BIOCHEMICAL
14, 117-124 (1975)
MEDICINE
Isolation
and from
Characterization the
HIROSHI Institute
of Clinical
of the
Glycopeptide
Urine of Myoclonus Epilepsy of Lafora-Body Type MASUDA Pathology,
AND MUTSUYA
TAKEUCHI
Kurume
School
University
of Medicine
AND AKIHITO Institute
of Brain
KUNITAKE Diseases, Kurume,
AND Kurume Fukuoka
SHIGEMI
ANRAKU
University School 830, Japan
of Medicine,
Received July 31, 197.5
Lafora-body’s disease (progressive myoclonus epilepsy with intraneuronal inclusion body of Lafora) is one of rare hereditary disorders (1). It can be characterized clinically by the onset, in adolescence, of myoclonus, convulsions, and progressive dementia with inexorable progression to death within 4 to 10 years. Of the few reported cases of this disease, most were diagnosed at autopsy, and studies restricted to histopathologic techniques (2, 3). In this paper, it was attempted from the biochemical aspect of this disease to isolate and characterize glycopeptides from the urine of the patient. METHODS Complete 24 hr urine samples, preserved with 0.1% sodium azide, were collected from one myoclonus epilepsy patient (Lafora-body type) of a 17-year-old boy, one myoclonus epilepsy patient (degenerative type) of a lPyear-old, and 11 healthy Japanese with ages ranging from 15-40 years. After measuring the volume of each pooled sample, they were stored in a deep freezer until further work was undertaken. lsolation of Crude Glycopeptides from Urine
Crude glycopeptides were isolated from the urine according to the procedure shown in Fig. 1. The urines were digested with papain (EC 3.4.4.10) in the presence of EDTA and cysteine at 65°C for 48 hr. The mixture was adjusted to pH 7.8 and then digested with trypsin (EC 3.4.4.4) at 37°C for 48 hr with simultaneous dialysis against 0.1 M phosphate buffer of pH 7.8. The digested solution was deproteinized with 5% trichloroacetic acid in the cold, and the supernatant was dialyzed against 117 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
MASUDA
Dissolved with (The Glycopeptide
ET AL.
deionized water and the Glycasamlnoglycans
mlXt”re’
FIG. 1. Isolation of the crude glycopeptide from the urine.
several changes of the cold deionized water for 72 hr. Then 4 vol of alcohol containing 0.1% acetic acid and 1% potassium acetate were added in order to precipitate the crude glycopeptide. The precipitate was washed with alcohol, dried in a desiccator over CaCl,, and then redissolved in 5 ml of deionized water. An aliquot (usually 0.5 ml) was used for hexose and sialic acid analyses. Electrophoresis of Glycopeptides on a Cellulose Acetate Strip.
The material was run in a horizontal type apparatus at constant current of 0.4 mA/cm for 40 min on an OXOID strip (8 x 1.2 cm) in barbital buffer, pH 8.6, p = 0.06. Five to 10 ~1 of aqueous suspensions of preparations in 1 mgimi were used in this study. After electrophoresis, the strip was stained with alcian blue and PAS in the normal way with human serum, hyaluronic acid, and chondroitin sulfate A as standard; hyaluronicacid and chondroitin sulfate A were purchased from Seikagaku Kogyo Co., Ltd, Japan; and hyaiuronic acid was derived from human umbilical cord and chondroitin sulfate A from whale cartilage, respectively. Fractionation of Glycopeptides by DEAE Sephadex A-50 Column chromatography
DEAE Sephadex A-50 (a product of Pharmacia, Uppsala, Sweden) was equilibrated with 0.1 M Tris-HCI buffer, pH 8.0, which was used as the starting buffer for a gradient elution system. Twenty milliliters of 2.5% dry crude material in the starting buffer was applied to a DEAE Sephadex A-50 column (2.5 x 45 cm). Elution was made with a linear gradient of 0.1-1.0 M Tris-HCI buffer. The total elution volume was 1 1. The eluant was collected in 15 ml fractions at a flow rate of 30 ml/hr. An aliquot (usually 0.5 ml, respectively) was used for hexose and peptide analyses. Isolated fractions were dialyzed against deionized water and used for chemical analyses.
MYOCLONUS
Analytical
EPILEPSY
119
Procedures.
