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[114] Chondrosulfatase from Proteus vulgaris
[114]
CttONDROSULFATASE FROM Proteus vulgaris
[ 114] Chondrosulfatase
663
f r o m P r o t e u s vulgaris
By ALU~ G. LLOYD T h e existence in certain "putrefactive" bacteria of enzyme systems capable of degrading cartilage chondroitin sulfate 1 both by depolymerization and liberation of inorganic sulfate was first noted by Neuberg and his co-workers2 -~ Following these early observations analogous systems have been shown to be widely distributed in bacteria and are also present in certain species of fungi and marine molluscs? The most extensively studied of these systems are those from the bacteria Proteus vulgaris N.C.T.C. 4636, P. vulgaris 31M, and Flavobacterium heparinum and the marine mollusc Charonia lampas. The enzyme responsible for the process of depolymerization has been termed "chondroitinase ''6 and t h a t catalyzing sulfate liberation "chondrosulfatase" (syn. chondroitin sulfatase; chondroitin sulfate sulfohydrolase EC 3.1.6.4). I t has been shown t h a t preliminary degradation of the polysaccharide chain is a prerequisite for sulfatase action. 7,s Assay
Principle. Chondrosulfatase m a y be assayed by following inorganic sulfate liberation 9 from either chondroitin 4-sulfate 1° or chondroitin 6-sulfate ~° providing chondroitinase is also present during the incubations. However, the determination of chondrosulfatase activity in enzyme l In much of the earlier work material termed "chondroitin sulfate" is employed extensively. This must be presumed to be an unfractionated mixture consisting largely of chondroitin 4-sulfate contaminated with varying amounts of chondroitin 6-sulfate and possibly keratan sulfate as well. The nomenclature of individual polysaccharides used for the present review is that recommended in "The Amino Sugars" (R. W. Jeanloz and E. A. Balazs, eds.). Academic Press, New York, in preparation. 2C. Neuberg and 0. Rubin, Biochem. Z. 67, 82 (1914). C. Neuberg and E. Hofman, Biochem. Z. 234, 345 (1931). ' C. Neuberg and E. Hofman, Naturwissenscha#en, 19, 484 (1931). :For the most recent general review, see K. S. Dodgson, in "The Amino Sugars" (R. W. Jeanloz and E. A. Balazs, eds.). Academic Press, New York, in preparation. It is preferable to use the name "chondroitinase" to distinguish the enzyme from the conventional bacterial hyaluronidases, which apparently have only limited action against sulfated glycuronoglycosaminoglycans. 7 K. S. :Dodgson and A. G. Lloyd, Biochem. J. ~)6, 532 (1957). 8Even the most recently recommended nomenclature for the sulfatase (see Report of the Commission on Enzymes of the International Union of Biochemistry, Pergamon Press, Oxford, 1964) appears to be based on the earlier belief that the enzyme was capable of hydroly~.ing ester sulfate linkages in the intact polymer.
664
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[114]
extracts devoid of chondroitinase requires the use of preparations of these polysaccharides which have been depolymerized to yield a mixture of sulfated oligosaccharides by prior treatment with testicular hyaluronidase. 11
Reagents Substrate solutions. (a) Potassium chondroitin 4-sulfate or potassium ehondroitin 6-sulfate each at a concentration of 0.25% (w/v) in 0.1 M Tris acetate buffer, pH 7.0. (b). Enzymatically degraded 12 chondroitin 4-sulfate or chondroitin 6-sulfate each at a concentration of 0.25% (w/v) in 0.1 M Tris acetate buffer, pH 7.0. Enzyme preparation in 0.1 M Tris acetate buffer, pH 7.0 Absolute ethanol Potassium hydrogen phthalate solution, 0.05 M, pH 4.0 Barium chloranilate reagent. 18 The reagent is prepared by suspending solid barium ehloranilate (100 mg) in 100 ml of 0.25M sodium acetate-acetic acid buffer, pH 4.0. Since the barium chloranilate is only slightly soluble and always present in excess, the addition of an exact amount is unnecessary. Standard sulfate solutions containing 10-80 ~g of SO, ~- ions in 0.2 ml of the buffer used in the enzyme assays.
Since it is now apparent that the true substrates are comparatively small oligosaccharides, revision of the nomenclature is obviously required. 9The turbidimetric method described for the assay of glycosulfatase is not applicable to the determination of chondrosulfatase as residual polysaccharide sulfates form a complex with the gelatin in the reagent to give dense cloudy solutions [K. S. I)odgson, Biochem. d. 78, 312 (1961)]. 1°Suitable preparations of chondroitin 4-sulfate and chondroitin 6-sulfate are obtained by the method of K. Meyer, E. I)avidson, A. Linker, and P. Hofman [Biochim. Biophys. Acta 21, 506 (1956) ]. See Vol. I, p. 166, for a suitable preparation. ~Enzymatically degraded chondroitin 4-sulfate and chondroitin 6-sulfate are prepared by treating solutions of the potassium salts of the polysaccharides (50 ml of a 0.50%, w/v, solution in 0.01 M sodium acetate buffer with testicular hyaluronidase at 37°). A total of 150 mg of hyaluronidase (3000 I.U./mg dry weight) is added in three 50-mg portions over a period of 48 hours. At the end of the incubation period the incubate is heated quickly to 100° and cooled rapid.ly. A protein precipitate which appears is removed by centrifuging and the clear supernatant diluted with an equal volume of 0.1 M Tris acetate buffer, p i t 7.2. 18It is recommended that barium chloranilate be freshly prepared from chloranilic acid (2:5-dichloro-3:6-dihydroxy-p-benzoquinone) and barium chloride by the procedure of R. J. Bertolacini and J. E. Barney [Anal. Chem. 29, 281 (1957)].
