht development of a scnsitivch and specific method for the detection of cu-L-fucosidase after st.arch-gel elect.rophoresis. Using this method, we have examined the enzyme in long-term lymphoid cell lines, cultured skin fibrohlasts, serum, plasma, and peripheral leucocytes. Materials and Methods cultured
cells
Long-term lymphoid cell lines were established after stimulation of pcripheral lymphocytes with phytohemagglutinin (PI-IA) and incubation with Epstein-Barr virus or with PHA only [lo]. Cells were grown in RPM1 1640 medium with the following additions: fetal calf serum, 20%; penicillin 100 U/ml; streptomycin, 100 &ml; 200 mM L-glutamine~ 1%. Cultures were fed with fresh medium 24 h before harvesting. Cells were harvested by eentrifugation at 840 X g for 15 min and washed 3 times with 0.9% saline. Cell pellets were stored at -96”. Skin fibroblasts were cultured in the same medium as above and similarly fed 24 h before harvesting. Cells were obtained after 1 min exposure to 0.25% trypsin and subsequently to 0.02% sodium EDTA in isotonic saline for 20 min. Cells were washed 3 times in 0.9% saline and stored at -96”. Serum, plasma and leucocytes Serum was derived from clotted blood. Plasma and leucocytes were prepared from freshly taken heparinized venous blood. For the preparation of leucocytes, 5 vols of whole blood were mixed with 1 vol. of 6% Dextran in 0.9% saline. The mixture was allowed to stand for 30 min at room temperature and the leucocyte-rich supernatant was then removed and centrifuged at 840 X g for 15 min. Contaminating erythrocytes were preferentially lysed with 0.183% NH4 Cl. Leucocytes were washed twice with 0.9% saline and stored as packed cells at -96” until required. Sample preparation All types of cells were lysed by freeze-thawing up to 10 times in 3-5 vols of distilled water using a dry ice-acetone bath. Debris was removed by centrifugation for 5 min at 1800 X g. Supernatants from lymphoid lines usually contained 6-12 mg protein/ml and those from leucocytes and cultured fibroblasts from 2-5 mg/ml. Elec trophoresis Horizontal starch-gel electrophoresis was used for all experiments. Gels containing 1170 starch (Connaught Labs) were prepared as described by
31
Smithies [ll]. Gels measuring 30 cm X 15 cm X 0.7 cm were used. Samples were inserted on small pieces of Whatman No. 3 or No. 17 filter paper. Gels were run for 20-22 h between copper cooling plates [ 12 ] through which water at +4” was circulated. A number of buffer systems was tested and the following two were found to give the best resolution of a-L-fucosidase. (1) Bridge buffer: 0.04 M sodium phosphate, pH 7.0; gel buffer: 1 in 8 dilution of bridge. This buffer system has been used for the separation of /3-galactosidase isozymes by vertical starch-gel electrophoresis [ 131. (2) Bridge buffer: 0.025 M disodium phosphate, 0.118 M citric acid pH 5.0; gel buffer: 1 in 100 dilution of bridge. In both systems a v-oltage gradient of 5 V/cm was used, giving a current of 15-18 mA. When using system 1, samples were inserted one-third of the way along the gel and in system 2, were inserted in the center of the gel. Staining The staining mixture consisted of 4-methylumbelliferyl-a-L-fucoside (Koch-Light Labs, Colnbrook, U.K.) at a concentration of 0.2 mg/ml (0.6 mM) in 0.1 M citrate-phosphate, pH 4.8. Gels were sliced into upper and lower halves and a piece of Whatman No. 3 filter paper was applied to the cut surface and soaked with the buffered substrate solution. Care was taken to expel air bubbles beneath the filter paper. Gels were then wrapped in thin plastic and incubated at 37” for 30-60 min. The filter paper was then removed and the gel surface flooded with 0.085 M glycine-carbonate buffer, pH 10.0. Under long-wave ultraviolet light, regions of a-L-fucosidase activity can be seen as fluorescent bands against a non-fluorescent background. Neuraminidase treatment