THE JOURNAL OF RIOLOGICAL CHEMISTRY Vol. 243, No. 7, Issue of April 10, pp. 1523-1535, 19G8

PTinted in U.S.A.

Purification and Properties of Bacterial Chondroitinases

and Chondrosulfatases”

(Received for publication, November G, 1967)

TATSUYA YAMAGAT~~, HIDEIIIKO SAITO, OSAMI HABUCHI, AID SAKMW SUZUKI

From the Department of Chemistry, Faculty oi’ Science, Nagoya University, Chikusa, Nagoya, Japan

SUMMARY

1. An enzyme, “chondroitinase-ABC,” has been purified to apparent homogeneity from extracts of Proteus vulgaris, NCTC 4636, which was adapted on a medium containing chondroitin sulfate C. It has the following properties. (a) At pH 8, it degrades chondroitin sulfates A, B, and C at greater rates than chondroitin and hyaluronic acid. It does not attack keratosulfate, heparin, or heparitin sulfate. (b) It carries out an elimination reaction, yielding A4,5-un- saturated disaccharides. (c) In the crude extract it is ac- companied by two different types of sulfatase, which are removed during purification.

2. The two sulfatases, “chondro-4-sulfatase” and “chon- dro-6-sulfatase,” have been separated from chondroitinase- ABC and from each other; both are required for the hydrolytic desulfation of chondroitinase products, A4, S-unsaturated disaccharide sulfates. They do not attack polymer chon- droitin sulfates, hexa-, penta-, tetra-, or trisaccharides derived from chondroitin sulfates A and C by digestion with crude testicular hyaluronidase, or acetylgalactosamine 4- and 6-sulfates.

One of these enzymes, chondro-4-sulfatase, catalyzes the conversion of A4,5-unsaturated disaccharide 4-sulfate (i.e. the product from the degradation of chondroitin sulfate A or B by chondroitinase-ABC) and its saturated analogue (i.e. acetylchondrosin 4-sulfate) to the corresponding nonsulfated disaccharides and inorganic sulfate, but does not attack A4,5-unsaturated disaccharide 6-sulfate (i.e. the product from the degradation of chondroitin sulfate C by chondroitin- ase-ABC) or its saturated analogue (i.e. acetylchondrosin 6-sulfate).

In contrast, chondro-6-sulfatase carries out the desulfation of the disaccharide 6-sulfates and acetylgalactosamine 4,6- disulfate at position 6 while it does not attack the disac- charide 4-sulfate isomers.

3. Another type of chondroitinase, “chondroitinase-AC,” has been purified also to apparent homogeneity from extracts of Flavobacterium heparinum, ATCC 13125, which was adapted on a medium containing chondroitin sulfate C. Its properties have been compared with those of chondroitinase-

* This study was supported in part by a research grant from the Ministry of Education, Japan.

ABC from P. vulgaris. (a) Unlike chondroitinase-ABC, it has no measurable activity with chondroitin sulfate B; like chondroitinase-ABC it carries out essentially the same re- actions with chondroitin sulfates A and C, chondroitin, and hyaluronic acid. (b) In the crude extract it is accompanied by an enzyme similar to chondroitinase-ABC, an enzyme similar to chondro-4-sulfatase, and a glucuronidase which hydrolyzes the fi-glucuronidic bond of unsaturated disac- charides but not the bond of saturated disaccharides. All these accompanying enzymes are removed during purification.

In several straiix of f+cvobacierium heparinro,z and Proteus vulgaris, enzyme systems have been detected which degrade chondroitin sulfates -1, II and C by an elimination mechanism rather than by hydrolysis (for a rcvicw of the literature, see References I and 2). The partially purified enzyme Ixel)arations from these bacteria cleave the endohesosaminyl groul) and produce A4,5-unsaturated disaccharides (3-j), unlike testicular hyaluronidase, which produces a series of .*aturatcd oligosac- charides. It is still not clear, however, whether the degradation of similar yet distinct chondroitin sulfate molecules by such preparations is a function solely of the same enzyme or whether indeed there are Feveral enzymes having different substrate specificities. To learn more about the q)rcificity of so-called chondroitinase, KC have ljurified the preparations from F. heparinum and P. vulgaris. The resulting I)urification indicated that enzyme extracts from E’. heparinurr~, *erWC 13125, contain at, least two distinct chondroitinase activities showing different substrate sl~ecificitien. The first of these enzymes act5 well on ChS-A,’ ChS-13, and ChS-C to form disaccharides. The second

1 The abbreviations used are: ChS-A, ChS-B, and ChS-C, chon- droitin sulfates A, B, and C, respectively; Alli-4S, 2-acetamido-2- deoxy-3-O-(p-n-gluco-4enepyranosyluronic acid)-i-O-sulfo-n-ga- lactose; ADi-GS, 2-acetamido-2-deoxy-3-0-(p-u-gl~~co-l-erre~~~ran- osyluronic acid)-6-0-sulfo-I>-galactose; Alli-OS, 2-acetamido-2- deoxy-3.0.(8.n-gluco-l-encpyrarlosyluic acid-n-galactose; chondroitinase-ABC and chorldroitirlase-AC; we suggest these trivial names to indicate that the latter enzyme differs from the former enzyme (which acts well on ChS-A, ChS-B, and ChS-C) in that it does not attack ChS-B and, also, because the enzymes

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1.524 ChondmZnases and Chondrosuljatascs Vol. 243, No. 7

ellzyme, henceforth rcfcrred to as “chondroitinase-AC,” differs from the fiwt enzyme in that it does not attack ChS-1%. In P. vrtlgaris, SCTC 4636, 011 the other hand, only one chondroitinase has been found. Like the first enzyme of F. heparinum, this enzyme attacks ChS-;\, ChS-B, and ChS-C with comparable facilit)- (this enzyme will be termed “chorldroitillase-ILIAC”). Iis a firsl; subject, this palxr describes how these enzymes may be ,sel)arated in a hi& state of llurity and summarizes their l)rol)- crtiw, inclliding substrate spwificit)-.

.\nother Ferics of cnz)-nm capable of degrading cshondroitiu sulfate by liberation of inorganic sulfate has becu reported to occur in bacteria, fungi, and marine n~olluscs (for a review of the literature, see ltefcrence 6). ?\mong t,heae, the KCTC 4636 strain of I’. vulgaris is known to be a 1)articularly potent source of the enzyme system responsible for the l~rocess of desulfation (7). .Ut,hough the system was termed “chondrosulfatase” (chontlroitin sulfate sulfohydrolasc, EC 3.1 .6.4) on the basis of the earlier belief that the elIa\-me was capable of hydrolyzing ester sulfate linkages in the intact polymer, subsequent n-ork showed that cliondrosulfatase was inactive toward polymer choiidroitin sulfate (8). More recently, it has been shown in this laboratory (9) that enzyme extracts from P. wlgaris contain two sulfat,ases wit,h diticrcnt, substrate. specificities. Olle of these is capable of hydrolyzing the sulfate linkage of AIX-4S, a cholldroitinase l)roduc? from ChS-i\, while the ot,her is capable of II>-tlrolyzing the sulfate linkage of Al%-GS, a chondroitinase Ixotlurt from ChS-C. This report describes, as a second subject, the tlctails of the 1)rowdure for purification of the sulfatases (to be rcfcrretl to as chontlro-4-sulfata~e and cxhoudro-6-sulfataw, resllectively) and certain of their lxopertics.

To date, a number of ctlzymei; for the degradation of mucol)oly sac~charidw have beell l)art,ially llurified from various sources, but wlatiwl~- little attclltioll has been given to their homogenrit! a11tl the 1)arameters dctcrmiiliiig their appawnt s~~ecificity. Ill-

formation regarding thcsc, (~IIZ~III(~S of tlefi~~tl slwcificity should bc \-aluablc, not only Iwau~c it may provide an insight into the mctalwlic fate of illric~ol)ol?-sac,c~haritlcs but also bccaux the CIIZJ,-~~CS with high substrate s;l)erificities may xr\-e as useful reagcilts in tlet~ermilrill~ the structure and quantity of muco- l)ol!-~ac~cliarides\\-illi a high &~~rce of precisioll. The ac*coml)anJ.- illg I\VO llaljers (10, 11) are colrwrlred with ali al)l)lication of the rnz\-me 1)rel)aratioirs i’i~iii I’. v~tlgaris aid F. hapaGzzc?x to

mi~i~otletcrl~~illatioll of isomcric c+iondroitiii sulfates and to itlctltificatioll of ol-rrsulfatt~tl cshonclroitin sulfates from various

.llateria/s

Cells and Crude I&zYracls-Proteus vulgaris, strain KCT(’ 4636, was grown at 30” with aeration (about I ..!I liters per mill) in a medium coiitaiiiiilg 15 g of l)eptone (I)ai#o Kagaku Corn- pall>-, Osaka), 4.5 g of meat extract (Xikuni C’hrmic~al Company, TokJ-o), 1.5 R of SaCl, and 1 g of ChS-C per liter. The medium was atljusted to pH 7 with SaOH. The cells were harvested by

dcgr:rtle hyaluronic acid only at extremely rctinccd rates, which clc:lrl~- sets these o~lzyrnc~s :~put from hgaluronidases (from testis ;tlld lxrctcria). Since the enzynles &polymerize chondroiliu sulf:ltcs hy all climill:11 ion mechanism, more systematic names are cholltlroitiu sulfates A, 13, x11(1 C lyase and cholltlroitin sulf:rtes ;Z anal C lynse, respect ivrly. The shorter nomcllclatlne Gil, how- cvcr, he used in the test.

cciitrifugation at 3” at the VII(~ of the logarithmic I)hase (usually bctwcw 8 and 10 hours), and ww washed with cold water. ~\j~~~roximatcl~- 350 g of wlls, n-c,t \I-ci,&t, were obtained from a 50.liter c:rJtivatioil.

