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Degradation of mucopolysaccharides by hepatic lysosomes
312
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 2 5 5 0 2
DEGRADATION OF MUCOPOLYSACCHARIDES BY HEPATIC LYSOSOMES* FERENC
HUTTERER
Department of Pathology, The Mount Sinai Hospital, New York, N . Y . (U,S.A,) (Received J u l y 261h, 1965)
SUMMARY
I. Hepatic lysosomes degraded highly polymerized mucopolysaccharides. The degradation products as shown by high-voltage electrophoresis were tetra- and oligosaccharides, indicating the presence of endohexosaminidase in the liver. 2. The role of bacterial contamination, and non-specific oxido-reductive degradation of mucopolysaccharides were excluded. 3. The activity of hepatic mucopolysaccharase (hyaluronate glycanohydrolase, EC 3.2.1.35 ) was related to the turnover rate of hepatic connective tissue. The enzyme activity was increased in reversible fibrosis and greatly diminished in irreversible fibrosis.
INTRODUCTION
Connective tissue excess in parenchymal organ upsets the normal structure and interferes with biochemical functions. Removal of the excess connective tissue demonstrated after ethionine-induced hepatic fibrosis in rats leads to the restoration of normal structure and function 1. Similar recoveries were noted in serial biopsies of human liver after hepatitis and early cirrhosisE The mechanism of the removal of the connective-tissue elements, collagen and mucopolysaccharides, is not well understood, since specific depolymerases of these macromolecules were not demonstrated conclusively in the liver, or in any other organ3, 4, with the exception of the hyaluronidase in the testis. Recently, BOLLETT et al. 5 presented suggestive evidence for the presence of hyaluronidase in the kidney and other tissues. It has previously been proposed that the initial step in collagen removal in the liver is a non-enzymatic denaturation of collagen, preceded by the disappearance of its cellular framework, and followed by partial digestion by non-specific proteinases and by phagocytosis 6. By contrast, this paper describes a specific enzyme for the degradation of mucopolysaccharides, localized exclusively in the lysosomal fraction of the liver. Analysis of the degradation products indicates that the enzyme is an endohexosaminidase. Its activity with different mucopolysaccharide-substrates and the changes of activity in different stages of hepatic fibrosis were also studied. * P r e l i m i n a r y r e p o r t s on this i n v e s t i g a t i o n a p p e a r e d in a b s t r a c t f o r m in: Federation Proc., 23 (1964) 334; Federation Proc., 24 (1965) 557.
Biochim. Biophys. Acta, 115 (I966) 312-319
313
MUCOPOLYSACCHARIDE DEGRADATION IN LIVER
MATERIALS AND METHODS
Hyaluronic acid grade I from umbilical cord was obtained from Sigma Co., St. Louis, Mo., with a relative viscosity of ten. Chondroitin sulfates A, B and C and heparitin sulfate were obtained from Dr. K. MEYER, Columbia University, New York, N.Y., and crystalline tetrasaccharide from hyaluronic acid from Dr. B. WEISSMANN, University of Illinois, Chicago, Ill.
Experimental animals Livers were obtained from Sprague-Dawley female rats with an average body weight of 17o g. Acute injury and reversible and irreversible hepatic fibrosis were produced by treatment with ethionine or carbontetrachloride as describedl,L The criterion for reversibility was the resorption of hepatic collagen from a fibrotic liver on a recovery diet.
Preparation of enzyme extract Livers were perfused with cold 0.25 M sucrose, and the subcellular particles were separated by differential ultracentrifugation s. The mitochondria were resuspended in o.25 M sucrose, layered on a Spinco Model L ultracentrifuge in a swinging bucket rotor for 15o min at 25ooo rev./min. The lysosomal fraction was obtained in the layer D = 1.28. The fraction was washed five times with physiologic NaC1 solution to remove sucrose, resuspended in 5 ml physiologic NaC1 solution, and treated with ultrasound. Optimum liberation of particle-bound enzyme without loss of activity was obtained at 9 A for 9osec. After centrifugation at 3oooo rev./min, the enzyme was recovered in the supernatant. It could be kept at --2o ° for I day without loss of activity.
Mucopolysaccharase assay* The incubation mixture consisted of 0.5 ml enzyme extract containing 0.53.o mg protein, and 0.5 ml o. I M acetate buffer containing o. 5 mg mucopolysaccharides. The p H of the acetate buffer was varied according to the optimum p H found for each substrate, 3.9 for hyaluronic acid and chondroitin sulfates A, B and C, and 4.8 for heparitin sulfate. Incubation was carried out at 37 ° under toluene for various intervals. The enzyme activity was measured by rate of liberation of N-acetylglucosamine endgroups. Aliquot of 0.5 ml of the incubation system was boiled with o.I ml of o.8 M potassium tetraborate (pH 9.1) and after cooling 3.o ml of 1% p-dimethylaminobenzaldehyde in glacial acetic acid containing 1.2 ml io N HC1 per ioo ml was added, and a color was developed for 2o min at 37 °. The absorbance was read in a Beckman DU spectrophotometer at 585 m/~ (ref. 9). If precipitation or turbidity developed, the samples were centrifuged before spectrophotometric measurement.