The following reaction mixture was normally used; 50 1.11sample, 50 ~1 neuraminidase (500 U/ml) and 20 ~1 5 mM citrate-phosphate buffer, pH 4.8. The mixture was incubated for 3-5 h at 37”. Controls contained distilled water in place of neuraminidase and were similarly incubated. Neuraminidase was obtained from the following sources; Behringwerke ORKD (500 U/ml), Calbiothem (B grade from Vibrio cholerae, 500 units/ml) and Sigma (from Cl. perfringens, Type V 0.12 units (NAN-Lactose)/mg and Type VI 1.55 units/mg). All types of neuraminidase tested gave very similar results and that from Calbiochem was normally used. Results The electrophoretic pattern of a-L-fucosidase was examined in 28 different lymphoid cell lines from phenotypically normal individuals. The enzyme could be resolved into as many as 6 discrete bands merging, in some cases, with a more anodal and less clearly resolved region of activity. The number of bands and their reiative staining intensities varied from one cell line to another. The range of this variation is illustrated in Fig. 1 which shows the electrophoretic patterns of 4 different cell lines using both the pH 5.0 and pH 7.0 buffer systems. The pattern shown by a given cell line remained relatively constant
from one harvest to another. .I similar variatio~i in c~lC~c~tro~)llort~ti~. I)nt.tc>rn occurred among fibroblast cultures although activity of thrl (‘enzyme \vas far lower in fibroblasts than in lymphoid cells and thr isozymrs \vt’r(’ l)oorly Ksolved (Fig. 2). Electrophoretic patterns of a-L-fucosidasc> in pc>riphcral l~~~~~~o~yl~~s and plasma are shown in Fig. 3. The canzyme from leucocytcs st,aillr>tl strongly and
65432,or *igin
l-
Fig.
2.
m-I,-Fucosidasr
electrophowsis
at
from pH
7.0.
a
culturrd
lymphoid
cell
lint,
and
from
diffc,rent
fibrobldst
rultuws
dftm
33
-or gin
5
--_ 6
Fig. 3. The electrophoretic pattern of u-L-fucosidasc from the following leucocytes; 3, lymphoid line from patient with fucosidosis: 4 and 5. normal
sources: lymphoid
1, plasma; lines.
2 and 6,
seemed to consist of a similar series of isozymes to those found in lymphoid lines. The enzyme from plasma or serum had an electrophoretic pattern quite different to that of the cell types studied. A diffuse region of activity was seen with an anodal mobility greater than that of the discrete cellular isozymes. None of the different isozymes was detected in lymphoid cell lines (Fig. 3), skin fibroblasts or serum from a child with fucosidosis. Fucosidase isozymes were detectable in the culture medium of lymphoid cell lines only after a lo-fold concentration of the medium by ultrafiltration (Amicon, UM-10). The isozymes seen corresponded to those of cell extracts. The enzyme was not detected in fibroblast culture medium after similar concentration. The effect of neuraminidase on the electrophoretic properties of Q-Lfucosidase was studied using the phosphate buffer system, pH 7.0. This was found to give better isozyme resolution after treatment than the pH 5.0 system. Treatment of extracts of cultured lymphoid lines with neuraminidase caused a loss of activity of the more anodal bands with a concomitant increase in the staining intensity of bands 1-3 (Fig. 4). It was possible to divide the 28 cell lines into two groups on the basis of the electrophoretic pattern after neuraminidase treatment. In 17 of the lines the treated enzyme consisted predominantly of anodal bands 2 and 3 together, in some cases, with a less active band 4. In the remaining 11 lines bands l-3 predominated after treatment. Increasing the amount of neuraminidase used or the incubation time did not alter these patterns. Among the 11 lines in which band 1 was seen after treatment this band was also detected in at least some untreated extracts of each line. On no occasion was band 1 seen in any of the other 17 lines although multiple preparations of most lines were examined.