The followina operations RX’W Iwrformctl at O-3”. The xashed wlls, 1 volume, \vcre huslw11(1~1 irl 3 volumes of 0.02 M ’15s.HC1 buffer, I)H 7.2, and disruptc~tl with the use of t,hc Fwuch ;xe:s (12). The suspe~~sion result illg 1'10111 this t wat merit n-as cell- tril’ugeti at 17,500 X g for 30 min. The sulwrnatant fluid thus obtained w8.s stored at -1X” (“bide extract”).

~lavobncferi~m heparincrm , strain ;\‘i’CC 13125, leas growl at 30” with aeration (about 0.X liltw per min) in a medium coutain- ing 7 g of ‘l’ryljticasc, 6 a ol Phytone (both from 13altimore Biological Laboratory), 1 g of &.ICOSC, 3 R of SaCI, 1 a of K2HP04, and 1 g of W-C per liter. The medium was atljuetcd to pH 7 with HCl. The cells were harvwtcd by cscntrifugation at 3” at the end of the logarithmic phase (usually between 10 and 12 hours), and were lx-a&d with rold water. -\l)l)rosimatcly 410 g (n-et weight) of cells were obtained from a 50.liter cultivation. The cells Jvere suspendctl in 3 volumes of cold 0.02 JI Tris-HCl, pH 7.2, and n-ere treatctl in the Kubota lo-kc sonic oscillator for 5 min at O-5”. The supcrnatant fluid obtained by centrifugation was stored at - 18” (“crude cstract”).

Cl~ertticaZ~-~~ chondroitin sulfate (c*alcium salt) n-as lwpared from bovine nasal srl)ta awording to the l~rocwlurc: of I;inbillder and Schubert (13) (“midfract ioIl”). A1fter it was dissolved in water, the solution n-as pass-cd over a l>ows 50-H+ wlumn (a column, 2 X 5 cm, of 200 lo 400 mesh 8X rwiii was Iibecl for 100 ma of l~olysacchai~itlc). ‘I’hc effluent was iicwl ralized with 10:; SaOH and lyol)hilizc~tl. The Ixcparatioil hat1 a sl)ecific optic*al rotation of [a] :’ -80” (c, 2.5, in water) and an aiialysis of gal:tc:tosaiiiille, 29.1 i( ; ~lucuroiiic acid, 32.05; ; and sulfur, 6.04’ ; 011 an auhydmus basis. Siwc the identity of this conl- lwulitl with choiidroitiii sulfate ‘1 preparations obtaiiicd 1~ gcncrallv acwptcd mc~thods ((:I’. I~cfcrcxcc 14) is iiirlic*atcd by the ol)tical rotation and inf‘rawtl spcctrru~~ (HOI slmw), this l)rc’para- tioii is refrrrctl to as <‘hS-.i in this and accoinl)aiiJ-iilg paper,< (IO, 11).

‘I’hc c~al(Gm salt of ChS-Il was prepared from boviltc lung 1)~ the nw~hod of Marbet a11t1 \\‘illtcwtc+~ (Is), and furlhw purified awortlill~ to the proc.c~durc of JIr)-cr et nl. (14). ‘1’11~ following anal\-ws were obtained 011 ail althydrous basis: ~alac~losaiiiine, 36.7’; ; nroiiic acid by 111~ owiilol assay (l(i), 36.3’~, ; ~iroi~ir acid d by the c~arbazolc assay (li), lS.2’,; ; alld 5ulfur, 6.73’ ; ; [a] b \vas -32.3” (r, 1, in water).

(:(w~1.011’; ,qifts of the I’ollo\G~g I)ol~sa~c~haritl(,s aw acaknon-l- c~tlactl: hcyl)aritiii suli’atc (hunla1i aorta) from 111,. I<. .\Ieyer, Ywhiva I-niwrsity, Scn- York; chondroitin (squid &in) (18) from 1)r. Ii. Akimbo, Ochanomizu ITniversity, Tokyo; and <‘hS-C, chc~lnic*ally tlcsuli’atctl ChS-(‘, and krl,atosulfatc (bhark rart ilage) (19) f’~nni 111,. T. Fui~uhaslii, Srikagaku KOQ-0 (‘onil)a~i~-, TOE;)-0.

l’wl)aration of lJC-labc~l(~tl choiithwitiii sulfate‘ \vas rarrird out by incubation of 1 g (n-cxt weight) f 1 o t IC tibiofcmoral epiph,wral caartilaac+ from 12-clay-old chick c~~nl~f~~os in 20 ml of a motlificd Kwbs-I<iii~cr medium (20), \\-hich contaiiierl 0.36 pniolc of laC!- glucow (uniformly labc~kd; slwific activity, 1.8 X 10” (‘pin pci pmol~) illstead of the unlabc~lctl &cose iii the ori~iiial tlescril)- tion, 300 units of 1xGcillin C, and 1 iiig of strc~ptoniyc~iii sulfate .-1ftrr iuc~ubation with shakilig at 37” for 1X hours, the chontlroitin sulfate n-a> isolated according to the procedure of l’crlmaii, ‘I’clser, and 1)orfman (21) \vith the csceptions that further di#cs-

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Issuc of April 10, 1968 Yamagata, Saito, Habuchi, and Suzuki 1.525

tion \\ith Pronace P (Kakcu Kagaku Company, Tokyo), l)an- erratic ribonuclcase (Worthington), and pancreatic dcoxy- ribonucleasc (Worthington) was carried out, after the J)apain digestion. The yield was 21 ~molcs as glucuronic acid or 2 x

10’ cpm as measured in a Packard liquid scintillation spectrom- eter. On exhaustive digestion with F. heparirLwn chondroitinase- alC (for techniques, Fee “Results”), the preparation (I I.tmole) was converted to AIX4S and Alli-6S in the yields of 0.38 pmole or 5.9 x 10” c‘lq and 0.62 pmolc or 3.6 x IO5 cpm, respectively. The ‘“C-chondroitin sulfate prel)aration may, therefore, be a 4:6 (w/w) misture 01’ *%XhS-h with specific activity of 1.5 X

10F qnn 11cr pmole of glucuronic acid and Y-ChS-C with qwific activity of 5.8 X 10” cpni per ~niolc of glucuronic acid.

Preparation of %labeled chondroitin sulfate was carrictl out by incubation of 4.6 g (R-et weight) of slices of the tibiofcmoral epil)hy.xeal cartilages from newly hatched male chicks in 100 ml of a modified Krebs-Ringer medium, which contained 24 mg of l\IgCIZ .6Hr0 and 4 mC of 3B-XasS04 (carrier-free) instead of the RIgS04.7H20 in the original description, 2000 units of 1)enicillin G, and 5 mg of streptomycin sulfate. After incubation with shaking at. 37” for 18 hours, the chondroitin sulfate was iholated as described above. The yield was 240 pmoles as glucuronic acid or 5 x lo8 cpm as measured in a Packard liquid scintillation sl)ectronietcr. The analysis with the UF’C of P’. heparinw~z chondroitina?e--1C (see belom)2 indicated that the lxeparation is a 4:6 (w/w) mixture of WChSZ1 with sl!ecific activity of 4 X 10” clm~ per ~.tiiiole of glucuronic acid and % ChS-C with spcrific activity of 1.0 X IO6 cpm per ~mole of glucuronic acid.

To prel)are %labeled unsaturated disaccharides on a large scale, a cholldroitinase-Al-3C digest of 30 pmoles (as glucuronic acid) of the Y!-chondroitin sulfate preparation was applied as a zone on thrw sheets (60 x 60 cm) of ‘To)-0 So. 50 filter paper which wrrc then chromatographed by descending technique in Solvent I (for holvcnt systems Bee below), in which :%Al)i-4S has a larger RF value than ““S-Al%6S (4). The unsaturated disaccharides were located by ultraviolet photography with a I\Iatsuda ultraviolet lamp, I x GL-15W, with Riken filter 2537. The ultraviolet light-absorbing materials were elutcd from the leaper with water and xparatcly subjected to a recond chromatog- raphy in the ?ame ,Golvent. ;\fterwards, each of the saml)les n-as further purified by successive chromatography in Solvrnt II, chromatogral)hy in Solvent III (for desalting), and paper elcctro- phoresis ill 0.05 M ammonium acetate buffer, I)H 5.0 (WC bclo~~). The zones foulId on the rlcctroJ)horetic strips \YCIYJ clutcd as above. The disaccharide solutions thus obtained contain a small amount of ammonium acetate, which, if desired, ran bc removed by paper chromatography in Solvent III. ““S-AIX4S (specific activity, 3.8 x 10” cpm per pmolc) and “%Al>i-6S (specific activity, 1.0 X 10G q)m Ijer pmole) were thus obtained in the yields of 3.6 ~moles and 6.6 pmoles, rcslwctiwly, as estimated from absorbance at 232 ml* of the aqueous solutions at pH 2.0 (4).

The followi-illg commercial mat trials wrc uwd : hyaluronic acid, tc,sticular hyaluronida?c, and bovine lircr /3-glucuronida~e from Sigma; hcl)arin from Calbiochcm; Hypatite C (hytlrosyl- allatitc susprnsiou) from (Xarkson Chemical Company, Inc., ~~illiams~~ort, Pennsylvania; Sephades G-200 from l’hwmacia, Ul)lwala; tlieth~lamillorth~l cellulose from I%rown Company,

2 A more det,:tiled description of these analyses is presented in the accompmlyillg paper (10).

Iiecne, Scn llamlwhirc; and ~~l~os~~l~ocellulosc from Scrva (Entwicklungslabor, Heidclbcrg). ‘rhr ccllulox ion cschangc materials were Ixepared for use by t’reatmrnt with large volumes of the following solutions in the indicated sequcncc: for I)]<:.-\E- celluloar, 1 .O >r SaCI, water, 0.5 M SaOH, water, 0.1 JI HCl, water, 0.5 nr SaOH, and water; for I)hosJ)l~oc’Cll11losr, 0.25 RI SaCI, water, 0.25 M SaOH, and water. Chemicals othrr than those listed abow wcrc obtained from Sigma, Kako Chcrnical Company, Osaka, and other commcwial sources.