Separation of the degradation products with high-voltage electrophoresis After incubation, the protein was precipitated with trichloroacetic acid (final concentration was lO%). Excess trichloroaeetic acid was extracted with ether and the slightly acidic aqueous phase was evaporated in vacuo. The residue was redissolved in * " M u c o p o l y s a c c h a r a s e " (hyaluronate glycanohydrolase, EC 3.2.1.35) is used as trivial n a m e instead of " h y a l u r o n i d a s e " because the s u b s t r a t e specificity of the liver enzyme is not established
Biochim. Biophys. Acta, 115 (1966) 312-319
314
F. HUTTERER
a small volume of water and applied to a Whatman No. 3MlX{ paper. Electrophoretic separation was carried out in a Savant H.V. electrophoretic system in pyridine acetic acid-water (I : lO:9O, v/v) for 3o min at 3ooo V. The paper was dried at 13 °° for 3 ° min, and the spots were visualized with p-dimethylaminobenzaldehyde. Since the buffer system and impurities in pyridine interfere with the color development, the solvents, especially pyridine, had to be redistilled. After drying, the paper was dipped into o.5 °/;o alcoholic K O H and transferred into o.5% alcoholic K O H containing 1.o ml acetylacetone per IOO ml; 3 min later the paper was put in an oven at IO5 ° for IO min, then dipped into IO % alcoholic HE1, and trangferred into IO % alcoholic HC1 containing I g p-dimethylaminobenzaldehyde per IOO ml. The paper, protected from light, was dried at room temperature. Without these precautions the color development was inhibited, or the color was very unstable.
Analysis Succinate dehydrogenase (succinate: (aeceptor) oxido-reductase, EC 1.3.99,I) 1°, /3-glucuronidase (fl-D-glucuronide glucuronohydrolase, EC 3.2.i.3i) 11, and protein ~2 were measured as quoted. RESULTS
SubcelluIar localization of mucopolysaccharase activity The mitoehondrial-lysosomal fraction exhibited more than 80% of the total activity, while approx. 12% was unsedimentable. The nuclear and microsomal fractions were inactive. The mitochondrial-lysosomal fraction was further separated by density-gradient ultracentrifugation on a sucrose gradient. The highest mucopolysaccharase activity, similarly to /3-glucuronidase, was found at the density of 1.28; succinate dehydrogenase activity was localized at the density of 1.24 (Fig. I). DIFFERENTIAL U LTRACEN TRIFUGATION
DENSITY GRADIENT ULTRACENTRIFUGATION
MUCOPOLYSACCHARASE
~ >--d
I
v-ul '~ ~
i~ 1'°1
J
.
1 ,8-GLUCURONIDASE
~o
L~V-
2
¢'~ (3
O.B
1 SUCCINATE DEHYDROGENASE
>~ jO w~ S
N M+L
ER
1.16 1.20 1.24 1.28 SUCROSE DENSITY
Fig. i. Localization of inucopolysaccharase activity in the subcellular fractions of liver. Fractions : S, soluble; N, nuclear; M + L, mitochondrial-lysosomal; Er, microsomal.
Release of particle-bound activity The sucrose layer ( D = I . 2 8 ) containing the highest enzyme activity was separated, diluted to isotonicity, washed repeatedly with and resuspended in physiologic Biochim. Biophys. Acta, 115 (1966) 312-319
315
MUCOPOLYSACCHARIDE DEGRADATION IN LIVER
N aC1. Ultrasonic treatment, 20 kcycles/sec, resulted in a complete release of the enzyme activity into the supernatant within 90 sec (Fig. 2).
3o
E
TIME
6b
9'0
OFULTRASONICTREATMENT,20 Kcycles/sec
I~ig. 2. I~elease of mucopo]ysaccharase activity from hepatic lysosomes by ultrasonication.
Optim~tm conditions for the enzyme determination The pH optimum in 0.02 M acetate buffer was 3.9 for hyaluronic acid. The limited solubility of high-viscosity hyaluronic acid, and the known inhibition of enzyme activity by high concentration of hyaluronic acid ~ did not permit detailed kinetic studies. The best compromise was a substrate concentration of 0.5 mg hyaluronic acid per I.O ml incubation mixture, when the enzyme activity was directly proportional to the protein concentration over a 6-fold range, and to the incubation time over an 8-h period (Fig. 3). pH
50L
E 20~
*oI 0
[ __k ~
2
2140 03050 SU /B *S/TRA./'°~'" TE CONC ~'~. ENTRAT •O IN
/"'X
40
.,
3 pM
4
5
IO
" '//I
I
0
0.2
I
I
I
~--
C.4 0 6 SUBSTRATE ( m9 )
<[ ENZYME PROTEINCONCENTRATO I Nd
1I20 00140
.J
ooL
L~J ,
~or-
./
- 2oI
60 4Gf 20
J
J0' I //" ~ [ L _ ~ 0
TIM~
I 04
u~
L
08 12 P R O T E I N { rag)
t
16
L --
0
~° / " °~° 2
4
6
8 I0 12 14 TIME (HOURS)
16 i~
Fig. 3. Effect of pH, substrate and e n z y m e concentrations, and t i m e of i n c u b a t i o n on hepatic m u c o p o l y s a c c h a r a s e a c t i v i t y . I n c u b a t i o n s y s t e m consisted of o. 5 ml l y s o s o m a l extract in 0.9% NaC] and h y a l u r o n i c acid in 0. 5 ml buffer. E n z y m e a c t i v i t y was expressed as mffmoles of liberated N - a c e t y l g l u c o s a m i n e . Boiled e x t r a c t served as control.