Peptide content was determined by the Lowry method (4) with human serum (Hyland Laboratory, U.S.A.) as a standard, and hexose content by tryptophane method of Shetlar et aE. (5), using a mixture of mannose and galactose as a standard. Sialic acid content was estimated by the Warren’s periodic oxidation-thiobarbituric acid method (6), with N-acetylneuraminic acid as a standard. Neutral sugars were separated and indentified by paper chromatography after hydrolysis in N HCl for 3 hr at 100°C in a sealed ampoule; S & S filter paper No. 598 was used as a supporting medium; the solvent system was n-butanol-pyridine-0.1 N HCl (5:3:2 v/v/v). Chromatogram obtained by descending paper chromatography, and double development techniques was stained with AgNO,-NaOH reagent. The separation and quantitative determination of glucosamine and galactosamine were carried out with a Hitachi KLA-3 Amino Acid Analyzer, with a 50 cm column in citrate buffer of pH 5.28 (Na:0.35 N) after hydrolysis in 4 N HCl for 6 hr at 100°C in the sealed ampul. RESULTS Results of the hexose and sialic acid analyses of the glycopeptide from urines of 11 normal persons, one myoclonus epilepsy patient (Lafora-body type), and one myoclonus epilepsy patient (degenerative type) are summarized in Table 1. The sialic acid:hexose ratio was 0.23 + 0.110 in normal, 0.30 + 0.098 in myoclonus epilepsy of Laforabody type, and 0.27 + 0.055 in myoclonus epilepsy of degenerative type, respectively. No difference was found among normal and diseases, except slightly higher ratio of sialic acid to hexose in the patient of Lafora-body type than in the others. Densitograms after electrophoresis of the crude material in the barbital buffer, pH 8.6 are shown in Fig. 2. When stained with alcian blue, both materials had four components which moved towards the anode. This is in agreement with the results of Manley et al. (7) who reported one glycoprotein and three glycosamino-glycans from the normal urine, that is, heparan sulfate, chondroitin sulfate, and hyaluronic acid, respectively. When stained with PAS, both materials had one component which moved slightly towards the anode. This component corresponds to the alcian blue stainable spot which migrated towards the anode more slowly than hyaluronic acid. Any significant difference, however, was scarcely found among normal and the diseases. The elution patterns of the DEAE Sephadex A-50 column chromatography of the crude material are depicted in Fig. 3. The materials from normal and the patient of degenerative type were separated into five hexose peaks at the molarity of Tris-HCI buffer ranging from 0.3 to 0.7 M, whereas that from the patient of Lafora-body type were separated
120
MASUDA
URINARY
OF LAFORA-BODY TYPE EPII.EPSY OF DEGENERATIVE
Myoclonus
epilepsy
16.7 29.6 ?O.O 24.4 16.6
6.7 3.8
1 I.2 20.4
6.0 7.0
7.8 IO.8 X.1
4.7 2.6
II & SD
3.1 t I.84
4.4
epilepsy
Sialic Hexose
3.7 7.1 5.6
Mean
ONCJS
type”
2. I
x 9 I0
MYOC~
MYO~LONL~S TYPE”
of degenerative
type” Sialic acid
IN AND
Myoclonu\
of Lafora-body
Subject
AL.
TABLE I OF GLYCOPEPTID~
EXCRETION
EPILEPSY
ET
15.9
9.4 -+ 6.84
acid
Hexose
4.0 2.3 3.3 6.8
17.1 IS.5 19.3 23.1
6.’ 6.3
23 ! --. I 7’
6.4 5.9
7 I .(I I’.!
5.4 x!z I .42
19.9
-+- 2.87
(’ In milligrams h The patient
per was
24 hr. a l7-year-old
boy,
who
was
examined
twice
a week
for
h weeks.
” The
was
a
boy,
who
was
examined
twice
a week
for
4 weeks.
patient
” Materials
were
collected
l9-year-old from
l I healthy
Japanese
with
ages
ranging
from
1 Y-40
years.
into three hexose peaks at the molarity of Tris-HCl buffer ranging from 0.4 to 0.7 M. Again, column chromatography of the urinary crude material on DEAE Sephadex A-50 showed three distinct elution diagrams. The first was characteristic of Lafora-body type who excreted fraction IV predominantly (A); the second was characteristic of degenerative type who excreted fractions I and II predominantly; and the third diagram was characteristic of the normal control (C and D). Therefore, the fractions corresponding to these peaks were pooled and dialyzed against deionized water for 2 days; the insoluble materials were centrifuged out, and the supernatants were further applied to chemical analyses. Each of the fractions from all materials were subjected to analyses for hexose, sialic acid, hexosamine, and peptide; the results are summarized in Table 2. They were characterized by an increase in peptide and sialic acid as the molarity of Tris-HCl buffer increased. Hexose was main component, and peptide was the next in each fraction from all materials. Sialic acid was contained three to four times more in amount in the fraction V of the material from the patient of Lafora-body type than from the others. The paper chromatography also revealed the presence of galactose. mannose. and fucose in each fraction from the materials.
