Paper disk assay for glycosaminoglycan sulfotransferases

ANALYTlCALBIOCHEMISTRY166,404-412(1987)

Paper Disk Assay for Glycosaminoglycan KAZLJYLJKI

SUGAHARA,

TAKAKO

Sulfotransferases’

ISHII. AND IKCIO YAMASHINA'

Received April 7. 1987 A method is described for the assay ofsulfotransferascs. which transfer sulfate from .l’-phosphoadenosine-5’-phosphosulfate (PAPS) to glycosaminoglycan acceptors. Following the sulfation reactions, the [3SS]sulfate-labeled products arc precipitated and then separated from a sulfate donor ([%]PAPS) and its degradation products by a paper disk method, and then the radioactivity remaining on the paper disk is subsequently determined by liquid scintillation counting. The rapidity and simplicity of the method arc advantageous for multiple assays and have allowed us to establish assay conditions for serum sulfotransferascs which introduce sulfate at position 6 of the internal hracetylgalactosamine units of chondroitin. position 2 (amino group) of the glucosaminc units of heparan sulfate and sugar units of kcratan sulfate. rcspcctively. The assay method will be applicable with modification to the assay ofother glycosaminoglycan sulfotransferases and glycoprotein sulfotransferases. 't. lYK7 Awdem,c Prcns. Inc KEl' WORDS: glycosaminoglycan: sulfotransfcrase: sulfation: chondroitin sulfate: kcratan sulfate: heparan sulfate

Recently obtained information regarding the structure-function relationships of sulfated glycosaminoglycans (GAGS)” has provided ample evidence of the biological importance of their sulfation ( 1.3). Defective sulfation of GAGS results in growth defects (3,4) or pathological conditions (5.6). After specific sulfation of the monosaccharide units of GAGS, these polysaccharides exhibit a variety of biological activities (2,7).

’ This work was supported by Grants-in-Aid for Scicntitic Research from the Ministv of Education and Culture of Japan. ’ To whom correspondence should be addressed. ’ Abbreviations used: GAG. glycosaminoglycan; PAPS. 3’phosphoadenosinc-5’-phosphosulfate; thn. chondroitin; KS. keratan sulfate: HS. heparan sulfate; DMP. 2.3-dimercaptopropan-l-01; TCA. trichloroacetic acid: Solvent A, n-butyric acid: 0.5 hl ammonia (5:3. v/v); Heprs. 4-(Z!-hydroxyethyl)-l-piperdzincethanesulfomc acid: IDi-OS. ADi-4s. and IDi-6s. CL,‘-I’-glucuronosyl-( I .3)-h’acetyl-D-galactosamine O-sulfate. 4sulfate. and h-sulfate. respectivel).

During the biosynthesis of proteoglycans. GAG chains are formed by sequential addition of the respective monosaccharides to the precursors. and sulfation of the growing or fully elongated chains is considered to be accomplished by a number of specitic sulfotransferaseslocated in the Golgi apparatus (8). For the sulfation of chondroitin (Chn) or chondroitin sulfate, four distinct enrymes have been so far demonstrated; those synthesize GalNAc 4-sulfated. GalNAc A-sulfated. and internal and terminal GalNAc 4.6-bissulfated structures, respectively (reviewed in Ref. (9)). At least two speciesof sulfotransferasesseem to be required for the sulfation of keratan sulfate (KS); those preferentially transfer sulfate either to GlcNAc or to Gal residues ( IO). There have been very few detailed investigations specifically of hcparan sulfate (HS) synthesis, and it has been inferred that the basic moditication stepsof HS synthesis occur as in heparin synthesis (2). where at least one h’-sulfotransferase and