[114]
CHONDROSULFATASE FROM Proteus vulgaris
665
Procedure. 14 The enzyme solution (0.1 ml) contained in a tapered Pyrex tube (40 mm X 5 ram), is preincubated for 3 minutes at 37 ° before the addition of 0.1 ml of the appropriate substrate solution, previously warmed to 37 ° . After incubation at 37 ° for the required period the reaction is stopped by the addition of 0.8 ml of ethanol, and after mechanical mixing, 15 the whole is allowed to stand at 0 ° for 10 minutes. Precipitated protein, polysaccharide, and oligosaccharides where present, are removed by centrifuging at 3500 g and 0 ° for 15 minutes. A portion (0.8 ml) of the clear ethanolie supernatant is withdrawn and transferred to a fresh tapered Pyrex tube. To the solution is added 0.2 ml of aqueous potassium hydrogen phthalate followed by 0.5 ml of the barium chloranilate reagent} 6 The tube is capped with Parafilm and the mixture is agitated ~ briefly at 1-minute intervals for a total of 20 minutes to keep the barium chloranilate particles in suspension. At the end of this period excess barium chloranilate and precipitated barium sulfate are removed by centrifugation at 4000 g and 0 ° for 10 minutes. All determinations are performed in duplicate and are accompanied by controls made by mixing enzyme and substrate only immediately before the addition of ethanol. The amount of chloranilate ion released into solution is measured against a reagent blank in quartz cuvettes of 1-cm light path at 350 m~. The extinctions are converted to inorganic S Q 2- ion content by reference to a calibration curve prepared in a manner similar to the above but using 0.2-ml portions of standard sulfate solutions. Purification Procedure
Step 1. Growth o] Bacteria and Preparation o] Acetone-Dried Cells. Proteus vulgaris (N.C.T.C. 4636) is conveniently grown in a medium containing the following in deionized water: 1% (w/v) 0xoid Peptone; 0.3% (w/v) 0xoid Lab Lemco; 0.1% (w/v) NaC1. The medium is adjusted to pH 7.0 by the addition of N NaOH prior to sterilization by autoclaving at 15 psi for 20 minutes. For the preparation of large quantities of cells, a shake culture of P. vulgaris (N.C.T.C. 4636) is prepared by inoculating 300 ml of the above medium in a 1-1 fiat-bottom flask and incubating the whole for 18 hours at 25 ° on a horizontal shaker. A portion (200 ml) of this culture 14A. G. Lloyd, Biochem. J. 72, 133 (1959). ,3Because of the narrow bore of the tubes it is best to use mechanical mixing in which the tube is held against the rapidly rotating (100 rpm) flattened spindle of an electric motor. '~Particulate barium chloranilate must be in suspension when the reagent is transferred.
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ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[114]
is used to inoculate 320 1 of the growth medium contained in a jacketed stainless steel fermenter. The large-scale culture is maintained at 28 ° and aerated at a rate of 3 cubic feet per minute without the addition of anti-foam. Optimum yields of cells are usually obtained after 5 days' incubation. At the end of this time the whole culture (320 l) is cooled to 0 ° and held thus for 18 hours before being passed through a large Sharples centrifuge at 2 °. The clear effluent is discarded. The packed cells are mechanically dispersed in distilled water (150 l) at 2 °, and the smooth suspension is passed through the Sharples centrifuge again. The clear effluent is once again discarded. The washed cells are macerated mechanically in absolute acetone (4 l) at --5 ° and then filtered at the pump using Green's 904 filter paper. The cake is resuspended in absolute acetone (5 l) at --5 °, refiltered and washed with absolute acetone (5 1). Residual acetone is removed as far as possible at the pump, and the process is completed in vacuo. Yields of acetone-dried cells in the range 0.75-1.0 g/1 of original medium are usually obtained. The activity of the acetone-dried cells is stable for several years when the preparation is stored at --10% Step 2. Enzyme Extraction. The acetone-dried cell preparation (40 g) is suspended in 800 ml of 0.2 M sodium acetate solution (pH 7.0) using a macerator. Portions (20 ml) of the suspension are then homogenized for 2-3 minutes in a Potter-Elvehjem homogenizer with a close-fitting ground glass pestle and mortar at 2 °. The finely dispersed homogenate is then readjusted to pH 7.0 and incubated at 37 ° for 2 hours. At the end of this period the cell suspension is centrifuged at 18,000 g (av.) and 2 ° for 20 minutes. The pale gold supernatant is collected and stored at 2 °, and the cell debris is resuspended as above in 400 ml of 0.2 M sodium acetate solution pH 7.0. The suspension is centrifuged as before, the supernatant is collected, and the cellular debris is discarded. The pooled supernatants are then dialyzed over a period of 16 hours against several changes of distilled water at 2 °. During the course of the dialysis a fine precipitate appears, and this is removed by centrifugation at 18,000 g (av.) and 2 ° for 15 minutes. The resulting preparation may be used immediately for further purification or lyophilized to give a white powder (average yield 0.1 g per gram of acetone-dried cells). The latter is stable for several years when kept at --10 °. Step 3. pH Precipitation. The dialyzed cell-free extract is cooled to 2 ° before the addition of a 5% (w/v) aqueous solution of a commercial preparation 1~ of the sodium salt of yeast nucleic acid previously adjusted to pH 7.4. The nucleic acid solution is added in the proportions 17Koch-Light Laboratories Ltd., Colnbrook, Bucks, England.