MYOCLONUS Alcian
Blue
121
EPILEPSY
Stain
PAS Stain
ChS-A
Ponceau-S
0
FIG. 2. Densitograms inoglycans on cellulose
after electrophoresis acetate strip.
of the urinary
glycopeptide
Stain
Anode
and glycosam-
DISCUSSION
In the present stage, opinions differ on the cause of myoclonus epilepsy of Lafora-body type. Harriman (8) and Schwarz (2) reported that it might be due to the disorders of glycoprotein or mucopolysaccharide metabolism, according to the histochemical study on the Lafora-body. Janeway (9) informed that it could arise from the affection of lipid metabolism, depending upon lipid and ganglioside analyses performed on specimens of the cerebral cortex biopsy. Yokoi (10) and Schnabel (11) accounted that the disease might be involved in the disformity of glycogen metabolism, based on the fact that obtained from the chemical analyses of Lafora-body, and histochemical study of Lafora-body, respectively. It is of interest that the intraneuronal inclusion body of Lafora is found in the brain of the myoclonus epilepsy of Lafora-body type and well stained with both alcian blue and PAS, but not in the brain of the myoclonus epilepsy of the degenerative (12); and that the sialic acid rich glycopeptide was isolated from the urine of the myoclonus epilepsy of Lafora-body type. Neither lipid nor glycogen may probably show positive stainability with both alcian blue and PAS. These facts may probably support Harriman (8) and Schwarz (2). But the reason why the sialic acid rich glycopeptide from Lafora-body type was eluted at the same
122
MASUDA
ET .4L.
FIG. 3. Chromatograms of a DEAE Sephadex A-50 elution: --concentration Tris-HCI buffer (M); --------- hexose content: -.-.-*-.-.- absorbance at 280 nm.
of
molarity of Tris-HCl buffer in DEAE Sephadex A-50 column chromatography as materials from normal and from degenerative type were eluted is still unknown. DEAE Sephadex A-50 column chromatography is surely involved in
MYOCLONUS TABLE ANALYSES
OF FRACTIONS
Fraction W
2
FROM DEAE CHROMATOGRAPHY~
Fraction UIP
123
EPILEPSY
SEPHADEX
Fraction (III?
A-50 COLUMN Fraction (IV)
Fraction W)
Myoclonus epilepsy of Lafora-body type Hexose Peptide Sialic Acid Glucosamine Galactosamine
50.9 13.7 2.8 5.6 0.7
56.4 19.2 5.4 11.9 1.9
45.2 19.0 15.1 8.0 2.5
Myoclonus epilepsy of degenerative type Hexose Peptide Sialic Acid Glucosamine Galactosamine
63.5 9.8 3.2 15.7 3.4
65.5 16.4 2.8 15.8 3.2
44.0 18.8 3.4 8.8 2.2
43.2 15.1 4.2 6.6 1.4
45.5 24.3 5.1 6. I 3.9
Normal Hexose Peptide Sialic Glucosamine Galactosamine
51.8 zk 8.2 11.5 -c 2.1 trace 8.5k3.3 3.4kO.8
41.8 k 7.3 17.6 k 3.8 trace 6.2 k 3.4 2.1 + 0.5
40.9 k 5.1 17.4 2 2.8 trace 6.1 f 1.2 2.2 +I 0.4
49.5 k 19.2 k 4.0 f 6.2 f 1.9k
8.3 4.1 0.9 3.0 0.3
50.0 -c 6.1 22.7 -r- 4.0 5.8 f 1.0 6.62 2.1 2.1 & 0.6
0 Percentage of weight. b Uranic acid was detected in the fractions I through III from all materials, though the carbazole reaction was not so typical of uranic acid as usual, but with a color of brown.
both ion exchange chromatography and gel filtration, and therefore further study especially on the third order structure of the glycopeptide will be necessary. If it is possible, the question whether sialic acid is located in the inside of the glycopeptide or on the surface will be clear, and then the reason will also be clear. Uranic acid was detected even in the fraction I over III from the all materials, though the carbazole reaction was not so typical of uranic acid as usual, but with a color of brown. This may suggest that glycosaminoglycan could possibly be contaminated into these fractions, as mentioned before. Further purification of those will surely be necessary, and accordingly it is in progress by the authors. If it is possible, biochemical feature of the myoclonus epilepsy (Lafora-body type) could be more clear; and thereby knowledge of the disease would be extended.
