Glycosulphatase from Pseudomonas carrageenovora

Transcription

1 Eur. J. Biohem. 11, (1979) Glyosulphatase from Pseudomonas arrageenovora Purifiation and Some Properties Maitland W. MLEAN and Frank B. WILLIAMSON Department of Biohemistry, University of Aberdeen (Reeived May 16, 1979) A glyosulphatase present in the soluble fration of disrupted Pseudomonas arrageenovora has been purified 5-fold by gel filtration on Sepharyl S-2 and ion-exhange hromatography on DEAE-Sepharose CL-6B. By dodeylsulphate/polyarylamide gel eletrophoresis the enzyme is pratially homogeneous and has a moleular weight of 55. Conditions of optimal sodium hloride onentration and ph at 25 "C were mol dm-3 and ph 7. respetively. The purified enzyme was inhibited by inorgani phosphate. Preparation is desribed of neoarrabiose 4--[35S]sulphate and neoarratetraose 4--[35S]- sulphate from labelled Chondrus rispus. The purified glyosulphatase is ative against both these substrates although only one of the two sulphate esters in the tetrasaharide is hydrolysed. Analysis of the reation produts was by gel filtration, eletrophoresis and I3C nulear magneti resonane spetrosopy. The results are onsistent with the produts of desulphation being respetively neoarrabiose and neoarratetraose 4--monosulphate with the sulphate ester proximal to the reduing end [ 3,6-anhydro-a-~-galatopyranosyl-( 1 + 3)-j3-~-galatopyranosyl-( )- 3,6-anhydro-a- D-galatopyranosyl-( )-~-galatose 4--sulphate]. Sulphatases have been deteted ubiquitously in organisms [l- 31 pertinent examples being bateria [4,5] and algae [6,7]. In the degradation of hondroitin sulphate by Proteus vulgaris [4] only disaharide oligomers are suseptible to the relevant sulphatases. This ontrasts with the algal sulphatases [6,7], whih release sulphate at the polymer level from galatose 6--sulphate residues with onomitant formation of the 3,6-anhydro derivative. In the initial desription of a glyosulphatase from Pseudomonas arrageenovora by Weigl and Yaphe [5], the partially purified enzyme was tested with several sugar sulphates. We onsider this enzyme to be potentially appliable in the study of arrageenan fragments, and present a satisfatory purifiation method. Preparation of neoarratetraose 4--[35S]sulphate and neoarratetraose 4--[35S]sulphate of high radiohemial purity allowed the development of a pratial assay similar to that desribed for holine sulphatase [8]. These oligosaharides were isolated after x-arrageenase digestion of [35S]arrageenan extrated from Chondrus rispus ultured in the presene of inorgani [35S]sulphate. Abbreviation. NMR, nulear magneti resonane. Enzymes. x-carrageenanase (EC ); glyosulphatase (EC ); a-galatosidase (EC ); p-galatosidase (EC ). MATERIALS AND METHODS Chemials, unless speified, were produts of BDH Ltd (Poole, England). Sephadex, Sepharyl S-2 and DEAE-Sepharose CL-6B were manufatured by Pharmaia (Uppsala, Sweden). Reagents for polyarylamide gel eletrophoresis and AG 1x8 hloride (2-4 mesh) were supplied by Bio-Rad Laboratories (Rihmond, U.S.A.). [35S]Sulphate, arrier-free was a produt of the Radiohemial Centre (Amersham, England). 3 MM filter paper and GF/C glass-fibre paper were supplied by Whatman Ltd (Maidstone, England). %-Carrageenan from Chondrus rispus (Danagel CX) was a gift from Litex (Glostrup, Denmark). Protein standards and nitrophenyl galatosides were from Sigma (St Louis, U.S.A.). >I-Carrageenase was purified as desribed previously [9]. Visking size 1 dialysis tubing was obtained from Mediell International Ltd (London). Sulphatase Assay Routine assay was buffer (1 mm3,.5 mol dm-3 sodium hloride,.2 mol dm-3 Tris, ph 7.9, sample (1 mm3) and neoarrabiose 4--[35S]~~lphate (1 mm3, 25 ounts min-', 23 nmol galatose

2 498 Glyosulphatase from Pseudomonas arragrenovora ontent, in deionized water). After inubation (15 min, 25'C) the reation was terminated by the addition of aqueous trihloroaeti aid (1 %, w/v). Carrier (1 mm3,.2 mol dm-3 sodium sulphate) was added and then barium hloride solution (5 mm3, 1. mol drnp3). The preipitated barium sulphate was retained on a glass-fibre dis (2.5 m diameter). Diss were then washed with water (1 m3), dehydrated with ethanol and dried. Radioativity was measured by immersion in sintillation fluid [lo] and ounting in the I4C hannel of a liquid sintillation spetrometer (Pakard model 3255). Control aliquots of substrate solution (1 mm3, 25 ounts min-l, 23 nmol galatose ontent) were inubated without