ASSAY

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three distinct 0-sulfotransferases have been suggested to bz involved (7). However, only a few sulfotransferases have been partially purified (reviewed in Ref. (9)). and available information about acceptor specificities of GAG sulfotransferases is limited. Most of the work on GAG sulfotransferases has been carried out with a tissue extract or a microsomal fraction (for a review, see Ref. (8)). The major problem appears to be the limitations of the conventional timeconsuming assays. Existing methods include the separation of the substrate from the product by precipitation/centrifugation (11) or by paper chromatography ( 13). Although gel filtration by means of high-performance liquid chromatography can also be used ( 10). it requires elaborate equipment. Recently. we developed a rapid and simple assay- method for Chn sulfotransferase through application of the paper disk method used for the assay of serum glycoprotein glycosyltransferases ( 13) and demonstrated that the level of Chn sulfotransferase activity changes during development: it is considerably higher in fetal than in adult bovine serum (14). This enzyme transfers sulfate from PAPS to position 6 of the internal nonsulfated GalNAc residues in Chn. namely. PAF’S:Chn (internal GalNAc) 6-0sulfotransferase.’ In the present study, we demonstrated the broad applicability of our new method to other GAG sulfotransferase assays and established assay conditions for KS sulfotransferase and HS (GlcNH?) Z-,V-sulfotransferase in addition to Chn (internal GalNAc) 6-0sulfotransferase in serum. In this paper. the enzymes are referred to as KS sulfotransferase, HS sulfotransferase. and Chn sulfotransferase. respectively. The assays are useful for systematic studies on GAG sulfotransferases.

’ The specificities of the sulfotransferascs nated as the acceptors. the monosaccharide the positions to be sulfated.

were desigunits. and

405

SULFOTRANSFERASES

MATERIALS

AND

METHODS

RfarcvQ1.s. [““SIPAPS (2.2 Ci/mmol) and nonradioactive PAPS were purchased from New England Nuclear and Sigma. respectively. International standards. bovine corneal KS (KS-I) (average 121,= 35.000; the molar ratio of sulfate to hexosamine = I .48), and human costal cartilage KS (KS-II) (molecular weight is unknown: molar ratio of sulfate to hexosamine = 1.96) were generous gifts from Drs. M. B. Mathews and A. L. Horwitr (Univ. of Chicago). Chondroitinase ABC, ~~lu~~ohuc~tc~~illlll heparitinase. P.scwdo/)?o~?us keratanase, Chn (a chemically desulfated derivative of whale cartilage Chn sulfate A). and bovine kidney HS were purchased from Scikagaku Kogyo Co., Tokyo. Each GAG preparation gave a single Alcian blue-positive spot on cellulose acetate strip electrophoresis. Disaccharide analysis of the Chn preparation by the method of Sugahara L’I al. (I 5) showed ADi-OS, ADi-4S, and ADi-6S in the molar ratio of 74:1 I :5. Analysisof a heparitinase digest of the HS preparation by high-performance liquid chromatography showed nonsulfatcd, Xmonosulfated. 0-monosulfated. iv/O-disulfated. and trisulfated disaccharides in the molar ratio of 60:12:7:8:3.’ 2.3-Dimercaptopropan-l-01 (DMP) was provided by Wako Pure Chemical Industries. Ltd., Osaka. Fetal calf scra were purchased from GIBCO. The other sem were prepared from rat and chicken blood obtained from Wistar rats and white leghorn chickens and from human blood donated by volunteers. PL~~cv. t/i.\/< I~YI.Y/I~/Z~ IIICTIIOLI.For the paper disk assayreported for glycoprotein glycosyltransferases (I 3), the washing procedure is carried out with IO’; trichloroacetic acid (TCA). This procedure can be applied to the assayof sulfotransferaseswith endogcnous or exogenous proteoglycans with low G.AG ’ Wc than!, Dr. K. Yashida. the personal communication.