3 I
0
1 5 4 3 2 atigin
1
Cl
b 1
c f t a
b
c
2
Fig. 4. Tht vffect of nwraminidasc on a-I,-fucosidaw from two cultured lymphold cell lmes as tlt~acrlbl~d ,n tt1v k.XL. a: h‘o nwraminidasr: samples wc‘rc’ incubatcbd for 5 II at 37 nwraminidnsc; r: 25 units ncilrailiiliici;ts~,.
(1 dtnd 2). All II: IO units
Identical treatment of plasma or serum with neuraminidase resulted in a cathodal shift in the mobility of the singlth, broad band. The treated enzyme had a mobility similar to that of bands 2 and 3 of the enzyme from lyn~~~hoid lines. Discussion The results demonstrate a considerable complexity in the electrophoretic properties of human a-L-fucosidase. Up to 6 clearly distinguishable isozymes were seen in cultured lymphoid cell lines. The same isozymes occurred iu fibroblasts and peripheral leucocytes. Elimination of the more anodal components by treatment with neuraminidase suggests that these forms of the enzyme contain sialic acid residues. Part of the electrophoretic variation found to occur among cultured cell lines would therefore seem to be due to differences in the amount of sialic acid bound to the enzyme. However, even after treatment with neuraminidase two or three prominent isozymes still remained. In some cell lines these corresponded to bands 1-3 (1 and 2 being the most prominent) and in others to bands 2 and 3 together with a weaker band 4. It seems therefore that variation in the enzyme other than that due to sialic acid occurs among cells from different individuals, particularly regarding the presence or absence of band 1. Whether this variation represents a genetic polymorphism or an as yet unexplained secondary enzyme variation can only be determined by family studies. Lymphoid cell lines, at the present time, do not readily lend themselves to the investigation of a sufficiently large number of families. Since peripheral leucocytes show a similar series of a-L-fucosidase isozymes to lymphoid lines it may be possible to investigate this problem by using these cells. a-L-Fucosidase in serum was quite different electrophoretically to the intracellular enzyme. It showed a single broad band with a greater anodal
35
mobility than the clearly resolved cellular isozymes. Treatment of serum with neuraminidase converted the enzyme into a form with a mobility in the region of the neuraminidase treated cellular isozymes, although the serum band remained broad and poorly resolved. It is therefore possible that the serum isozyme is derived from the intracellular forms by complexing with sialic acid prior to secretion. However, the serum isozyme was not secreted by lymphoid cell lines. The possibility that the serum and cellular isozymes are the products of separate gene loci cannot be excluded, but the absence of enzyme activity in tissues [1,5,6], serum [7] and cultured cells [9] from individuals with fucosidosis suggests that the various isozymes have at least one subunit in common. Acknowledgements Supported by U.S. Public Health Service Grant I-ID-002552 and Clinical Genetics Center Grant GM-19443. K.H. is a Career Scientist of the Health Research Council of the City of New York (I-513). References 1
F. Van
2
D. Robinson
Hoof
3
G.Ya.
and
H.G.
and
R.
Hers.
Eur.
Thorpe,
Wiederschain,
E.L.
J. Biochem.,
Clin,
Chim.
Rosenfeld.
7 (1968)
Acta,
A.I.
47
34
(1973)
Brusilovsky
403
and
L.G.
Kolibaba
, Clin. Chim.
Acta,
35
(1971)
99 4
P. Duxand,
M. Philippart,
5
P. Durand,
C. Bortone
6
I. Matsuda,
7
K.
Zielke.
8
V.
Pat&
9
K.
Zielke.
10
N.G.
11
0.
S. Arashima, S. Okada M.L.
Beratis
P.J.
13
M.W.
McAlpine,
Biochem.
and
Ege and
75 Med.,
Zeman.
Science.
176
(1972)
O’Brien,
J. Exp.
Mamm. (1955)
Clin.
and
Chrom.
Pediat. (1969)
I. Hay&a.
Clin.
Hirschhorn,
O’Brien,
Cella,
J. Pediat.,
J. Lab.
J.S.