enzyme to obtain the zero ount, or heated with hydrohlori aid (1 m3, 2. mol dm-3, llo'c, 17 h) in a sealed tube and the released [3sS]sulphate assayed as total available ounts. For purifiation purposes an arbitary unit of ativity was defined as the quantity of enzyme required to release 1% of the radioativity in the assay desribed. x-carrageenuse Asmy Samples were assayed for arrageenase ativity by a reduing-power method [9] using Danagel CX as substrate. a-galatosidase and P-Gnlutosidase Assays The respetive 4-nitrophenyl u-galatoside (.2 m3, 5. mm~ldm-~), buffer (.4 m3,.5 mol dm-3 sodium hloride,.2 mol dm-3 Tris, ph 7.5) and sample (.2 m3) were inubated together (15 min, 25 "C). Aqueous sodium arbonate (3.2 m3,.5 rnol dm-3) was added to terminate the reation and the released 4-nitrophenol estimated from the A45 ",. Misellaneous Assays Protein was estimated by the method of Lowry et al. [Ill with bovine serum albumin as standard, and total arbohydrate by the phenol/sulphuri aid proedure of Dubois et al. [12] with galatose as standard. 3,6-anhydro sugar ontent was assayed by the resorinol/hydrohlori aid method of Yaphe and Arsenault [13] taking methyl 3,6-anhydro-a-~-galatopyranoside as standard, and inorgani phosphate by the molybdate tehnique of Fiske and Subbarow ~41. Dodeylsulphatel Polyarylamide Gel Eletrophoresis Protein speies were examined by the gel eletrophoresis tehnique of Weber and Osborn [15]. Arylamide onentration was 7.4 % (w/v) and urea (4. mol dmp3) was inluded in the gel mixture. After eletro- phoresis gels were stained with Coomassie brilliant blue. Moleular weight standards were bovine serum albumin (66 ), ovalbumin (45 ), trypsinogen (24) and P-latoglobulin (184). High- Voltage Paper Eletrophoresis A flat-bed apparatus was used (Multiphor, LKB, Sweden). Samples were applied as spots to Whatman 3MM paper wetted with buffer (.5 mol dmp3 ammonium aetate, ph 5.) and then eletrophoresed (3 V m-l, 75 min). Completed eletrophoretograms were air-dried and sugars deteted by the silver nitrate tehnique of Trevelyan et al. [16]. Radioativity was visualized by photography in a spark hamber (Birhover Instruments Ltd., Hithin, England) or exposure to X-ray film (Blue Brand Medial X-ray, Kodak, England). Neoarrahiose 4--(3s SISulphate and Neoavratetraose 4--(3sS]Sulphate Chondrus rispus fronds (4. g) were gathered loally (Collieston) and ultured under the onditions previously desribed for Porphyra umbilialis [17]. Medium omposition was essentially that of Imada [18], with the onentration of inorgani sulphate reredued to 1. mmol dmp3. Carrier-free 35SOi- (93 MBq) was added as the soure of label in a medium volume of 4 m3. Uptake of label was estimated by the net loss of radioativity from the medium [19]. After inubation (75 h), with 35 :d of the label residing in the medium, the fronds were rinsed with sodium hloride solution (.5 mol dm-3) and then disrupted by two passages in an X-Press (Biote, Stokholm, Sweden) at -2 'C. Carrageenan was extrated twie from the homogenate by a hot aqueous tehnique (.5 niol dm-3 sodium hloride, 15 m3 g-' wet plant, 9"C, 2 h) and the suspension larified by entrifugation (16 x g, 15 min). Crude arrageenan was preipitated from the supernatant with ethanol (3 vol.), and subsequently washed with aliquots of aqueous ethanol (75 % v/v) and finally absolute ethanol before drying under redued pressure. The dried rude arrageenan was dissolved in aqueous buffer (1 m3,.5 rnol dm-3 sodium hloride,.1 mol dm-3 sodium phosphate, ph 7.5) and x-arrageenase (1.O mg) added. During inubation (25"C, 2 h), degradation of the arrageenan as estimated by the reduing power assay [9] was omplete by 3 min. High-moleular-weight material was preipitated (2 "C) by the addition of ethanol (1.5 vol.) and removed by entrifugation (16 x g, 15 min). After onentration of the supernatant by rotary evaporation (bath temperature, 4 "C) it was dialysed (Visking size 1, 3 m) against water (.5 dm3, 5 "C,