Seikagaku

Kogyo

Co.. for

without the exogenous acceptor. respeccontents as sulfate acceptors, but not with tively. Thr latter \aluc is higher than the zero GAGS or proteoglycans with high GAG contents. which are soluble in 10’; TCA. Thrrctime ~aluc. which is usually less than IO0 fore. this washing solvent was replaced b> cpm. indicating no significant precipitable ?I-butyric acid/O.5 M ammonia (5:3, v/l,) radioactive materials in the PAPS prepara(Solvent A), which has been long used for tion per SC. The radioactive products obpaper chromatographic GAG sulfotransfertained by standard incubations (see below) ase assays and with which sulfated GAGS re- with respective exogenous GAGS were isomain on the paper at the origin. indicating lated by the method of Sugahara rf (I/. ( 14) by that they are insoluble in the solvent. The HS 5% TCA extraction and subsequent gel tilsulfotransferase described in this manuscript tration. The net transfer of [?j]sulfate to introduces sulfate to the amino groups of each isolated exogenous GAG was also calglucosamine units of HS (see below). Alculated by subtraction of the blank value due though sulfamide linkages are acid labile, it to the sulfate transfer to endogenous accepwas confirmed that HS labeled with [‘%Itors and was comparable to the net transfer obtained above by the paper disk method. sulfate at amino groups did not release [“%Isulfate in Solvent A (pH 3.5), at least for 24 confirming quantitative precipitation of each h, and that the method could also be applied “S-labeled GAG in Solvent A of the latter to the HS sulfotransferase assays. method. One unit of each enzyme was deFollowing incubation of a serum preparafined as the amount that catalyzed the incortion with [35S]PAPS and a suitable sulfate poration of 1 pmol of sulfate per minute. acceptor (GAG) in a total volume of 60 ~1 Stutdtrrd iwdmcliorl cwzclitiot1.v. In order to determine the standard incubation condiunder the appropriate conditions described tions with exogenous GAGS as sulfate accepbelow, incorporation of [35S]sulfate into tors. the effect of their concentrations was GAGS was determined by the paper disk method. At zero time and the indicated time investigated. As shown in Fig. 1, the apparpoints, 5 to 25-ptl aliquots were withdrawn ent K,,, values for Chn of Chn sulfotransferand spotted onto 2.5-cm Whatman No. 1 ase. KS-I of KS sulfotransferase, and HS of HS sulfotransferase were 0.17.0.63. and 0.36 paper disks. which were immediately. without drying. dipped and left in Solvent A in a mg/ml, as sodium salts. respectively. For the glass beaker at room temperature for 30 min standard incubations. fmal concentrations of 3.0. 0.33. and 0.50 mg/ml were used. respecto terminate the sulfation reactions. The disks were then rinsed three times in fresh tively. Relatively low concentrations of KS-I Solvent A. They were finally washed with and HS were used because of their expenethanol/ether (2: 1. v/v) and then with ether, siveness. The apparent k;,, for PAPS of Chn air-dried, and placed in 5 ml of toluene consulfotransferase was 7.4 PM. A final PAPS taining 12 g of 2.5-diphenyloxazole/liter. and concentration of 10 FM was tentatively used then the radioactivity remaining on the disks for the standard incubations of all the GAG was counted with a liquid scintillation sulfotransferases. counter (Beckman LS-7500). The washing The effects of buffers, pH, NaCl. and metal ions were investigated under the stansteps were carried out under a well-ventilated dard conditions described below and the rehood because of the rancid odor of Solsults are presented under Results and Disvent A. The net transfer of [35S]sulfate to endogecussion. nous acceptors and each exogenous acceptor The assay mixture for Chn sulfotransferase contained 20 PI of fetal calf serum appropriwas calculted by substraction of the zero ately diluted with IO mM Hepes-NaOH. pH time value and the blank value obtained

ASSAY

FOR

GLYCOSAMINOGLYCAN

2

4 Acceptor

407

SULFOTRANSFERASES

0.4

0.4

0.8

concentration

(mg

0.E

I ml)

FIG. 1. Effects of acceptor concentrations on the rate of sulfate transfer to GAGS from PAPS. Leji. sulfate transfer to e:iogenous Chn by Chn sulfotransfcrase in fetal calf serum corresponding to 20 fig protein. ~\lr~/N’lc~. sulfate transfer to exogenous KS by KS sulfotransferase in fetal calfserum corresponding to 800 ~g protein. Righr. sulfate transfer to exogenous HS by HS sulfotransferase in fetal calfserum corresponding to 390 ~g protein. Incubations were carried out under the conditions which are given below as the standard conditons for each enzyme (see Materials and Methods) except that the concentration of the exogenous acceptor was varied. The insets show Lineweaver-Burk plots of the same data.