124
MASUDA
ET
AL..
SUMMARY
Urinary glycopeptides in one myoclonus epilepsy patient of Laforabody type, one myoclonus epilepsy patient of degenerative type, and 11 normal humans were assayed by determining the carbohydrate content of materials which remained after proteolysis. Fractionation of nondialyzable glycopeptide from the patient of Lafora-body type by DEAE Sephadex A-SO column chromatography yielded three; from the patient of degenerative type, five; and from normal, five fractions, respectively: they were generally characterized by an increase in peptide and sialic acid as the molarity of Tris-HCI buffer increased. Hexose was the main component, and peptide was the next in each fraction from all the materials. The sugar components consisted of galactose, mannose, fucose, sialic acid. glucosamine. and galactosamine with an almost near content distribution in the all groups except sialic acid. which was contained three to four times more in amount in the material from the patient of Lafora-body type than from the patient of degenerative type and the normal. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9.
Lafora, G. R.. Bull. Gal,. Hosp. Ima& 3, 96 (191 I ). Schwarz, G. A.. and Yanoff. M., Arch. Nrrrrd. 12, 172 (1965). Diebold, K., Arch. Psych&. Nervenkr. 215, 362 (1972). Lowry, 0. H., Rosebrough, N. J., Farr. A. L.. and Randall. R. J.. 265 (1951). Shetlar, M. R., Foster, J. V.. and Everett, M. R.. Pr(~c,. SOC. Erp, (1948). Warren, I-.. J. Biol. Cl~rn~. 234, 1971 (1959). Manley. G.. Severn. M., and Hawksworth. J.. J. C/in. Putho/. 21, Harriman. D. G. F.. and Millar, J. H. D.. Brain 78, 325 (1955). Janeway. R.. Ravens, J. R., Pearce. L. A.. Odor. D. L.. and Naurol.
16, 565
J. Biol. Biol.
Clrrm. Met/.
193, 67, 175
339 (1968). Suzuki,
K..
(1967).
10. Yokoi. S.. Austin. J.. Witmer, F.. and Sakai, M., Arch. Nrurol. 19, 15 (1968). 11. Schnabel. M., and Gootz. M.. Nvuroparhologv 18, 17 (1971). 12. Anraku, S.. and Kawasaki, H.. Folia Psychicd. Neural. Jup. 20, 33 (1966).
Arch.
14, 117-124 (1975)
MEDICINE
Isolation
and from
Characterization the
HIROSHI Institute
of Clinical
of the
Glycopeptide
Urine of Myoclonus Epilepsy of Lafora-Body Type MASUDA Pathology,
AND MUTSUYA
TAKEUCHI
Kurume
School
University
of Medicine
AND AKIHITO Institute
of Brain
KUNITAKE Diseases, Kurume,
AND Kurume Fukuoka
SHIGEMI
ANRAKU
University School 830, Japan
of Medicine,
Received July 31, 197.5
Lafora-body’s disease (progressive myoclonus epilepsy with intraneuronal inclusion body of Lafora) is one of rare hereditary disorders (1). It can be characterized clinically by the onset, in adolescence, of myoclonus, convulsions, and progressive dementia with inexorable progression to death within 4 to 10 years. Of the few reported cases of this disease, most were diagnosed at autopsy, and studies restricted to histopathologic techniques (2, 3). In this paper, it was attempted from the biochemical aspect of this disease to isolate and characterize glycopeptides from the urine of the patient. METHODS Complete 24 hr urine samples, preserved with 0.1% sodium azide, were collected from one myoclonus epilepsy patient (Lafora-body type) of a 17-year-old boy, one myoclonus epilepsy patient (degenerative type) of a lPyear-old, and 11 healthy Japanese with ages ranging from 15-40 years. After measuring the volume of each pooled sample, they were stored in a deep freezer until further work was undertaken. lsolation of Crude Glycopeptides from Urine
Crude glycopeptides were isolated from the urine according to the procedure shown in Fig. 1. The urines were digested with papain (EC 3.4.4.10) in the presence of EDTA and cysteine at 65°C for 48 hr. The mixture was adjusted to pH 7.8 and then digested with trypsin (EC 3.4.4.4) at 37°C for 48 hr with simultaneous dialysis against 0.1 M phosphate buffer of pH 7.8. The digested solution was deproteinized with 5% trichloroacetic acid in the cold, and the supernatant was dialyzed against 117 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
MASUDA
Dissolved with (The Glycopeptide
ET AL.
