THE JOURNAL OP I~IOLOGIC.\L CIIP\IISTRY Vol. 213, No. 17, Issue of September 10, pp. 4494-4499, 1968 Printed in U.S.A. Catabolism of Mucopolysaccharides by Rat Liver LY sosomes in Viva* KATHAN N. ARONSON, JR..$ AND EUGENE A. DAVIDSON~ (Received for publication, February 19, 19G8) From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina SUMMARY Rats treated by injection of the radioactive mucopolysac- charides, %-labeled chrondroitin 4- or d-sulfate and tritium- labeled chondroitin sulfate B, have been shown to be capable of concentrating about 1% of these materials within their liver lysosomes. The chondroitin 4-sulfate, which is a substrate for lysosomal hyaluronidase, was depleted from the lysosomes 4 days after injection, whereas, after this same time period, one-third of the chondroitin sulfate B remained in these cellular particles. The role of this phe- nomenon in Hurler’s syndrome, a connective tissue disorder involving storage of chondroitin sulfate, is discussed. The mucopolysaccharides of the ground substance of con- nective tissue have been shown to turn over in vivo, by assess- ment of the incorporation of either r4C or 3% into these compounds and the subsequent observation of their specific radioactivity as a function of time (1). Workers in several laboratories have reported studies aimed at elucidating the synthetic mechanisms responsible for the formation of these polysaccharides, but little information is available about the nature and locale of t.he degradative systems involved in mammalian mucopolysaccharide metabolism. Recent reports from this laboratory have described the properties of a hyaluronidase isolated from rat liver lyso- somes (2, 3). Since the lysosomes contain this mucopolysac- charidase together with other degradative enzymes, such as /I-glucuronidase and P-N-acetylhexosaminidase (4, 5)) both of which are capable of attacking oligosaccharide substrates, the current investigation was carried out to show the possible involvement of these organelles in the breakdown of connective tissue components. * This work was supported by Grants AM 02903.08 and AM 04315-06 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service, Bethesda, Mary- land. 3 Present address, The Rockefeller University, New York, New York 10021. 0 To whom inquiries should be addressed. Present address, Department of Biological Chemistry, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033. EXPERIMENTAL PROCEDURE Preparation OJ” Labeled Polysaccharides-Chondroitin sulfate l3 was prepared from pig skin (6). The polysaccharide was labeled with tritium by exposure to tritium-labeled water in the presence of platinum catalyst at an elevated temperature. Some degradation of the material occurred during this process, and the polysaccharide was initially repurified by exhaustive dialysis followed by fractionation of the calcium salt with ethanol. Examination of this preparat,ion by electrophoresis in 0.05 M acetate buffer, pH 5.0, for 18 hours at 60 volts indicated that t.he material staining with toluidinc blue had essentially the same mobility as that containing the radioactive label. Xl- though most of the radioactivity coincided with the t,oluidine blue stain, some remained at the origin and smeared slightly along the electrophoretogram. The nature of this apparent contaminant is not known, but presumably it represents degraded material formed during the tritiation process. Final purification, carried out by preparative electrophoresis under the above con- ditions, yielded a product which was essentially homogeneous on electrophoresis or Sephadex G-25 chromatography. The tritium-labeled material had a specific activity of 2.0 x lo6 cpm per pmole of uranic acid. %-Labeled chondroitin 6-sulfate was prepared by the use of carrier chondroitin B-sulfate as a sulfate acceptor in a cell-free sulfating system prepared from chick embryo cartilage according to the procedure of Meezan and Davidson (7). The specific activity of this preparation was 0.95 X lo6 cpm per pmole of uranic acid. In order to obtain such high radioactivity, the substrate was partially desulfated (8), and the 3B-labeled polysac- charide isolated from a first incubation was used as carrier polysaccharide in a second incubation with the cartilage enzyme system. Analyses of this material have been reported (9). Chondroitin 4-sulfate was prepared by treating lo-day-old chick embryos by injection of 50 PC of carrier-free 3%sulfate. At the end of 14 days, the embryos were dissected to obtain the long bones and cartilage, and the chondroitin 4-sulfate was isolated as previously described (9). Standard chondroitin was prepared chemically from chondroitin 4-sulfate by the pro- cedure of Kantor and Schubert (8). Isolation and Fractionation of Subcellular Components--In all cases, Osborne-Mendel rats of approximately 190 g were given intravenous injections in the tail vein of the appropriate labeled 4494 This is an Open Access article under the CC BY license.
Catabolism of Mucopolysaccharides by Rat Liver Lysosomes in Vivo
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Catabolism of Mucopolysaccharides by Rat Liver Lysosomes in VivoTHE JOURNAL OP I~IOLOGIC.\L CIIP\IISTRY Vol. 213, No. 17, Issue of September 10, pp. 4494-4499, 1968
Printed in U.S.A.
LY sosomes in Viva*
(Received for publication, February 19, 19G8)
From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina
SUMMARY
Rats treated by injection of the radioactive mucopolysac- charides, %-labeled chrondroitin 4- or d-sulfate and tritium- labeled chondroitin sulfate B, have been shown to be capable of concentrating about 1% of these materials within their liver lysosomes. The chondroitin 4-sulfate, which is a substrate for lysosomal hyaluronidase, was depleted from the lysosomes 4 days after injection, whereas, after this same time period, one-third of the chondroitin sulfate B remained in these cellular particles. The role of this phe- nomenon in Hurler’s syndrome, a connective tissue disorder involving storage of chondroitin sulfate, is discussed.