_ I nalytical I’ro~edlrres--lliol.gallic hulfate was drtcrmilwtl 1)~ the method of l)otlgson (22), uranic acid by the carbazole method (17) and by the oricinol method (16)) hcsosamilrr by the l)orate-catal~zcd Elsoll-Morgan mrthods (23), Icducing J,oi\-er by the mcthotl of Park and ,Johwon (24), and l)rotein by the method of Lowry et al. (25). For mcasurcmcnt of’ radioactivity, sample solutions a-we alq)lird on discs (2.4 cm in diameter) of Toyo No. 51 A palm, which were then dried in an oven at 60”. The radioactivities of the discs were detwmincd irr a l’ackard liquid scintillation spcctrometcr with the solvent I)rr\-iouslJ- described (26).

Paper Chromatography and Paper l~lectrophoresis--l’he l)al)cl

chromatographic solvent systems uactl, with descending chroma- tography on Toyo Xo. 50 and Xo. 51 .-I filter pal)cr (60 CJJJ long), n-we Solvent I, I-butyric acid-O..5 K ammonia, 5:3 (v/v) ; Molwnt II, 1 -butanol-acetic acid-l N SHaOH, 2:3: 1 (v/v/v) ; alld Sol- vent III, I -butanol-cthallol-n-ater, 52:32: 16 (v/v/v).

Paper electrophorwis \vas carried out on BO-cm strilw of ‘I’oyo Ko. 51.1 paler in the apparatus dewribrd by Markham and Smith (27) at, a potential gradicwt of 30 volts I)er cm. The buffer system wed leas 0.05 M ammonium acetate-acetic acid, pH 5.0.

011 a ]xcyaratiw .scalc, the saJll]Jk was a]q)licd as a thin zOJJe 011 the chromatogral)hic or elcrtrol)horetic strip). I f ncwssarv guide stril)s wre cut and stained. ‘I’lJe c~onipounds were th,J;

cluted from the remainder of the I)alwr with water. *ill the reducing sugars tlescribctl in this paper could bc tle-

tected with the aniline hydrogen 1)hthalatc spra>- (28). The disaccharides containing A4,5-glucuronic~ acid were detcctcd also by ultraviolet ab.qorption l)hotography (we ahovc) or hy viewing under a Mincralight, S-2537. Inorganic sulfate was located by s])raying the l~aCl,-rhoclizoliat(, J?a#cJJtS (29).

.Issay of Chondroitinase (-1 ssfqs I, 2, and S)-‘l’hrec~ assay methods were used.

.\ssay 1, bawd on the formation of cthallol-solul)le lxotlucta from 14C-labrlrtl chondroitin sull’atc (.\ + (‘), was 1)rimarily uwd for thr purification eslwimcwts. Routine ilrcuhation mistures contained, in final volumc~s of 50 ~1, ‘Y-cholltlwitin sulfate (-1 + C), 10 mpJJwh as glucwonic acid (10” cpm); ‘I’ris- HCl, pH 8.0, 2.5 p~no]es; sodium acetate, 3 ~molrs; bol-inc .wrum albumin, 5 pg; and 0.0001 to 0.0005 unit of enqmc (i’or the defiliitioii of ciizyir unit, see lwlon-). Controls contaiwtl heat- inactivated cwzymr. ,Zftrr incubation for 10 min at 37”, the reaction misturrs were hcatcd at 100” for 1 min. Ilnrcactcd 14C-ChS was ~nwil~italctl by qurntial addition of 50 ~1 of a carrier solutioii containing 3.5 nig of (‘IIS-A\ alltl 3.5 nig of (‘IiS-C

per ml, and 0.2 1111 of ethanol containing 1 (,‘I potassium awlatc. ‘I’hr mixture was allowed to stand at 0” for 30 mill. ‘1‘110 Iwr- cil)itate was rcmovccl by cciitrifugation. ‘I’hr radioac.tivi(!. of soluble products in the sqwnatant solutioi~~ was ~IWJJ JJ~~YL~~~J~N~ by the method described above (set “-\nalytical l’ro(~(~(1111.(~~“).

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15% Chondroitinases and Chondrosulfatases Vol. 243, No. 7

The assay was linear with time and amount of enzyme under the conditions for routine assay. One unit of enzyme was defined as the quantity that catalyzed the release of 1 pmole (as unsaturated disaccharide) of soluble products per min under the conditions described above. One advantage of this assay is that it is not affected by the pre~encc in enzyme preparations of sulfatases and glucuronidases, which can degrade the product disaccharides to smaller fragments. All the chondroitinases could be assayed by this method when it was desirable that the values obtained with different purification grades of enzyme preparations be comparable.

Assay 2 measured the conversion of a chondroitin sulfate that is inactive in the Morgan-Elson reaction to a form that is active in t,he color reaction. Routine incubation mixtures contained, in final volumes of 50 ~1, ChS-al, ChS-B, or ChS-C, 0.05 pmole as uranic acid; Tris-HCl, pH 8.0 for chondroitinase-ilBC and l)H 7.3 for chondroitinase-AC, 2.5 pmoles; sodium acetate, 3 ~moles; bovine Serum albumin, 5 pg; and 0.0005 to 0.0025 unit of enzyme. When ChS-A or ChS-B was used as substrate, 0.01 unit of chondro-4-sulfatase (see below) was also added to the mixture. Controls contained heat-inactivated enzyme. ,ifter incubation for 10 min at 37”, the reaction mixtures were heated at 100” for 1 min and centrifuged if necessary (only with the crude extracts), and the entire sample or & aliquot of the super- natant solution was analyzed by the borate-catalyzed hlorgan- Elson method (23).

The assay was linear with time and amount of enzyme under the conditions for routine assay. One unit of enzyme was de- fined as the quantity that catalyzed the release of 1 pmole (as ADi-OS or ADi-6s) of product per min under the conditions de- scribed above. This assay has the advantage over iissay 1 that the measurements ran be made with the individual chondroitin sulfate isomers.

-issay 3, based on the formation of A4,5-unsaturated disac- charides that have a marked absorption in the ultraviolet region, n-as primarily used for the experiments in which effects of various coml)ounds and conditions on the lnuified chondroitinase prep- arations were tested. Routine incubation mixtures contained, in fiual volumes of 50 pl, mucopolysaccharide substrate, 0.1 ~mole as uranic acid or hexosamine; the buffcrs indicated in t,he individual experiments, 2.5 pmoles; Fodium acetate, 3 pmoles; bovine swum albumin, 5 pg; and 0.001 t,o 0.005 unit of enzyme. Controls contained heat-inactivated enzyme. After incubation at 3i” for 10 min, reaction was stopped by diluting the mixture with 0.45 ml of 0.05 M KCl-HCl buffers, pH 1.8. It n-as then centrifuged at 10,800 x g for 10 min, and absorption of the super- nai;ant of each sample was measured at 232 111~ against the corre- sponding blank mixture. The amount of uusaturaled disac- charide lmducts was calculated from the change in absorbance with the use of 5.7, 5.1, and 5.5 as millimolar absorption co- efficients of ADi-OS, AX-4S, and ADi-BS, respect.ively.

The assay was linear with time and amount of enzyrne under the conditions for routine assay. One unit of enzyme was de- fined as the quantity that catalyzed the rclcase of I. pmolc (as unsaturated disaccharide) of product) 1:~ min under the con- ditions described above. -Is will become evident, crude enzyme prrl)arations, particularly those of E’. heparinunz, exhibit glucu- ronida?e activities which interfere with this assay by hydrolyzing the products to compounds with little absorbance at 232 nip. It is ncccssary n-ith such preparat,ions to we ;\ssay 1 or 2.

Assay of Chrondrosulfatases (Assay /,)--This assay measures

the conversion of ““S-labeled AIX4S or AlX6S to 35S-inorganic sulfate by electrophoretic separation. Routine incubation mix- tures contained, in final volumes of 50 ~1, WADi-4s or %- ADi-6S, 0.01 pmole (about 5 x 10” cpm); Tris-acetate, pH 7.5, 2.5 pmoles; bovine serum a!bumin, 5 pg; and 0.0001 to 0.0005 unit of enzyme. Controls contained heat-inactivated enzyme. After incubation for 10 min at 37”, the reaction mixtures were heated at 100” for 1 min and centrifuged if necessary, and a lo- ,~l portion of the entire sample or the supernatant Jvas applied to Toyo ?;o. 51A paper (60 cm long) together with 0.03 pmole of unlabeled NaZS04 as an internal marker. Elcctrophoresis was carried out in 0.05 3% ammonium acetate, pH 5.0, at 30 volts per cm for 20 min. The area corresponding to sulfate ion was located by spraying the BaClz-rhodizouate reagents (29), cut out, and counted in a Packard liquid scintillation spectrometer.

The assay was linear with time and amount of enzyme under the conditions for routine assay. OJIC unit of enzyme was de- fined as the quantity that catalyzed the rcleaFc of I pmole of inorganic sulfate per min under the conditions described above.

Assay of Ii’. heparinwn Glucuronidase (it ssaM 5)-ADi-OS is hydrolyzed to acetylgalactosamine and cu-kcto acid in the pres- ewe of F. heparinum “glucuronidase.” The rate of decompo- sition of ADi-OS is measured by the dccreasc in absorbance at 232 mp. Routine incubation mixtures contained, in final vol- umes of 50 ~1, ADi-OS, 0.02 pmole; T&HCl, pH 7.2, 2 pmoles; bovine serum albumin, 5 pg; and 0.0002 to 0.0015 unit of enzyme. Controls contained heat-inactivated enzyme. After incubation for 10 min at 37”, the mixtures were diluted with 0.45 ml of 0.05 RI KCl-HCl buffers, l)H 1.8. They were then centrifuged at 10,800 x g for 10 min, and absorbance of the supernatant of each sample n-as mrasurcd at 232 nip against the blank mixture.

