Biochemistry and biology of mucopolysaccharides

Biochemistry

and Biology

of

Mucopolysaccharides* KARL

MEYER,

M.D., PH.D.

AJeze, York, New York Jfucopolysaccharides are glycosamino-glycans, i.e., heteropolysaccharides composed of hexosamines and non-nitrogenous sugars linked by glycosidic bonds; some also contain various substituent groups. The mucopolysaccharides of mammalian tissues may be classified as (1) polycarboxylates (hyaluronic acid, chondroitin), (2) polysulfates (keratan sulfates), and (3) polycarboxy-sulfates (chondroitin 4- and 6-sulfates, previously designated chondroitin sulfate A and C, respectively; dermatan sulfates; and heparitin sulfates). The structure of these various mucopolysaccharides and the nature of their protein linkages is discussed. The functional role of the various mucopolysaccharides in connective tissues is still largely presumptive. They occur in different proportions in different tissues, and the pattern of distribution in the same tissue changes with maturation and aging. The importance of disturbances in the natural distribution of mucouolvsaccharides is indicated bv the clinical abnormalities characterizing the various mucopolysaccharidoses. I

d

T

HE STUDY of the connective tissues started with descriptive anatomic details, then went on to ever refined histologic data and to increasingly complex descriptive chemical investigations involving the dynamic and functional aspe’cts of the cells and tissues. Thus evolved the concept of connective tissue as an composed organ, the largest in the mammal, of a variety of cells, of fibrous elements and of intercellular and interfibrillar ground substances. The latter were visualized microscopically by typical staining procedures, the basis of which was the anionic character of the
All mucopolysaccharides of connective tissue are polyanions, the negatively charged groups being either carboxy groups, sulfate half ester groups or a combination of both. Accordingly we divide mucopolysaccharides into polycarboxylates, polysulfates and polycarboxysulfates [I]. The mucopolysaccharides of mammalian tissues are listed in Table I. In the tissues, mucopolysaccharides occur bound to protein in stable linkage, with the probable exception of hyaluronic acid. From these tissues the mucopolysaccharides, especially of cartilage, are extracted by salt solutions as protein complexes or, after proteolytic digestion, covalently linked to small peptides. The oldest method, extraction with alkali, is now rarely used since alkali leads to significant degradation. The ease of extraction and subsequent fractionation varies considerably with the types of polysaccharides present and with the tissues investigated. Chemical studies of mucopolysaccharides of different tissues as a rule start with proteolytic digestion of the tissues, followed by fractionation of the polysaccharides by a variety of procedures. In the

* From the Department of Chemistry, Belfer Graduate School of Science, Yeshiva University, New York, New York. The studies have been supported by grants from the National Institute of Arthritis and Metabolic Diseases and the National Institute of Child Health and Human Development. 664

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Biochemistry

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of Mucopc)l~sac~haricles

ttcxxt step tftt, plzrity of the isolated components 11~s to 1~ tejtetl by chemical, physical and en,\m;ttic methods. The detailed chemical struct lit-e of tl~c components has been ascertained 1)~ chemical antl enzymatic degradation, meth;t11(1 prriodate oxidation, and other ylation p~-o~e(!ure\ t15ecl in r-arl)ohydrate and protein ( lienii~l r) FOV xtudics of the protein complexes, the tissues are extracted under the mildest possible colltlitions with aqueous solutions. These solutionj ;I,(: either fractionated by absorption on ion cxtllange rclsins followed by gradient elution or 1~ tlensity gradient centrifugation, a nlcthoc! tlevc~lopetl in nucleic acid chemistry. I II? extraction of a protein-complex of dernl;lt;rll sulfate was not possible until recently. ~r-om cerlaill tissues such as skin, tendon 01 ht:;li-t valves, the complex could be brought i1110 solution only wit11 concentrated urea at rlc~ttc~l temperatures [?I. Whether this refractoriness oi the dermatan sulfate-protein is ~,~mctl by strong physical bonds or by covalent (.ll(~ni( al linkages presumably to collagen is not known. 111 stutliva on the types of mucopolysaccIl:uitlcs ~x~se~l~ in different tissues and in their cIlenlic;~I and pll)sical properties, the proteasestill tligestctl l~ol~s;rccharitles are isolated, linkrtl co\2lently with a pepticle remnant of III(~ original protein-polysaccharides, since tile eyIr;i( 1ion 2nd ( Ilarxteri7ation of the formei is 1nor.v simple aI1cI more quantitative. In fact, n~ost of out’ present kno\%.ledge of structure, distriblttiotl, biosynthesis and degradation of tllcsc. ~on~pou~~tls has bc,en obtained with pepti(lo-l)ol~Si~c~liarides. l‘he only exception is hy;~ltlronate, which can be obtained from most tissues anti fiuicls in high yield without proteolysi\ .11td in wllich the existence of a stable Iink;rgcl to pl.otcsin is at present still question;I I)le.