7.4, IO p1 of 13uffer 1. 10 ~1 of 60 PM [3’S]PAPS (4-6 X IO4 cpm). 10 ~1 of 2.4 mg/ml polylysine. and IO ~1 of 12 mg/ml Chn (sodium salt). Buffer I contained 30 mM EDTA. 60 mM MgCi:!, and 30 mM DMP in 60 mM Hepes-NaOH, pH 7.4. The assay mixture for KS sulfotransferase contained 10 /*l of 0.3 M Hepes-NaOH. pH 7.0, 5 ~1 of 60 mM DMP/90 mM CaCI1. 5 ~1 of 4 mg/ml KS-I (sodium salt), 10 ~1 of 60 FM [3’S]PAPS (4--6 X lo4 cpm). 10 ~1 of water, and 20 /*l of diluted serum in a total volume of 60 ~1. It should be noted that the KS-I used as an exogenous acceptor had been treated with chondroitinase ABC and then with nitrous acid (I 6) to ensure the complete removal of possible trace amounts of contaminating Chn and HS. A commercial preparation of bovin’e cornea1 KS-I from Seikagaku Kogyo Co. served equally well as an acceptor. The assay mixture for HS sulfotransferase contained 10 ~1 each of 0.3 M Tris-HCI, pH 7.5, 30 mM DMP/60 mM CaC12/0.3 M NaCI, 3 mg/ml HS ‘(sodium salt). 60 PM [35S]PAPS ( 1-3 X IO5 cpm), water. and diluted serum.

nptc’rrninciti(,rl

(?i’ ~.‘.-1I’S-d~~~~rudin~~r

mYi\‘-

PAPS degradation was determined according to Yoshida et (I/. (I 7). An aliquot of the reaction mixture was mixed with 1.0 ml of water, 50 PI of 0.2 M Na$O,. and 40 mg of active charcoal (Darco G60). left to stand for IO min at room temperature. and then centrifuged to remove the charcoal which had adsorbed [“SIPAPS. An aliquot of the supernatant was counted to determine the radioactivity due to inorganic sulfate. OI~ICJVr~~ctl7od.s. Isolation of GAG sulfotransferase reaction products was carried out as previously reported ( 14), and digestions ot the isolated products with chondroitinase, heparitinase, and keratanase were performed according to the suppliers’ manuals, respectively. Paper electrophoresis for determination of inorganic sulfate was performed on a 60-cm strip of Toyo No. 5 1A paper with a potential gradient of 78 V/cm for 30 min in pyridine/acetate buffer. pH 5.3 (pyridine: acetic acid:water, 3: 1:397. v/v). it!,.

RESULTS

AND DISCUSSION

Using the new rapid and simple paper disk method, the assay conditions for three GAG

408

SUGAHARA.

ISHII,

SAND Y.AMASHlNA

sulfotransferases in fetal calf serum were established as follows. In the first place, cam was taken to prevent the degradation of the sulfate donor, PAPS. Studies on GAG sulfotransferases in a variety of animal tissues have been hampered by the rapid degradation of PAPS (IS), which has been prevented to some extent by the use of a phosphate buffer ( 18-20) and/or by the addition of fluoride (10.19-33) and AMP (923). Recently, Fukui ef ul. (34) demonstrated that PAPS is rapidly degraded by nucleotide pyrophosphatase, which is widely distributed in various tissues of rats, while Faltynek & a/. (35) have shown that sugar nucleotide degradation by Zn’+-requiring nucleotide pyrophosphatase and phosphatase can be effectively inhibited by the addition of the Zr?+-specific chelator, DMP. In view of these observations, we investigated the ability of DMP to prevent PAPS degradation in a serum Chn sulfotransferase assay system ( 14). Following the incubation of fetal calf serum with [35S]PAPS under the conditions described in the legend to Fig. 2,