deionized water and the Glycasamlnoglycans
mlXt”re’
FIG. 1. Isolation of the crude glycopeptide from the urine.
several changes of the cold deionized water for 72 hr. Then 4 vol of alcohol containing 0.1% acetic acid and 1% potassium acetate were added in order to precipitate the crude glycopeptide. The precipitate was washed with alcohol, dried in a desiccator over CaCl,, and then redissolved in 5 ml of deionized water. An aliquot (usually 0.5 ml) was used for hexose and sialic acid analyses. Electrophoresis of Glycopeptides on a Cellulose Acetate Strip.
The material was run in a horizontal type apparatus at constant current of 0.4 mA/cm for 40 min on an OXOID strip (8 x 1.2 cm) in barbital buffer, pH 8.6, p = 0.06. Five to 10 ~1 of aqueous suspensions of preparations in 1 mgimi were used in this study. After electrophoresis, the strip was stained with alcian blue and PAS in the normal way with human serum, hyaluronic acid, and chondroitin sulfate A as standard; hyaluronicacid and chondroitin sulfate A were purchased from Seikagaku Kogyo Co., Ltd, Japan; and hyaiuronic acid was derived from human umbilical cord and chondroitin sulfate A from whale cartilage, respectively. Fractionation of Glycopeptides by DEAE Sephadex A-50 Column chromatography
DEAE Sephadex A-50 (a product of Pharmacia, Uppsala, Sweden) was equilibrated with 0.1 M Tris-HCI buffer, pH 8.0, which was used as the starting buffer for a gradient elution system. Twenty milliliters of 2.5% dry crude material in the starting buffer was applied to a DEAE Sephadex A-50 column (2.5 x 45 cm). Elution was made with a linear gradient of 0.1-1.0 M Tris-HCI buffer. The total elution volume was 1 1. The eluant was collected in 15 ml fractions at a flow rate of 30 ml/hr. An aliquot (usually 0.5 ml, respectively) was used for hexose and peptide analyses. Isolated fractions were dialyzed against deionized water and used for chemical analyses.
MYOCLONUS
Analytical
EPILEPSY
119
Procedures.
Peptide content was determined by the Lowry method (4) with human serum (Hyland Laboratory, U.S.A.) as a standard, and hexose content by tryptophane method of Shetlar et aE. (5), using a mixture of mannose and galactose as a standard. Sialic acid content was estimated by the Warren’s periodic oxidation-thiobarbituric acid method (6), with N-acetylneuraminic acid as a standard. Neutral sugars were separated and indentified by paper chromatography after hydrolysis in N HCl for 3 hr at 100°C in a sealed ampoule; S & S filter paper No. 598 was used as a supporting medium; the solvent system was n-butanol-pyridine-0.1 N HCl (5:3:2 v/v/v). Chromatogram obtained by descending paper chromatography, and double development techniques was stained with AgNO,-NaOH reagent. The separation and quantitative determination of glucosamine and galactosamine were carried out with a Hitachi KLA-3 Amino Acid Analyzer, with a 50 cm column in citrate buffer of pH 5.28 (Na:0.35 N) after hydrolysis in 4 N HCl for 6 hr at 100°C in the sealed ampul. RESULTS Results of the hexose and sialic acid analyses of the glycopeptide from urines of 11 normal persons, one myoclonus epilepsy patient (Lafora-body type), and one myoclonus epilepsy patient (degenerative type) are summarized in Table 1. The sialic acid:hexose ratio was 0.23 + 0.110 in normal, 0.30 + 0.098 in myoclonus epilepsy of Laforabody type, and 0.27 + 0.055 in myoclonus epilepsy of degenerative type, respectively. No difference was found among normal and diseases, except slightly higher ratio of sialic acid to hexose in the patient of Lafora-body type than in the others. Densitograms after electrophoresis of the crude material in the barbital buffer, pH 8.6 are shown in Fig. 2. When stained with alcian blue, both materials had four components which moved towards the anode. This is in agreement with the results of Manley et al. (7) who reported one glycoprotein and three glycosamino-glycans from the normal urine, that is, heparan sulfate, chondroitin sulfate, and hyaluronic acid, respectively. When stained with PAS, both materials had one component which moved slightly towards the anode. This component corresponds to the alcian blue stainable spot which migrated towards the anode more slowly than hyaluronic acid. Any significant difference, however, was scarcely found among normal and the diseases. The elution patterns of the DEAE Sephadex A-50 column chromatography of the crude material are depicted in Fig. 3. The materials from normal and the patient of degenerative type were separated into five hexose peaks at the molarity of Tris-HCI buffer ranging from 0.3 to 0.7 M, whereas that from the patient of Lafora-body type were separated