Degradation products of hyaluronic acid after incubation with hepatic lysosomal extract With high-voltage electrophoresis, glucuronic acid (GlcUA), which gave a dark brown color after p-dimethylaminobenzaldehyde staining, served as reference. Crystalline tetrasaccharide from hyaluronic acid emerged with an RGletTA of 0.94, and N-acetylglucosamine with a n RGle UA of 0.03. Both gave a pink color after staining, which was distinguishable from the brown spot of glucuronic acid. After incubation of hyaluronic acid with the lysosomal extract for 8 h, tetrasaccharides and several oligosaccharides of unknown chain length were demonstrated, but neither free N-acetylglueosamine nor free glucuronic acid were found (Fig. 4). Biochim. Bio#hys. Acta, 115 (1966) 3 1 2 - 3 1 9
3 I6
F. HUTTERER
IIII
M U C O P O L Y S A C C H A R A S E ACTIVITY --"--
UNSEDIMENTABLE
p GLUCURONIDASE
" '~
100 -
OCONTROL
ACUTE
REVERSIBLE
IRREV.
INd.
F~~ O S I S
FIBROSIS
Fig. 4. Separation of degradation products of hyaluronic acid by high-voltage electrophoresis after digestion witla lysosomal extract of the liver, incubation systenl contained o. 5 ml lysosomal extract, 0. 5 ml substrate (0. 5 rag) in acetate buffer (pH 3-9)- G, glucuronie acid; H, N acetylglucosamine ; T, crystalline tetrasaccharidc from hyaluronic acid ; X, hepatic digest showing tetraand oligosaccharides and traces of disaecharides. (Recently, similar results have been obtained by applying Dowex-L-X*o column chromatography followed by paper chromatographic sepa ration2L) Fig. 5. Hepatic nlucopolysaccharasc and fl glucuronidase activity in acute injury and during reversible and irreversible fibrosis. TABLE 1 HEPATIC
MUCOPOLYSACCItARASE
ACTIVITY
WITH
DIFFERENT
SUBSTRATES
Incubation system: o. 5 ml lysosomal extract in o.9°o NaC1, o. 5 mg substrate in o.o2 M acetate buffer (pH 3.9), except with heparitin sulphate (pH 4.5)-
Subslrates
Enzyme aeti~,ity N-A cetylhe.rosamine liberaled (re#moles per mg protein)
Hyaluronic acid Chondroitin sulfate A Chondroitin sulfate B Chondroitin sulfate C Heparitin sulfate
6 h
z8 h
34.4 4.8 None 4.5 5.8
84-o 13.o None 12.8 14.2
Biochim. Biophys. dcla, 1i 5 (t966) 312 319
MUCOPOLYSACCHARIDE
D E G R A D A T I O N IN L I V E R
317
Degradation of different mucopolysaccharides Besides hyaluronic acid, chondroitin sulfates A, B and C, and heparitin sulfate were used as substrates. Chondroitin sulfates A and C and heparitin sulfate were cleaved six to seven times more slowly than hyaluronic acid. Chondroitin sulfate B was not degraded by the lysosomal extract (Table I).
Pathological alterations In acute liver injury produced b y ethionine, hepatic mucopolysaccharase activity was lower than in the control, while the relative proportion of unsedimentable to sedimentable activity was higher. In reversible fibrosis, exemplified by subacute ethionine intoxication, the enzyme activity had risen over that of the control. In irreversible fibrosis, exemplified by chronic CC14 intoxication, the enzyme activity was greatly diminished. Lysosomal ~-glucuronidase had shown a similar pattern of activity (Fig. 5).
Hepatic mucopolysaccharase in gerrnzfree rats Lysosomal extracts of livers of six germ-free rats liberated 37.1 ± 4.2 mlzmoles of N-acetylglucosamine from hyaluronic acid, as compared to 34.4 ~ 6.6 m~moles by conventionals. The results statistically are not significantly different. DISCUSSION
The short half life of mucopolysaccharides 13 and the lack of specific hyaluronidases in mammalian tissues 3 led to the proposition of a series of indirect mechanisms of mucopolysaccharide degradation. These included bacterial contamination 14, non-specific oxido-reductive degradation ~s, and the combined action of exohexosaminidase and glucuronidase at the chain end 16. Bacteria are rich sources of hyaluronidases, and the exogenous or endogenous contamination of rat liver m a y produce misleading results a4. To exclude the role of contamination, germ-free rats were used in some experiments. Their livers degraded hyaluronic acid at the same rate as the conventional controls, eliminating the possibility that the demonstrated mucopolysaccharase activity of the liver is of bacterial origin. Non-specific oxido-reductive degradation of mucopolysaccharides was observed in the presence of ascorbic acid, hydroquinone, cysteine, Fe z+, Cu 2+. It was suggested that protein m a v also mediate this reaction ~5. The localization of enzyme activity exclusively in the lysosomal fraction tends not to support this possibility. At present, there is no indication that the lysosomal fraction is a better mediator of the oxidoreductive degradation than the mitochondrial fraction. N-Acetyl-/3-glucosaminidase and /3-glucuronidase are both localized in the lysosomes 17. Their combined action at the chain-end of hyaluronic acid would result in the accumulation of free N-acetylglucosamine and glucuronic acid as degradation products. Neither of these could be demonstrated by high-voltage electrophoresis. Instead, tetrasaccharides and oligosaecharides were shown indicating the presence of an endohexosaminidase in hepatic lysosomes. Further degradation of tetrasaccharides was prevented by acetate, which is an inhibitor of exohexosaminidase is. The lysosomal extract also cleaved chondroitin sulfates A and C and heparitin sulfate, though at a Biochim. Biophys. Acta, 115 (1966) 3 1 2 - 3 1 9