no lnhlbitor

t 5mM

PAPS degradation and [“Slsulfatc incorporation into endogenous acceptors were determined. Since considerable amounts of undersulfated chondroitin sulfate proteoglycans arc present in serum (36), a certain level of Chn sulfotransferase activity can be detected without exogenous Chn. It has been previously shown that more than 9OS,of the “S-labeled endogenous macromolecules were sensitive to chondroitinase AC and that over 75%, of the chondroitinase-produced oligosaccharides was ADi-6S ( 14). As shown in Fig. 2, 5 mtvt DMP was more effective than a high concentration of NaF; there were linear and higher incorporations of sulfate into endogenous acceptors. Although DMP is an SH reagent. it seemsto act as a chelator rather than an SH reagent in this case. PAPS degradation was also prevented by EDTA, but not by mercaptoethanol (data not shown). Although cytidine diphosphate (CDP)-choline. a competitive inhibitor for PAPS degradation, was also effective. it is not practical because of its expensiveness.

DMP

+C&frh:ne

+&mM

NaF

/d

[

30 Incuboton

60

30

60

Time ( nun)

FIG. 2. Effects of various reagents on PAPS degradation and sulfate incorporation into endogenous acceptors in serum. The reaction mixture contained 10 ~1 of60 mM Hepes-NaOH, pH 7.4. 5 ~1 of60 IIIM EDTA. 2 ~1 of 300 mM MgCl?, 30 ~1 of fetal calf serum (40 mg protein/ml) diluted with IO mM Hepes-NaOH, pH 7.4. IO /II of 60 pM [%]PAPS (1.165 X 10” cpm). and 3 ~1 of water, 300 mM DMP. 75 IIIM NaF. or 300 mM CDP-choline. At zero time. and after 30 and 60 min. 5- and IO-~1 aliquots were withdrawn to determine PAPS degradation (see Materials and Methods) and sulfate incorporation into endogenous acceptors, respectively. Sulfate incorporation was determined as described under Materials and Methods except that 10% TCA was used instead of Solvent A.

ASSAY

10

Chn (endDsems

FOR

GLYCOSAMINOGLYCAN

I

t

Chn (erogmovs

1

FIG. 3. Effects of buffers and pH on the rate of sulfate transfer. The effect of pH on the sulfate transfer to endogenous Chn (~rp~~er I&), exogenous Chn (~OWW k/i), exogenous KS (q~pt~r r~~yht). and exogenous HS (k~cr ri&/) was determined using the standard assay for each sulfotransferase with different buffers at a final concentration of 0.05 M. The buffers used were Na acetate (0 - q ), Na phosphate (W - n ), Mes-NaOH (h - a). Tris-HCI (0 - 3). and Hepes-NaOH (0 - 0). In the case of Chn suifotransferase assays, 0.01 M HepesNaOH buffer (0 --- l ) was also employed.

Thus, DMP was included for the following sulfotransferase assays. It should be noted that in these preliminary experiments washing of the paper disks was carried out with 10%)TCA, and hence 10-30%) less [35S]sulfate incorporation was observed as compared to that in the case of washing with Solvent A, presumably due to the loss of some TCA-soluble products (see Materials and Methods). Thus. in the following experiments. 10% TCA was replaced by Solvent A eken for the determination of endogenous products. &&cts qf’ salts, hfirs, and p1-I. Figure 3 shows the elTects of buffers and pH on the various GAG sulfotransferase activities in fetal calf serum. The maximal introduction of sulfate into endogenous acceptors was obtained with 10 mM Hepes-NaOH, pH 7.4,