120
MASUDA
URINARY
OF LAFORA-BODY TYPE EPII.EPSY OF DEGENERATIVE
Myoclonus
epilepsy
16.7 29.6 ?O.O 24.4 16.6
6.7 3.8
1 I.2 20.4
6.0 7.0
7.8 IO.8 X.1
4.7 2.6
II & SD
3.1 t I.84
4.4
epilepsy
Sialic Hexose
3.7 7.1 5.6
Mean
ONCJS
type”
2. I
x 9 I0
MYOC~
MYO~LONL~S TYPE”
of degenerative
type” Sialic acid
IN AND
Myoclonu\
of Lafora-body
Subject
AL.
TABLE I OF GLYCOPEPTID~
EXCRETION
EPILEPSY
ET
15.9
9.4 -+ 6.84
acid
Hexose
4.0 2.3 3.3 6.8
17.1 IS.5 19.3 23.1
6.’ 6.3
23 ! --. I 7’
6.4 5.9
7 I .(I I’.!
5.4 x!z I .42
19.9
-+- 2.87
(’ In milligrams h The patient
per was
24 hr. a l7-year-old
boy,
who
was
examined
twice
a week
for
h weeks.
” The
was
a
boy,
who
was
examined
twice
a week
for
4 weeks.
patient
” Materials
were
collected
l9-year-old from
l I healthy
Japanese
with
ages
ranging
from
1 Y-40
years.
into three hexose peaks at the molarity of Tris-HCl buffer ranging from 0.4 to 0.7 M. Again, column chromatography of the urinary crude material on DEAE Sephadex A-50 showed three distinct elution diagrams. The first was characteristic of Lafora-body type who excreted fraction IV predominantly (A); the second was characteristic of degenerative type who excreted fractions I and II predominantly; and the third diagram was characteristic of the normal control (C and D). Therefore, the fractions corresponding to these peaks were pooled and dialyzed against deionized water for 2 days; the insoluble materials were centrifuged out, and the supernatants were further applied to chemical analyses. Each of the fractions from all materials were subjected to analyses for hexose, sialic acid, hexosamine, and peptide; the results are summarized in Table 2. They were characterized by an increase in peptide and sialic acid as the molarity of Tris-HCl buffer increased. Hexose was main component, and peptide was the next in each fraction from all materials. Sialic acid was contained three to four times more in amount in the fraction V of the material from the patient of Lafora-body type than from the others. The paper chromatography also revealed the presence of galactose. mannose. and fucose in each fraction from the materials.
MYOCLONUS Alcian
Blue
121
EPILEPSY
Stain
PAS Stain
ChS-A
Ponceau-S
0
FIG. 2. Densitograms inoglycans on cellulose
after electrophoresis acetate strip.
of the urinary
glycopeptide
Stain
Anode
and glycosam-
DISCUSSION
In the present stage, opinions differ on the cause of myoclonus epilepsy of Lafora-body type. Harriman (8) and Schwarz (2) reported that it might be due to the disorders of glycoprotein or mucopolysaccharide metabolism, according to the histochemical study on the Lafora-body. Janeway (9) informed that it could arise from the affection of lipid metabolism, depending upon lipid and ganglioside analyses performed on specimens of the cerebral cortex biopsy. Yokoi (10) and Schnabel (11) accounted that the disease might be involved in the disformity of glycogen metabolism, based on the fact that obtained from the chemical analyses of Lafora-body, and histochemical study of Lafora-body, respectively. It is of interest that the intraneuronal inclusion body of Lafora is found in the brain of the myoclonus epilepsy of Lafora-body type and well stained with both alcian blue and PAS, but not in the brain of the myoclonus epilepsy of the degenerative (12); and that the sialic acid rich glycopeptide was isolated from the urine of the myoclonus epilepsy of Lafora-body type. Neither lipid nor glycogen may probably show positive stainability with both alcian blue and PAS. These facts may probably support Harriman (8) and Schwarz (2). But the reason why the sialic acid rich glycopeptide from Lafora-body type was eluted at the same
122
MASUDA
ET .4L.