The mucopolysaccharides of the ground substance of con- nective tissue have been shown to turn over in vivo, by assess- ment of the incorporation of either r4C or 3% into these compounds and the subsequent observation of their specific radioactivity as a function of time (1). Workers in several laboratories have reported studies aimed at elucidating the synthetic mechanisms responsible for the formation of these polysaccharides, but little information is available about the nature and locale of t.he degradative systems involved in mammalian mucopolysaccharide metabolism. Recent reports from this laboratory have described the properties of a hyaluronidase isolated from rat liver lyso- somes (2, 3). Since the lysosomes contain this mucopolysac- charidase together with other degradative enzymes, such as /I-glucuronidase and P-N-acetylhexosaminidase (4, 5)) both of which are capable of attacking oligosaccharide substrates, the current investigation was carried out to show the possible involvement of these organelles in the breakdown of connective tissue components.
* This work was supported by Grants AM 02903.08 and AM 04315-06 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service, Bethesda, Mary- land.
3 Present address, The Rockefeller University, New York, New York 10021.
0 To whom inquiries should be addressed. Present address, Department of Biological Chemistry, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033.
EXPERIMENTAL PROCEDURE
Preparation OJ” Labeled Polysaccharides-Chondroitin sulfate l3 was prepared from pig skin (6). The polysaccharide was labeled with tritium by exposure to tritium-labeled water in the presence of platinum catalyst at an elevated temperature. Some degradation of the material occurred during this process, and the polysaccharide was initially repurified by exhaustive dialysis followed by fractionation of the calcium salt with ethanol. Examination of this preparat,ion by electrophoresis in 0.05 M
acetate buffer, pH 5.0, for 18 hours at 60 volts indicated that t.he material staining with toluidinc blue had essentially the same mobility as that containing the radioactive label. Xl- though most of the radioactivity coincided with the t,oluidine blue stain, some remained at the origin and smeared slightly along the electrophoretogram. The nature of this apparent contaminant is not known, but presumably it represents degraded material formed during the tritiation process. Final purification, carried out by preparative electrophoresis under the above con- ditions, yielded a product which was essentially homogeneous on electrophoresis or Sephadex G-25 chromatography. The tritium-labeled material had a specific activity of 2.0 x lo6 cpm per pmole of uranic acid.
%-Labeled chondroitin 6-sulfate was prepared by the use of carrier chondroitin B-sulfate as a sulfate acceptor in a cell-free sulfating system prepared from chick embryo cartilage according to the procedure of Meezan and Davidson (7). The specific activity of this preparation was 0.95 X lo6 cpm per pmole of uranic acid. In order to obtain such high radioactivity, the substrate was partially desulfated (8), and the 3B-labeled polysac- charide isolated from a first incubation was used as carrier polysaccharide in a second incubation with the cartilage enzyme system. Analyses of this material have been reported (9). Chondroitin 4-sulfate was prepared by treating lo-day-old chick embryos by injection of 50 PC of carrier-free 3%sulfate. At the end of 14 days, the embryos were dissected to obtain the long bones and cartilage, and the chondroitin 4-sulfate was isolated as previously described (9). Standard chondroitin was prepared chemically from chondroitin 4-sulfate by the pro- cedure of Kantor and Schubert (8).
Isolation and Fractionation of Subcellular Components--In all cases, Osborne-Mendel rats of approximately 190 g were given intravenous injections in the tail vein of the appropriate labeled
4494
This is an Open Access article under the CC BY license.
Issue of September 10, 1968 N. N. Aronson, Jr. and E. A. Davidson
polysaccharide, dissolved in 0.15 M sodium chloride. The rats were killed by decapitation and their blood was collected in 0.1 volume of 5y0 EDTA. Cells were removed by centrifugation and the plasma was retained for determination of radioactivity. Initial studies with chondroitin B-sulfate were carried out with the use of Triton WR-1339-filled lysosomes isolated according to the procedure of Wattiaux, Wibo, and Baudhuin (10).
All subsequent isolations were carried out by the following modification of t.heir procedure. Homogenization was carried out in 0.25 M sucrose with a Potter-Elvehjem homogenizer and the nuclear fraction was removed by centrifugation at 1,000 X g for 10 min. The supernatant fluid resulting from this frac- tionation was centrifuged at 25,000 rpm in a Spinco model L 2 centrifuge with the use of the 30 rotor for a period of 8 min and 20 sec. The pellet resultin, 0‘ from this centrifugation was washed once by suspension in 0.25 M sucrose and recentrifugation. The combined supernatants from this procedure were centrifuged for 2 hours at 144,000 x g to yield a microsomal pellet and a final supernatant fraction. The pellet from the 25,000 rpm centrifugation represents a combined lysosomal and mitochon- drial fraction which was furt,her resolved by sucrose density gradient centrifugation. The pellet was suspended in sucrose of density 1.21 with the use of about 1 ml per g of the starting liver, wet weight. This fraction was transferred to an SW 25.2 centrifuge tube, and on top of this were layered 20 ml of 1.155 density sucrose and 10 ml of 1.06 density sucrose. The tubes were centrifuged at 24,000 rpm for 2 hours in the Spinco 25.2 rotor and fractionated by displacement with sucrose of density 1.30. In all experiments, the separation of the mito- chondria and lysosomes was facilitated by preliminary treat- ment of the rats with Triton WR-1339. Triton-filled lysosomes float at the interface between the 1.06 and 1.155 density sucrose, whereas the mitochondrial fraction remains in the bottom of the tube. The above method for isolating Triton-filled lysosomes is the method of Trouet (11) as modified by Leighton et al. (12) J
Assays-Hyaluronidase was assayed as previously described, except that sodium acetate buffer, pH 3.5, and a hyaluronate concentration of 1.2 mg per ml were used (7). In experiments involving uptake of chondroitin sulfate B, 20 pg of protamine sulfate were added to assay mixtures to prevent any inhibition. All counting of radioactivity was done in a Packard scintillation counter. An appropriate sample of each tissue fraction was diluted to 0.5 ml and counted in a mixture of Cabosil gel powder and 7.5 ml of 1,4-dioxane-anisole-l , 2-dimethoxyethane solution (6 : 1: 1) containing 2,5-diphenyloxazole and 1,4-bis[2-(5-phen- yloxazolyl)]benzene (40: 1) as scintillators. A counting effi- ciency of 60% for % and 30% for tritium was obtained. The number of counts reported are corrected for background.
