- Email: [email protected]
On the metabolic role of β-glucuronidase
SHORT COMMUNICATIONS
137
viously 7. It is of interest to note t h a t the values for heparin fluctuate in the same direction as those for hyaluronic acid. This investigation was supported b y grants from The National F o u n d a t i o n and the National Institute of Arthritis and Metabolic Diseases of the United States Public Health Service (AM 05996). The authors are grateful to Dr. D. PETERSON of the Upjohn C o m p a n y for gifts of hydrocortisone and prednisolone.
La Rabida-University of Chicago Institute and the Departments of Biochemistry and Pediatrics, University of Chicago, Chicago, Ill. (U.S.A.) I 2 3 4 5 6 7
SARA SCHILLER* NELLIE BLUMENKRANTZ ALBERT DORFMAN
S. SCHILLERAND A. DORFMAN,Endocrinology, 60 (1957) 376. S. SCHILLER,G. A. SLOVERAND A. DORFMAN,J. Biol. Chem., 236 (I96I) 983. Z. DISCH~, J. Biol. Chem., I67 (1947) 189. S. SCHILLERAND A. DORFMAN,Biochim. Biophys. Acta, 78 (1963) 371. Z. FEDIAYAND M. M. CLAY, Nature, 202 (1964) 907. D. KAPLANAND B. FISHER, Biochim. Biophys. Acta, 83 (I964) lO2. S. SCHILLER,G. A. SLOVERAND A. DORFMAN,Biochim. Biophys. Acta, 58 (1962) 27.
Received October 2Ist, 1964 * Present address: Departments of Pathology and Biochemistry, New York Medical College, New York, U.S.A.
Biochim. Biophys. Acta, IOI (1965) 135-137
sc 83018
On the metabolic role of~3-glucuronidase fl-Glucuronidase (fl-D-glucuronide glucuronohydrolase, EC 3.2.1.31) is an ubiquitous enzyme whose specific role in intermediary metabolism has not been ascertained, as yet. It has been implicated in the biosynthetic p a t h of L-ascorbic acid, since one of the products of the reaction is w-glucuronic acid, a clearly established precursor of vitamin C. However, a different p a t h of D-glucuronic acid formation has been suggested where fl-glucuronidase does not intervene:. Evidence for the participation of fl-glucuronidase stems from the fact t h a t the administration of drugs capable of stimulating L-ascorbic acid synthesis produces an increase of glucuronyl transferase, an enzyme responsible for the formation of fl-glucuronides, substrates of fl-glucuronidase:. To test the possibility t h a t fl-glucuronidase functions as postulated, the urinary excretion of L-ascorbic acid was measured 2 in two strains of mice, one (DBA/2j) with high and the other (C3H/Hej) with low enzyme activity3, 4 after the administration of chloretone, a c o m p o u n d causing augmentation of vitamin C excretion x. Fig. I illustrates the results of this experiment. It can be seen t h a t the two strains behaved in a similar manner. I t was further proved t h a t the corresponding levels of liver flglucuronidase in the two mice strains (4.3o and o.13/,moles of p r o d u c t per min per m g of protein) was not modified, as assayed b y the m e t h o d of LEVVY~, after extraction
Biochim. Biophys. Acta, ioi (1965) 137-14o
138
SHORT COMMUNICATIONS
" •1.0"o
E
°
1
v "c3 o
o_ x~ 0.5tn
o~ w
0
I
I
I
Time (clays) Fig. I. Urinary excretion of L-ascorbic acid by mice following a single intraperitoneal injection of 4.0 mg of chloretone (arrow). O - - © , DBA/2j; Q---Q, C3H/He j.
of the tissue with water to ensure complete release of the enzyme from the particles. These results indicate that the participation of/~-glucuronidase in the vitamin C biosynthetic path is rather questionable. fi-Glucuronidase has also been implicated in the catabolism of mucopolysaceharides 5, a fact that is in agreement with its location in lysosomes 6. Because hyaluronic acid can be completely hydrolyzed in vitro by the sequential action of hyaluronate lyase (formerly known as hyaluronidase, EC 4.2.99.1), fi-glucuronidase and another lysosomal enzyme 7, fl-N-acetyl glucosaminidase s, it was decided to measure the two latter activities in processes characterized by a drastic tissue catabolic breakdown. The systems chosen for this purpose were the diminution of liver mass in the rat produced b y starvation 9 and the reabsorption of the tadpole's tail (Rana montezumae) during metamorphosis, fl-N-Acetyl glucosaminidase was assayed after FINDLAY et al. lo.