3 I8
F. HUTTERER
much slower rate. Chondroitin sulfate B was not susceptible to cleavage by the enzyme. The cleavage of heparitin sulfate is of particular interest, because heparitin sulfate is resistant to testicular hyaluronidase ~9. A lack of enzyme may play a role in the accumulation of heparitin sulfate in the liver in some pathologic conditions, such as the Hurler syndrome ~° and amyloidosis 21. The physiologic role of the enzyme is not yet clear. Two mechanisms have been suggested for the catabolism of mucopolysaccharides: one is phagocytosis by mesenchymal cells followed by intracellular degradation; the second is direct excretion of the polymer into the urine after dissociation of the mucopolysaccharide-protein complex. The relative importance of the two pathways depends upon the type of mucopolysaccharide undergoing degradation, its molecular size, and its digestibility 22. The intracellular degradation of nmcopolysaccharides following phagocytosis may be initiated by mucopolysaccharase and brought to completion in vivo by exohexosaminidase 23 and glucuronidase. This would imply that the mucopolysaccharase is contained in mesenchymal cells, a possibility supported by the observation that the enzyme activity changes parallel with the number of mesenchymal cells '4. The pathologic role of the enzyme is similarly obscure. In acute liver injury, the increase in the unsedimentable portion of the lysosomal enzymes is an indication of proceeding cell disruption due to necrosis "a. In reversible fibrosis, in which the half life of insoluble collagen is less than 25 days 2G, mucopolysaccharase activity was increased. In the irreversible phase, in which the half life of insoluble collagen is more than 25o days 26, mucopolysaccharase activity was greatly diminished. The parallel nature of the changes in mucopolysaccharase and fl-glucuronidase activity reflects lysosomal alterations rather than specific changes in connective tissue metabolism. The variations in enzyme activity may therefore be a simple expression of the number and activity of the lysosome-rich hepatic mesenchymal cells, which, by their phagocytic and hydrolytic capacities, influence the turnover of connective tissue in the reversible and irreversible phases of fibrosis. ACKNOWLEDGEMENTS
This work was supported by Grants No. DA-49-oo7-MD-79oo3 from the U.S. Army Medical Research and Development Command, and No. AM-o3846 from the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service.
REFERENCES I H. HUTTERER, E. RUBIN, E. J. SINGER AND H. POPPER, Cancer Res., 21 ( i 9 6 i ) 205. 2 H. POPPER AND F. SCHAFFNER, Liver: Structure and Function, The B l a k i s t o n Division, Mc G ra w H i l l Book C o m p a n y , New York, 1957, p. 777. 3 M. SCHUBERT, Pfoe. Syrup. N . Y . Heart A SHOe., New York, 1964, Li t t l e , B r o w n a n d Co., Boston, 1964, p. 124. 4 J- MANDEL, Advan. Enzymol., 23 (1961) 557. .5 i . J. ]~OLLETT, ~r. M. BONNER, JR. AND J. L. NANCE, ft. Biol. Chem., 238 (1963) 3522. '6 F. HUTTERER, E. RUBIN AND H. POPPER, Exptl. Mol. Pathol., 3 (1964) 215. 7 E. I~UBIN, F. HUTTERER AND H. POPPER, ,4rn. J. Pathol., 42 (1963) 715 . 8 W. C. SCHNEIDER AND G. H. HOGEBOOM, J. Biol. Chem., 183 (1956) 123. 9 J. C. REISSIG, J. L. STROMINGER AND L. F. LELOIR, J. Biol. Chem., 217 (1955) 959. I o T. BARKA AND G. DALLNER, J. Histoehem. Cytoehem., 6 (1958) 174. i i P. TALALAY, \V. H. PISHMAN AND C. HUGGINS, J. Biol. Chem., 166 (1946) 757.
Biochim. Biophys. Acta, 115 (1966) 312-319
MUCOPOLYSACCHARIDE DEGRADATION IN LIVER
319
12 O. H. LOWRY, l'q. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . 13 E. A. DAVlDSON AND W. SMALL, Biochim. Biophys. Acta, 69 (1963) 445. 14 E. CHAIN AND E. S. DUTHIE, Brit. J. Exptl. Pathol., 21 (194 o) 324 . 15 W. PIGMAN, W. HAWKINS, E. GRAMLING, S. RIZVI AND H. L. HOLLEY, Arch. Biochem. Biophys., 89 (196o) 184. 16 A. LINKER, K. MEYER AND ]3. ~vVEISSMAN,J. Biol. Chem., 213 (1955) 237. 17 O. Z. SELLINGER, H. ]3EAUFAY, P. JACQUES, A. DOYEN AND C. DE DUVE, Bioehem. J., 74 (196o) 45 ° • 18 D. PUGH, D. H. LEABACK AND P. G. WALKER, Biochem. J., 65 (1957) 464 • 19 P. G. WALKER, in F. CLARK AND F. U. GRANT, The Biochemistry of Mucopolysaeeharides of Connective Tissue, Cambridge University Press, 1961, p. lO9. 20 K. MEYER, P. HOFFMAN, A. LINKER, 3/I. M. GRUMBACH AND P. SAMPSON, Proc. Soe. Exptl. Biol. ivied., lO2 (1959) 587. 21 A. LINKER, P. t-IOFFMAN, P. SAMPSON AND K. MEYER, Bioehim. Biophys Aeta, 29 (1958) 44322 D. KAPLAN AND l(. MEYER, J. Clin. Invest., 41 (1962) 743. 23 ]3. \VEISSMAN, S. HADJIIOANNOU AND J. TORNHEIM, J. Biol. Chem., 59 (1964) 239. 24 E. RUBIN, V. HUTTERER AND H. POPPER, Federation Proe., 24 (1965) 431. 25 H. ]3EAUFAY, E. VAN CAMPENHOUT AND C. DE DUVE, Bioehem. J., 73 (1959) 617. 26 F. HUTTERER AND H. POPPER, Federation Proe., 23 (1964) 334. 27 N. N. ARONSON AND E. A. DAVIOSON, J. Biol. Chem., 240 (1965) 3222.