‘Tile majority of the mucopolysaccharides of ~)nnc( live tissues are unbranched polymers colnposetl of disaccharide units typical for the \pt=cirs. The disaccharide units of hyaluronic alrtl chondl-oitin 4- and &ulfate, dermatan sulfate and heparitin sulfate consist of a hexuronicle linked to a hexosamine which, with the partial exception of heparitin sulfate, is N-acet)-latetl. The disaccharide repeating units arc linked via the hexosaminidic bond to the

---;llqw

(IO.5

* Originally chondroitin 4- and h-sulfates werr called chondroitin sulfate :\ and C, resprctivcly. Keratan sulfate arc synonymous with kerato-sulfate ; dvrm;ltall sulfatr with chondroitin-snlfatr B; and hrparititr sulfate with hvparall sulfatr.

hexuronidic group of the next follorving disaccharide so that the carbohydrate chains consist of alternating uronidic and hexosaminiclic moieties [l’]. In keratan sulfate, which contains no uranic acid, galactosyl groups are linked to N-acetylglucosamine. ,4s a rule the hexosaminyl groups on the sulfated mucopolysaccharides are esterified with sulfate either in the 4 position or in the 6 position. In heparitin sulfate the glucosaminyl groups are in part Nacetylated, in part X-sulfated, and in rhis as well as in other properties show their chemical relatiorl~liip to heparin. The repeating trnitz of the disaccharides of the polysaccharides ‘Ire structures ~1101\.11 in representctl 1)) the 1:igul.e I. The similarity of the carbon skeleton of h)aluronate, cliondroitin 4- and 6-jilltare 2nd of tlermatan sulfate is quite obvious. In the first four of these the uronidic linkage is p l-+3, the hexosamindic p l-+-l. ?‘he ltexosamine of hyaluronate is D-glucosamine; in chontlroitin -I- and 6-sulfate and in dermatan bulfates it is n-galactosamine. In dermat;rn sull-ate:, the [Ironidic moiety is L-iduronic- ;I
Biochemistry

666

and Biology

of Mucopolysaccharides-Meyer

Ch 6-S

1

n

n

Hep - S

‘# R

0 R’= &CH3 R&=H

KS

or

or

SOi

SOT

Ch-S-

B

b0 R=H

or

SO:

I

C”3

n FIG. 1.

heparitin sulfate is not firmly established. From the available evidence both the glucosaminidic and ‘the glucuronidic bonds are a! 1+4. In the main fractions of heparitin sulfate half of the glucosaminine is N-sulfated, half is N-acetylated. The O-sulfate ester groups may be located either in the 6 position of the

N-acetylated disaccharides or in the 6 position of the N-sulfated units [8]. The repeating units of the disaccharide sulfates are simplified structures which do not represent completely the true structure of the macromolecules. The linkage region of the polysaccharide chains of chondroitin 4- and

Biochemistry

and

Biology

of Mucopolysaccharides---rlfeypr

tjhulfate ;srid of dermatan sulfate starts with 3 xylosyl~~ligalactosyl-glucuronosyl sequence IV!. Tile xylosyl - mucin and blood group substances, i< N-acetylgalactosamine. This was demon\OL.