[114]
CHONDROSULFATASE FROM Proteus vulgaris
667
0.2 ml of solution per 20 ml of cell-free extract. The pH of the mixture is then adjusted to 4.0 by the addition of glacial acetic acid and the whole allowed to stand for 30 minutes at 2 ° . Precipitated material is collected by centrifuging at 4500 g and 2 ° for 30 minutes, and the clear supernatant is discarded. The precipitate is resuspended in a volume of water corresponding to one-fifth of the original volume of extract and is dissolved by the addition of N NaOH to pH 7.4. Step 4. Removal o] Nucleic Acid. The enzyme solution is adjusted to pH 6.7 by the addition of 1 N HCI. A 2% (w/v) solution of protamine sulfate, ~ previously adjusted to pH 6.7 by the addition of 1 N Na0H, is then added in 2-ml portions. The addition of the protamine sulfate solution is then continued until a portion of the treated enzyme solution, clarified by centrifugation at 18,000 g (av.) and 2 ° for 30 minutes, no longer gives a precipitate on adding further protamine sulfate solution. A slight excess of protamine sulfate solution (corresponding to onetwentieth of the volume of the original enzyme solution) is then added to the bulk enzyme solution and the whole dialyzed against several changes of distilled water (corresponding to 40 volumes in all) at 2 ° for 48 hours. Material which precipitates during the dialysis is removed by centrifugation at 4000 g (av.) and 0 ° for 20 minutes. The supcrnatant solution is adjusted to pH 8.0 with 1 N NaOH, held at 2 ° for 20 minutes and then clarified by centrifuging at 18,000 g (av.) and 0 ° for 15 minutes. The supernatant solution is redialyzed at 2 ° against several changes of water. The resulting clear solution is lyophilized to give a white powder (yield 0.1 g per 10 g of acetone-dried cells). The preparation at this stage still contains both chondroitin sulfatase and chondroitinase. Step 5. Adsorption on Calcium Phosphate Gel. TM A portion (200 mg) of the lyophilized powder from step 4 is dissolved in water (50 ml), and the solution is adjusted to pH 6.5 by the addition of glacial acetic acid. To this solution is added 16 ml of an aqueous suspension of calcium phosphate gel (containing 8 mg dry weight of gel per milliliter) previously adjusted to pH 6.5 with glacial acetic acid. After cautious mixing by inversion, the whole is then diluted by the addition of 34 ml of water and the suspension is mixed before standing at 2 ° for 20 minutes. The gel tends to settle during this period and should be kept in suspension by inversion of the container at 5-minute intervals. The gel-enzyme complex is collected by centrifuging at 2500 g (av.) and 0 ° for 10 minutes, and the clear supernatant is discarded. The complex is washed by suspension 1~The gel should be freshly prepared [D. Keilin and E. F. Hartree, Proc. Roy. Soc. B124, 397 (1938)] and not aged.
668
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[114]
in 100 ml of water at 2 ° using a glass homogenizer and then recentrifuged. The washing procedure is repeated three times with water and then twice more using 0 . 0 0 5 M sodium acetate solution (pH 7.2). The washed gel-enzyme solution is finally sedimented and enzyme elution is commenced by suspending the complex in 5 ml of 2 M sodium acetate solution (pH 8.0) with a glass homogenizer. The suspension is kept for 15 minutes at 2 ° before it is centrifuged at 2500 g and 0 ° for 10 minutes. The clear supcrnatant is retained and the sedimented gelenzyme complex is reextracted in an identical fashion with 12 further separate portions (5 ml) of 2 M sodium acetate solution, with intermediate centrifugation, the clear supernatants being held separately on each occasion. Assays of supernatant fractions consistently demonstrate the complete elution of chondroitinase, contaminated with chondrosulfatase, by the end of third washing, while chondrosulfatase devoid of chondroitinase continues to be eluted in diminishing amounts up to the final washing. Supernatants 4-12 are combined, dialyzed against several changes of distilled water (100 volumes in all) at 0 ° to remove sodium acetate and then freeze-dried. 19 The purification procedure is summarized in the table. PURIFICATION PROCEDURE
Purification stage 1. 2. 3. 4. 5.
Acetone-dried powder Enzyme extraction pH precipitation Removal of nucleic acid Calcium phosphate gel
Chondrosulfatase activityc (units~)
Chondroitinase activity c (units~)
Nucleic acid (%)
8.7 19.0 65.0 69.4 i. 1.1 ii. 49.5 ~
16.1 37.0 107.8 117.6 ---
-5 3 ~1 ~1 ~1
a Condrosulfatase activity is expressed in terms of micrograms of inorganic SO42ions liberated from the appropriate substrate per hour per milligram of protein. b Chondroitinase units are arbitrary and are expressed in terms of micrograms of reducing substance corresponding to D-glucosestandards, estimated by the method of K. S. Dodgson, A. G. Lloyd, and B. Spencer [Biochem. J. 6§~ 131 (1957)] liberated by 1 mg of protein during the incubation of the enzyme preparation with chondroitin 4-sulfate. c Assays were performed with polymer preparations of chondroitin 4-sulfate except as noted. d Assays were made with hyaluronidase-treated preparations of chondroitin 4-sulfate ~gH. I. Nakada and J. B. Wolfe [Arch. Biochem. Biophys. 94, 244 (1961)] have reported the separation of chondroitinase and chondrosulfatase from P. vulgaris 31 M by stepwise elution from a DEAE-cellulose column.