The assay was linear with time and amount of enzyme under the conditions for routine assay. One unit of enzyme n-as de- fined as the quantity that catalyzed the decomposition of 1 pmole of Al&OS or a dccrcase of 11.4 in absorbance at 232 rnp per min under the conditions dcscribcd above.

IIESULTS AKD DISCUSSION

Chondroiiinase jiom P. vulgaris

It has been reported by Maycs and Hansen (30) that the in duction of a chondroitinasc specific for a given chondroitin sulfate did not ouxw whether P. vzrlgaris was induced 0~1 ChS-,LZ, ChS-I$ or hyaluronic acid. In accord with this observation, our pre- liminary rxpcriments haw indicated that all the activities for ChS--1, ChS-T$ and ChS-C were cwhanccd about 150.fold n-hcther the organism n-as growl ill the lwesence of ChS-A or ChS-C. The eridcnw lweyentcd below indicates that the adal)ted cells contain a chondroitinasc n-hich catalyzes the dcg- radation of all the chondroitin sulfates.

I-‘vriJica2ion-Uillcss otherwii;e specified, all operations were conducted between 0” and 4’. All caentrifugat,ions were at 17,500 X g for 30 min.

To jO0 ml of the crude extract of I’. vulgaris (WC “Methods”) were added, with stirring, 125 ml of a 5y; st,rcptomycin sulfate solution in 0.02 JI Tris-HCl, pH i.2. -\l’ter standing for 1 hour, the suspension \vas centrifuged. The supernatant was allowed to stand for 12 hour>, and the resultant l)recipitate was removed by centrifugalion.

To the supernatant fluid (5i3 ~111) were added, with stirring, 140 g of solid ammonium sulfate. The suspension was allowed to

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stand for 1 hour after the ammonium sulfate had dissolved, and then it was centrifuged. To the supernatant fluid wcrc added, with stirring, an additional 84 g of ammonium sulfate. The sus- pension was allowed to stand and was centrifuged as before. The resulting precipitate was dissolved in 50 ml of 0.02 M Tris- HCl huffcr, pH 7.2. This solution was dialyzed for 24 hours against five 2-liier changes of the same buffer.

--I column (4.5 x 40 cm) of DE.1E-cellulose was prepared and equilibrated with 6 liters of 0.02 M Tris-HCl buffer, $I 7.2. The dialyzed amm&ium sulfate fraction (65 ml) was applied to the column at the rate of 60 ml per hour. The adsorbent was then washed with 1 liter of 0.02 M ‘I’ris-HCl buffer, $1 7.2. ;\pprosi- mately 65(/l of the activity applied to the column was recovered in the mash (the 500.ml fraction, ree Fig. 11, below), whereas approsimately 95’); of the protein 1Ta.s retarded by the column.

II column (2.1 x 15 cm) of phosphocellulo~e was equilibrated with 0.5 liter of 0.02 M Tris-HCl buffer, l)H 7.2. The I)IUE- cellulose wash, 500 ml, was applied to the column at the rate of 30 ml l)er hour, and the adsorbent was washed with 100 ml of 0.1 RI XaCl in 0.02 hf Tris-NC1 buffer, pH 7.2. A linear gradient of elution was applied with 0.1 M and 0.6 M KaCl in the same buffer as limiting concentrations. The total volume of the gradient x-as 600 ml. The flow rate was 30 ml per hour, and 5-ml fractions were collected. X single-peak was *obPervcd, and the fractions containing the major portion of this peak (tubes 20 to 40) wrr pooled. The pooled phosphocellulo~e fraction (105 ml) n-as concentrated to 6 ml by pressure dialysis against 3 liters of 0.02 JI Tris-WC1 buffers, pH 7.2 (choatlroitinase-l~~C). ;1 sum- mary of the purification procedure is shown in Table 1. The acrylamidr gel clectrophoresis pattern of a fraction at the final stage of purification is shown in Fig. I &arently, the phos- phoccllulose fraction contained no pro1 ein impurity detectable by the elect,rol,Eloi,esis. The enzyme (4.4 rnff of protein per ml) retailled about 9Oc/, and 50% of its activity for 2 and 9 months, respcctiT-cly, n-hen stored in an ice bath. The fresh cxzyme so- lution could bc lyophilized without any sipiificant loss of activity.

St&ies of Xature OJ” neaction--Treatmeiit of ““S-labeled choa- droitin sulfate (a mixture of ChS-LL and ChS-C) with the purified enzyme resulted in the disappearance of the polysaccharide frac- tion, lyith corresponding appearance of a sin,rrle fraction behaving

T.\BLE: I

PuTi,fir&ion c?f c~hmadroitinasc-A UC from P. ozdgu~~i.s, SCTC 4636

Artivitics were rne:~sru’ed by Assay 1. 111 ihis assay, the iJlitia1

concentration of srJbstrale is very low (0.2 m&Z 14C-(~hoJJtlroitin sulfate, as IJroJlic acid) so that. differences in the sl~bstratc concew Iration would have a coJJsidcrablc effect on I he appawnt vcloci-

ties. A value of 130 was, in fact, obtained for the specific activity of Step 5 enzyme, when i1 was rneaslwcd by Assay 2 wilh a ChS-C concentr:ttioJl of 1 rn~. 111 1 his and i he accompanying papers

(10, II), the :~nloJmt of choJldroitilluse~AUC is rcprcsnJrlcd bv the VRlIIP ObtxiJJc~tl with Assay 2.

step -

1. CJ’lldk? CXtI’Xt

2. S1 J~eplomyciJr

3. AtnmoJJirJrn sulfate, after tlialvsis

4. I)IL4ti-cellulose colurnJ~.

5. I’hosI~hoc~c~llrll~lsc collJmll.

Total activity

3800 2500

990

Specitic actkity

7lbole.~/?zg/llli~

0.28

0.45

0.80

11.0

37.5

Purification

-fold

I

l.G

2.8 39

131

FIG. 1. hcrylamide gel electrophoresis of aliquots (about 10 pg) of the most highly purified fractions of 1, chondroitinnse-ABC (P. vulgaris) ; 9, chondroitinase-AC (F. heparinum) ; 3, chondro-4- sulfatase (P. vulgaris) ; and 4, chondro-C,-sulfat:tse (P. vulgaris). The electrophoresis was carried out according to the method of Ornstcin (31). The origin is indicated by the arr0w.s.

as unsaturated disaccharides (e.g. AIL4s or ADi-6s) on chroma- tography on Sephadcs G-50 (Fig. 2).3 011 exhaustive digestion, about 10” cpm of Wchontlroitin sulfate were converted quan- titatively to the disaccharide fraction. When a testicular hy- aluronidase digest of the “%chondroitin sulfate sample was chromatographcd, it showed a markedly different distribution of S”S-l>roducts, t,he hyaluronidase products behaving as higher oligosaccharides than the chondroitina?e tlisawharide. The product from degradation of %-chondroitin sulfate by chon- droitinawM3C was further examined by chromatography on paper in Solvent I and by electrophorcsis on leaper at pH 5, with the USC ol’ Na2S04, acet,ylgalactosamine 4-sulfate, acetglgalac- tosamine B-sulfate, Al L4S, and ADi-6S as intcwal markers (for RF values Fee I?cl’c~~cnw 4). 111 both cases, all of the radioac- tivity applied to the l)al)er was rccovcred as AlL4S and ADi-6s. The alwencc of ““S-containing material in the inorganic sulfate reg:ion indicates that the enzyme is devoid of chondrosulfatase activity.

It should also Ix noted here that the most highly purified enzyme sholT-ed no AIL@-degrading: activity under the con- ditions of’ .2ssay 5 for glucuronidase, although the crude extract of P. vlrlgaris shon-etl a n-cak but significant activity.

To obtain a 1aJg’Cr aJnouJrt of the disawharidc produrts, 5 g of sodium ChS-11 WJ~C iJJcuhatetl for 2 hours with 1000 units of choildroitiliasc-~~I~~, :t~Jd the misturc mw desalted with the use of a C(!lite-l)arco G60 cwlumi~ (4), the11 scrcencd successively by paper chroniato~ral)hy in Solrcnt I (1~ cycles), l)apcr chroma- tograllhy in Solvent 11, paper c~hroruato~ral)ll~ in Solvent III, palmer c,lectrol)horcsis at pH 5.0, and paprr chromatogral)hy in Solvent III, csscntially as tlescribed for the lweparation of ““S- labeled disaccharides (see “Chemicals”). ‘1’1w tlisaccharitle product was c~lutetl from the last chromatopams with water. .\ftcr the eluates acre tlricd in a vaculml over P,O,, the compound was further lmrifictl by precipitating the compound as the sodium

3 When instead of the 3%.labeled chondroilin sIllfate, a 3%. chondroitinslJlf:Lte-prot,eill complex (obtaiJJed from the ““S-labeled cartilage without the [EC of alkali and Pronase I’ (33)) was treated under 1 he same conditions, the snbslrute was tIcgraded to yield the disaccharide fraction at about the saJnc rate. Apparently, the protein linked to ~hc chondroitin sulfate chain does not tcud to retard degradation.

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152s

salt with acetone from aqueous

was 3 g.