37,

NOVEMBER

1969

(56’7

stratecl 1,). tfie disappearance of X-acetylgalactosamine exclusively on alkaline elimination followed by acid hydrolysis, by the parallel formation of a typical chromogen in the alkaline elimination and the formation of a t.ypical reduction product on treatment wit11 alkali and borohydricle [I?]. Further evidence of the similarity of both types of keratosulfates to glycoproteins is that both keratosulfates contain mannose, which had been believetl to be typically 3 constituent of glycoproteiris. In fact, from ihe quantitative estimation of a mannose derivative by gas chromatography thr: average molecular weight of the keratosulfate chains is 10,800, assuming 1 mole of mannose per chain; this is in good agreement with the average molecular weights recorded in I he :iiterature [PI. In histologic technics the method of catalytic desulfation in anhydrous methanol catalyzed by hydrogen ion has been applied to isolated sulfated polysaccharides [U]. This procedure, however, leads not only to removal of the sulfate ester Lgroups but also ro methanolysis of glycosidic bonds. For example, when applied to keratosulfate concomitant with the removal of the sulfate ester groups all the sialic acid and fucose, and about 20 per cent of the galactose, is split off and becomes dialysable. From this and otller expcriments we have concluded that KS1 and to a greater degree KS11 have galactosidic groups which branch off the main chain ol the Nacetyllactosamine repeating units [7]. Identification of the different types of mucopolysacrharides, and in large part elucidation of their structure, was immensely facilitated and indeed partly based on the finding and mode of action of enzymes. foremost among them the various hyaluronidases and other enzymes mainly of microbial origin. The classes of hyaluronidases, their substrates and their mode of action are outlined in Table II [14]. A type similar to testicular hyaluronidase has also been demonstrated in a number of snake venoms. Lysosomal hyaluronidase has been obtained from liver [18], from fibroblasts of healing wounds [IS] and bone [20]. but has not been found in cartilage. The rate of deGyradation by the testicular type of hyaluromdases is greatly decreased by sulfate ester groups. Tll1.1~ the degradation of hyaluronate

Biochemistry

668

and

Biology

HYALURONIDASES,

Substratcs

Type Testicular (lysosomal) Pneumococcal, staphylococcal, streptococcal Proteus vulgaris, flavobacterium Leech

THEIR

of Mucopolysaccharides-Meyer

SUBSTRATES

Mode

Hyaluronic acid, chondroitin, Ch 4-S, Ch 6-S, dermatan sulfate* Hyaluronic acid, chondroitin Hyaluronic acid, Ch 4-S, Ch G-S, dermatan sulfate Hyaluronic acid only

AND

of Action

Endo-fl-hexosaminidase, transglycosylation Endo-P-hexosaminidase by elimination reaction Endo+hexosaminidase by elimination reaction Endo-P-glucuronidasc

MODE

OF ACTION

Main

Products

01.rgosaccharides,

tetrasaccharide (major), disaccharide (minor) > Unsaturated? disaccharides

Remarks End products after exhaustive digestion

Unsaturated disaccharides,1 either desulfated or sulfated Mainly tetra-, with glucuranic acid at reducing end

* Dermatan sulfate split only at few sites containing n-glucuronosyl groups; iduronosyl sites arc resistant [ 7.51. t p-elimination by migration of H from C5 of the glucuronosyl group to the O-atom of the hexosaminidic bond, thus leading to splitting of the bond with formation of A4-5 unsaturated glucuronosyl l-t3 N-acetylhexosamine. $ Yamagata et al. [ 761 have purified the bacterial extracts and separated specific 4- and 6-sulfatases. They also described other methods by which Ch 4-S could be distinguished from dermatan sulfate. The unsaturated disaccharides formed from Ch 4-S and dermatan sulfate are identical however, due to the conversion of C5 of the uronosyl group to the same symmetrical -C=Cgrouping [ 771.

and chondroitin is about fifteen times faster than that of chondroitin 4- and 6sulfates under similar conditions [14]. Transglycosylation by testicular hyaluronidase has been elegantly demonstrated with mixtures of hyaluronate and chondroitin sulfate [Z]. The hydrolysis products yielded oligosaccharides corresponding in their composition to their respective macromolecules, and in addition hybrid oligosaccharides containing, in the same molecule, repeating units of hyaluronic acid and of chondroitin sulfate. It would not be surprising if such hybrids occurred in the urine. In addition to possessing hyaluronidases among them a flavosome microorganisms, have been induced to degrade bacterium, heparitin sulfate and heparin specifically by growing the organism on either of these substrates [ZZ]. Apparently the primary enzyme in this case is also an eliminase leading to unsaturated disaccharides [23]. (Recently, however, degradation by extracts of the same organism have been reported which yield normal oligosaccharides [24].) From a coccobacillus we have obtained a purified enzyme which appears to degrade keratosulfates specifically by splitting endo-p-galactosidic bonds. HETEROGENEITY