[114]
CLIONDROSULFATASE FROM Proteus vulgaris
669
Properties
Specificity. The factors determining the suitability of carbohydrate sulfates as substrates for bacterial chondrosulfatases remain incompletely clarified, but preliminary suggestions may be made in the light of accumulated experimental evidence. Extracts of P. vulgaris (N.C.T.C. 4636) chondrosulfatase devoid of chondroitinase are without measurable activity against high polymer preparations of bovine or whale chondroitin 4-sulfate, bovine chondroitin 6-sulfate, shark chondroitin sulfate (sulfated chondroitin 6-sulfate), dermatan sulfate, keratan sulfate, heparan sulfate, heparin, fucoidin, ;~- and K-carrageenin5 ° On the other hand, when each of the chondroitin sulfates is depolymerized to sulfated oligosaccharides (mainly di- and tetra-saccharides), by the action of either testicular hyaluronidase or P. vulgaris chondroitinase, hydrolysis of O-sulfate linkages by chondrosulfatase can be observedJ ,2°-~2 The chondrosulfatases of P. vulguris 31M and F. heparinum exhibit similar properties. 19,2~ Of the remaining polysaccharides it has been reported that dermatan sulfate can also be depolymerized and desulfated by enzyme extracts from P. vulgaris and F. heparinum53-~5 However, there has been no quantitative study of the hydrolysis of sulfate ester linkages during the action of the bacterial enzymes on this polymer. Extensive degradation of heparin and heparan sulfates, involving depolymerization and hydrolysis of 0-sulfate and N-sulfate (sulfamate) groups has only been demonstrated with enzyme extracts from cells of F. heparinum previously cultured on these polymers. 26-29 The extracts retain the ability to depolymerize and desulfate chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate. It is still not clear whether the degradation of heparin and heparan sulfates by such extracts is solely a function of the induction of an N-sulfatase (sulfamidase) 2s working jointly with constitutive chondrosulfatase and chondroitinase or whether indeed there is also an induction of additional O-sulfatase and depolymerase activity. Proteus chondrosulfatase is inactive against a range of monosac~°A. G. Lloyd, unpublished results. ~S. Suzuki and J. A. Strominger, J. Biol. Chem. 235, 2768 (1960). uS. Suzuki, J. Biol. Chem. 235, 3580 (1960). A. Linker, P. l=Ioffman, K. Meyer, P. Sampson, and E. D. Korn, J. Biol. Chem.
~, up. ~J. ~A. *' E. E. "A.
306i (1960). Hoffman, A. Linker, V. Lippman, and K. Meyer, J. Biol. Chem. 235, 3066 (1960). S. Maces and R. G. ttansen, Anal. Biochem. 10, 15 (1965). N. Payza and E. D. Korn, J. Biol. Chem. 223, 853 (1956). D. Korn and A. N. Payza, J. Biol. Chem. 223, 859 (1956). D. Kom, J. Biol. Chem. 226, 841 (1957). Linker and P. Sampson, Biochim. Biophys. Acta 43, 366 (1960).
670
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[115]
charide 0-sulfates and N-sulfates (sulfamates) and in particular Nacetyl-D-glucosamine 6-0-sulfate, N-acetyl-D-galactosamine 6-0-sulfate, N-acetyl-D-galactosamine 4-0-sulfate, UDP-N-acetyl-D-galactosamine 4-0-sulfate, and 2-deoxy-2-sulfoamino-D-glucose (D-glucosamine N-suN fate) .20 On the basis of this accumulated information it may be deduced tentatively that the substrates for bacterial chondrosulfatase are oligosaccharides containing saturated or unsaturated uronide linked N-acetylhexosamine sulfates. It appears that ester sulfate groups attached to primary (position 6) or secondary (position 4) hydroxyls on the hexosamine residues may be hydrolyzed. It is not known at what degree of polymerization chondrosulfatase action ceases. Inhibitors. Proteus chondrosulfatase is inhibited by phosphate, fluoride, hydroxylamine, and cobalt ions. 7 pH Optimum. A broad optimum pH is a characteristic of P. vulgaris chondrosulfatase, maximum activity being observed in .the range 6.7-7.3. 7 Kinetic Properties. A kinetic treatment of chondrosulfatase action has not been made owing to difficulties involved in purifying the substrates for the enzyme in quantities sufficient for routine analysis. Note Added in Proof: Recent studies (A. G. Lloyd, A. H. 01avesen, and K. S. Dodgson, unpublished results) have established the suitability of N-acetylchondrosin 6-0-sulfate (isolated following the chemical sulfation of N-acetylchondrosin) as a substrate for kinetic studies on P. vulgaris chondrosulfatase in the absence of chondroitinase. Evidence has also been presented that enzyme extracts from P. vulgaris contain two "chondrosulfatases" having different substrate specificities [T. Yamagata, Y. Kawamura, and S. Suzuki, Biochim. Biophys. Acta 115, 250 (1966)]. Under arbitrary conditions of pH and substrate concentration one of these specifically cleaved the ester sulfate linkage of biosynthetically prepared N-acetylchondrosin 6-0- [35S] sulfate (or its A,4:5-unsaturated derivative). The other appeared to hydrolyze preferentially the ester sulfate group of biosynthetic N-acetylchondrosin 4-0-[a5S] sulfate (or its A,4:5-unsaturated derivative).