J.. 61
A.
G. Della
O’Brien,
D.A. Hopkinson
Ho and J.S.
and C&a,
M. Anakura,
and W.
Veath
and K.
G. Della
and J.S.
I. Watanabe
Smithies,
12
C. Borrone and
Med.,
Res..
Tohoku
79
1 (1967)
416
665
(1972)
J. Exp.
Med.,
109
34 (1970)
61
164
426
136
Newsl.,
(1972) 14
(1973)
197 114
629 H. Harris,
Chim.
Acta,
Ann. 32
Hum.
(1971)
443
C&net.
Land.,
(1973)
41
Clinica Chimica
@ Elsevier
Acta,
Scientific
57 (1974) 29-35 Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6629
ISOZYMES OF HUMAN (Y-L-FUCOSIDASE GEL ELECTROPHORESIS
B.M. TURNER,
N.G. BERATIS,
Division of Medical the City University
(Received
VIRGINIA
DETECTABLE
S. TURNER
BY STARCH
and K. HIRSCHHORN
Genetics, Department of Pediatrics, Mount Sinai School of New York, New York, N.Y. 10029 (U.S.A.)
of Medicine
of
May 14, 1974)
Summary Methods are described for the electrophoresis in starch-gel of human O-Lfucosidase and for the detection of the enzyme using the fluorogenic substrate 4-methylumbelliferyl-a-l,-fucopyranoside. The electrophoretic pattern of the enzyme was examined in leucocytes, serum, cultured fibroblasts and long-term lymphoid cell lines. The enzyme from cultured lymphoid cell lines was found to consist of up to 6 clearly resolved electrophoretic isozymes plus a diffuse, more anodal region of lower staining intensity. The enzyme from leucocytes and cultured skin fibroblasts was less clearly resolved, but these cell types appeared to have components corresponding in mobility to the isozymes of lymphoid lines. In contrast, the enzyme from serum (or plasma) showed only a diffuse region of activity, with an anodal mobility slightly greater than that of the most anodal lymphoid line isozymes. Evidence is presented which indicates that the electrophoretic heterogeneity of a-L-fucosidase is due in part to the binding of sialic acid to the primary gene product. None of the isozymes was detectable in lymphoid cell lines, serum or cultured fibroblasts from a patient with fucosidosis, an inborn error of metabolism.
Introduction The enzyme oc-L-fucosidase has been detected in a wide range of human tissues [l-3] by assay methods employing the artificial substrate p-nitrophenyl fucoside. The properties of the human enzyme have not yet been extensively studied, but evidence has been presented that human liver contains at least two forms of a-L-fucosidase which differ in charge, molecular weight and thermostability [2]. Interest in the enzyme has been stimulated by the description of an inborn error of metabolism, fucosidosis, involving the excessive accumulation of fucose-containing glycolipids and glycoproteins [ 4-61. This accumulation is
30
accompanied by severe’ progressive> cerebral degeneration. loss 01’ muscles strength and increasing respiratory difficulties, leading to death in early <*hiltlhood [5]. A complete absence or marked deficiency of a-L-fucosidasc> activity has been reported in a number of tissues [1,5--Y 1, urine and k:ucoc:ytt~s [ 8 1 and cultured fihroblasts [9] from patients. The recent availability of the fluorogenic substrate 4-n~t~thylumt~ellifery1cr-L-fucopyranoside has made possible tht development of a scnsitivch and specific method for the detection of cu-L-fucosidase after st.arch-gel elect.rophoresis. Using this method, we have examined the enzyme in long-term lymphoid cell lines, cultured skin fibrohlasts, serum, plasma, and peripheral leucocytes. Materials and Methods cultured
cells