AND

VARIATIONS

OF

MUCOPOLYSACCHARIDES

Mucopolysaccharides,

as isolated

from mam-

malian tissues, show a degree of heterogeneity and variation far greater than that found in proteins. The same presumably applies to the carbohydrate chains of some glycoproteins although in glycoproteins heterogeneity is even more difficult to determine than in the mucopolysaccharides. The heterogeneity presumably is explained by the circumstan’ce that, unlike proteins, there is no direct transcription from a code, thus permitting a greater degree of variation. In some instances it is impossible to decide whether the observed differences in exosubstituents such as sulfate ester groups, sialic acid or methylpentose are due to “errors” in biosynthesis or are caused secondarily by partial enzymatic hydrolysis in the extracellular spaces. No variations have been detected in hyaluronate thus far. Hyaluronate fractions, even after the mildest methods of extraction and purification, have varying degrees of polydispersity, i.e., they vary greatly in molecular weight. Whether or not the traces of galactosamine found in many samples of hyaluronate of different origin substitute for glucosamine in the same chain, or are due to admixture with other mucopolysaccharides, cannot be decided. Chondroitin 4- and 6-sulfates occur together in most tissues, in varying proportions. It is not known at present (and it would be imAMERICAN

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irl tact to establish) whether both I\ pes of sulfate ester groups in cartilage can chain and be 01 ( 111 ill one polysaccharide litlkvtl to OIW protein backbone. Variations ill the tlegree of sulfation occur, however, in 111~chontlroitin sulfates as well as in dermatan srllfa~es. ‘l’llese fraction> and also the kcrato\~rlf;ttc~. ma! contain a sulfate to hexosamine f-;tlio < 1, 1 or 3 1. Cllondroitin sulfates of a low degrrc of sulfation have been isolated tiont rapidly growing cartilage, such as epiphyseal plate or chondrocytes in tissue culture. ()\~erslilfat ion occurs physiologically in all Iliucol)olys;lccllaricles in a variety of fishes [25], and in tile dermatan sulfates of mammals [2(7]. 111all tliese instances the extra sulfate appears to bc attached to the L’- or S-position of the uronos!l moieties. Even in the highly oversulf;ttetI cIlondroitin 6-sulfate of the shark, bowe\vr, tl~e esibtence of sulfate gaps in multiples of tlis;tccharide units has been demonstrated itI frac,tions isolated after digestion wit11 hysluronitlascs (which does not contain a sulf;~tasc). In both KS1 and especially KSII. sulf;lte c5ters have been isolated (after acid hytlrolysih) esterified in the 6-position of both possible

giucosamine and of galactose. Other variations i11 KS11 relate to the degree of substituents v itI1 sialic acid and with fucose [II]. KS11 of rpipll\~eal cartilage has been obtained with o\el- 7 l)er cent sialic acid, in KS11 of Alarfan’s s\9itlromc bvitll o\er 5 per cent. 1’11~ I1yl)ritl nature of dermatan sulfate was rcaportcd from our laboratory. Fransson ant1 K0tl~11 [I ij II;IIT confirrnecl and elaborated on tiiis ftlitliyg. 411 dermatan sulfate fractions (oiitain l)oth I,-itluroiiic acid and n-glucuronic :I( id nmieties. X‘lic ratio of I.-iduronit to n-glu(c)l-onic acid of the major fraction of tlernlatan \t~lfa~c of bull llide or pig skin is approxiin;rtcl\ I: I. ,\Iinor fractions of various ratios lvitll ;I maximllnl of 1: 1 have been obt;rinetl /?;I. .\ niinw portion of tile glucuronic acid is Iocxle(l ilr tile linkage region [15]. The clistCI)iltioil 01’ rlie major portion of the gluciiroiios\l ~ro~11x is unknown. Apparently I.illuronic acitl illso is present in heparin and Irep;tritin sulfates 1281. The hybrid nature of l~c~p;nitin sulfate is also evident from its \xyilig content of N-acetylglucosamine and iY-sulI;lLetl glucosamine. From the results of ervyrrlaric tlegradations and mild acid hydrolysis [?9,30l and reaction with nitrous acid [28] it i\ obvious that (1) the ‘two substituents occui‘