[ 115] G l y c o s u l f a t a s e s f r o m M o l l u s c s
By ALvN G. LLOYD Glycosulfatases (sugar-sulfate sulfohydrolases, EC 3.1.6.3) capable of hydrolyzing ester sulfate linkages in a variety of mono-, di-, and trisulfate substituted monosaccharides and disaccharides, have been demon-
CttONDROSULFATASE FROM Proteus vulgaris
[ 114] Chondrosulfatase
663
f r o m P r o t e u s vulgaris
By ALU~ G. LLOYD T h e existence in certain "putrefactive" bacteria of enzyme systems capable of degrading cartilage chondroitin sulfate 1 both by depolymerization and liberation of inorganic sulfate was first noted by Neuberg and his co-workers2 -~ Following these early observations analogous systems have been shown to be widely distributed in bacteria and are also present in certain species of fungi and marine molluscs? The most extensively studied of these systems are those from the bacteria Proteus vulgaris N.C.T.C. 4636, P. vulgaris 31M, and Flavobacterium heparinum and the marine mollusc Charonia lampas. The enzyme responsible for the process of depolymerization has been termed "chondroitinase ''6 and t h a t catalyzing sulfate liberation "chondrosulfatase" (syn. chondroitin sulfatase; chondroitin sulfate sulfohydrolase EC 3.1.6.4). I t has been shown t h a t preliminary degradation of the polysaccharide chain is a prerequisite for sulfatase action. 7,s Assay
Principle. Chondrosulfatase m a y be assayed by following inorganic sulfate liberation 9 from either chondroitin 4-sulfate 1° or chondroitin 6-sulfate ~° providing chondroitinase is also present during the incubations. However, the determination of chondrosulfatase activity in enzyme l In much of the earlier work material termed "chondroitin sulfate" is employed extensively. This must be presumed to be an unfractionated mixture consisting largely of chondroitin 4-sulfate contaminated with varying amounts of chondroitin 6-sulfate and possibly keratan sulfate as well. The nomenclature of individual polysaccharides used for the present review is that recommended in "The Amino Sugars" (R. W. Jeanloz and E. A. Balazs, eds.). Academic Press, New York, in preparation. 2C. Neuberg and 0. Rubin, Biochem. Z. 67, 82 (1914). C. Neuberg and E. Hofman, Biochem. Z. 234, 345 (1931). ' C. Neuberg and E. Hofman, Naturwissenscha#en, 19, 484 (1931). :For the most recent general review, see K. S. Dodgson, in "The Amino Sugars" (R. W. Jeanloz and E. A. Balazs, eds.). Academic Press, New York, in preparation. It is preferable to use the name "chondroitinase" to distinguish the enzyme from the conventional bacterial hyaluronidases, which apparently have only limited action against sulfated glycuronoglycosaminoglycans. 7 K. S. :Dodgson and A. G. Lloyd, Biochem. J. ~)6, 532 (1957). 8Even the most recently recommended nomenclature for the sulfatase (see Report of the Commission on Enzymes of the International Union of Biochemistry, Pergamon Press, Oxford, 1964) appears to be based on the earlier belief that the enzyme was capable of hydroly~.ing ester sulfate linkages in the intact polymer.
664
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[114]
extracts devoid of chondroitinase requires the use of preparations of these polysaccharides which have been depolymerized to yield a mixture of sulfated oligosaccharides by prior treatment with testicular hyaluronidase. 11
Reagents Substrate solutions. (a) Potassium chondroitin 4-sulfate or potassium ehondroitin 6-sulfate each at a concentration of 0.25% (w/v) in 0.1 M Tris acetate buffer, pH 7.0. (b). Enzymatically degraded 12 chondroitin 4-sulfate or chondroitin 6-sulfate each at a concentration of 0.25% (w/v) in 0.1 M Tris acetate buffer, pH 7.0. Enzyme preparation in 0.1 M Tris acetate buffer, pH 7.0 Absolute ethanol Potassium hydrogen phthalate solution, 0.05 M, pH 4.0 Barium chloranilate reagent. 18 The reagent is prepared by suspending solid barium ehloranilate (100 mg) in 100 ml of 0.25M sodium acetate-acetic acid buffer, pH 4.0. Since the barium chloranilate is only slightly soluble and always present in excess, the addition of an exact amount is unnecessary. Standard sulfate solutions containing 10-80 ~g of SO, ~- ions in 0.2 ml of the buffer used in the enzyme assays.
Since it is now apparent that the true substrates are comparatively small oligosaccharides, revision of the nomenclature is obviously required. 9The turbidimetric method described for the assay of glycosulfatase is not applicable to the determination of chondrosulfatase as residual polysaccharide sulfates form a complex with the gelatin in the reagent to give dense cloudy solutions [K. S. I)odgson, Biochem. d. 78, 312 (1961)]. 1°Suitable preparations of chondroitin 4-sulfate and chondroitin 6-sulfate are obtained by the method of K. Meyer, E. I)avidson, A. Linker, and P. Hofman [Biochim. Biophys. Acta 21, 506 (1956) ]. See Vol. I, p. 166, for a suitable preparation. ~Enzymatically degraded chondroitin 4-sulfate and chondroitin 6-sulfate are prepared by treating solutions of the potassium salts of the polysaccharides (50 ml of a 0.50%, w/v, solution in 0.01 M sodium acetate buffer with testicular hyaluronidase at 37°). A total of 150 mg of hyaluronidase (3000 I.U./mg dry weight) is added in three 50-mg portions over a period of 48 hours. At the end of the incubation period the incubate is heated quickly to 100° and cooled rapid.ly. A protein precipitate which appears is removed by centrifuging and the clear supernatant diluted with an equal volume of 0.1 M Tris acetate buffer, p i t 7.2. 18It is recommended that barium chloranilate be freshly prepared from chloranilic acid (2:5-dichloro-3:6-dihydroxy-p-benzoquinone) and barium chloride by the procedure of R. J. Bertolacini and J. E. Barney [Anal. Chem. 29, 281 (1957)].