Bioehim. Biophys. Aeta, i i 5 (1966) 312-319
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 2 5 5 0 2
DEGRADATION OF MUCOPOLYSACCHARIDES BY HEPATIC LYSOSOMES* FERENC
HUTTERER
Department of Pathology, The Mount Sinai Hospital, New York, N . Y . (U,S.A,) (Received J u l y 261h, 1965)
SUMMARY
I. Hepatic lysosomes degraded highly polymerized mucopolysaccharides. The degradation products as shown by high-voltage electrophoresis were tetra- and oligosaccharides, indicating the presence of endohexosaminidase in the liver. 2. The role of bacterial contamination, and non-specific oxido-reductive degradation of mucopolysaccharides were excluded. 3. The activity of hepatic mucopolysaccharase (hyaluronate glycanohydrolase, EC 3.2.1.35 ) was related to the turnover rate of hepatic connective tissue. The enzyme activity was increased in reversible fibrosis and greatly diminished in irreversible fibrosis.
INTRODUCTION
Connective tissue excess in parenchymal organ upsets the normal structure and interferes with biochemical functions. Removal of the excess connective tissue demonstrated after ethionine-induced hepatic fibrosis in rats leads to the restoration of normal structure and function 1. Similar recoveries were noted in serial biopsies of human liver after hepatitis and early cirrhosisE The mechanism of the removal of the connective-tissue elements, collagen and mucopolysaccharides, is not well understood, since specific depolymerases of these macromolecules were not demonstrated conclusively in the liver, or in any other organ3, 4, with the exception of the hyaluronidase in the testis. Recently, BOLLETT et al. 5 presented suggestive evidence for the presence of hyaluronidase in the kidney and other tissues. It has previously been proposed that the initial step in collagen removal in the liver is a non-enzymatic denaturation of collagen, preceded by the disappearance of its cellular framework, and followed by partial digestion by non-specific proteinases and by phagocytosis 6. By contrast, this paper describes a specific enzyme for the degradation of mucopolysaccharides, localized exclusively in the lysosomal fraction of the liver. Analysis of the degradation products indicates that the enzyme is an endohexosaminidase. Its activity with different mucopolysaccharide-substrates and the changes of activity in different stages of hepatic fibrosis were also studied. * P r e l i m i n a r y r e p o r t s on this i n v e s t i g a t i o n a p p e a r e d in a b s t r a c t f o r m in: Federation Proc., 23 (1964) 334; Federation Proc., 24 (1965) 557.
Biochim. Biophys. Acta, 115 (I966) 312-319
313
MUCOPOLYSACCHARIDE DEGRADATION IN LIVER
MATERIALS AND METHODS
Hyaluronic acid grade I from umbilical cord was obtained from Sigma Co., St. Louis, Mo., with a relative viscosity of ten. Chondroitin sulfates A, B and C and heparitin sulfate were obtained from Dr. K. MEYER, Columbia University, New York, N.Y., and crystalline tetrasaccharide from hyaluronic acid from Dr. B. WEISSMANN, University of Illinois, Chicago, Ill.
Experimental animals Livers were obtained from Sprague-Dawley female rats with an average body weight of 17o g. Acute injury and reversible and irreversible hepatic fibrosis were produced by treatment with ethionine or carbontetrachloride as describedl,L The criterion for reversibility was the resorption of hepatic collagen from a fibrotic liver on a recovery diet.
Preparation of enzyme extract Livers were perfused with cold 0.25 M sucrose, and the subcellular particles were separated by differential ultracentrifugation s. The mitochondria were resuspended in o.25 M sucrose, layered on a Spinco Model L ultracentrifuge in a swinging bucket rotor for 15o min at 25ooo rev./min. The lysosomal fraction was obtained in the layer D = 1.28. The fraction was washed five times with physiologic NaC1 solution to remove sucrose, resuspended in 5 ml physiologic NaC1 solution, and treated with ultrasound. Optimum liberation of particle-bound enzyme without loss of activity was obtained at 9 A for 9osec. After centrifugation at 3oooo rev./min, the enzyme was recovered in the supernatant. It could be kept at --2o ° for I day without loss of activity.
Mucopolysaccharase assay* The incubation mixture consisted of 0.5 ml enzyme extract containing 0.53.o mg protein, and 0.5 ml o. I M acetate buffer containing o. 5 mg mucopolysaccharides. The p H of the acetate buffer was varied according to the optimum p H found for each substrate, 3.9 for hyaluronic acid and chondroitin sulfates A, B and C, and 4.8 for heparitin sulfate. Incubation was carried out at 37 ° under toluene for various intervals. The enzyme activity was measured by rate of liberation of N-acetylglucosamine endgroups. Aliquot of 0.5 ml of the incubation system was boiled with o.I ml of o.8 M potassium tetraborate (pH 9.1) and after cooling 3.o ml of 1% p-dimethylaminobenzaldehyde in glacial acetic acid containing 1.2 ml io N HC1 per ioo ml was added, and a color was developed for 2o min at 37 °. The absorbance was read in a Beckman DU spectrophotometer at 585 m/~ (ref. 9). If precipitation or turbidity developed, the samples were centrifuged before spectrophotometric measurement.