RESULTS
After injection of 35S-labeled chondroitin 4- or 6-sulfate2 into the animals, the urine collected during the following 48-hour period contained 35 to 50% of the injected radioactivity; a small proportion appeared as inorganic sulfate. Similar observations have previously been reported by Dziewiatkowski (13) and Dohlman (14). An examination of the blood of the animals throughout this period revealed that essentially no uptake of
1 We are indebted to Dr. C. de Duve for the details of this pro- :edure prior to publication.
2 Substantially identical results were obtained with chondroitin 4-sulfate and chondroitin B-sulfate.
Fraction No.
FIG. 1. Distribution of 0, specific radioactivity; and n , ref- erence enzyme, hyaluronidase, after centrifugation of a lysosomal fraction of rat liver through a linear sucrose-water gradient (from 1.10 to 3.42 molal). The rat had been given an injection of a%- chondroitin sulfate A 15 min before it was killed. Centrifugation was for 24 hours at 39,000 rpm in an SW 39 swinginK bucket rotor with a Spinco model L 2 ultracentrifuge. The lyiosimal fraction, prepared bv the procedure of Wattiaux, Wibo. and Baudhllin ClOj (equivalent to-O.65 g of original tissuej, was layered above the gradient after suspension in 0.65 ml of 0.25 M sucrose. Fractions were collected and assayed after the bottom of the tube had been punctured. Relative hyaluronidase concentration refers to t.he ratio of the observed activity to that which would have been found if the enzyme had been homogeneously distributed throughout the gradient. The control for radioactivity, 0, was carried out by adding an equivalent amount of radioactive polysaccharide to the liver homogenate of a rat not treated with radioactive ma- erial. This was then fractionated as above. The top of the entrifuge tube is at the right. See t;he text for additional details.
polysaccharide occurred in the cellular fraction and that all of the radioactivity was present in the plasma.
%-Labeled chondroitin g-sulfate was injected, the animal
was killed 15 min later, and the blood was fractionated as de- scribed above. This procedure showed about 1% of the total injected counts to be present in both normal lysosomes and the tritosomes3 The density gradient separation of the labeled material from normal liver is illustrat.ed in Fig. 1. The radio- activity along the gradient coincided with the hyaluronidase activity, which served as the lysosomal marker enzyme. The small peak at Fraction 6 coincides with the mitochondrial frac- tion, and may represent lysosomal particles which have not taken up the Triton WR-1339. The amount of material present was insufficient to permit reisolation and chemical identification of the polysaccharide, but electrophoresis of the labeled fractions showed that the radioactive component had the same mobility as chondroitin 4- or B-sulfate and was readily distinguished from inorganic sulfate. The distribution of radioactivity in other cellular fractions for Triton-treated animals is summarized in Table I. Since chondroitin 4- and B-sulfates are substrates for
3 Defined as Triton WR-1339-filled lysosomes. It should be noted that the use of Triton WR-1339 permits excellent separation of lysosomal and mitochondrial particles. Control experiments carried out with animals not receiving the detergent showed essentially the same amount of uptake in the lysosomal fraction as that of treated animals. However, this fraction is contam- inated with mitochondria and the l’riton procedure was therefore routinely used.
Mucopolysaccharide Catabolism Vol. 243, No. 17
TABLE I
Subcellular distribution of injected polusaccharides
Animals treated 4 days previously by injection of Triton WR-1339 were given intravenous injections of labeled polyssc- charide 15 min prior to death. Liver fractions were isolated
according to the procedure of Leighton et al. (12). The total amount of radioactivity administered was 9.5 X IO5 cpm for the 35Schondroitin 4-sulfate and 2.5 X IO6 cpm for the 3H-chondroitin
sulfate B. After addition of either polysaccharide to liver homogenates of normal animals, over 90% of the radioactivity is located in the supernatant fraction and less than 0.1% in any of
the particulate fractions. See the text for details.
Fraction
< 100 200 7,200 21,000 1,060 6,250
375 1,470 1,800 7,300
19,000 < 100
108,000 300
Fraction No.