[114]
CHONDROSULFATASE FROM Proteus vulgaris
665
Procedure. 14 The enzyme solution (0.1 ml) contained in a tapered Pyrex tube (40 mm X 5 ram), is preincubated for 3 minutes at 37 ° before the addition of 0.1 ml of the appropriate substrate solution, previously warmed to 37 ° . After incubation at 37 ° for the required period the reaction is stopped by the addition of 0.8 ml of ethanol, and after mechanical mixing, 15 the whole is allowed to stand at 0 ° for 10 minutes. Precipitated protein, polysaccharide, and oligosaccharides where present, are removed by centrifuging at 3500 g and 0 ° for 15 minutes. A portion (0.8 ml) of the clear ethanolie supernatant is withdrawn and transferred to a fresh tapered Pyrex tube. To the solution is added 0.2 ml of aqueous potassium hydrogen phthalate followed by 0.5 ml of the barium chloranilate reagent} 6 The tube is capped with Parafilm and the mixture is agitated ~ briefly at 1-minute intervals for a total of 20 minutes to keep the barium chloranilate particles in suspension. At the end of this period excess barium chloranilate and precipitated barium sulfate are removed by centrifugation at 4000 g and 0 ° for 10 minutes. All determinations are performed in duplicate and are accompanied by controls made by mixing enzyme and substrate only immediately before the addition of ethanol. The amount of chloranilate ion released into solution is measured against a reagent blank in quartz cuvettes of 1-cm light path at 350 m~. The extinctions are converted to inorganic S Q 2- ion content by reference to a calibration curve prepared in a manner similar to the above but using 0.2-ml portions of standard sulfate solutions. Purification Procedure
Step 1. Growth o] Bacteria and Preparation o] Acetone-Dried Cells. Proteus vulgaris (N.C.T.C. 4636) is conveniently grown in a medium containing the following in deionized water: 1% (w/v) 0xoid Peptone; 0.3% (w/v) 0xoid Lab Lemco; 0.1% (w/v) NaC1. The medium is adjusted to pH 7.0 by the addition of N NaOH prior to sterilization by autoclaving at 15 psi for 20 minutes. For the preparation of large quantities of cells, a shake culture of P. vulgaris (N.C.T.C. 4636) is prepared by inoculating 300 ml of the above medium in a 1-1 fiat-bottom flask and incubating the whole for 18 hours at 25 ° on a horizontal shaker. A portion (200 ml) of this culture 14A. G. Lloyd, Biochem. J. 72, 133 (1959). ,3Because of the narrow bore of the tubes it is best to use mechanical mixing in which the tube is held against the rapidly rotating (100 rpm) flattened spindle of an electric motor. '~Particulate barium chloranilate must be in suspension when the reagent is transferred.
666
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[114]
is used to inoculate 320 1 of the growth medium contained in a jacketed stainless steel fermenter. The large-scale culture is maintained at 28 ° and aerated at a rate of 3 cubic feet per minute without the addition of anti-foam. Optimum yields of cells are usually obtained after 5 days' incubation. At the end of this time the whole culture (320 l) is cooled to 0 ° and held thus for 18 hours before being passed through a large Sharples centrifuge at 2 °. The clear effluent is discarded. The packed cells are mechanically dispersed in distilled water (150 l) at 2 °, and the smooth suspension is passed through the Sharples centrifuge again. The clear effluent is once again discarded. The washed cells are macerated mechanically in absolute acetone (4 l) at --5 ° and then filtered at the pump using Green's 904 filter paper. The cake is resuspended in absolute acetone (5 l) at --5 °, refiltered and washed with absolute acetone (5 1). Residual acetone is removed as far as possible at the pump, and the process is completed in vacuo. Yields of acetone-dried cells in the range 0.75-1.0 g/1 of original medium are usually obtained. The activity of the acetone-dried cells is stable for several years when the preparation is stored at --10% Step 2. Enzyme Extraction. The acetone-dried cell preparation (40 g) is suspended in 800 ml of 0.2 M sodium acetate solution (pH 7.0) using a macerator. Portions (20 ml) of the suspension are then homogenized for 2-3 minutes in a Potter-Elvehjem homogenizer with a close-fitting ground glass pestle and mortar at 2 °. The finely dispersed homogenate is then readjusted to pH 7.0 and incubated at 37 ° for 2 hours. At the end of this period the cell suspension is centrifuged at 18,000 g (av.) and 2 ° for 20 minutes. The pale gold supernatant is collected and stored at 2 °, and the cell debris is resuspended as above in 400 ml of 0.2 M sodium acetate solution pH 7.0. The suspension is centrifuged as before, the supernatant is collected, and the cellular debris is discarded. The pooled supernatants are then dialyzed over a period of 16 hours against several changes of distilled water at 2 °. During the course of the dialysis a fine precipitate appears, and this is removed by centrifugation at 18,000 g (av.) and 2 ° for 15 minutes. The resulting preparation may be used immediately for further purification or lyophilized to give a white powder (average yield 0.1 g per gram of acetone-dried cells). The latter is stable for several years when kept at --10 °. Step 3. pH Precipitation. The dialyzed cell-free extract is cooled to 2 ° before the addition of a 5% (w/v) aqueous solution of a commercial preparation 1~ of the sodium salt of yeast nucleic acid previously adjusted to pH 7.4. The nucleic acid solution is added in the proportions 17Koch-Light Laboratories Ltd., Colnbrook, Bucks, England.