The effect of starvation on specific activity of the enzymes (#moles of product per rain per mg protein) is expressed as the change in percentage, from the controls, of the ratio liver weight/body weight, since no correlation could be established between the length of food deprivation and the specific activity of the enzymes studied. Rats of 20-40 g were employed (Fig. 2). It can be seen that only those animals where the mentioned ratio decreased, showed an elevation in the activity of both enzymes. The total activity (/zmoles of product per rain per whole liver) was not changed b y starvation which indicates that the enzymes are preferentially spared while other proteins are being destroyed. Rats of larger size (65-25o g) failed to show the enzyme elevation under the same conditions and they showed no change in the ratio liver weight/body weight. Fig. 3 presents the change in the specific activity of the enzymes assayed in the tadpole tail during different stages of metamorphosis, expressed as a change in the ratio tail length/hind-limb length. It can be observed that the specific activity increases as the tail shortens during the process. The total activity was not determined because the limits of the tail are not precise. I t should be mentioned, as an interBiochim. Biophys. Acta, IOI (1965) 137-14o
z39
SI'IORT COMMUNICATIONS
c
o
A
B
c_ 3 0 -
o
E fi
-lOO
o
o
o
Ck
20-
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o
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I
100 Liver
Ratio
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I
I
50
100
weigth~Body weigt h
Fig. 2. Specific activity of fl-glucuronidase (A) and fl-N-acetyl glucosaminidase (B) in the liver of control (0) and starved ( 0 ) rats (see text for details).
esting incidental finding, that the morphological alterations observed during the reabsorption of the tadpole tail, very closely resemble those present in muscular dystrophy, a degenerative disease, where an increase in fl-glucuronidase has also been reported n. This implies that the investigation of biochemical changes occurring in the tadpole tail during metamorphosis, might be a suitable model for such studies. A
3.0-
150-
B
k k E'O0-
~ 2.0-
U
E
8
m
k
-6
1.o-
E 50"1
"6 E
.>
-2-
E w
b
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*
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lb
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Ratio: tail l e n g t h / h i n d limb l e n g t h
Fig. 3. Specific activity of fl-glucuronidase (A) and fl-N-acetyl glucosaminidase (B) in the tadpole tail during metamorphosis. Biochim. Biophys. Acta, zoi (i965) r37-r4o
SHORT COMMUNICATIONS
14 °
The evidence presented here is a compatible with the functioning of fl-glucuronidase and fl-N-acetyl glucosaminidase in the catabolic breakdown of nmcopolysaccharides. This work was supported with a research grant from the Rockefeller Foundation (RF62oo5). The authors are grateful to Dr. LEVVY for the small amount of p-nitrophenyl-N-acetyl-fl-glucosaminide for testing the quality of the product synthesized by them.
Departamento de Bioqulmica, Instituto Nacional de la Nutricidn, Mdxico, D.F. (Mdxico)
j . MORA L. CANEDO (;, SOBERON
I S. HOLLMANN AND O. TOUSTER, Biochim. Biophys. Acta, 62 (1962) 338. 2 T h e A s s o c i a t i o n of V i t a m i n Chemists, lnc., Methods of V i t a m i n assay, 211(1 ed., l n t e r s c i e n c e , New York , 1951 , p. 76 . 3 A. C,. MORROW, E. M. (;REENSPAN AND I). M. CARROLL, dr. Natl. Cancer 1J*st., 1o (1949) 6574 K. PAIGEN, Proc. Natl. Acad. Sci. U.S., 47 (1961) I641. 5 G. A. LEVVY AND C. A. MARSH, Advan. Carbohydrate Chem., 14 (1959) 38~ 6 C. DE DUVE, B. C. PRESSMAN, R. GIANETTO, R. WATTIAUX AND F. APPELMANS, Biochem. J., 60 (1955) 604. 7 0 . Z. SELLINGER, H. BEAUFAY, r'. JACQUES, A. DOYEN AND C. DE DUVE, Biochem. J., 74 8 9 IO II
(196o) 45 ° . A. LINKER, K. MEYER AND B. WEISSMANN, J . Biol. Chem., 213 (1955) -'37. G. SOBER6N AND Q. E. S,~NCHEZ, J. Biol. Chem., 236 (1961) 16o2. J. FINDLAY, G. A. LEVVY AND C. A. MARSH, Biochem. J., 69 (19.58) 467 . H. ZALKIN, A. L. TAPPEL, }{. A. CALDWELL, S. SHIBKO, ]. D. DESAI AND T. A. HOLLIDAY, J. Biol. Chem., 237 (1962) 2678.