CHONDROITINASE

Chondroiiinases and Chondwsulfatases Vol. 243, No. 7

methanol solution. The yield

HYALURONIDASE

0 20 LO 60 0 20 40 60

ELUATE VOLUME (ml1

FIG. 2. Gel filtration of chondroit.inase-+BC digests (left) and testicular hyaluronidase digests (right) on Sephadex G-50. Reac- tion mixtures for chondroitinase-ABC were those in Assay 1, ex- cept that the substrate was 2 mrmoles of chondroitin 3%sulfate and that mixtures 1,2, and 3 contained boiled enzyme, 0.0001 unit of enzyme, and 0.03 unit of enzyme, respectively. Incubations were conduct.ed for 10 min at 37”. Reaction mixtures for testicular hyaluronidase contained, in 50 ~1, 2 mpmoles of chondroitin 3%- sulfate; 2.5 pmoles of acetate buffer, pH 5.0; 0.1 mg of gelatin; 0.45 mg of NaCl; and .J, boiled enzyme; 5, 25 turbidity-reducing units (32) of enzyme; or 6, 250 turbidity-reducing units of enzyme. Incubations were conducted for 30 min (Mizture 5) or 10 hours (Mixlures .J and 6) at 37”. Each mixture was applied to a Sepha- dex G-50 column, 1.2 X 68 cm, equilibrated with 0.02 M Tris-HCl, pH 7.5. Elution was accomplished at room temperature with the same buffer. Fractions of 1 ml were collected and counted. The position of the ADi-6S standard is indicated by the uc1’~ows.

g 0.3- Z

2 f5

0.2 -

: a 0.b a

PH

FIG. 3. Effect of pR on rate of degradation of various muco- polysaccharides with chondroitinase-ABC. The conditions of the experiment were those in Assay 3, except for the pH of the buffers. The bufl’ers used were 2.5 pmoles of Tris-HCl (PI-I 7.2 to 8.8) and sodium acetate (pH 5.0 to 7.1) per incubation mixture. Incuba- tion was carried out for 10 min with 0.004 unit of enzyme. The substrates used were 0, ChS-A; A, ChS-B; 0, ChS-C; A, chon- droitin (squid) ; and X, hyaluronic acid.

Likewise, 3.9 g of the disaccharide product were obtained from 5 g of sodium ChS-C.

Linker et al. (3) have indicated t.hat an enzyme prepared ac- cording to the method of 1)odgson and Lloyd (8) from f’. vulgaris NCTC 4636 l~*oduces an oc,/3-unsat,urated disaccharide and a sulfated unsaturated disaccharide from chondroitin sulfate.

Suzuki (4), on the other hand, has identified the sulfated umatu-

rated disaccharides from ChS-.-I (or ChS-1%) and C’hS-C (or chondroitin sulfate from shark caltilage) as ADi-4s and ADiBS, respectively. Such formulations are supported by the following evidence: (a) an absorption maximum at 232 mp, (b) the COW sumption of 1 mole of bromine, (c) the cquimolar proportion of galactosamine to uranic acid to sulfate, (d) the isolation of acetyl- galactosamine 4-sulfate (from Al%4s) or acetylgalactosamine 6-sulfate (from ADi-6s) after mild acid hydrolysis, and (e) the formation of a nonsulfatcd unsaturated disaccharide (the analy- sis fitted the formula C14H21011?;) from both AT%4S and Alli- 6S after desulfation with a crude chondrosulfatase preparation.

Similar measurements were performed with the products oh- tained with the purified chondroitina?e-ABC to ascertain whether the compounds from ChS-11 and ChS-C would be identical with ADi-4S and ADi-6S, respectively, and led to the conclusion that the products are, in fact, ADi-4s and ADi-6% The millimolar absorption coefficients of these newly prepared products at 232 mp were 5.1 (for ADi-4s) and 5.5 (for ADi-6s) and those at 585 mp in the borate-catalyzed MorganWon reaction (23) were 0 (for ADi-4s) and 20.0 (for ADi-6S), the values used for ana- lytical purposes in this and accompanying papers (10, 11). The structure of ADi-6S has been substantiated by our fiuding that depadat,ion by I?. heparinum glucuronidase results in the quail- tit,ative hydrolysis of the compound to an cy-keto acid and acetyl- galactosamine B-sulfate (see below).

Properties of Chondroitinase-A4 BC-Chondroitinase-XTX’, rc- covered from phosphocellulo?e and free of contaminants as judged by acrylamide gel electrophoresis, formed a single activity zone at a density of 10.5% sucroc‘e when examined by centrifugation in a sucrose densit,y gradient (density range, 5 to 2OYL). This den&y was far lower than that of a catalase sample (Tokyo Kasei Company, Tokyo; mol wvt 240,000, assayed by the method of Chance and Maehly (34)) and slightly lower than t,hat of a yeast alcohol dehydrogenase sample (Boehringcr und Scehnc; mol wt 150,000, assayed by the method of Racker (35)) which were added before centrifugation. -11~0 to be noted is the fact that chondroitinase-ABC was eluted near the void volume from the col- umn of Sephadex G-150, whereas it was retarded by the column of Sellhades G-200. These behaviors are consistent with those of a protein with a molecular weight slight,ly lower than 150,000.

Fig. 3 shows the effect of l)H 011 the rates of deg-radatioll of hyaluronic acid, chondroitin, ChS-:\, ChS-15, and ChS-C by the purified preparation of choildroitillase-;\I~C. The ol)tirnal pH for chondroitin sulfates A, 13, and C was found t,o be approxi- matcly 8.0, based on exl)criments with 0.05 IV Tris-HCI buffel solutions. In contrast, t,he maximal rates of degradation of chondroitin and hyaluronic acid oc~rrtd near pH 6.2 and pH 6.8, rcspect,ively, in 0.05 M sodium acetate buffer. The differ- ence in pH optima for the sulfated and the nollsulfatcd polysac- charides may be interpreted as being due to a pH effect on the

polysaccharides (i.e. maintcnancc of the .substrate molecules in the proper ionic state for clizpic~ action).

To determine the rate and extent of reaction, ChS-=1, ChS-U, ChS-C, chondroitin from squid skin, hyaluronic acid, kcrato-

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sulfate from shark cartilage, hcparitin sulfate from human aorta, and helwin wre incubated in 0.05 M ‘I’ris-HCl, pH 8.0, with the 1)urifirtl lwparat~ioll of chondt.oitillasr-~\l~C. .I11 substrates were used at 0.1 pmole (a.s uranic acid or hesosamillc) 1~ 50 ~1, and the inrreasr ill abi;orl)lion at 232 mp was measured undw the conditions of -\.ssay 3. The results are shown in Fig. 4. The degradation of ChS-12 and ChS-C proceeded linearly over 10 min. ‘I’hc init,ial rates of degradat’ion of ChS-E, chondroitin, and hyalurollic ac4i.d were virtually 40yc/,, 20f/L, and 2 yi, respectively, of that of ChS-L1 and ChS-C under these conditions. -2fter l)rolollged incubation, the level of unsaturated disaccharides in the (‘hS-.\, C’hS-13, and C’hS-C digests, estimated with the use of the millimolar alxwytion coefficients of unsaturated disaccha- ridrs (see above), rose to 1009; of the total uranic a&l iu the poly- sac<charitles. From kcratosulfatc, heparitin sulfate, and heparin, on the other hand, wither ultra\-iolet-altsorl)iilg material nor re- ducing material (as detected by the l’ark-Johnsol method (24)) wa.s l~roduc~ed.

The rxlxriments rcl)ortctl in the accompanying l)al)er (I I) have l)rovidcd additional information regarding the specificit of cllondroilillase-Alec; i.e., as can be xen from the dala oh- tairwtl with c*hondroitin sulfa& from shark cartilage (Type 1)) and from squid csartilage (?‘yl)c E), the occurrence of estra sul- fate in the glucruollic acid moieties of a ‘I’ylw C chain has IIO sig- nificallt cflcct of either rate or extent of the reaction, but the oc- twwnce of two sulfate rwidues in the same ac~ct~lgalactosn~~ii~~e moicatich of a I)ol!-s;nccllaritIc chain tends to l)rotluce a rcdwtion in initial rate.

The ol)t imal tcqwraturc for this cwzyme n-as found to he al)l)roximatel>- 37”. Thus, the rate of degradation of ChS-C at 30” was SO’,; of 1 hat ob.wwd al 37”.

Cllondroilinases and Relaied lhz,qmesJrom F. heparimm

I,ilrkcr et nl. (3) and Hoffman et al. (36) haw reported that cstracts of /:. heparincLm (grown on a glu~oso-‘l‘r~I)ticasc- Phytolwsalt.~ medium) tlqqadc ChS-:1 and ChS-C at a relativtl3 ral)itl rate and ChS-13 at a blowcr rate, r, Gving both sulfated and tlcsulfatctl unsaturatctl tlisac&arides. -1 glwuronitlasr which degrades the nonsulfated disaccharide to monouacBcharid(: and a-kcto acid has also 1x:en found in the cstracts. These cwzyine actix-ities haw been ahowl to Ix enhancwl n-hen the organism is first adal)tcd to ChS-,\ or ChS-13. .lcwrding to our lxeliminary ,sur~~~y, (111 the other hantl, the activities with ChS-I3 and (M-C Tvel’e illc>rcased 9- and 4-fold, wq)ectively, if the organism was growl in the l~rtwnce of <‘hS-C. Furthcrmow, the cvidenw l)rc- scntctl Ix~low indicates t,hat the adaljted cell contains (o,) an enzyme similar to cholrtlroitillasc-;213C which acts well on ChS- -1, ChS-13, and C”hS-C, (b) a c~holltlroitillare (cElolldroitillas(~-.~~) which acts on ChSL\ ant1 ChS-C, and not on ChS-I%, (c) a su- fatasc which is similar to l’. vltlqaris chondro-4-sl~lfatasc (we below) nith rcslxxt to its suhstratc specificity, ant1 (d) a glucu- ronitlasc which acts ou ADi-OS and ADi-6% The wcontl choll-

tlroitinax (~hoiltlroitinasc-hC) n-as not induced xhcn the 01’. ganixm was grown in the presence of ChS-13, unlike the first chondroitinaw, xhich n-as indwed n-ith ChS-Is to the same cs- tent as with (‘hS-C.

Pl/rZ/icatioll-r:11le~s otherwiw specified, all olwat iolls wrc conducted hctwccn 0” and 4”. -111 ccntrifugations wcrc at 17,500 X g for 30 mill.

‘I’0 500 ml of the crude extract of P. heparinzim (we “lrelh- oda”) Iwre added, with stirring, 125 ml of SC/; streptomycin sul-

1.2

1 i

100%

2 hl I- 0.8 a

w g 0.6 a

0 20 LO 60

fate: in 0.02 11 Tris-IICl, 1% i.2. .\fter stantlillg for 4 holvs, the susl)eiision was wntrifugcd.