00”

in one molecule and (2) the two moieties oc( ur in bunched sequences. 1Ve have interpreted these data to imply a branched c~arboliytlrate in which the inner core is N-sulfated and tile outer branches are I\‘-acetylated. The lligh tlegree of licterogeneity and Variation of t Iif: heparitin sulfates suggests that heparirin
clioti~liuititi sulfate to one protein b;ickl~~rn~ lvac ol)t;iine~l I)): Sciio ct xl. [IT]. The) (leixionstrale(l, lvitli isolatctl ~~e~~titlo-~~ol~c;1c~c~li;~~~ide tliat tlic l~ol~s;i~~~li;~i~itlesmigrated in ai1 elecrric field as ;I single c.oniponent althougli 2ftt.i ;ilkalilic elimination they separatetl into 11~0 l,ol)‘s;l~cllal.itles Tvhich on isolation %;I\c KSTI and c~hontlroi~ill fi-sulfate. From tllc simultaneotls increase in thontlroitin ti-sulfate and KS11 in car1 ilagc of tlifferent sources, lvc (‘onclutle thaL the protein backbone collLainir,g both pol~sacc~liarides is distinct from tliaL Iiaving cliontli-oitin .*-sulfate side chains. FlIrthermore we lla\,e postulated that KS11 ;tl~d peptides or proccin are linked to KS11 by a second alkali stable bond, i.e., that KS11 crosslinks two sites of the same protein or crosslitlk5 two sparate protein chains.

670

Biochemistry

and Biology

of Mucopolysaccharides-Meyer

BIOLOGY

Mucopolysaccharides occur in different connective tissues in defined typical patterns [31]. These patterns change with maturation and aging. Production of hyaluronic acid appears to be typical for the young fibroblast in vivo and in tissue culture. In embryonic pig skin the content of hyaluronic acid is 78 per cent, of dermatan sulfate between 5 and 12 per cent. In adult pig skin the hyaluronic acid content is 30 per cent whereas dermatan sulfate is -60 per cent. About 20 per cent of the mucopolysaccharides of embryonic skin was found to be chondroitin 6-sulfate whereas in adult skin the total of chondroitin 4- and 6-sulfate is only a few per cent [32]. The content of hyaluronic acid probably decreases in all connective tissues in the course of maturation and aging, thus contributing to the loss in water content and turgor of the tissues. The changes in the mucopolysaccharide pattern of cartilage with aging are among the most extensively studied. These changes vary quantitatively in cartilage in different locations but appear to be qualitatively similar. (1) The total mucopolysaccharide content decreases. (2) From birth to old age the content of chondroitin 4-sulfate decreases whereas chondroitin 6-sulfate increases. (3) Concomitant with the increase in chondroitin 6-sulfate there is an increase of KS11 [33]. It should be mentioned that, according to Mathews, embryonal cartilage contains mainly chondroitin 6-sulfate, which is replaced up to birth by chondroitin 4-sulfate. The changes with aging that occur in the mucopolysaccharide pattern in vascular tissues are controversial and complicated by the time-dependent increase in atheromatosis and atherosclerosis. Hyaluronic acid, chondroitin 4- and 6-sulfate, dermatan sulfate and heparitin sulfate have been isolated from human, bovine and pig aortas. The simultaneous occurrence of dermatan sulfate and heparitin sulfate as major constituents presumably is typical for all vascular tissues. In aging human aorta, according to our investigation, hyaluronic acid and chondroitin 4- and 6-sulfate decrease, dermatan sulfate increases slightly and heparitin sulfate increases considerably [34]. The increase in heparitin sulfate from the fourth decade on may depend on the degree of atherosclerosis, and specifically on the proliferation of the intima. A similar correlation