409

SULFOTRANSFERASES

although a second optimum was found at pH 4.3. It should be noted that a phosphate buffer, often used to prevent PAPS degradation. was strongly inhibitory toward Chn sulfotransferase. With exogenous Chn as an acceptor. a few hundred-fold greater activity was again obtained with two optimal pH values, 5.0 and 7.4. However, the maximal activity at pH 7.4 was observed only when 10 mM Hepes-NaOH buffer was used, the reason for which remains to be clarified. It is possiblethat the difference in pH profiles between the endogenous and exogenous acceptors resulted from the difference in their sulfation degrees: presumably, the endogenous Chn sulfate is sulfated to a higher extent than the exogenous Chn. As to KS sulfotransferase with a Hepes buffer, maximum activity was seen at pH 6.5-7.4 with a second optimum at pH 4.3. HS sulfotransferase showed a single optimum pH. 7.0. Figure 4 showsthe effects of NaCl on the GAG sulfotransferase activities. The Chn sulfotransferase and KS sulfotransferase activities were strongly in-

0

0.1 0.2 Nacl cmentration

0.3 (molll)

0.4

FIG. 4. Effect of the NaCl concentration on the sulfotransferase activities. The rate of sulfate transfer to exogenous Chn (0). KS (0). and HS (0) was determined using the standard assay conditions for each sulfotransferase except that the concentration of NaCl was varied. Fetal calf serum corresponding to 40 pg protein (for Chn sulfotransferase) and 400 pg protein (for KS and HS sulfotransferases) was used as the enzyme source. The results are expressed as percentage activities.

410

SUGAHAR.4.

ISHII

hibited by NaCI. while the HS sulfotransferase activity was enhanced: the highest activity was seen at 50 mM NaCI. &/jkts qf’vletrtl ioas. Chn sulfotransferasc activity was greatly enhanced by Mn”, Mg’+, and Ca’+ and strongly inhibited by Zn’+ (Fig. 5). A greater stimulatory effect was seenwith a combination of 5 mM EDTA and 10 mM Mg’+, but not Mn”. presumably because EDTA bound inhibitory divalent cations such as Zn’+ in the serum preparation. However, since this effect of 5 mrvl EDTA was seen even in the presence of the Zr?+-specific chelator, DMP, EDTA seems to function through binding to inhibitory divalent cations other than Zn’+. Therefore. for the standard incubation for Chn sulfotransferase assays, IO mM MgC12 and 5 mM EDTA and DMP were included. KS and HS sulfotransferases were also stimulated by Mg2+ and Ca’+. but to lesser extents than Chn sulfotransferase. and strongly inhibited bv Zn2+. For the standard incubations for KS and HS sulfotransferase assays, 7.5 and 10

Chn

5-j I”$*, 10 20 Cmcentrot~on

] 30 ( mM 1

FIG. 5. Effects of metal ions on the sulfotransferase activities. The Chn (rap). KS (vzidclle). and HS (hotfont) sulfotransferase activities were measured under the standard assay conditions except that the concentrations of EDTA and/or chloride salts of divalent cations were varied. EDTA (A-A). MnClz (0 - 0). ZnClz (B - n), CaClz (0 - 0) MgClz (0 - 0). EDTA plus M&l2 (0 --- Cl), EDTA plus MnC12 (0 --- 0).

\ND

\‘.IM~ISHINA

l”ClJbO,lO”

nme

(ml”)

FIG. 6. Effect of the incubation time on the rate of sulfate transfer. The rate of sulfate transfer to exogenous Chn (/e/r). KS (~rrirltr’lc,). and HS (r/,q/~/) as a function of the incubation time was dctermincd using the standard assay mixture on a twofold scale. Fetal calfserum used as the enzyme source corresponded to 80. lh00. and 800 ~g protein for the Chn. KS, and HS sulfotransfemse assays. respectively.