Long-term lymphoid cell lines were established after stimulation of pcripheral lymphocytes with phytohemagglutinin (PI-IA) and incubation with Epstein-Barr virus or with PHA only [lo]. Cells were grown in RPM1 1640 medium with the following additions: fetal calf serum, 20%; penicillin 100 U/ml; streptomycin, 100 &ml; 200 mM L-glutamine~ 1%. Cultures were fed with fresh medium 24 h before harvesting. Cells were harvested by eentrifugation at 840 X g for 15 min and washed 3 times with 0.9% saline. Cell pellets were stored at -96”. Skin fibroblasts were cultured in the same medium as above and similarly fed 24 h before harvesting. Cells were obtained after 1 min exposure to 0.25% trypsin and subsequently to 0.02% sodium EDTA in isotonic saline for 20 min. Cells were washed 3 times in 0.9% saline and stored at -96”. Serum, plasma and leucocytes Serum was derived from clotted blood. Plasma and leucocytes were prepared from freshly taken heparinized venous blood. For the preparation of leucocytes, 5 vols of whole blood were mixed with 1 vol. of 6% Dextran in 0.9% saline. The mixture was allowed to stand for 30 min at room temperature and the leucocyte-rich supernatant was then removed and centrifuged at 840 X g for 15 min. Contaminating erythrocytes were preferentially lysed with 0.183% NH4 Cl. Leucocytes were washed twice with 0.9% saline and stored as packed cells at -96” until required. Sample preparation All types of cells were lysed by freeze-thawing up to 10 times in 3-5 vols of distilled water using a dry ice-acetone bath. Debris was removed by centrifugation for 5 min at 1800 X g. Supernatants from lymphoid lines usually contained 6-12 mg protein/ml and those from leucocytes and cultured fibroblasts from 2-5 mg/ml. Elec trophoresis Horizontal starch-gel electrophoresis was used for all experiments. Gels containing 1170 starch (Connaught Labs) were prepared as described by
31
Smithies [ll]. Gels measuring 30 cm X 15 cm X 0.7 cm were used. Samples were inserted on small pieces of Whatman No. 3 or No. 17 filter paper. Gels were run for 20-22 h between copper cooling plates [ 12 ] through which water at +4” was circulated. A number of buffer systems was tested and the following two were found to give the best resolution of a-L-fucosidase. (1) Bridge buffer: 0.04 M sodium phosphate, pH 7.0; gel buffer: 1 in 8 dilution of bridge. This buffer system has been used for the separation of /3-galactosidase isozymes by vertical starch-gel electrophoresis [ 131. (2) Bridge buffer: 0.025 M disodium phosphate, 0.118 M citric acid pH 5.0; gel buffer: 1 in 100 dilution of bridge. In both systems a v-oltage gradient of 5 V/cm was used, giving a current of 15-18 mA. When using system 1, samples were inserted one-third of the way along the gel and in system 2, were inserted in the center of the gel. Staining The staining mixture consisted of 4-methylumbelliferyl-a-L-fucoside (Koch-Light Labs, Colnbrook, U.K.) at a concentration of 0.2 mg/ml (0.6 mM) in 0.1 M citrate-phosphate, pH 4.8. Gels were sliced into upper and lower halves and a piece of Whatman No. 3 filter paper was applied to the cut surface and soaked with the buffered substrate solution. Care was taken to expel air bubbles beneath the filter paper. Gels were then wrapped in thin plastic and incubated at 37” for 30-60 min. The filter paper was then removed and the gel surface flooded with 0.085 M glycine-carbonate buffer, pH 10.0. Under long-wave ultraviolet light, regions of a-L-fucosidase activity can be seen as fluorescent bands against a non-fluorescent background. Neuraminidase treatment The following reaction mixture was normally used; 50 1.11sample, 50 ~1 neuraminidase (500 U/ml) and 20 ~1 5 mM citrate-phosphate buffer, pH 4.8. The mixture was incubated for 3-5 h at 37”. Controls contained distilled water in place of neuraminidase and were similarly incubated. Neuraminidase was obtained from the following sources; Behringwerke ORKD (500 U/ml), Calbiothem (B grade from Vibrio cholerae, 500 units/ml) and Sigma (from