FIG. 2. Distribution of 0, specific radioactivity; and n , ref- erence enzyme, hyaluronidase, after centrifugation of a lysosomal fraction of rat liver through a linear sucrose-water gradient (from 1.10 molal to 3.42 molal). The rat had been treated with %- chondroitin sulfate A 4 days before death. Centrifugation, frac- tionation of gradient, and expression of data are the same as those given in legend for Fig. 1. The top of the centrifuge tube is at the right.
lysosomal hyaluronidase, it might be expected that ingested
polymer would be lost by degradation to oligosaccharides and possibly further to monosaccharides, because of the activity of the glucuronidase and hexosaminidase present. As is shown in Fig. 2, a rat treated with the same amount of labeled material as above has almost no radioactivity remaining in the lysosomes when killed 4 days later. Hyaluronidase activity remained the same, both in amount and in location along the gradient. The subcellular distribution is summarized in Table II. It should be noted that only 35% of the injected radioactive label could be accounted for in the urine.
As has been shown previously, chondroitin sulfate B is a potent competitive inhibitor of lysosomal hyaluronidase (3),
but it is not known to serve as a substrate for any mammalian enzyme system. This polysaccharide was injected into a rat previously treated wit.h Triton, and the animal was killed in 15 min; approximately 1 TO of the radioactivity was recovered in the lysosomal fraction after the sucrose gradient fractionation. Results are summarized in Fig. 3 and Table I. The major radioactive peak corresponded exactly to the bulk of the hya- luronidase-positive material, and the minor peak to the mito- chondrial fraction. The association of some hyaluronidase
TABLE II
Subcellular distribution of injected polysaccharides
Animals were treated as described in Table I, except that they were killed 96 hours after injection of polysaccharide. See
the text for details.
Liver Nuclei
500 <lOO
I 3 5 7 9 II 13 15 17 IS 21 23 25 :
Fraction No,
FIG. 3. Distribution of l , specific radioactivity; and q , ref- erence enzyme, hyaluronidase, after centrifugation of a lysosomal fraction equivalent to 1.5 g of rat liver through a sucrose-water gradient. The rat had been treated with 3H-chondroitin sulfate B (2.5 X lo6 cpm) 15 min before death. Control for radioactivity, centrifugation time, and expression of data are the same as given in the legend for Fig. 1, except that 20 pg of protamine per ml were added to hyaluronidase assays to avoid inhibition by chon- droitin sulfate B (4). Preparation of the lysosomes and the gradient used are described in the text. The smaller peak coin- cides with the mitochondrial fraction, and may represent lyso- somes which did not accumulate the Triton WR-1339. In this experiment fractions were collected by displacement of the tube contents with sucrose of density 1.30. The top of the centrifuge tube is at the left.
Issue of September 10, 1968 hr. N. Aronson, Jr. and E. A. Davidson 4497
I 3 5 7 9 II 13 15 17 19 21 23 25 i
Fraction No.
FIG. 4. Distribution of l , specific radioactivity; and n , ref- erence enzyme, hyaluronidase, after centrifugation of a lysosomal fraction of rat liver through a linear sucrose-water gradient (from 1.10 molal to 3.42 molal). The rat had been treated with 3H- chondroitin sulfate B (2.5 X lo6 cpm) 4 days before death. Con- trol for radioactivity, centrifugation, fractionation of gradient, enzyme assay, and expression of data are the same as in the legend for Fig. 3. The top of the centrifuge tube is at the left.
b > .- E
KF ‘0 * -
l CHONDROITIN
D/stance (inches)
FIG. 5. Characterization of injected 3H-chondroitin sulfate B CSB) and isolated radioactivity from lysosomes. The lysosomal
fraction from the rat treated 4 days previously with 3H-chondro- itin sulfate B was deproteinized with 1O70 trichloracetic acid, dialyzed overnight against running tap water and then against distilled water, lyophilized, and finally dissolved in a small volume of water and precipitated with 2.5 volumes of ethanol. This material was spotted on Whatman No. 3MM paper and subjected to electrophoresis for 18 hours at 0.1 volt per cm2 in 0.05 M sodium acetate buffer, pH 5.0. One-half inch strips were cut and counted in toluene in a scintillation counter. The upper portion of the figure represents toluidine blue staining of 30 pg of standard chon- droitin and chondroitin sulfate B along with the injected 3H- chondroitin sulfate B.
w&h this latter peak suggests the presence of some lysosomal particles at this position in the gradient. Since the chondroitin sulfate B is not a substrate for either purified lysosomal hya- luronidase or int,act lysosomes, the radioactivity incorporated under these conditions should remain in these particles unless there is some process by which the filled lysosomes can expel the polysaccharide in a macromolecular state. As is shown in Fig. 4 and Table II, approximately 0.40% of the counts which were injected remained in the lysosomes of the rat killed 4 days after injection of tritium-labeled chondroitin sulfate B.
The radioactive material from four rats treated 4 days pre- viously with tritium-labeled chondroitin sulfate B was reisolated from the pooled lysosomal fraction by precipitation of the pro- tein with trichlorncetic acid, dialysis, and ethanol precipitation of the polysaccharide. As is shown in Fig. 5, the bulk of the reisolat,ed material showed the same electrophoretic mobility as the injected tritium-labeled chondroitin sulfate B, and thus there appears to be relatively little degradation of this material in the lysosomes. Significant loss of sulfate apparently does not occur, since this would result in radioactive material which moved with a mobility intermediate between a desulfated standard (chondroitin) and the chondroitin sulfate B. Ap- proximately 8Ooj, of the radioactivity was recovered in the area corresponding to the mobility of the injected mat,erial.