may exist between the quantity of heparitin sulfate and intimal hypertrophy in Hurler’s syndrome [35]. Information on the biologic function of mucopolysaccharides and their protein complexes is scanty. Hyaluronic acid in the vitreous humor presumably maintains the turgor of this organ and serves as a shock absorber. In the articular spaces it presumably serves a similar function; its function as a lubricant in rapid movement has become doubtful in recent years. However, in articular spaces, in elastic tissues and in tendon sheaths it probably facilitates the gliding of surfaces due to its high viscoelasticity. (The physical properties of hyaluronate solutions and their biologic consequences are discussed extensively in the Proceedings of the Seminar in Biophysics and Physical Chemistry of Connective Tissue (Fed. Proc., 2.5, May-June 1966).) The protein complexes of chondroitin 4- and 6-sulfates and of dermatan sulfate presumably function in the extracellular assembly and architecture of the fibrous proteins, foremost of collagen. From the absence of dermatan sulfate in cornea, cartilage and bone, and its association with coarse-bundled collagen in skin and other tissues, we concluded that dermatan sulfate was somehow related to the formation of coarse collagen. It was pointed out that chondroitin 4- was associated with mediumsized and chondroitin 6-sulfate with the finest collagen fibers. We postulated that the geometry and intergroup distances of the anionic sites in the chains were the determinants for the fibril arrangement extracellularly [36]. No experimental evidence is available to test this hypothesis. It is unfortunate that no conclusive data are available as to the submicroscopic anatomy of the protein-polysaccharides relative to the fiber structures. Electron microscopic resolution of the anionic groups stained with ruthenium red or bismuth salts is too low to permit interpretation of the pictures. The resolution presently available will not permit distinction between different polyanions. The reversible drooping ear experiment of L. Thomas [38], after papain injection, is visible proof of the contribution of the proteinpolysaccharides to the structural properties of cartilage, but the mechanism of the interaction is not known. Total absence of any of the polysaccharides AMERICAN

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in connective tissues has not been observed. A LOWlevel of r,honclroitin 4-sulfate in the limbs 01 txtnomelic chick embryos has been reported bl, Mathews /_?$I and it &gilt be expected that minor quantitaLi~~e or even qualitative deletions 01 some OF the biosynthetic enzyme systenls M.ill I)e detected in lethal traits of eml)i,)os ;It eariy stages. No infol-mation is available regarding the function, biosynthesis and even the exact histologic sites of the heparitin sulfates in the tissues. Their chemical similarity to heparin h;lh led to the conclusion by some workers that it is either a precursor or degradation product 01 heparin. In our opinion the differences in tl~e clistribntion of the J)olysaccharides in various organs, the absence of blood coagulation ahnormalities in the mucopolysaccharitloses speak against this biologic relationship of the two ~nuc.oJ’olysarcharides.

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:!.

.?.

!.

i.

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!,?l

of Mucopolysa~charic~es~~-.tl~~:yrr

und Biologie der Polysaccharidsulfate irn Bindegewebe. In: Struktur und Stoffwechsel des Bindgewebes, p. 12. Edited by Hauss, 1960. Georg M’. I-T. and I,ossc, H., Stuttgart Thieme Vcrlag. TOOLE, B. P. and LOIVFTHER, D. A. Dermatan sulfate-protein: isolation from and interaction with collagen. ilrch. BiocAern., 128: 567, 1968. HOFFMAN, P. and MEYER, K. Structural studies of mucopolysaccharides of connective tissues. Fed. Proc., 21: 1064, 1962. HOFFMAN, P., LINKER, A. and MEYER, K. The acid mucopolysaccharides of connective tissue. III. The sulfate linkage. niochim. et biophys. ~/a, 30: 184, 1!)58. 131~~0. S., HOFFMAN, P. and MEYER, K. The structrlre of keratosulfate of bovine cornea. J. Organ. C~UWZ.,26: 5064, 1961. BHAVANANDAN, V. P. and MEYER, K. Studies on keratosulfates. Methylation and partial acid hydrolysis of bovine cornea1 keratosulfate. J. Bio[. Chew., 2Q: 4352, 1967. BHAVANANDAN, V. P. and MEYER, K. Studies on kraratosulfates, methylation, desulfation, and acid hydrolysis studies on old human rib cartilage. I Biol. Chew., 243: 1052, 1968. CIFONELLI, J. A. Reaction of heparitin sulfate with nitrous acid. Carbohydrate Res., 8: 233, 1968. RoI)~:N, L. Linkage of acid mucopolysaccharides to protein. In: Biochemistry of Glycoproteins and Related Substances, p. 185. Edited by Rossi, E. and St&l, E., Basel/Ncw York 1968. S. Karger. ANDERSON, B., SENO, h’., SAMPSON, P., RILEY, J. G., HOFFMAN, P. and MEYER, K. Threonine and scrine linkages in mucopolysaccharides and glycoprotl,ins. I. Viol. Chem., 239: 2761, 1964. &NO, I\‘., MEYER, K., ANDERSON, B. and HOFFMAN,