Separation of the degradation products with high-voltage electrophoresis After incubation, the protein was precipitated with trichloroacetic acid (final concentration was lO%). Excess trichloroaeetic acid was extracted with ether and the slightly acidic aqueous phase was evaporated in vacuo. The residue was redissolved in * " M u c o p o l y s a c c h a r a s e " (hyaluronate glycanohydrolase, EC 3.2.1.35) is used as trivial n a m e instead of " h y a l u r o n i d a s e " because the s u b s t r a t e specificity of the liver enzyme is not established
Biochim. Biophys. Acta, 115 (1966) 312-319
314
F. HUTTERER
a small volume of water and applied to a Whatman No. 3MlX{ paper. Electrophoretic separation was carried out in a Savant H.V. electrophoretic system in pyridine acetic acid-water (I : lO:9O, v/v) for 3o min at 3ooo V. The paper was dried at 13 °° for 3 ° min, and the spots were visualized with p-dimethylaminobenzaldehyde. Since the buffer system and impurities in pyridine interfere with the color development, the solvents, especially pyridine, had to be redistilled. After drying, the paper was dipped into o.5 °/;o alcoholic K O H and transferred into o.5% alcoholic K O H containing 1.o ml acetylacetone per IOO ml; 3 min later the paper was put in an oven at IO5 ° for IO min, then dipped into IO % alcoholic HE1, and trangferred into IO % alcoholic HC1 containing I g p-dimethylaminobenzaldehyde per IOO ml. The paper, protected from light, was dried at room temperature. Without these precautions the color development was inhibited, or the color was very unstable.
Analysis Succinate dehydrogenase (succinate: (aeceptor) oxido-reductase, EC 1.3.99,I) 1°, /3-glucuronidase (fl-D-glucuronide glucuronohydrolase, EC 3.2.i.3i) 11, and protein ~2 were measured as quoted. RESULTS
SubcelluIar localization of mucopolysaccharase activity The mitoehondrial-lysosomal fraction exhibited more than 80% of the total activity, while approx. 12% was unsedimentable. The nuclear and microsomal fractions were inactive. The mitochondrial-lysosomal fraction was further separated by density-gradient ultracentrifugation on a sucrose gradient. The highest mucopolysaccharase activity, similarly to /3-glucuronidase, was found at the density of 1.28; succinate dehydrogenase activity was localized at the density of 1.24 (Fig. I). DIFFERENTIAL U LTRACEN TRIFUGATION
DENSITY GRADIENT ULTRACENTRIFUGATION
MUCOPOLYSACCHARASE
~ >--d
I
v-ul '~ ~
i~ 1'°1
J
.
1 ,8-GLUCURONIDASE
~o
L~V-
2
¢'~ (3
O.B
1 SUCCINATE DEHYDROGENASE
>~ jO w~ S
N M+L
ER
1.16 1.20 1.24 1.28 SUCROSE DENSITY
Fig. i. Localization of inucopolysaccharase activity in the subcellular fractions of liver. Fractions : S, soluble; N, nuclear; M + L, mitochondrial-lysosomal; Er, microsomal.
Release of particle-bound activity The sucrose layer ( D = I . 2 8 ) containing the highest enzyme activity was separated, diluted to isotonicity, washed repeatedly with and resuspended in physiologic Biochim. Biophys. Acta, 115 (1966) 312-319
315
MUCOPOLYSACCHARIDE DEGRADATION IN LIVER
N aC1. Ultrasonic treatment, 20 kcycles/sec, resulted in a complete release of the enzyme activity into the supernatant within 90 sec (Fig. 2).
3o
E
TIME
6b
9'0
OFULTRASONICTREATMENT,20 Kcycles/sec
I~ig. 2. I~elease of mucopo]ysaccharase activity from hepatic lysosomes by ultrasonication.
Optim~tm conditions for the enzyme determination The pH optimum in 0.02 M acetate buffer was 3.9 for hyaluronic acid. The limited solubility of high-viscosity hyaluronic acid, and the known inhibition of enzyme activity by high concentration of hyaluronic acid ~ did not permit detailed kinetic studies. The best compromise was a substrate concentration of 0.5 mg hyaluronic acid per I.O ml incubation mixture, when the enzyme activity was directly proportional to the protein concentration over a 6-fold range, and to the incubation time over an 8-h period (Fig. 3). pH
50L
E 20~
*oI 0
[ __k ~
2
2140 03050 SU /B *S/TRA./'°~'" TE CONC ~'~. ENTRAT •O IN
/"'X
40
.,
3 pM
4
5
IO
" '//I
I
0
0.2
I
I
I
~--
C.4 0 6 SUBSTRATE ( m9 )
<[ ENZYME PROTEINCONCENTRATO I Nd
1I20 00140
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ooL
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~or-
./
- 2oI
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J0' I //" ~ [ L _ ~ 0
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I 04
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L
08 12 P R O T E I N { rag)
t
16
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0
~° / " °~° 2
4
6
8 I0 12 14 TIME (HOURS)
16 i~
Fig. 3. Effect of pH, substrate and e n z y m e concentrations, and t i m e of i n c u b a t i o n on hepatic m u c o p o l y s a c c h a r a s e a c t i v i t y . I n c u b a t i o n s y s t e m consisted of o. 5 ml l y s o s o m a l extract in 0.9% NaC] and h y a l u r o n i c acid in 0. 5 ml buffer. E n z y m e a c t i v i t y was expressed as mffmoles of liberated N - a c e t y l g l u c o s a m i n e . Boiled e x t r a c t served as control.
Degradation products of hyaluronic acid after incubation with hepatic lysosomal extract With high-voltage electrophoresis, glucuronic acid (GlcUA), which gave a dark brown color after p-dimethylaminobenzaldehyde staining, served as reference. Crystalline tetrasaccharide from hyaluronic acid emerged with an RGletTA of 0.94, and N-acetylglucosamine with a n RGle UA of 0.03. Both gave a pink color after staining, which was distinguishable from the brown spot of glucuronic acid. After incubation of hyaluronic acid with the lysosomal extract for 8 h, tetrasaccharides and several oligosaccharides of unknown chain length were demonstrated, but neither free N-acetylglueosamine nor free glucuronic acid were found (Fig. 4). Biochim. Bio#hys. Acta, 115 (1966) 3 1 2 - 3 1 9
3 I6
F. HUTTERER
IIII
M U C O P O L Y S A C C H A R A S E ACTIVITY --"--
UNSEDIMENTABLE
p GLUCURONIDASE
" '~
100 -
OCONTROL
ACUTE
REVERSIBLE
IRREV.