Cl. perfringens, Type V 0.12 units (NAN-Lactose)/mg and Type VI 1.55 units/mg). All types of neuraminidase tested gave very similar results and that from Calbiochem was normally used. Results The electrophoretic pattern of a-L-fucosidase was examined in 28 different lymphoid cell lines from phenotypically normal individuals. The enzyme could be resolved into as many as 6 discrete bands merging, in some cases, with a more anodal and less clearly resolved region of activity. The number of bands and their reiative staining intensities varied from one cell line to another. The range of this variation is illustrated in Fig. 1 which shows the electrophoretic patterns of 4 different cell lines using both the pH 5.0 and pH 7.0 buffer systems. The pattern shown by a given cell line remained relatively constant
from one harvest to another. .I similar variatio~i in c~lC~c~tro~)llort~ti~. I)nt.tc>rn occurred among fibroblast cultures although activity of thrl (‘enzyme \vas far lower in fibroblasts than in lymphoid cells and thr isozymrs \vt’r(’ l)oorly Ksolved (Fig. 2). Electrophoretic patterns of a-L-fucosidasc> in pc>riphcral l~~~~~~o~yl~~s and plasma are shown in Fig. 3. The canzyme from leucocytcs st,aillr>tl strongly and
65432,or *igin
l-
Fig.
2.
m-I,-Fucosidasr
electrophowsis
at
from pH
7.0.
a
culturrd
lymphoid
cell
lint,
and
from
diffc,rent
fibrobldst
rultuws
dftm
33
-or gin
5
--_ 6
Fig. 3. The electrophoretic pattern of u-L-fucosidasc from the following leucocytes; 3, lymphoid line from patient with fucosidosis: 4 and 5. normal
sources: lymphoid
1, plasma; lines.
2 and 6,
seemed to consist of a similar series of isozymes to those found in lymphoid lines. The enzyme from plasma or serum had an electrophoretic pattern quite different to that of the cell types studied. A diffuse region of activity was seen with an anodal mobility greater than that of the discrete cellular isozymes. None of the different isozymes was detected in lymphoid cell lines (Fig. 3), skin fibroblasts or serum from a child with fucosidosis. Fucosidase isozymes were detectable in the culture medium of lymphoid cell lines only after a lo-fold concentration of the medium by ultrafiltration (Amicon, UM-10). The isozymes seen corresponded to those of cell extracts. The enzyme was not detected in fibroblast culture medium after similar concentration. The effect of neuraminidase on the electrophoretic properties of Q-Lfucosidase was studied using the phosphate buffer system, pH 7.0. This was found to give better isozyme resolution after treatment than the pH 5.0 system. Treatment of extracts of cultured lymphoid lines with neuraminidase caused a loss of activity of the more anodal bands with a concomitant increase in the staining intensity of bands 1-3 (Fig. 4). It was possible to divide the 28 cell lines into two groups on the basis of the electrophoretic pattern after neuraminidase treatment. In 17 of the lines the treated enzyme consisted predominantly of anodal bands 2 and 3 together, in some cases, with a less active band 4. In the remaining 11 lines bands l-3 predominated after treatment. Increasing the amount of neuraminidase used or the incubation time did not alter these patterns. Among the 11 lines in which band 1 was seen after treatment this band was also detected in at least some untreated extracts of each line. On no occasion was band 1 seen in any of the other 17 lines although multiple preparations of most lines were examined.
3 I
0
1 5 4 3 2 atigin
1
Cl
b 1
c f t a
b
c
2
Fig. 4. Tht vffect of nwraminidasc on a-I,-fucosidaw from two cultured lymphold cell lmes as tlt~acrlbl~d ,n tt1v k.XL. a: h‘o nwraminidasr: samples wc‘rc’ incubatcbd for 5 II at 37 nwraminidnsc; r: 25 units ncilrailiiliici;ts~,.