DISCUSSION
Both Dohlman (14) and Dziewiatkowski (13) showed that rats were able to metabolize exogenous chondroitin 35S-sulfate, as evidenced by the appearance of urinary inorganic sulfate after injection of labeled polysaccharide. However, the majority of the mucopolysaccharide was excreted in macromolecular form, although physicochemical data obtained were insufficient to permit assessment of slight amounts of degradation. Kaplan and Xteyer (15) also showed that approximately one-half of the injected mucopolysaccharides was excreted in the urine without degradation, although a portion did appear as inorganic sulfate. They suggested that two mechanisms exist for the disposal of mucopolysaccharides that reach the bloodstream: degradation somewhere within tissue, or excretion by the kidneys in the absence of breakdown. These reports still left uncertain the locality of the degradative system or the nature of the en- zymes involved, and they did not give any information on the breakdown of the carbon chain of the mucopolysaccharides. As is shown by this report, rat liver lysosomes are at least one site for the degradation of chondroitin 4- and 6-sulfate, although only 1 y0 of the injected material is taken up by these organelles in a 15-min period. Since %-labeled material is used and thus only the sulfate label could be shown to be metabolized, it might be concluded that these experiment,s offer no proof for the break- down of the carbon chain but only represent the result of sul- fatase activity. The arguments against this are the following. (a) Hyaluronidase, one of the substrates of which is chondroitin 4- or 6-sulfate, is known to be present in liver lysosomes. (b) Although experiments from this laboratory have indicated the presence of low levels of chondrosulfatase activity in the ly- sosomes, it is important to note that the sulfatase is inactive on polymeric substrat.es and requires intermediate size oligosac- charides (16). Similar results have been obtained by Dodgson and Lloyd (17), who showed that high molecular weight chon- droitin sulfate is not a substrat’e for a bacterial sulfa&e and that, instead, smaller oligosaccharides serve as the best sub-
4498 Mucopolysaccharide Catabolism Vol. 243, No. 17
skates. This implies that carbon chain depolymerization must precede sulfate hydrolysis. (c) The fact that tritium-labeled chondroitin sulfate B was apparently not significantly degraded, as evidenced by the electrophoret,ic mobility, suggests that the 4-sulfate of +labeled chondroitin 4-sulfat.e, which has the same configurstion as t’he 4 axial sulfate of…
Printed in U.S.A.
LY sosomes in Viva*
(Received for publication, February 19, 19G8)
From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina
SUMMARY
Rats treated by injection of the radioactive mucopolysac- charides, %-labeled chrondroitin 4- or d-sulfate and tritium- labeled chondroitin sulfate B, have been shown to be capable of concentrating about 1% of these materials within their liver lysosomes. The chondroitin 4-sulfate, which is a substrate for lysosomal hyaluronidase, was depleted from the lysosomes 4 days after injection, whereas, after this same time period, one-third of the chondroitin sulfate B remained in these cellular particles. The role of this phe- nomenon in Hurler’s syndrome, a connective tissue disorder involving storage of chondroitin sulfate, is discussed.
The mucopolysaccharides of the ground substance of con- nective tissue have been shown to turn over in vivo, by assess- ment of the incorporation of either r4C or 3% into these compounds and the subsequent observation of their specific radioactivity as a function of time (1). Workers in several laboratories have reported studies aimed at elucidating the synthetic mechanisms responsible for the formation of these polysaccharides, but little information is available about the nature and locale of t.he degradative systems involved in mammalian mucopolysaccharide metabolism. Recent reports from this laboratory have described the properties of a hyaluronidase isolated from rat liver lyso- somes (2, 3). Since the lysosomes contain this mucopolysac- charidase together with other degradative enzymes, such as /I-glucuronidase and P-N-acetylhexosaminidase (4, 5)) both of which are capable of attacking oligosaccharide substrates, the current investigation was carried out to show the possible involvement of these organelles in the breakdown of connective tissue components.
* This work was supported by Grants AM 02903.08 and AM 04315-06 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service, Bethesda, Mary- land.
3 Present address, The Rockefeller University, New York, New York 10021.
0 To whom inquiries should be addressed. Present address, Department of Biological Chemistry, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033.
EXPERIMENTAL PROCEDURE
Preparation OJ” Labeled Polysaccharides-Chondroitin sulfate l3 was prepared from pig skin (6). The polysaccharide was labeled with tritium by exposure to tritium-labeled water in the presence of platinum catalyst at an elevated temperature. Some degradation of the material occurred during this process, and the polysaccharide was initially repurified by exhaustive dialysis followed by fractionation of the calcium salt with ethanol. Examination of this preparat,ion by electrophoresis in 0.05 M
acetate buffer, pH 5.0, for 18 hours at 60 volts indicated that t.he material staining with toluidinc blue had essentially the same mobility as that containing the radioactive label. Xl- though most of the radioactivity coincided with the t,oluidine blue stain, some remained at the origin and smeared slightly along the electrophoretogram. The nature of this apparent contaminant is not known, but presumably it represents degraded material formed during the tritiation process. Final purification, carried out by preparative electrophoresis under the above con- ditions, yielded a product which was essentially homogeneous on electrophoresis or Sephadex G-25 chromatography. The tritium-labeled material had a specific activity of 2.0 x lo6 cpm per pmole of uranic acid.