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1’. \‘atiations in keratosulfatcc. .I. Bioi. Chem., 240: 1005, 1965. AXDUW~N BRAY, B., LIEBERMAN, R. and MEYER. I(. Structure of human skeletal keratosulfate. The linkage region. I. Biol. Chern., 242: 33i3, 1967. KAWOR, T. A. and SCHUBERT, M. A method for t(lc dcsulfation of chondroitin sulfate. 1. :l?n C~WU. SOL., 79: 152, 19,X. MEYER, K., HOFF&IAN, P. and LIIVKER, A. Hvaluroni. dases. In: The Enzymes, vol. 4, p. 447. Xew York. 1960. Acatlemic Press, Inc. FRANSSON, I.. A. and ROD&, I,. Structure 01 tlrrma. tan sulfate. 11. Characterization of products oh tained bv hyaluronidase digestion of tlc,rmatnll sulfate. 1. Biof. Chem., 242: 4170, 1967. YAMACT.\, T., SAITO, H., HABUCRI, 0. and Suzur;r. S. Purification and properties of bacterial thotldroitinnses and chontlrosulfatases. J. Biol. Che~l.. 243: 1523. 11168. HOFFMAN. P.. LIUKER, A., I.IPPMAN, V. and MEYER, K. Tllc, structure of chondroitin sulfate 1% front studies Tvith Llavobacterium enzvmrs. I. Biol Cllt-Ill. , 2%: 3066, 1960. ARONSON, N. A. and DAVIDSON, E. A. L~sosomal hyaluronidase from rat liver. I. Biol. Chem., 242. 437, 1967 GROSS, J. Personal communication. \‘AES, G. 1IX;aluronidasc activity in lysoson~c~~ of bnnt. tissue. ninche?,r j., 103: 802, 1967. HOFFMAN, P., MOYER, K. and LINKER, A. Transply. cosylation during the mixed digestion ot hyalllronic acid and chondroitin sulfate by tcsticul:li h~?luronitlase. J. Biol. Chem., 219: 653, 19.56. HOFF~WN, I’., LINKER, A., SAMPSON, P., MEYFR, 1; and ICORN, E. D. The degt-adation of hyaluron!t(,. the chondroitin sulfates and heparin by hacterizl I enqmcs (flavobacterinm). Biochim. et hiophyt. itctn, 25: f>58. 19.57. LINKER. A. and HOWNCH, P. The enzymatic tlegra~l;l. tion of heparin and heparitin sulfate. I. ‘f’hc fractionation of a crude heparinase from flavobacteria. J. Biol. Chem., 240: 3724, 1965. DIF.TRICH, 1.. P. Novel heparin degradation product\ l\olation and characterization of novel disaccharidcs and oligosaccharides produced frorrl heparin by bacterial degradation. Biochem. J , 108: 647, 1968. MA ~HEIVS, M. B. Macromolecular evolution of con. nectivc tissue. Biol. Rev., 42: 499, 1967. SUTLIKI, S., 3~1~0, H., YAMAC.ATA,T., ANNO, K., SENO, h’. and KAWAI, Y. Formation of three types of disulfate(1 disaccharides from chondroitin sulfates I)y chondroitinase, I. Biol. Chcm., 243: 1548, 1968. HOFFMAN, I’., L,INKER, A. and MEYER, K. The acitl nlucopolysaccharides of connective tissues. II. Further experiments of chondroitin sulfate IX. Arch.

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of heparitin sulfate wit}1 28. CIFONELI.I, J. A. Reaction nitrous acid. Carbohydr. Res., 8: 233, 1968. 29. LINKER, A.. HOFFMAN, P., SAMPSON, P. and MEYER, K. Heparitin sulfate. Biochim. et biophys. nctn, 29: 443, 1958.

30. LINKER, A. and

SAMPSON, P. The

enzymic

degrada-

Biochemistry

672 tion

of heparitin

sulfate.

and Biology

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et

of Mucopolysaccharides-Meyer

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