To the sulwrnatant fluid (580 ml) ~VCIY atltlctl, Gth stirring, 181 g of &tl an~monirm~ hulfate. The suslwnsion was allowed to stand for I hour after the ammol~im~z sulfate had dissolved, and then it n-as c~cwtril’ugctl. ‘1’0 the qwnataiit fluid ww added, with stirrilig, an additioiial 145 g of ammonium hulfate. The suspension was allowd to stand and was wntrifugetl as before. The rrsulting prwipitatc~ was &sol\-ed ill 30 ml of 0.02 M ‘I’ris- HCI buficr, 111-1 7.2. This solution was dialyzetl for 24 hours against five 2.liter changw of the same l)riffrr.

X w1um11 (2.5 X 30 (am) of ~~hosl)horollulo~c~ was cquilibratcd with 2 lit (w of 0.02 JI ‘I‘&H(‘1 l)df’er, 1)1-I 7.2. ‘I‘hc dialyzrd ammonium .sulfatc frad ion \~a:: al)l)lietl to th(> c~~lumn at the rate of 60 ml l)rr hour, and lhc atleorlwiit n-as washed with 400 ml of 0.02 M ‘I’&-H(‘1 buffer, 111-I 7.2. The column was tle~c~lol~cd by linear gratlicnt elulion with 400 ml of 0.02 RI ‘I’ris-HCl, l)H 7.2, in the misillg flask and 400 ml of 0.5 11 SaC’l in 111~ same buffer in the resclvoir. The flow rate was 60 ml lwr hour, and fractions of 8 ml n-err collected.

Chondroitinasc was assayetl by 11~ formation of tlisaccharidcs (&say 2) with u.be of the substrates, ChS-.\, (W-1$ and C’hH-C. JIost of the activit!- with C’hS-13 was fount1 ill ihr fracatiolls from tubes 30 to 45 (Fig. 5) as a sin& peak, n-hc~lcas tllc activities with ChS--\ and ChS-C wrc cxacll separattcl into two wmponwt~s, the earl?- peak Iwing cluted at the same region as the (X-I% peak and the late peak h&g sq)arated with some o\-erlap with t,hat region. In all fractions of the late peak, the ratio of the ChS-*-1 to ChS-C actirit ios was constant, buggesting that the degradation of ChS-*-I and ChS-C is c*atalyzetl I,!- the same enzyme.

In addition, the same ammonium sulfate fraction +ldcd two

TIME (MINI

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1530 Chondroztinases and ChondrosulJatases Vol. 243, No. 7

20 LO 60 60 100

TUBE NO.

FIG. 5. Separation of chondroitinases, chondrosulfatase, and glucuronidase of F. heparinrlm by phosphocellulose chromatogra- phy. The procedures for chromatography are described in the text. Fractions were assayed for chondroitinase activities by Assay 2 with the use of ChS-A (O---O),.ChS-B (a-----n), and ChS-C (O--O) as substrates; for chondrosulfatase activity by Assay 4 with the use of 35S-ADi-4S (A- - -A) as substrate; and for glucuronidase activity by Assay 5 with the LISC of Alli-OS (X.. . .X) as substrate. When 3B-Al)i-6S was used as the sub- strate instead of 38S-AI>i-4S, no release of inorganic “+-sulfate was observed with any of the fractions. Note the difference in the scale of the ordinate for the curves of sulfatase (units X lo3 per ml), of chondroitinase for ChS-B andglucuronidase for ADi-OS (units X 10 per ml), and of the others (llnits per ml).

peaks which appeared chromatogral)hically homogeneous and which had chondro-4-sulfatasa activity and glucuronidaye ac- t,ivity, respectively, running at slightly slower rates than the early chondroitinase peak (Fig. 5).

The late peak fractions of chondroitinase (tubes 55 to 80) were pooled and concentrated to 2 ml by pressure dialysis against 2 liters of 0.02 M Tris-HCl buffer, pH 7.2. A l-ml portion of the concentrated fraction was applied to the top of a Sephades G-200 column (1.8 x 63 cm) equilibrated with 0.1 M XaCl in 0.02 RI Tris-HCl buffer, pH 7.2. Elution with the same salt solution was carried out at the rate of 10 ml per hour with collection of 2-ml fractions. The void volume of the column was about 50 ml, and the total elution of all materials on the column required 140 ml. h single peak \vas observed (Fig. 6). The fractions (tubes 38 to 47) which were devoid of glucuronidase activity were pooled and concentrated to 2.5 ml by pressure dialysis against 2 liters of 0.02 M Tris-HCI buffer, pH 7.2. =\pparently, the choa- droit,inase-AC preparation contains no protein impurity, as seen from the electrophoresis on acrylamide gel (Fig. 1). A summary of t,he purification procedure is shown in Table II.4 The enzyme

4 In order to prepare chondroitinase-AC free from contaminat- ing glucuronidase, a thermal inactivation procedure has recently been devised in this laboratory. A portion of the combined phos- phocellulose fraction with the protein concentration of 0.G mg per ml was incubated with vigorous shaking for 1 min in a water bath at 50”. The thermal inactivation destroyed 457, of the chondroit- inase activity, but reduced the glucuronidase activity below a detectable level in the test system used. The preparation had an activity of 105 units per mg of protein.

(0.8 mg of protein per ml) was stable for at least 6 months when stored in an ice bath.

Properties of Chondroitinase-AC-In the presence of sufficiently high concentrations of cnzymc (0.003 unit/50 ~1 of standard re- action mixture), ChS-A, ChS-C, and chondroitin (chemically desulfated ChS-C and squid skin chondroitin) were converted very rapidly to unsaturated disaccharides, the level of which in the digests rose to nearly 1005<, of the total glucuronic acid in the substrates (Fig. 7). Hyaluronic acid, which is degraded at an extremely reduced rate by P. vulgaris chondroitinasc-ABC (see Fig. 4), was degraded at a considerably more rapid rate. C”hS-B, in contrast, was not attacked even by very high concentrations of enzyme. Kcratosulfatc from shark cartilage, heparitin sulfate from human aorta, and heparin were also inactive with this enzyme.

The products present at the end of &gradation in digests of squid cshondroitin, ChS-L\, and ChS-C were identified as AIL OS, AIL48, and AIX6S, respectively, by paper chromatography

30 LO 50 60 70

TUBE NO. FIG. F. Separation of chondroitinase and glucuronidase of F.

heparinum by Sephadex G-200 chromatography. The procedures for chromatography were described in the text, and those for en- zyme assay were as described in the legend to Fig. 5. The sub- strates used were O---O, ChS-A (for chondroitinase); O--O, ChS-C (for chondroitinase) ; A- - -A, 3%ADi-4S (for chondro- sulfatase); and X.. X, ADi-OS (for glucuronidase). Kate the difference in the scale of the ordinate for the curves of chondroitin- ase (units per ml), of glllcuronidase (units X lo2 per ml), and of sulfatase (units X lo3 per ml).

T.WI,E II

PlLrij(;alion of chontlroitinose-.4C from 1'. heparinurn, ATCC 13125 Activities were nlensllretl by Assay 2 with ChS-C as srtbxlrate.

step

1. Crrlde extract,. 2. Streptomycin..

3. Ammonillrn sIllfate, after dialysis

4. Phospllocclllllose colrlrnll.

5. Scphadex G200 colurn~l.

1220 944 170

0.75 5 3 130 927 43 287

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and paper electrophoresis (the procedures were the same as those described for chondroitinase-ABC; see above)

On treatment of AILOS, AlL6S, and ADi-4S (2 pmoles of each) with an excess of the purified chondroitinase-AC (1 unit), none of these disaccharides was converted to monosaccharide or desulfated disaccharide, as judged by paper chromatography in Solvent I and paper electrophoresis at pH 5. The preparation must therefore be free from glucuronidase and chondrosulfatase present in the. crude extracts.

ChS-13 was found to be a potent inhibitor of chondroitinase- AC. A detailed examination of the kinetics of inhibition by ChS-B showed it to be a competitive inhibitor of the enzyme (Fig. 8). The dissociation constant of the enzyme-inhibitor complex (Ki) calculated from Fig. 8 is 1.0 X IOh M (expressed as moles of ChS-B uranic acid). This is to be comparcd with a value of 9.4 X 10-j M, which is the R, observed fo< ChS-C (expressed as moles of ChS-C glucuronic acid).

Since ChS-B, as well as ChS-C, is a polyelectrolyte which should respond to the ionic cnvironmcnt, the level of inhibition may vary as the pH and ion concentration of the reaction mixture are varied. In fact, as shown in Fig. 9 (daslzed line), there is a difference between the pH optimum with ChS-C in the absence of ChS-B and that in the presence of ChS-I$ where the optimum pH is markedly high. Also to be noted is the iaact that the ChS-B inhibition was reduced to the 509; level by the presence of 0.05 BI KaCl in the reaction mixture.

Like ChS-B, heparin (an inhibitor of P. vulgaris chondroitinase; see Ifeferencc 5), heparitin sulfate, and keratosulfate (O.O6yi, W/V) inhibited the chondroitinase-XC reaction 6Oy& 357;, and 80yc, respcctivcly, under the conditions of Assay 2.

Several biralent cations, Mn++, Mg++, Ca++, and Isa++, activated the chondroitinasc-dC system (about 1.3. to 1.5.fold at 0.001 M), while %n++, Fe++, and Cu++ inhibited the system,

i

--I I

,lOO% DEPOLYMERIZATION

0 40 80 120 240

TIME (MINI

FIG. 7. Rate and extent of degradation of various mucopoly- saccharides with chondroitinase-AC. The conditions of the ex- periment were those in Assay 3 with 0.003 unit of enzyme and the following compounds as substrates: 0, ChS-A; l , ChS-C; A, chondroitin (squid) ; A, oversulfated chondroitin sulfate (Type D) (11) from shark cartilage; X, hyaluronic acid; and 0, ChS-B, heparin, heparitin sulfate, or keratosulfate. At the time indi- cated by the arrow, 0.05 unit of enzyme was added to each 50 ~1 of the incubation mixtures of hyaluronic acid.