%-Labeled chondroitin 6-sulfate was prepared by the use of carrier chondroitin B-sulfate as a sulfate acceptor in a cell-free sulfating system prepared from chick embryo cartilage according to the procedure of Meezan and Davidson (7). The specific activity of this preparation was 0.95 X lo6 cpm per pmole of uranic acid. In order to obtain such high radioactivity, the substrate was partially desulfated (8), and the 3B-labeled polysac- charide isolated from a first incubation was used as carrier polysaccharide in a second incubation with the cartilage enzyme system. Analyses of this material have been reported (9). Chondroitin 4-sulfate was prepared by treating lo-day-old chick embryos by injection of 50 PC of carrier-free 3%sulfate. At the end of 14 days, the embryos were dissected to obtain the long bones and cartilage, and the chondroitin 4-sulfate was isolated as previously described (9). Standard chondroitin was prepared chemically from chondroitin 4-sulfate by the pro- cedure of Kantor and Schubert (8).
Isolation and Fractionation of Subcellular Components--In all cases, Osborne-Mendel rats of approximately 190 g were given intravenous injections in the tail vein of the appropriate labeled
4494
This is an Open Access article under the CC BY license.
Issue of September 10, 1968 N. N. Aronson, Jr. and E. A. Davidson
polysaccharide, dissolved in 0.15 M sodium chloride. The rats were killed by decapitation and their blood was collected in 0.1 volume of 5y0 EDTA. Cells were removed by centrifugation and the plasma was retained for determination of radioactivity. Initial studies with chondroitin B-sulfate were carried out with the use of Triton WR-1339-filled lysosomes isolated according to the procedure of Wattiaux, Wibo, and Baudhuin (10).
All subsequent isolations were carried out by the following modification of t.heir procedure. Homogenization was carried out in 0.25 M sucrose with a Potter-Elvehjem homogenizer and the nuclear fraction was removed by centrifugation at 1,000 X g for 10 min. The supernatant fluid resulting from this frac- tionation was centrifuged at 25,000 rpm in a Spinco model L 2 centrifuge with the use of the 30 rotor for a period of 8 min and 20 sec. The pellet resultin, 0‘ from this centrifugation was washed once by suspension in 0.25 M sucrose and recentrifugation. The combined supernatants from this procedure were centrifuged for 2 hours at 144,000 x g to yield a microsomal pellet and a final supernatant fraction. The pellet from the 25,000 rpm centrifugation represents a combined lysosomal and mitochon- drial fraction which was furt,her resolved by sucrose density gradient centrifugation. The pellet was suspended in sucrose of density 1.21 with the use of about 1 ml per g of the starting liver, wet weight. This fraction was transferred to an SW 25.2 centrifuge tube, and on top of this were layered 20 ml of 1.155 density sucrose and 10 ml of 1.06 density sucrose. The tubes were centrifuged at 24,000 rpm for 2 hours in the Spinco 25.2 rotor and fractionated by displacement with sucrose of density 1.30. In all experiments, the separation of the mito- chondria and lysosomes was facilitated by preliminary treat- ment of the rats with Triton WR-1339. Triton-filled lysosomes float at the interface between the 1.06 and 1.155 density sucrose, whereas the mitochondrial fraction remains in the bottom of the tube. The above method for isolating Triton-filled lysosomes is the method of Trouet (11) as modified by Leighton et al. (12) J
Assays-Hyaluronidase was assayed as previously described, except that sodium acetate buffer, pH 3.5, and a hyaluronate concentration of 1.2 mg per ml were used (7). In experiments involving uptake of chondroitin sulfate B, 20 pg of protamine sulfate were added to assay mixtures to prevent any inhibition. All counting of radioactivity was done in a Packard scintillation counter. An appropriate sample of each tissue fraction was diluted to 0.5 ml and counted in a mixture of Cabosil gel powder and 7.5 ml of 1,4-dioxane-anisole-l , 2-dimethoxyethane solution (6 : 1: 1) containing 2,5-diphenyloxazole and 1,4-bis[2-(5-phen- yloxazolyl)]benzene (40: 1) as scintillators. A counting effi- ciency of 60% for % and 30% for tritium was obtained. The number of counts reported are corrected for background.
RESULTS
After injection of 35S-labeled chondroitin 4- or 6-sulfate2 into the animals, the urine collected during the following 48-hour period contained 35 to 50% of the injected radioactivity; a small proportion appeared as inorganic sulfate. Similar observations have previously been reported by Dziewiatkowski (13) and Dohlman (14). An examination of the blood of the animals throughout this period revealed that essentially no uptake of
1 We are indebted to Dr. C. de Duve for the details of this pro- :edure prior to publication.
2 Substantially identical results were obtained with chondroitin 4-sulfate and chondroitin B-sulfate.
Fraction No.