INd.
F~~ O S I S
FIBROSIS
Fig. 4. Separation of degradation products of hyaluronic acid by high-voltage electrophoresis after digestion witla lysosomal extract of the liver, incubation systenl contained o. 5 ml lysosomal extract, 0. 5 ml substrate (0. 5 rag) in acetate buffer (pH 3-9)- G, glucuronie acid; H, N acetylglucosamine ; T, crystalline tetrasaccharidc from hyaluronic acid ; X, hepatic digest showing tetraand oligosaccharides and traces of disaecharides. (Recently, similar results have been obtained by applying Dowex-L-X*o column chromatography followed by paper chromatographic sepa ration2L) Fig. 5. Hepatic nlucopolysaccharasc and fl glucuronidase activity in acute injury and during reversible and irreversible fibrosis. TABLE 1 HEPATIC
MUCOPOLYSACCItARASE
ACTIVITY
WITH
DIFFERENT
SUBSTRATES
Incubation system: o. 5 ml lysosomal extract in o.9°o NaC1, o. 5 mg substrate in o.o2 M acetate buffer (pH 3.9), except with heparitin sulphate (pH 4.5)-
Subslrates
Enzyme aeti~,ity N-A cetylhe.rosamine liberaled (re#moles per mg protein)
Hyaluronic acid Chondroitin sulfate A Chondroitin sulfate B Chondroitin sulfate C Heparitin sulfate
6 h
z8 h
34.4 4.8 None 4.5 5.8
84-o 13.o None 12.8 14.2
Biochim. Biophys. dcla, 1i 5 (t966) 312 319
MUCOPOLYSACCHARIDE
D E G R A D A T I O N IN L I V E R
317
Degradation of different mucopolysaccharides Besides hyaluronic acid, chondroitin sulfates A, B and C, and heparitin sulfate were used as substrates. Chondroitin sulfates A and C and heparitin sulfate were cleaved six to seven times more slowly than hyaluronic acid. Chondroitin sulfate B was not degraded by the lysosomal extract (Table I).
Pathological alterations In acute liver injury produced b y ethionine, hepatic mucopolysaccharase activity was lower than in the control, while the relative proportion of unsedimentable to sedimentable activity was higher. In reversible fibrosis, exemplified by subacute ethionine intoxication, the enzyme activity had risen over that of the control. In irreversible fibrosis, exemplified by chronic CC14 intoxication, the enzyme activity was greatly diminished. Lysosomal ~-glucuronidase had shown a similar pattern of activity (Fig. 5).
Hepatic mucopolysaccharase in gerrnzfree rats Lysosomal extracts of livers of six germ-free rats liberated 37.1 ± 4.2 mlzmoles of N-acetylglucosamine from hyaluronic acid, as compared to 34.4 ~ 6.6 m~moles by conventionals. The results statistically are not significantly different. DISCUSSION
The short half life of mucopolysaccharides 13 and the lack of specific hyaluronidases in mammalian tissues 3 led to the proposition of a series of indirect mechanisms of mucopolysaccharide degradation. These included bacterial contamination 14, non-specific oxido-reductive degradation ~s, and the combined action of exohexosaminidase and glucuronidase at the chain end 16. Bacteria are rich sources of hyaluronidases, and the exogenous or endogenous contamination of rat liver m a y produce misleading results a4. To exclude the role of contamination, germ-free rats were used in some experiments. Their livers degraded hyaluronic acid at the same rate as the conventional controls, eliminating the possibility that the demonstrated mucopolysaccharase activity of the liver is of bacterial origin. Non-specific oxido-reductive degradation of mucopolysaccharides was observed in the presence of ascorbic acid, hydroquinone, cysteine, Fe z+, Cu 2+. It was suggested that protein m a v also mediate this reaction ~5. The localization of enzyme activity exclusively in the lysosomal fraction tends not to support this possibility. At present, there is no indication that the lysosomal fraction is a better mediator of the oxidoreductive degradation than the mitochondrial fraction. N-Acetyl-/3-glucosaminidase and /3-glucuronidase are both localized in the lysosomes 17. Their combined action at the chain-end of hyaluronic acid would result in the accumulation of free N-acetylglucosamine and glucuronic acid as degradation products. Neither of these could be demonstrated by high-voltage electrophoresis. Instead, tetrasaccharides and oligosaecharides were shown indicating the presence of an endohexosaminidase in hepatic lysosomes. Further degradation of tetrasaccharides was prevented by acetate, which is an inhibitor of exohexosaminidase is. The lysosomal extract also cleaved chondroitin sulfates A and C and heparitin sulfate, though at a Biochim. Biophys. Acta, 115 (1966) 3 1 2 - 3 1 9
3 I8
F. HUTTERER
much slower rate. Chondroitin sulfate B was not susceptible to cleavage by the enzyme. The cleavage of heparitin sulfate is of particular interest, because heparitin sulfate is resistant to testicular hyaluronidase ~9. A lack of enzyme may play a role in the accumulation of heparitin sulfate in the liver in some pathologic conditions, such as the Hurler syndrome ~° and amyloidosis 21. The physiologic role of the enzyme is not yet clear. Two mechanisms have been suggested for the catabolism of mucopolysaccharides: one is phagocytosis by mesenchymal cells followed by intracellular degradation; the second is direct excretion of the polymer into the urine after dissociation of the mucopolysaccharide-protein complex. The relative importance of the two pathways depends upon the type of mucopolysaccharide undergoing degradation, its molecular size, and its digestibility 22. The intracellular degradation of nmcopolysaccharides following phagocytosis may be initiated by mucopolysaccharase and brought to completion in vivo by exohexosaminidase 23 and glucuronidase. This would imply that the mucopolysaccharase is contained in mesenchymal cells, a possibility supported by the observation that the enzyme activity changes parallel with the number of mesenchymal cells '4. The pathologic role of the enzyme is similarly obscure. In acute liver injury, the increase in the unsedimentable portion of the lysosomal enzymes is an indication of proceeding cell disruption due to necrosis "a. In reversible fibrosis, in which the half life of insoluble collagen is less than 25 days 2G, mucopolysaccharase activity was increased. In the irreversible phase, in which the half life of insoluble collagen is more than 25o days 26, mucopolysaccharase activity was greatly diminished. The parallel nature of the changes in mucopolysaccharase and fl-glucuronidase activity reflects lysosomal alterations rather than specific changes in connective tissue metabolism. The variations in enzyme activity may therefore be a simple expression of the number and activity of the lysosome-rich hepatic mesenchymal cells, which, by their phagocytic and hydrolytic capacities, influence the turnover of connective tissue in the reversible and irreversible phases of fibrosis. ACKNOWLEDGEMENTS
This work was supported by Grants No. DA-49-oo7-MD-79oo3 from the U.S. Army Medical Research and Development Command, and No. AM-o3846 from the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service.