0 05 1.0

‘/[ChS Cl. lo-’ M

FIG. 8. Effect of ChS-C concentration on inhihit~ion of chon- droitinase-AC by ChS-B. Incubation was carried out with 0.00055 unit of chondroitinase-AC as described for Assay 2, except that varying amounts of ChS-C were used and the indicated amounts of ChS-I3 were added. The velocity, 0, is expressed as millimicro- moles of AIli-6S formed in 10 min.

0.5

E’ 0.4 : hl

2 0.3 W

ki

2 0.2

i5

2 Q 0.1 a

L

I I / 1 I I 4 5 6 7 8 9

PH

FIG. 9. Effect of p1-I on the rate of degradation of various muco- polysaccharides with chondroitinase-AC. The conditions of the experiment were those in Assay 3, except that 2.5 pmoles of Tris- HCI (pH 7.3 to 9.1), potassium phosphate (pI1 5.8 to 7.5), and potassium acetate-acetic acid (pH 3.6 to 5.9) per incubation mix- ture were used. Incubation was carried out for 10 min with 0.003 unit of enzyme and 0, ChS-A; 0, ChS-C; or A, chondroitin as the substrate. X, curve for hyaluronic acid, obtained by the use of 0.03 unit of enzyme. - - -, pTI-activity curve for ChS-C in the presence of 50 mpmoles (as uranic acid) of ChS-B (0.03 unit of enzyme was used).

half-inhibition being obtained at 5 X lo-” 31, 10P3 RI, and lo+ 11, respectively. These results arc quite similar to those reported by Kakada and Wolfe (5) for the chondroitinai;e system of P. vulgaris.

Fluoride and phosphate, which were shown to activate the chondroitinase system of I’. vulgaris (5) and which have been shown to inhibit the ehondrosulfatase system (set below), had little effect on the ChS-C : chondroitinasc-AC system.

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1532 Chondroitinuses and Chond~osulJafases Vol. 243, So. 7

40 60 80 100 120

TUBE NO.

FIG. 10. Separation of glucuronidase and chondroitinase of F. heparinurn by hydroxylxpatite chromatography. The procedures for chromatography- were described in the text;, and those for en- zyme assay were as described in the legend to’Fig. 5. The sub- strates used were 0, ChS-C (for chondroitinase); 0, ADi-OS (for glucuronidase). The elution profile of rechrornatography of the glucuronidase fraction (tubes 75 to 95) is shown in the inset.

As also shown in Fig. 9, t,he maximal rat,es of degradation of t,he chondroitin sulfates, hyaluronic acid, and chondroitin OP curred near pH 6.6 (in 0.05 M potassium phosphate), pH 6.0 (in 0.05 31 potassium phosphate), and pH 5.0 (in 0.05 M potassium acetatc), respectively.

Cortments on Sulfatase, Chondroitinase (Bady Peak Fraction),

and Glr*curonidase-The occurrence in P’. heparinum of a glu- curonidase which hydrolyzed A4, S-unsaturated disaccharides at the glucuronidic linkage was first recognized by Linker et al.

(3). Sot much is known regarding the specificity of the glu- curonidase for its substrates, but t.he small amount of informa- tion available suggest:: that the enzyme is specific for the ml- saturated uranic acid moiety.

,1s described above, the ChS-A- and ChS-C-degrading activity was resolved into two peaks by phosl~hocellulose chromatography (Fig. 5). In addition, the phosphocellulose fractions with a peak at tube 45 had an activity degrading ADi-OS under the condi- tions of Assay 5 for glucuronidasc and those with a peak at tube 40 an activity degrading ADi-4s under the conditions of Assay 4 for chondro-4-sulfatase.

;2lthough the close association of the chondroit,inase (early peak), glucuronidase, and sulfatase was apparent when attempts were made to separate the three activities, some degree of yepara- tion of t,he glucuronidase from the chondroitirlase was obtained by chromat~ography on hydrosylapatite (Hypatite C) (Fig. 10). In this srl)aration experiment, portions (128 ml) of the phos- phocellulose fractions from tubes 30 to 45 were concentrated to 2 ml by pressure dialysis against 2 liters of 0.01 M phosphate buffer, pH 6.8, and applied to the top of a hydrosylapatite column (1.2 x 8.4 cm) equilibrated with the same buffer. A linear gradient of elution from 0.01 M to 0.5 M potassium phos- phate, p1-I 6.8, was then applied to the column. The total gra- dient volume was 120 ml. The flow rate was 5 ml per hour, and l-ml fractions wcrc collcct)ed.

As can be seen from Fig. 10, the chondroitinase activity (tubes 40 to 60) could be obtained free from the glucuronidase activit,y,

although the latter activity was still accompanied by the chon- droitinase activity. Ak glucuronidase preparation devoid of chondroitinase act,ivity could be obtained by rechromatography of the fractions from tubes 75 to 95 on a hydroxylapatite column (Fig. 10, inset). The glucuronidase preparation, however, still exhibited the sulfatase activity after concentration of the pooled fractions by pressure dialysis against 2 liters of 0.02 M Tris-HCl pH 7.2. Specific activity of the sulfatasc was 0.08 unit per mg of protein in the second hydroxylapatitc preparation.

The pH optimum of the purified glucuronidase was between pH 5.5 and pH 7.0, as measured by a decrease in the absorption of ADi-OS at 232 nip (Assay 5). The glucuronidase preparat,ion (the second hydroxylapatite fraction) had a specific activity of about 2.2 units per mg of protein and was l)ure enough to give no product other than acetylgalactosamine and or-keto acid from ADi-OS as judged by paper electrophoresis at, pH 5 followed ba staining with t,he aniline hydrogen phthalate reagent (28) for sugars and with the o-phenylenediamine reagent (37) for cr-keto acids. 11s will be discussed in the accompanying paper (ll), the enzyme also catalyzes a quant,itative hydrolysis of ADi-6S to ol-keto acid and acetylgalactosamine 6-sulfate. A1 decrease in the ultraviolet absorption was observed when the most highly puri- fied fraction was incubated with ADi-4s (the apparent rate was

about 0.59; of that of AIKOS under the conditions of :2ssay 5), but this phenomenon requilcs comment. When the reaction misturcs were examined by paper chromatography in Solvent I, acetSlgalactosailiiiie, but never acct~ylgalactosaminc 4-sulfate, was tlrtrcted on t,he chromatogram. In addition, acetylgalac- tosamine 4-sulfate, treated with the glucuronidase preparation under the same conditions, remained urlaffected. It can be assumctl, therefore, that ADi-4s itself may not be attacked b!; glucuronidayc but may be convertrd, under the influence of the sulfatase present in the preparation, to ADi-OS, which is an active substrate for glucuronidasr. h more det,ailed description of the?{, esl)eriments is presented in an accompanying leaper (I 1).

When the most highly purified glucuronidasc was examined for substrate specificity under the conditions of dssay .5 (all substrates wcrc used at final concentrations of 0.02 pmole/50 pl), it T\-as shoTTn either by a apectrophotomctric assay for pmnitro- 1)henol and phenolphthalein or by determination of reducing sugar that the following coml)ounds were not hydrolyzed : P-n-(p-llitrol)hen3-l)-glucuroaide, fl-D-(phenolphthalein) +$ucu- ronide, ai~D~phell~lglucuroIlidc, chondrosin (i.e. 2-amino-2-deosy- 3-0-(P-n-glucol)~rallos~luronic~ acid)-r-galactose), and iv-acctyl- chondrosin. It is therefore clear that the glucuronic acid unit in the substrate molecules must he unsaturated glucuronic acid.

Although the F. heparinzm sulfa&c is distinct from thr P. vulgaris sulfatases (sre belon) in its chromatographic properties, the former and one of the latter sulf’atases (chondro-4-sulfatase, see below) have a similarity with regard to the substrate specific ity. Thus, t,he F. heparinum preljaration did not rclcase %- sulfate from %Al)i-6H (&say 4), but acted on ADi-4s to give inorganic sulfate and a mixture of acetylgalactosamine and 01. keto acid (detected by paper elcctrol)horesis at l)H 5). &)par- cntly, the results can be interpreted as coming from a concerted action of an enzyme similar to chondro-4-sulfatase and the glucuronidase described above, i.e. ADi-4s may first be con vertrd to ADi-OS 1)~. the sulfatase, the ADi-OS then being hydrolyzed by the glucuronidase present in the preparation to acetylgalactosamine and cr-keto acid. Since the P. heparin.um

sulfatase was recovered in much smaller yields than the P.

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vulgaris sulfatases, further investigation of the enzyme from F. heparinum has not been carried out. For the same reason, the early peak chondroitinase (Fig. 5) has not been studied extcn- sively. It. may be noteworthy, however, that the temperature optimum of this chondroitinase was between 25” and 30”. The value is significantly lower than that of P. vulgaris chondroi- tinase-dRC (see above).

Chondrosulfatases from P. vulgaris

We have briefly reported the occurrence in P. vulgaris, XCTC 4636, of two separate enzymes capable of liberating sulfuric acid from either ADi-4S or ADi-6s (9). Available data indicated that one of the sulfatases has a high degree of specificity for the 4.sulfate ester, and the other for the B-sulfate ester. Keither sulfatase attacked ChS-A, ChS-C, sulfated hexa-, penta-, tetla-, and trisaccharides prepared by degrading ChS-il and ChS-C with a crude testicular hyaluronidase preparation, acetylgalac- tosamine 4.sulfate, or acetylgalactosamine 6-sulfate. Our sub- sequent work has shown that both activities were largely eil- hanced (35. to 380.fold) when the organism was grown on a medium containing either ChS-A or ChS-C. Purification of the enzymes from the ChS-C-adapted cells and propert,ies of the purified enzymes are described below.