FIG. 1. Distribution of 0, specific radioactivity; and n , ref- erence enzyme, hyaluronidase, after centrifugation of a lysosomal fraction of rat liver through a linear sucrose-water gradient (from 1.10 to 3.42 molal). The rat had been given an injection of a%- chondroitin sulfate A 15 min before it was killed. Centrifugation was for 24 hours at 39,000 rpm in an SW 39 swinginK bucket rotor with a Spinco model L 2 ultracentrifuge. The lyiosimal fraction, prepared bv the procedure of Wattiaux, Wibo. and Baudhllin ClOj (equivalent to-O.65 g of original tissuej, was layered above the gradient after suspension in 0.65 ml of 0.25 M sucrose. Fractions were collected and assayed after the bottom of the tube had been punctured. Relative hyaluronidase concentration refers to t.he ratio of the observed activity to that which would have been found if the enzyme had been homogeneously distributed throughout the gradient. The control for radioactivity, 0, was carried out by adding an equivalent amount of radioactive polysaccharide to the liver homogenate of a rat not treated with radioactive ma- erial. This was then fractionated as above. The top of the entrifuge tube is at the right. See t;he text for additional details.
polysaccharide occurred in the cellular fraction and that all of the radioactivity was present in the plasma.
%-Labeled chondroitin g-sulfate was injected, the animal
was killed 15 min later, and the blood was fractionated as de- scribed above. This procedure showed about 1% of the total injected counts to be present in both normal lysosomes and the tritosomes3 The density gradient separation of the labeled material from normal liver is illustrat.ed in Fig. 1. The radio- activity along the gradient coincided with the hyaluronidase activity, which served as the lysosomal marker enzyme. The small peak at Fraction 6 coincides with the mitochondrial frac- tion, and may represent lysosomal particles which have not taken up the Triton WR-1339. The amount of material present was insufficient to permit reisolation and chemical identification of the polysaccharide, but electrophoresis of the labeled fractions showed that the radioactive component had the same mobility as chondroitin 4- or B-sulfate and was readily distinguished from inorganic sulfate. The distribution of radioactivity in other cellular fractions for Triton-treated animals is summarized in Table I. Since chondroitin 4- and B-sulfates are substrates for
3 Defined as Triton WR-1339-filled lysosomes. It should be noted that the use of Triton WR-1339 permits excellent separation of lysosomal and mitochondrial particles. Control experiments carried out with animals not receiving the detergent showed essentially the same amount of uptake in the lysosomal fraction as that of treated animals. However, this fraction is contam- inated with mitochondria and the l’riton procedure was therefore routinely used.
Mucopolysaccharide Catabolism Vol. 243, No. 17
TABLE I
Subcellular distribution of injected polusaccharides
Animals treated 4 days previously by injection of Triton WR-1339 were given intravenous injections of labeled polyssc- charide 15 min prior to death. Liver fractions were isolated
according to the procedure of Leighton et al. (12). The total amount of radioactivity administered was 9.5 X IO5 cpm for the 35Schondroitin 4-sulfate and 2.5 X IO6 cpm for the 3H-chondroitin
sulfate B. After addition of either polysaccharide to liver homogenates of normal animals, over 90% of the radioactivity is located in the supernatant fraction and less than 0.1% in any of
the particulate fractions. See the text for details.
Fraction
< 100 200 7,200 21,000 1,060 6,250
375 1,470 1,800 7,300
19,000 < 100
108,000 300
Fraction No.
FIG. 2. Distribution of 0, specific radioactivity; and n , ref- erence enzyme, hyaluronidase, after centrifugation of a lysosomal fraction of rat liver through a linear sucrose-water gradient (from 1.10 molal to 3.42 molal). The rat had been treated with %- chondroitin sulfate A 4 days before death. Centrifugation, frac- tionation of gradient, and expression of data are the same as those given in legend for Fig. 1. The top of the centrifuge tube is at the right.
lysosomal hyaluronidase, it might be expected that ingested
polymer would be lost by degradation to oligosaccharides and possibly further to monosaccharides, because of the activity of the glucuronidase and hexosaminidase present. As is shown in Fig. 2, a rat treated with the same amount of labeled material as above has almost no radioactivity remaining in the lysosomes when killed 4 days later. Hyaluronidase activity remained the same, both in amount and in location along the gradient. The subcellular distribution is summarized in Table II. It should be noted that only 35% of the injected radioactive label could be accounted for in the urine.
As has been shown previously, chondroitin sulfate B is a potent competitive inhibitor of lysosomal hyaluronidase (3),
but it is not known to serve as a substrate for any mammalian enzyme system. This polysaccharide was injected into a rat previously treated wit.h Triton, and the animal was killed in 15 min; approximately 1 TO of the radioactivity was recovered in the lysosomal fraction after the sucrose gradient fractionation. Results are summarized in Fig. 3 and Table I. The major radioactive peak corresponded exactly to the bulk of the hya- luronidase-positive material, and the minor peak to the mito- chondrial fraction. The association of some hyaluronidase
TABLE II
Subcellular distribution of injected polysaccharides
Animals were treated as described in Table I, except that they were killed 96 hours after injection of polysaccharide. See
the text for details.
Liver Nuclei
500 <lOO
I 3 5 7 9 II 13 15 17 IS 21 23 25 :
Fraction No,
FIG. 3. Distribution of l , specific radioactivity; and q , ref- erence enzyme, hyaluronidase, after centrifugation of a lysosomal fraction equivalent to 1.5 g of rat liver through a sucrose-water gradient. The rat had been treated with 3H-chondroitin sulfate B (2.5 X lo6 cpm) 15 min before death. Control for radioactivity, centrifugation time, and expression of data are the same as given in the legend for Fig. 1, except that 20 pg of protamine per ml were added to hyaluronidase assays to avoid inhibition by chon- droitin sulfate B (4). Preparation of the lysosomes and the gradient used are described in the text. The smaller peak coin- cides with the mitochondrial fraction, and may represent lyso- somes which did not accumulate the Triton WR-1339. In this experiment fractions were collected by displacement of the tube contents with sucrose of density 1.30. The top of the centrifuge tube is at the left.