FIG. 3. Chromatograms of a DEAE Sephadex A-50 elution: --concentration Tris-HCI buffer (M); --------- hexose content: -.-.-*-.-.- absorbance at 280 nm.
of
molarity of Tris-HCl buffer in DEAE Sephadex A-50 column chromatography as materials from normal and from degenerative type were eluted is still unknown. DEAE Sephadex A-50 column chromatography is surely involved in
MYOCLONUS TABLE ANALYSES
OF FRACTIONS
Fraction W
2
FROM DEAE CHROMATOGRAPHY~
Fraction UIP
123
EPILEPSY
SEPHADEX
Fraction (III?
A-50 COLUMN Fraction (IV)
Fraction W)
Myoclonus epilepsy of Lafora-body type Hexose Peptide Sialic Acid Glucosamine Galactosamine
50.9 13.7 2.8 5.6 0.7
56.4 19.2 5.4 11.9 1.9
45.2 19.0 15.1 8.0 2.5
Myoclonus epilepsy of degenerative type Hexose Peptide Sialic Acid Glucosamine Galactosamine
63.5 9.8 3.2 15.7 3.4
65.5 16.4 2.8 15.8 3.2
44.0 18.8 3.4 8.8 2.2
43.2 15.1 4.2 6.6 1.4
45.5 24.3 5.1 6. I 3.9
Normal Hexose Peptide Sialic Glucosamine Galactosamine
51.8 zk 8.2 11.5 -c 2.1 trace 8.5k3.3 3.4kO.8
41.8 k 7.3 17.6 k 3.8 trace 6.2 k 3.4 2.1 + 0.5
40.9 k 5.1 17.4 2 2.8 trace 6.1 f 1.2 2.2 +I 0.4
49.5 k 19.2 k 4.0 f 6.2 f 1.9k
8.3 4.1 0.9 3.0 0.3
50.0 -c 6.1 22.7 -r- 4.0 5.8 f 1.0 6.62 2.1 2.1 & 0.6
0 Percentage of weight. b Uranic acid was detected in the fractions I through III from all materials, though the carbazole reaction was not so typical of uranic acid as usual, but with a color of brown.
both ion exchange chromatography and gel filtration, and therefore further study especially on the third order structure of the glycopeptide will be necessary. If it is possible, the question whether sialic acid is located in the inside of the glycopeptide or on the surface will be clear, and then the reason will also be clear. Uranic acid was detected even in the fraction I over III from the all materials, though the carbazole reaction was not so typical of uranic acid as usual, but with a color of brown. This may suggest that glycosaminoglycan could possibly be contaminated into these fractions, as mentioned before. Further purification of those will surely be necessary, and accordingly it is in progress by the authors. If it is possible, biochemical feature of the myoclonus epilepsy (Lafora-body type) could be more clear; and thereby knowledge of the disease would be extended.
124
MASUDA
ET
AL..
SUMMARY
Urinary glycopeptides in one myoclonus epilepsy patient of Laforabody type, one myoclonus epilepsy patient of degenerative type, and 11 normal humans were assayed by determining the carbohydrate content of materials which remained after proteolysis. Fractionation of nondialyzable glycopeptide from the patient of Lafora-body type by DEAE Sephadex A-SO column chromatography yielded three; from the patient of degenerative type, five; and from normal, five fractions, respectively: they were generally characterized by an increase in peptide and sialic acid as the molarity of Tris-HCI buffer increased. Hexose was the main component, and peptide was the next in each fraction from all the materials. The sugar components consisted of galactose, mannose, fucose, sialic acid. glucosamine. and galactosamine with an almost near content distribution in the all groups except sialic acid. which was contained three to four times more in amount in the material from the patient of Lafora-body type than from the patient of degenerative type and the normal. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9.
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