Received December 7th, 1964 Biochim. Biophys. Acla, i o i (1965) I 3 7 - I 4 o
137
viously 7. It is of interest to note t h a t the values for heparin fluctuate in the same direction as those for hyaluronic acid. This investigation was supported b y grants from The National F o u n d a t i o n and the National Institute of Arthritis and Metabolic Diseases of the United States Public Health Service (AM 05996). The authors are grateful to Dr. D. PETERSON of the Upjohn C o m p a n y for gifts of hydrocortisone and prednisolone.
La Rabida-University of Chicago Institute and the Departments of Biochemistry and Pediatrics, University of Chicago, Chicago, Ill. (U.S.A.) I 2 3 4 5 6 7
SARA SCHILLER* NELLIE BLUMENKRANTZ ALBERT DORFMAN
S. SCHILLERAND A. DORFMAN,Endocrinology, 60 (1957) 376. S. SCHILLER,G. A. SLOVERAND A. DORFMAN,J. Biol. Chem., 236 (I96I) 983. Z. DISCH~, J. Biol. Chem., I67 (1947) 189. S. SCHILLERAND A. DORFMAN,Biochim. Biophys. Acta, 78 (1963) 371. Z. FEDIAYAND M. M. CLAY, Nature, 202 (1964) 907. D. KAPLANAND B. FISHER, Biochim. Biophys. Acta, 83 (I964) lO2. S. SCHILLER,G. A. SLOVERAND A. DORFMAN,Biochim. Biophys. Acta, 58 (1962) 27.
Received October 2Ist, 1964 * Present address: Departments of Pathology and Biochemistry, New York Medical College, New York, U.S.A.
Biochim. Biophys. Acta, IOI (1965) 135-137
sc 83018
On the metabolic role of~3-glucuronidase fl-Glucuronidase (fl-D-glucuronide glucuronohydrolase, EC 3.2.1.31) is an ubiquitous enzyme whose specific role in intermediary metabolism has not been ascertained, as yet. It has been implicated in the biosynthetic p a t h of L-ascorbic acid, since one of the products of the reaction is w-glucuronic acid, a clearly established precursor of vitamin C. However, a different p a t h of D-glucuronic acid formation has been suggested where fl-glucuronidase does not intervene:. Evidence for the participation of fl-glucuronidase stems from the fact t h a t the administration of drugs capable of stimulating L-ascorbic acid synthesis produces an increase of glucuronyl transferase, an enzyme responsible for the formation of fl-glucuronides, substrates of fl-glucuronidase:. To test the possibility t h a t fl-glucuronidase functions as postulated, the urinary excretion of L-ascorbic acid was measured 2 in two strains of mice, one (DBA/2j) with high and the other (C3H/Hej) with low enzyme activity3, 4 after the administration of chloretone, a c o m p o u n d causing augmentation of vitamin C excretion x. Fig. I illustrates the results of this experiment. It can be seen t h a t the two strains behaved in a similar manner. I t was further proved t h a t the corresponding levels of liver flglucuronidase in the two mice strains (4.3o and o.13/,moles of p r o d u c t per min per m g of protein) was not modified, as assayed b y the m e t h o d of LEVVY~, after extraction
Biochim. Biophys. Acta, ioi (1965) 137-14o
138
SHORT COMMUNICATIONS
" •1.0"o
E
°
1
v "c3 o
o_ x~ 0.5tn
o~ w
0
I
I
I
Time (clays) Fig. I. Urinary excretion of L-ascorbic acid by mice following a single intraperitoneal injection of 4.0 mg of chloretone (arrow). O - - © , DBA/2j; Q---Q, C3H/He j.
of the tissue with water to ensure complete release of the enzyme from the particles. These results indicate that the participation of/~-glucuronidase in the vitamin C biosynthetic path is rather questionable. fi-Glucuronidase has also been implicated in the catabolism of mucopolysaceharides 5, a fact that is in agreement with its location in lysosomes 6. Because hyaluronic acid can be completely hydrolyzed in vitro by the sequential action of hyaluronate lyase (formerly known as hyaluronidase, EC 4.2.99.1), fi-glucuronidase and another lysosomal enzyme 7, fl-N-acetyl glucosaminidase s, it was decided to measure the two latter activities in processes characterized by a drastic tissue catabolic breakdown. The systems chosen for this purpose were the diminution of liver mass in the rat produced b y starvation 9 and the reabsorption of the tadpole's tail (Rana montezumae) during metamorphosis, fl-N-Acetyl glucosaminidase was assayed after FINDLAY et al. lo.