Issue of September 10, 1968 hr. N. Aronson, Jr. and E. A. Davidson 4497
I 3 5 7 9 II 13 15 17 19 21 23 25 i
Fraction No.
FIG. 4. Distribution of l , specific radioactivity; and n , ref- erence enzyme, hyaluronidase, after centrifugation of a lysosomal fraction of rat liver through a linear sucrose-water gradient (from 1.10 molal to 3.42 molal). The rat had been treated with 3H- chondroitin sulfate B (2.5 X lo6 cpm) 4 days before death. Con- trol for radioactivity, centrifugation, fractionation of gradient, enzyme assay, and expression of data are the same as in the legend for Fig. 3. The top of the centrifuge tube is at the left.
b > .- E
KF ‘0 * -
l CHONDROITIN
D/stance (inches)
FIG. 5. Characterization of injected 3H-chondroitin sulfate B CSB) and isolated radioactivity from lysosomes. The lysosomal
fraction from the rat treated 4 days previously with 3H-chondro- itin sulfate B was deproteinized with 1O70 trichloracetic acid, dialyzed overnight against running tap water and then against distilled water, lyophilized, and finally dissolved in a small volume of water and precipitated with 2.5 volumes of ethanol. This material was spotted on Whatman No. 3MM paper and subjected to electrophoresis for 18 hours at 0.1 volt per cm2 in 0.05 M sodium acetate buffer, pH 5.0. One-half inch strips were cut and counted in toluene in a scintillation counter. The upper portion of the figure represents toluidine blue staining of 30 pg of standard chon- droitin and chondroitin sulfate B along with the injected 3H- chondroitin sulfate B.
w&h this latter peak suggests the presence of some lysosomal particles at this position in the gradient. Since the chondroitin sulfate B is not a substrate for either purified lysosomal hya- luronidase or int,act lysosomes, the radioactivity incorporated under these conditions should remain in these particles unless there is some process by which the filled lysosomes can expel the polysaccharide in a macromolecular state. As is shown in Fig. 4 and Table II, approximately 0.40% of the counts which were injected remained in the lysosomes of the rat killed 4 days after injection of tritium-labeled chondroitin sulfate B.
The radioactive material from four rats treated 4 days pre- viously with tritium-labeled chondroitin sulfate B was reisolated from the pooled lysosomal fraction by precipitation of the pro- tein with trichlorncetic acid, dialysis, and ethanol precipitation of the polysaccharide. As is shown in Fig. 5, the bulk of the reisolat,ed material showed the same electrophoretic mobility as the injected tritium-labeled chondroitin sulfate B, and thus there appears to be relatively little degradation of this material in the lysosomes. Significant loss of sulfate apparently does not occur, since this would result in radioactive material which moved with a mobility intermediate between a desulfated standard (chondroitin) and the chondroitin sulfate B. Ap- proximately 8Ooj, of the radioactivity was recovered in the area corresponding to the mobility of the injected mat,erial.
DISCUSSION
Both Dohlman (14) and Dziewiatkowski (13) showed that rats were able to metabolize exogenous chondroitin 35S-sulfate, as evidenced by the appearance of urinary inorganic sulfate after injection of labeled polysaccharide. However, the majority of the mucopolysaccharide was excreted in macromolecular form, although physicochemical data obtained were insufficient to permit assessment of slight amounts of degradation. Kaplan and Xteyer (15) also showed that approximately one-half of the injected mucopolysaccharides was excreted in the urine without degradation, although a portion did appear as inorganic sulfate. They suggested that two mechanisms exist for the disposal of mucopolysaccharides that reach the bloodstream: degradation somewhere within tissue, or excretion by the kidneys in the absence of breakdown. These reports still left uncertain the locality of the degradative system or the nature of the en- zymes involved, and they did not give any information on the breakdown of the carbon chain of the mucopolysaccharides. As is shown by this report, rat liver lysosomes are at least one site for the degradation of chondroitin 4- and 6-sulfate, although only 1 y0 of the injected material is taken up by these organelles in a 15-min period. Since %-labeled material is used and thus only the sulfate label could be shown to be metabolized, it might be concluded that these experiment,s offer no proof for the break- down of the carbon chain but only represent the result of sul- fatase activity. The arguments against this are the following. (a) Hyaluronidase, one of the substrates of which is chondroitin 4- or 6-sulfate, is known to be present in liver lysosomes. (b) Although experiments from this laboratory have indicated the presence of low levels of chondrosulfatase activity in the ly- sosomes, it is important to note that the sulfatase is inactive on polymeric substrat.es and requires intermediate size oligosac- charides (16). Similar results have been obtained by Dodgson and Lloyd (17), who showed that high molecular weight chon- droitin sulfate is not a substrat’e for a bacterial sulfa&e and that, instead, smaller oligosaccharides serve as the best sub-
4498 Mucopolysaccharide Catabolism Vol. 243, No. 17
skates. This implies that carbon chain depolymerization must precede sulfate hydrolysis. (c) The fact that tritium-labeled chondroitin sulfate B was apparently not significantly degraded, as evidenced by the electrophoret,ic mobility, suggests that the 4-sulfate of +labeled chondroitin 4-sulfat.e, which has the same configurstion as t’he 4 axial sulfate of…
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