REFERENCES I H. HUTTERER, E. RUBIN, E. J. SINGER AND H. POPPER, Cancer Res., 21 ( i 9 6 i ) 205. 2 H. POPPER AND F. SCHAFFNER, Liver: Structure and Function, The B l a k i s t o n Division, Mc G ra w H i l l Book C o m p a n y , New York, 1957, p. 777. 3 M. SCHUBERT, Pfoe. Syrup. N . Y . Heart A SHOe., New York, 1964, Li t t l e , B r o w n a n d Co., Boston, 1964, p. 124. 4 J- MANDEL, Advan. Enzymol., 23 (1961) 557. .5 i . J. ]~OLLETT, ~r. M. BONNER, JR. AND J. L. NANCE, ft. Biol. Chem., 238 (1963) 3522. '6 F. HUTTERER, E. RUBIN AND H. POPPER, Exptl. Mol. Pathol., 3 (1964) 215. 7 E. I~UBIN, F. HUTTERER AND H. POPPER, ,4rn. J. Pathol., 42 (1963) 715 . 8 W. C. SCHNEIDER AND G. H. HOGEBOOM, J. Biol. Chem., 183 (1956) 123. 9 J. C. REISSIG, J. L. STROMINGER AND L. F. LELOIR, J. Biol. Chem., 217 (1955) 959. I o T. BARKA AND G. DALLNER, J. Histoehem. Cytoehem., 6 (1958) 174. i i P. TALALAY, \V. H. PISHMAN AND C. HUGGINS, J. Biol. Chem., 166 (1946) 757.
Biochim. Biophys. Acta, 115 (1966) 312-319
MUCOPOLYSACCHARIDE DEGRADATION IN LIVER
319
12 O. H. LOWRY, l'q. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . 13 E. A. DAVlDSON AND W. SMALL, Biochim. Biophys. Acta, 69 (1963) 445. 14 E. CHAIN AND E. S. DUTHIE, Brit. J. Exptl. Pathol., 21 (194 o) 324 . 15 W. PIGMAN, W. HAWKINS, E. GRAMLING, S. RIZVI AND H. L. HOLLEY, Arch. Biochem. Biophys., 89 (196o) 184. 16 A. LINKER, K. MEYER AND ]3. ~vVEISSMAN,J. Biol. Chem., 213 (1955) 237. 17 O. Z. SELLINGER, H. ]3EAUFAY, P. JACQUES, A. DOYEN AND C. DE DUVE, Bioehem. J., 74 (196o) 45 ° • 18 D. PUGH, D. H. LEABACK AND P. G. WALKER, Biochem. J., 65 (1957) 464 • 19 P. G. WALKER, in F. CLARK AND F. U. GRANT, The Biochemistry of Mucopolysaeeharides of Connective Tissue, Cambridge University Press, 1961, p. lO9. 20 K. MEYER, P. HOFFMAN, A. LINKER, 3/I. M. GRUMBACH AND P. SAMPSON, Proc. Soe. Exptl. Biol. ivied., lO2 (1959) 587. 21 A. LINKER, P. t-IOFFMAN, P. SAMPSON AND K. MEYER, Bioehim. Biophys Aeta, 29 (1958) 44322 D. KAPLAN AND l(. MEYER, J. Clin. Invest., 41 (1962) 743. 23 ]3. \VEISSMAN, S. HADJIIOANNOU AND J. TORNHEIM, J. Biol. Chem., 59 (1964) 239. 24 E. RUBIN, V. HUTTERER AND H. POPPER, Federation Proe., 24 (1965) 431. 25 H. ]3EAUFAY, E. VAN CAMPENHOUT AND C. DE DUVE, Bioehem. J., 73 (1959) 617. 26 F. HUTTERER AND H. POPPER, Federation Proe., 23 (1964) 334. 27 N. N. ARONSON AND E. A. DAVIOSON, J. Biol. Chem., 240 (1965) 3222.
Bioehim. Biophys. Aeta, i i 5 (1966) 312-319