[114]
CHONDROSULFATASE FROM Proteus vulgaris
667
0.2 ml of solution per 20 ml of cell-free extract. The pH of the mixture is then adjusted to 4.0 by the addition of glacial acetic acid and the whole allowed to stand for 30 minutes at 2 ° . Precipitated material is collected by centrifuging at 4500 g and 2 ° for 30 minutes, and the clear supernatant is discarded. The precipitate is resuspended in a volume of water corresponding to one-fifth of the original volume of extract and is dissolved by the addition of N NaOH to pH 7.4. Step 4. Removal o] Nucleic Acid. The enzyme solution is adjusted to pH 6.7 by the addition of 1 N HCI. A 2% (w/v) solution of protamine sulfate, ~ previously adjusted to pH 6.7 by the addition of 1 N Na0H, is then added in 2-ml portions. The addition of the protamine sulfate solution is then continued until a portion of the treated enzyme solution, clarified by centrifugation at 18,000 g (av.) and 2 ° for 30 minutes, no longer gives a precipitate on adding further protamine sulfate solution. A slight excess of protamine sulfate solution (corresponding to onetwentieth of the volume of the original enzyme solution) is then added to the bulk enzyme solution and the whole dialyzed against several changes of distilled water (corresponding to 40 volumes in all) at 2 ° for 48 hours. Material which precipitates during the dialysis is removed by centrifugation at 4000 g (av.) and 0 ° for 20 minutes. The supcrnatant solution is adjusted to pH 8.0 with 1 N NaOH, held at 2 ° for 20 minutes and then clarified by centrifuging at 18,000 g (av.) and 0 ° for 15 minutes. The supernatant solution is redialyzed at 2 ° against several changes of water. The resulting clear solution is lyophilized to give a white powder (yield 0.1 g per 10 g of acetone-dried cells). The preparation at this stage still contains both chondroitin sulfatase and chondroitinase. Step 5. Adsorption on Calcium Phosphate Gel. TM A portion (200 mg) of the lyophilized powder from step 4 is dissolved in water (50 ml), and the solution is adjusted to pH 6.5 by the addition of glacial acetic acid. To this solution is added 16 ml of an aqueous suspension of calcium phosphate gel (containing 8 mg dry weight of gel per milliliter) previously adjusted to pH 6.5 with glacial acetic acid. After cautious mixing by inversion, the whole is then diluted by the addition of 34 ml of water and the suspension is mixed before standing at 2 ° for 20 minutes. The gel tends to settle during this period and should be kept in suspension by inversion of the container at 5-minute intervals. The gel-enzyme complex is collected by centrifuging at 2500 g (av.) and 0 ° for 10 minutes, and the clear supernatant is discarded. The complex is washed by suspension 1~The gel should be freshly prepared [D. Keilin and E. F. Hartree, Proc. Roy. Soc. B124, 397 (1938)] and not aged.
668
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[114]
in 100 ml of water at 2 ° using a glass homogenizer and then recentrifuged. The washing procedure is repeated three times with water and then twice more using 0 . 0 0 5 M sodium acetate solution (pH 7.2). The washed gel-enzyme solution is finally sedimented and enzyme elution is commenced by suspending the complex in 5 ml of 2 M sodium acetate solution (pH 8.0) with a glass homogenizer. The suspension is kept for 15 minutes at 2 ° before it is centrifuged at 2500 g and 0 ° for 10 minutes. The clear supcrnatant is retained and the sedimented gelenzyme complex is reextracted in an identical fashion with 12 further separate portions (5 ml) of 2 M sodium acetate solution, with intermediate centrifugation, the clear supernatants being held separately on each occasion. Assays of supernatant fractions consistently demonstrate the complete elution of chondroitinase, contaminated with chondrosulfatase, by the end of third washing, while chondrosulfatase devoid of chondroitinase continues to be eluted in diminishing amounts up to the final washing. Supernatants 4-12 are combined, dialyzed against several changes of distilled water (100 volumes in all) at 0 ° to remove sodium acetate and then freeze-dried. 19 The purification procedure is summarized in the table. PURIFICATION PROCEDURE
Purification stage 1. 2. 3. 4. 5.
Acetone-dried powder Enzyme extraction pH precipitation Removal of nucleic acid Calcium phosphate gel
Chondrosulfatase activityc (units~)
Chondroitinase activity c (units~)
Nucleic acid (%)
8.7 19.0 65.0 69.4 i. 1.1 ii. 49.5 ~
16.1 37.0 107.8 117.6 ---
-5 3 ~1 ~1 ~1
a Condrosulfatase activity is expressed in terms of micrograms of inorganic SO42ions liberated from the appropriate substrate per hour per milligram of protein. b Chondroitinase units are arbitrary and are expressed in terms of micrograms of reducing substance corresponding to D-glucosestandards, estimated by the method of K. S. Dodgson, A. G. Lloyd, and B. Spencer [Biochem. J. 6§~ 131 (1957)] liberated by 1 mg of protein during the incubation of the enzyme preparation with chondroitin 4-sulfate. c Assays were performed with polymer preparations of chondroitin 4-sulfate except as noted. d Assays were made with hyaluronidase-treated preparations of chondroitin 4-sulfate ~gH. I. Nakada and J. B. Wolfe [Arch. Biochem. Biophys. 94, 244 (1961)] have reported the separation of chondroitinase and chondrosulfatase from P. vulgaris 31 M by stepwise elution from a DEAE-cellulose column.