CaC12were included. respectively. It remains to be determined whether the inhibitory effect of Zn’+ resulted from its direct binding to these GAG sulfotransferases or from the activation of nucleotide pyrophosphatase, which is responsible for PAPS degradation. As shown in Fig. 6, under the established standard assay conditions for each sulfotransferase, linear sulfate incorporation into the respective exogenous acceptor occurred over 60 min. that is. until approximately 20’%of the PAPS had been consumed. Sulfated products were isolated from each standard incubation by 5% TCA extraction of the reaction mixture and subsequent gel filtration (14) and then analyzed. The “?S-labeled products from a Chn sulfotransferase reaction were completely degraded by chondroitinase ABC. and over 98% of the produced radioactive oligosaccharides were LDi-6S as reported ( 14). Thus, the Chn sulfotransferase detected under the established assay conditons is PAPS:Chn (internal GalNAc) 60sulfotransferase. More than 98% of the “‘S-labeled products obtained from a standard HS sulfotransferase incubation were degraded on heparitinase digestion (15) or on nitrous acid treatment (16) as determined by Sephadex G-50 mM

ASSAY

FOR

GLYCOSAMINOGLYCAN

column chromatography. indicating that the produced labeled materials were indeed HS. The degradation products recovered on gel filtration of the nitrous acid-treated sample gave a single radioactive spot migrating with inorganic sulfate on paper electrophoresis (data not shown). suggesting that [-‘%]sulfate had been introduced at the amino groups of glucosamine residues. Thus, the HS sulfotransferase detected under the standard assay conditions is primarily PAPS:HS (GlcNH?) 3-h;-sulfotransferase activity. The reason for the failure of detection of 0-sulfotransferase activities is unknown. The exogenous acceptor may not satisfy the structural requirements for 0-sulfotransferases: the N-sulfation and subsequent epimerization reactions in HS synthesis proceed prior to 0-sulfation reactions which largely occur in regions of’ high N-sulfated glucosamine content (for a review. see Ref. (3)). Nineteen percent of the “S-labeled materials isolated from a standard KS sulfotransferase incubation were degraded on chondroitinase ABC digestion and were quantitatively equivalent to the labeled products obtained from a control incubation conducted without exogenous KS. thus representing endogenous Chn-derived materials. The %-labeled chondroitinase-resistant materials were completely degraded on keratanase digestion as judged by gel filtration, confirming that the net [35S]sulfate transfer calculated by subtraction of the control value represents KS sulfotransferase activity. Investigation of the sulfated sites of KS-I has been carried out and the data have indicated that sulfate groups are introduced largely, if not exclusively. to position 6 of the Gal units. The following observations have been made and the details will be published elsewhere (:!9). (i) Human costal cartilage KS-II, which is more sulfated at position 6 of the Gal resi8dues than KS-I, exhibited only one-fifth the acceptor activity obtained with KS-I. (ii) Keratanase digestion of the substrate (KS-l 1 yielded oligosaccharides. with

SULFOTRANSFERASES

411

GlcNAc (6-sulfate)-Gal as a major component, while the KS sulfotransferase reaction products failed to yield %-labeled disaccharides. The results can be interpreted as indicating that sulfation of Gal units rendered the galactoside linkages resistant to keratanase. which acts only on nonsulfated galactoside linkages. (iii) 3’S-Labeled KS tetrasaccharide. a major product isolated from a keratanase digest of [3’S]KS-I. yielded Gal 6-sulfate. but not GlcNAc 6-sulfate, on mild acid hydrolysis. To detect KS (CJICNAC) 6-O-sulfotransferase, different assay conditions or substrates may be required. Recently. lnoue of ctl. (9) reported the properties of G.4G sulfotransferases in human serum. including the Chn, KS, and HS sulfotransferases. The properties of the latter in general including the acceptor specificities seem to be similar to those of the enzymes in fetal calf serum reported in this paper. In addition. they reported the occurrence of these activities in sera from several animals including calf (fetus). In order to show the applicability of our method, the sulfotransferase activities were measured in sera from various animal species. The results arc summarized in Table I with those reported by lnoue et ~1. (9) for comparison. The activity levels we detected under the presently established conditions were considerably higher than those they reported: in the case of fetal calf serum. for example, activities were approximately 60-fold, 3.5-fold, and 5-fold higher for Chn, KS, and HS sulfotransferases, respectively. The use of our assay conditions in combination with the rapid and simple paper disk washing method allows sensitive and multiple assays. which are required for enzyme purification and screening for defects in GAG biosynthesis (6.27). Recently. using these assays, we successfully demonstrated developmental changes in GAG sulfotransferase activities in animal sera (29).