The effect of starvation on specific activity of the enzymes (#moles of product per rain per mg protein) is expressed as the change in percentage, from the controls, of the ratio liver weight/body weight, since no correlation could be established between the length of food deprivation and the specific activity of the enzymes studied. Rats of 20-40 g were employed (Fig. 2). It can be seen that only those animals where the mentioned ratio decreased, showed an elevation in the activity of both enzymes. The total activity (/zmoles of product per rain per whole liver) was not changed b y starvation which indicates that the enzymes are preferentially spared while other proteins are being destroyed. Rats of larger size (65-25o g) failed to show the enzyme elevation under the same conditions and they showed no change in the ratio liver weight/body weight. Fig. 3 presents the change in the specific activity of the enzymes assayed in the tadpole tail during different stages of metamorphosis, expressed as a change in the ratio tail length/hind-limb length. It can be observed that the specific activity increases as the tail shortens during the process. The total activity was not determined because the limits of the tail are not precise. I t should be mentioned, as an interBiochim. Biophys. Acta, IOI (1965) 137-14o
z39
SI'IORT COMMUNICATIONS
c
o
A
B
c_ 3 0 -
o
E fi
-lOO
o
o
o
Ck
20-
~o
o
o*
g~oo
o
El 5_
I
o
~.-..I
~o-
E c £o
I
Q.
o
oO~O°
"6
o
~5
°°o
° :':1 '
50
I
100 Liver
Ratio
E
I
I
50
100
weigth~Body weigt h
Fig. 2. Specific activity of fl-glucuronidase (A) and fl-N-acetyl glucosaminidase (B) in the liver of control (0) and starved ( 0 ) rats (see text for details).
esting incidental finding, that the morphological alterations observed during the reabsorption of the tadpole tail, very closely resemble those present in muscular dystrophy, a degenerative disease, where an increase in fl-glucuronidase has also been reported n. This implies that the investigation of biochemical changes occurring in the tadpole tail during metamorphosis, might be a suitable model for such studies. A
3.0-
150-
B
k k E'O0-
~ 2.0-
U
E
8
m
k
-6
1.o-
E 50"1
"6 E
.>
-2-
E w
b
**
*
-° %
lb
*°-*
2'o
0
o
° *
"~
*--?~*
20
Ratio: tail l e n g t h / h i n d limb l e n g t h
Fig. 3. Specific activity of fl-glucuronidase (A) and fl-N-acetyl glucosaminidase (B) in the tadpole tail during metamorphosis. Biochim. Biophys. Acta, zoi (i965) r37-r4o
SHORT COMMUNICATIONS
14 °
The evidence presented here is a compatible with the functioning of fl-glucuronidase and fl-N-acetyl glucosaminidase in the catabolic breakdown of nmcopolysaccharides. This work was supported with a research grant from the Rockefeller Foundation (RF62oo5). The authors are grateful to Dr. LEVVY for the small amount of p-nitrophenyl-N-acetyl-fl-glucosaminide for testing the quality of the product synthesized by them.
Departamento de Bioqulmica, Instituto Nacional de la Nutricidn, Mdxico, D.F. (Mdxico)
j . MORA L. CANEDO (;, SOBERON
I S. HOLLMANN AND O. TOUSTER, Biochim. Biophys. Acta, 62 (1962) 338. 2 T h e A s s o c i a t i o n of V i t a m i n Chemists, lnc., Methods of V i t a m i n assay, 211(1 ed., l n t e r s c i e n c e , New York , 1951 , p. 76 . 3 A. C,. MORROW, E. M. (;REENSPAN AND I). M. CARROLL, dr. Natl. Cancer 1J*st., 1o (1949) 6574 K. PAIGEN, Proc. Natl. Acad. Sci. U.S., 47 (1961) I641. 5 G. A. LEVVY AND C. A. MARSH, Advan. Carbohydrate Chem., 14 (1959) 38~ 6 C. DE DUVE, B. C. PRESSMAN, R. GIANETTO, R. WATTIAUX AND F. APPELMANS, Biochem. J., 60 (1955) 604. 7 0 . Z. SELLINGER, H. BEAUFAY, r'. JACQUES, A. DOYEN AND C. DE DUVE, Biochem. J., 74 8 9 IO II
(196o) 45 ° . A. LINKER, K. MEYER AND B. WEISSMANN, J . Biol. Chem., 213 (1955) -'37. G. SOBER6N AND Q. E. S,~NCHEZ, J. Biol. Chem., 236 (1961) 16o2. J. FINDLAY, G. A. LEVVY AND C. A. MARSH, Biochem. J., 69 (19.58) 467 . H. ZALKIN, A. L. TAPPEL, }{. A. CALDWELL, S. SHIBKO, ]. D. DESAI AND T. A. HOLLIDAY, J. Biol. Chem., 237 (1962) 2678.
Received December 7th, 1964 Biochim. Biophys. Acla, i o i (1965) I 3 7 - I 4 o