[114]
CLIONDROSULFATASE FROM Proteus vulgaris
669
Properties
Specificity. The factors determining the suitability of carbohydrate sulfates as substrates for bacterial chondrosulfatases remain incompletely clarified, but preliminary suggestions may be made in the light of accumulated experimental evidence. Extracts of P. vulgaris (N.C.T.C. 4636) chondrosulfatase devoid of chondroitinase are without measurable activity against high polymer preparations of bovine or whale chondroitin 4-sulfate, bovine chondroitin 6-sulfate, shark chondroitin sulfate (sulfated chondroitin 6-sulfate), dermatan sulfate, keratan sulfate, heparan sulfate, heparin, fucoidin, ;~- and K-carrageenin5 ° On the other hand, when each of the chondroitin sulfates is depolymerized to sulfated oligosaccharides (mainly di- and tetra-saccharides), by the action of either testicular hyaluronidase or P. vulgaris chondroitinase, hydrolysis of O-sulfate linkages by chondrosulfatase can be observedJ ,2°-~2 The chondrosulfatases of P. vulguris 31M and F. heparinum exhibit similar properties. 19,2~ Of the remaining polysaccharides it has been reported that dermatan sulfate can also be depolymerized and desulfated by enzyme extracts from P. vulgaris and F. heparinum53-~5 However, there has been no quantitative study of the hydrolysis of sulfate ester linkages during the action of the bacterial enzymes on this polymer. Extensive degradation of heparin and heparan sulfates, involving depolymerization and hydrolysis of 0-sulfate and N-sulfate (sulfamate) groups has only been demonstrated with enzyme extracts from cells of F. heparinum previously cultured on these polymers. 26-29 The extracts retain the ability to depolymerize and desulfate chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate. It is still not clear whether the degradation of heparin and heparan sulfates by such extracts is solely a function of the induction of an N-sulfatase (sulfamidase) 2s working jointly with constitutive chondrosulfatase and chondroitinase or whether indeed there is also an induction of additional O-sulfatase and depolymerase activity. Proteus chondrosulfatase is inactive against a range of monosac~°A. G. Lloyd, unpublished results. ~S. Suzuki and J. A. Strominger, J. Biol. Chem. 235, 2768 (1960). uS. Suzuki, J. Biol. Chem. 235, 3580 (1960). A. Linker, P. l=Ioffman, K. Meyer, P. Sampson, and E. D. Korn, J. Biol. Chem.
~, up. ~J. ~A. *' E. E. "A.
306i (1960). Hoffman, A. Linker, V. Lippman, and K. Meyer, J. Biol. Chem. 235, 3066 (1960). S. Maces and R. G. ttansen, Anal. Biochem. 10, 15 (1965). N. Payza and E. D. Korn, J. Biol. Chem. 223, 853 (1956). D. Korn and A. N. Payza, J. Biol. Chem. 223, 859 (1956). D. Kom, J. Biol. Chem. 226, 841 (1957). Linker and P. Sampson, Biochim. Biophys. Acta 43, 366 (1960).
670
ENZYMES OF COMPLEX SACCHARIDE UTILIZATION
[115]
charide 0-sulfates and N-sulfates (sulfamates) and in particular Nacetyl-D-glucosamine 6-0-sulfate, N-acetyl-D-galactosamine 6-0-sulfate, N-acetyl-D-galactosamine 4-0-sulfate, UDP-N-acetyl-D-galactosamine 4-0-sulfate, and 2-deoxy-2-sulfoamino-D-glucose (D-glucosamine N-suN fate) .20 On the basis of this accumulated information it may be deduced tentatively that the substrates for bacterial chondrosulfatase are oligosaccharides containing saturated or unsaturated uronide linked N-acetylhexosamine sulfates. It appears that ester sulfate groups attached to primary (position 6) or secondary (position 4) hydroxyls on the hexosamine residues may be hydrolyzed. It is not known at what degree of polymerization chondrosulfatase action ceases. Inhibitors. Proteus chondrosulfatase is inhibited by phosphate, fluoride, hydroxylamine, and cobalt ions. 7 pH Optimum. A broad optimum pH is a characteristic of P. vulgaris chondrosulfatase, maximum activity being observed in .the range 6.7-7.3. 7 Kinetic Properties. A kinetic treatment of chondrosulfatase action has not been made owing to difficulties involved in purifying the substrates for the enzyme in quantities sufficient for routine analysis. Note Added in Proof: Recent studies (A. G. Lloyd, A. H. 01avesen, and K. S. Dodgson, unpublished results) have established the suitability of N-acetylchondrosin 6-0-sulfate (isolated following the chemical sulfation of N-acetylchondrosin) as a substrate for kinetic studies on P. vulgaris chondrosulfatase in the absence of chondroitinase. Evidence has also been presented that enzyme extracts from P. vulgaris contain two "chondrosulfatases" having different substrate specificities [T. Yamagata, Y. Kawamura, and S. Suzuki, Biochim. Biophys. Acta 115, 250 (1966)]. Under arbitrary conditions of pH and substrate concentration one of these specifically cleaved the ester sulfate linkage of biosynthetically prepared N-acetylchondrosin 6-0- [35S] sulfate (or its A,4:5-unsaturated derivative). The other appeared to hydrolyze preferentially the ester sulfate group of biosynthetic N-acetylchondrosin 4-0-[a5S] sulfate (or its A,4:5-unsaturated derivative).
[ 115] G l y c o s u l f a t a s e s f r o m M o l l u s c s
By ALvN G. LLOYD Glycosulfatases (sugar-sulfate sulfohydrolases, EC 3.1.6.3) capable of hydrolyzing ester sulfate linkages in a variety of mono-, di-, and trisulfate substituted monosaccharides and disaccharides, have been demon-