PuriYcation-With 500 ml of the crude extract of P. vulgaris

as starting material, a preliminary concentration of the sulfatases as far as the stage for adsorption onto THESE-cellulose (see “Chondroitinase from P. vulgaris: l’urification”) was done. The subsequent purification step, involving the linear gradient chromatography with NaCl (9)) was modified slightly since the two enzymes were not Feparated completely from each other by t,he original technique unless the chromatography was repeated at least two times for the ADi-4s act)ivity and three times for the

ADi-6S activity. In the modified procedure, the adsorbent was eluted first with 3 liters of 0.07 M NaCl in 0.02 M Tris-HCl buffer, pH 7.2, and then with 3 liters of 0.15 M NaCl in the same buffer. The flow rate was 60 ml per hour, and 20.ml fractions were col- lected. As shown in Fig. 11, most of the activity for ADi-4s appeared in the 0.07 M NaCl part, and that for ADi-6S in the 0.15 M NaCl part. The activity for AN-4s was obtained com- pletely free from the ADi-6S activity. The fract,ions containing the major portion of the activity (tubes 95 to 170) were pooled and concentrated to 10 ml by pressure dialysis against 10 liters of 0.02 M Tris-HCl, pH 7.2 (chondro-4-sulfatase). The activity for ADi-6S, on the other hand, was still accompanied by the chondro-4-sulfatase activity. Therefore, the fractions (tubes 230 to 270) were pooled, concentrated as above, and subjected to a second chromatography on a column (2.1 x 24 cm) of I)E-1E- cellulose equilibrated with 0.02 M Tris-HCl buffer, pH 7.2. The column was washed with 650 ml of 0.07 M NaCl in the same buffer and t,heneluted with 1200 ml of 0.15 M NaCl in the same buffer. The elution profile is shown in the inset of Fig. II. The chondro- 4-sulfat,ase could be almost completely removed from t,he ADi-6S activity by this procedure. The fractions (tubes 100 to 150) were pooled and concentrated to IO ml by pressure dialysis against 10 liters of 0.02 RI Tris-HCl, pH 7.2 (chondro-6-sulfatase). .4 summary of the purification procedure is shown in Table III. Acrylamide gel electrophoresis patterns of the most highly puri- fied sulfatase fractions are shown in Fig. 1. Both preparations contain several protein impurities. The preparations (1.0 mg of protein per ml) could be stored in an ice bath for at least 2 months without significant loss of activity. Attempts at separation of

0 100 200 3?0

TUBE NO.

FIG. 11. Separation of chondrosulfatases of P. vulgaris by DEAE-cellulose chromatography. The procedures for chroma- tography were described in the text,. Activities were measured by Assay 4. The elution profile of rechromatography of the chon- dro-6-sulfatase fraction (tubes 230 to 270) is shown in the inset.

-, elution profile of chondroitinase-ABC (Assay 1).

TABLE III

Pur$calion of chondroslrlfatases from P. vulgaris, NCTC 46%

step

1. Crude extract..

2. Streptomycin. 3. Ammonium sulfate,

after dialysis.. 4. DEAE-cellulose col-

UIIlIl

Early peak.. _.

Late peak.. 5. Second DEAE-cellrt-

lose column, late peak.

Chondro-4-sulfatase i Chondro-h-sulfa&se

T Total Specific Purifi- Total

activity rctivity cation ctivitl ipecific Lctivitl

: I a

P II

-

Purifi- cation

pnoles/ min

4100 4460

1640

650 24

0

‘WkOltZS/ ng/min -fold

0.19 1 0.29 1.5

0.34 1.8

4.75 25.0 o.ozz

0

mnlcs/ mwz

1703 1660

630

0 386

220

moles/ ‘tg/min

0.08 0.11

0.13

0 0.5

1.7

-jda

1

1.4

1.6

6.3

21.3

the two enzymes by chromatography on Sephadex G-150 or on hydrosylapatite were unsuccessful.

Properties oj Chondro-4-sulfatase and Chondro-6-sulfaiase-

Incubation of ADi-4S with the purified cholldro&sulfatase re- sulted in the complete disappearance of ADi-4s and the appear- ance of a compound with the electrophoretic mobility of ADi-OS. Xtrr incubation for 30 min with 0.1 unit of the enzyme, 1 pmole

of ADi-4S was quantitatively converted to Al&OS, as measured directly in aliquots of the reaction mist,ure by the modified Morgan-Elson method (23). Inorganic sulfate, 1 pmole, was also formed in the mixture and was determined by the method of Dodgson (22). Neither Al&OS nor inorganic sulfate was formed when ADi-6s was substituted for AI%4S, indicating the absence of chondro-6-sulfatase in the enzyme preparation.

Wit’h the purified chondro&sulfatase, on the other hand, ADi-6s was converted quantitatively to ADi-OS and inorganic sulfate, whereas ADi-4s was not affect,ed at all. In these ex- periments,, the disappearance of ADi-6s and the formation of ADi-OS were directly determined in the reaction mixtures b3

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1534 Chondroitinases and Chond~osulfatases Vol. 243, No. 7

taking advantage of the fact that the millimolar absorption co- efficient at 585 rnp of ADi-6S in the modified Morgan-Elson re- action is significantly higher than that of AILOS (20.0 for ADi- 6s and 11.0 for ADi-OS).

Also, we have reported previously that a mixture of the chon- drosulfatases (Le. a crude sulfatase fraction which was freed from chondroit,inase by chromatography on phosphocellulose) does not attack Y-labeled ChS-?I and ChS-C, hexa-, penta-, tetra-, and trisaccharides prepared from %labeled ChS-A and ChS-C, acetglgalactosamine 4-35S-sulfate, or acetylgalactosamine 6.%- sulfate, but can hydrolyze acetylchondrosin 4-Wsulfate and acetylchondrosin 6-%-sulfate (i.e. saturated disaccharides) to give inorganic sulfate (9). In these tests, the reactions were followed by scanning the electrophoretograms of the reaction mixtures for 3% activity and making t,heir autoradiograms. At this point in the investigation, t,herefore, it was obvious that both chondro-4-sulfatase and chondro-6-sulfatase exhibit high selectivit,y for chain length of substrate molecules.

The list of active and inactive compounds has been extended by the present investigation. Thus, comparing the saturated disac- charides and the unsaturated disaccharides with respect to their activities as substrates for the purified chondrosulfatascs, it has been observed that chondro4-sulfat,ase hydrolyzed bot,h satu- rated and unsaturated disaccharide 4-sulfbtes at about the same speed, while it did not act on the saturated disaccharide 6-sulfate. With regard to the specificity of chondro-6-sulfatase, on the other hand, it has been shown that the saturated disaccharide 6-sulfat,e is an excellent substrate, whereas it,s 4-sulfate isomer is com- pletely inert.

The availabilit,y of acetylgalact,osamine 4,6-disulfate (38) has she\\-n that the disaccharide sbructure of the substrate molecules is not a critical factor; i.e. treatment of 0.3 pmole of acetyl galactosamine 4,6-disulfate with 0.16 unit of chondro-6-sulfa- tase for 1 hour converted the compound in a yield over 90% to a compound chromatographically and electrophoretically identical Fith acetylgalactosamine 4-sulfate. Under the same condition, the monosaccharide disulfate was not attacked by chondro-4- sulfatase at a11.5

Further information regarding the specificity of the chondro- sulfatases was given by the experiments reported in the accom- panying paper (11). In these experiments, three different types of unsat,urated disulfated disaccharides were prepared from ChS-B of bovine lung, a chondroitin sulfate from shark cartilage, and a chondroitin sulfate from squid cartilage, and the suscepti- bility of the sulfate bonds to the chondrosulfatases was deter- mined. Thus, one of the two sulfate bonds of the bovine and shark disaccharides aras shown to be completely resistant to the chondrosulfatases, a clue which led to the conclusion that the sulfate is linked to the glucuronic acid moiety in both com- pounds (II). In contrast, the two sulfate groups located at positions 4 and 6 of the acetylgalactosamine moiety of the squid disaccharide can be removed by chondro-4-sulfatase and chondro- 6-sulfatase, respectively.

Maximum chondro&sulfatase and chondro-6-sulfatase ac-

5 It is of interest in this connection to note that a sulfatase which is capable of catalyzing the quantitative conversion of acetyl- galactosamine 4,6-disulfate to acetylgalactosamine 6-sulfate has been found in commercial preparations of testicular hyaluronidase (T. Yamagata, T. Harada, and S. Suzuki, unpublished observa- tion).

tivities were obtained at pH 7.5 when measured from pH 7.2 through pH 8.5 by the use of Tris-HCl buffers in the presence of 0.06 M sodium acetate. The act,ivity of chondro-6-sulfatase at the optimal pH was considerably enhanced (about 3-fold) by the presence of acetate ion, while that of chondro+sulfatase was not. Citrate, sulfate, and chloride ion did not affect either activity under the same conditions. The sulfatase activities were also unaffected, unlike chondroitinase-4C, by the presence of heparin, heparit’in sulfate, keratosulfate, hyaluronic acid, chondroitin, or chondroitin sulfates A, B, and C in concentrations of 0.01 to 0.07 qo (w/v)

The emphasis in the foregoing discussion has been on the puri- fication and properties of the enzymes which catalyze the degra- dation of the various chondroitin sulfate constituents. The demonstration of the presence of chondroitinase-ABC, chondro- itinase-AC, chondro-4-sulfatase, chondro-6-sulfatase, and un- saturated disaccharide glucuronidase provides an enzymat,ic mechanism for the degradation of chondroitin sulfates by P.

vulyaris and F. hepurinum to a mixture of monosaccharides, keto acid, and inorganic sulfate. Another obvious feature of these enzymes that has not been alluded t,o is the use to which they may be put as reagents in studying t,he structure of chondroit,in sulfates as well as the composition of a given mixture of isomeric chondroitin sulfates. In the following two papers, several ex- amples will be presented to prove that the enzymes are, in fact, excellent reagents for these purposes.

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Tatsuya Yamagata, Hidehiko Saito, Osami Habuchi and Sakaru SuzukiChondrosulfatases

Purification and Properties of Bacterial Chondroitinases and

1968, 243:1523-1535.J. Biol. Chem. 

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