412

SUGAHARA. TABLE

DISTRIBUTION

.AND

1

OF GAG SULFOTRANSFERASES OF VARIOCIS ANIMAL SPECIES Specific

ISHII.

activity” determined exogenous acceptor (v units/ml)

Rod&n L. ( 1980) 01 The Biochemistry of Glycoproteins and Protcoglycans (Lennarr. W. J.. Ed.). pp. 367-37 1. Plenum. New Yorh. Inouc. H.. Otsu, li.. \ oneda. M.. tiimata. K.. Suluki. S., and Nahanishi. 1’. ( 1986) ./. /1io/ C’/~C~)I

IN SERA

with

an

261.4J60-4469.

10. Riitter,

Chn

(w/ml)

Human

89 k I (971’

Bovine fetus

l9i (1.0)

40 zk 7 (ND)’ 67 2 2

Rat

6

2790 k 390 (39) 27+ I

(85) 70 * 9 (56)

Chlcken

(11) 518-t

85

(4-i)

25

KS

HS

ndd (0.35)

38i (IX)

+ 0.2

(7.3) 2.0 f 0. I (1.1) 7.3 2 1.3 (2.7)

5

122 i 14 (251 239 I21 (71) 1372 15 (33)

a Values were determined wth three or four serum specimens from each species and expressed as the mean and the standard deviation. ‘Determined by the method of Lowry cr a/ (3) with hovine serum albumin as standard. ‘Values m parentheses were taken from Inoue tv ul (9). Note that they are expressed hy the enzyme umt defined in rhls report (see text). d nd. not detected. ’ ND. not determined.

The assay method will be applicable with modifications to assays for other GAG sulfotransferases and glycoprotein sulfotransferases.

We thank Misses Hayuru for their excellent secretarial

Sakai and Yurika assistance.

235,

C%cvn

257-166.

13. Baxter. A.. and Durham, J. P.. (1979) .lr?al Riod~vn 98, Y5-101. 13. Sugahara. K.. Shibamoto. S.. and Yamashina, I. (1985) F,!?LIS Lett. 183.43-46. IS. Sugahara. K.. Fukui. S., and Yamashina. I. ( 1985).J. Hioc,hcnr. (%&WI) 98, X75-885. 16. Lagunoff. D.. and Warren. G. (1966) .-lr&. Bioch(‘1n. Bity’//jY. 99, 3'35-400. 17. Yoshida. H.. Fukui. S.. Yamashina. I., Tanaka. T.. Sakano. T.. Usui. T. Shimotsuji. T.. Yabuuchl. H.. Owada. M.. and Kitagawa. T. ( 1982) Biodrnn. Biop/~~:~. Rc\. (‘o/~~r,tun. 107, 1 144- 1 150. J. B. (lY64) Bio&ivl. Hiophj~.v. -1ctu 83. 18. Adams. 127-129. 19. Foley. T.. and Baker. J. R. ( 1973) Uioc,lrcvn .J. 135, 187-192. 30. Hart. G. W. (1978) J H/I)/ (‘/ltvz 253, 337-353. 21. Suzuki. S.. and Strominger. J. L. (1960) 1. Biol. (‘hen1. 235, 257-266. ‘2. Giihler. D., Nieman. R.. and Buddecke. E. (1984) Eur. J. Bioc~hrwz. 138, 30 I-308. 23. Nahanishi. Y., Shimilu. M., Otsu. K.. Kato, S.. Tsuji. M.. and Suzuki, S. ( 198 1) J. Biol. C‘lruru 24.

Yakura

REFERENCES

50,

H. ( 1984) .J. H/o/. <‘/rcw

1 I. Hahuchi. 0.. Yamagata. T.. and Suzuki. S. ( I97 I ) J. Biol. (‘IMWI 246, 73.57-7365. 11. Suzuki. S.. and Stromlnger. J. L. (1960) J. Hit)/

25. 26.

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