(1 dtnd 2). All II: IO units
Identical treatment of plasma or serum with neuraminidase resulted in a cathodal shift in the mobility of the singlth, broad band. The treated enzyme had a mobility similar to that of bands 2 and 3 of the enzyme from lyn~~~hoid lines. Discussion The results demonstrate a considerable complexity in the electrophoretic properties of human a-L-fucosidase. Up to 6 clearly distinguishable isozymes were seen in cultured lymphoid cell lines. The same isozymes occurred iu fibroblasts and peripheral leucocytes. Elimination of the more anodal components by treatment with neuraminidase suggests that these forms of the enzyme contain sialic acid residues. Part of the electrophoretic variation found to occur among cultured cell lines would therefore seem to be due to differences in the amount of sialic acid bound to the enzyme. However, even after treatment with neuraminidase two or three prominent isozymes still remained. In some cell lines these corresponded to bands 1-3 (1 and 2 being the most prominent) and in others to bands 2 and 3 together with a weaker band 4. It seems therefore that variation in the enzyme other than that due to sialic acid occurs among cells from different individuals, particularly regarding the presence or absence of band 1. Whether this variation represents a genetic polymorphism or an as yet unexplained secondary enzyme variation can only be determined by family studies. Lymphoid cell lines, at the present time, do not readily lend themselves to the investigation of a sufficiently large number of families. Since peripheral leucocytes show a similar series of a-L-fucosidase isozymes to lymphoid lines it may be possible to investigate this problem by using these cells. a-L-Fucosidase in serum was quite different electrophoretically to the intracellular enzyme. It showed a single broad band with a greater anodal
35
mobility than the clearly resolved cellular isozymes. Treatment of serum with neuraminidase converted the enzyme into a form with a mobility in the region of the neuraminidase treated cellular isozymes, although the serum band remained broad and poorly resolved. It is therefore possible that the serum isozyme is derived from the intracellular forms by complexing with sialic acid prior to secretion. However, the serum isozyme was not secreted by lymphoid cell lines. The possibility that the serum and cellular isozymes are the products of separate gene loci cannot be excluded, but the absence of enzyme activity in tissues [1,5,6], serum [7] and cultured cells [9] from individuals with fucosidosis suggests that the various isozymes have at least one subunit in common. Acknowledgements Supported by U.S. Public Health Service Grant I-ID-002552 and Clinical Genetics Center Grant GM-19443. K.H. is a Career Scientist of the Health Research Council of the City of New York (I-513). References 1
F. Van
2
D. Robinson
Hoof
3
G.Ya.
and
H.G.
and
R.
Hers.
Eur.
Thorpe,
Wiederschain,
E.L.
J. Biochem.,
Clin,
Chim.
Rosenfeld.
7 (1968)
Acta,
A.I.
47
34
(1973)
Brusilovsky
403
and
L.G.
Kolibaba
, Clin. Chim.
Acta,
35
(1971)
99 4
P. Duxand,
M. Philippart,
5
P. Durand,
C. Bortone
6
I. Matsuda,
7
K.
Zielke.
8
V.
Pat&
9
K.
Zielke.
10
N.G.
11
0.
S. Arashima, S. Okada M.L.
Beratis
P.J.
13
M.W.
McAlpine,
Biochem.
and
Ege and
75 Med.,
Zeman.
Science.
176
(1972)
O’Brien,
J. Exp.
Mamm. (1955)
Clin.
and
Chrom.
Pediat. (1969)
I. Hay&a.
Clin.
Hirschhorn,
O’Brien,
Cella,
J. Pediat.,
J. Lab.
J.S.
J.. 61
A.
G. Della
O’Brien,
D.A. Hopkinson
Ho and J.S.
and C&a,
M. Anakura,
and W.
Veath
and K.
G. Della
and J.S.
I. Watanabe
Smithies,
12
C. Borrone and
Med.,
Res..
Tohoku
79
1 (1967)
416
665
(1972)
J. Exp.
Med.,
109
34 (1970)
61
164
426
136
Newsl.,
(1972) 14
(1973)
197 114
629 H. Harris,
Chim.
Acta,
Ann. 32
Hum.
(1971)
443
C&net.
Land.,
(1973)
41