3 M. W. MLean and F. B. Williamson h). Material permeating the sa was similarly onentrated and applied to Sephadex G-25 fine (77 x 1.45 m) equilibrated with aqueous buffer (.1 mol dm-3 sodium hloride,.25 mol dm-3 sodium aetate, ph 5.). Frations were assayed for total arbohydrate and radioativity, and the tubes ontaining the disaharide and tetrasaharide respetively pooled. After dilution with water (4 vol.) the disaharide pool was applied to a olumn of AG 1x8 hloride (7 x.6 m). The resin was washed with water (1 m3) and then eluted with ammonium arbonate solution (NH4HC3/NH2COONH4, 1.O rnol dm-3). Frations were assayed for radioativity and those ontaining the peak pooled and lyophilized. The freeze-dried material was dissolved in aqueous ammonium aetate (.1 mol dmp3, ph 8.8) and then hromatographed on Sephadex G-15 (98 x 1.45 m) equilibrated with the same buffer. Frations were assayed for arbohydrate, radioativity and phosphate, and those ontaining the disaharide pooled and lyophilized. By suitably altering the onditions of ounter-ion and ph from the initial frationation (on Sephadex G-25) any inorgani [35S]sulphate and inorgani phosphate are separated from the disaharide. The tetrasaharide pool from the Sephadex (3-25 frationation was lyophilized and hromatographed on Sephadex G-15 under similar onditions to those desribed for the disaharide. Frations were assayed for arbohydrate and radioativity, and those ontaining the tetrasaharide pooled and lyophilized. Neoarrabiose 4-O-Sulphate and Neoarratetraose 4-O-Sulphate from Commerial x-carrageenan %-Carrageenan (Danagel CX, 1 g, 58 % galatose, estimated by the phenol/sulphuri aid method [12]) was dissolved in buffer (1 dm3,.1 mol dm3 sodium phosphate, ph 7.5) and x-arrageenase (1. mg) added. Digestion was arried out in an orbital inubator (25 "C, 2 h, 1 rev. min-') before adding ethanol (1.5 vol.) to preipitate high-moleular-weight material. After entrifugation (14 x g, 15 min) the supernatant was onentrated by rotary evaporation and dialysed against deionized water (1 dm3, 5"C, 2 h). The dialysate was similarly onentrated and then frationated in two aliquots on a olumn of Sephadex (3-25 fine (125 x 3. m) equilibrated with aetate buffer (.25 mol dm-3 sodium aetate,.1 mol dm-3 sodium hloride, ph 5.). Frations were assayed for arbohydrate and phosphate. Those ontaining tetrasaharide and phosphate-free disaharide were respetively pooled. Typial yields (as apparent galatose ontent) were tetrasaharide, 75 mg and disaharide, 95 mg of whih 64 mg was phosphate-free. Rehromatography of the phosphateontaminated disaharide resulted in quantitative purifiation. Samples of the purified oligosaharides were subjeted to 13C-NMR spetrosopy. l3 C Nulear Magneti Resonane Spetrosopy Samples (1-2 mg galatose ontent) were taken up in deuterium oxide (2 m3) and spetra reorded on a Varian CFT-2 pulse spetrometer. Pulse width was 9 ps, aquisition time.65 s, 52 data points, number of aumulated transients 1 and temperature 34 "C. Chemial shifts were referened with respet to tetramethylsilane (external apillary). Purifiation of Glyosulphatase Pseudomonas arrageenovora (NCMB no. 32) was grown up as previously desribed [9] and the ells harvested in late stationary phase (2 x 1 dm3 ultures, 7 h) by entrifugation (14 x g, 3 min). The supernatant was disarded and the ell pellet washed by suspending in an aliquot of buffer (5 m3,.5 mol dm-3 sodium hloride,.2 rnol dm-3 Tris, ph 7.5). All further operations were performed at 4 ' C. After further entrifugation (25 x g, 15 min) the ells (6 g) were suspended in fresh buffer (25 m3) and passaged (15 MPa) in an Amino pressure ell (Amerian Instrument Co., Maryland, U.S.A.). Debris was removed by entrifugation (34 x g, 3 min) and supernatant hromatographed on Sepharyl S-2 (115 x 2.4 m) equilibrated with Tris buffer (.5 mol dm-3 sodium hloride,.2 mol dm-3 Tris, ph 7.5). Ultraviolet absorption (28 nm) was monitored ontinuously (Uviord 11, LKB, Sweden) and the frations assayed for sulphatase and x-arrageenase. Major frations of the sulphatase peak were pooled and olumn dialysed on Sephadex G-25 medium (56 x 3. m) equilibrated with low-ioni-strength Tris buffer (.1 mol dmp3, ph 8.). The peak of exluded material was applied to a olumn of DEAE-Sepharose CL-6B (7 x 1.1 m) equilibrated with the same buffer. After washing (1 m3) the olumn was eluted with a linear gradient of sodium hloride (.-.3 mol dm-3, 4 m3 total volume) superimposed on the starting buffer. Eluent was monitored at 28 nm and the frations assayed for sulphatase. Dodeylsulphate/polyarylamide gel eletrophoresis of frations omprising the sulphatase peak was arried out before pooling the appropriate tubes. Solid sodium hloride (1 mg ~ m-~) and bovine serum albumin (2 mg ~ m-~) were added to the enzyme solution before storing (- 2 "C). Ejfet of NaCI, ph and Phosphate. Determination of K, The effet of sodium hloride onentration was estimated in Tris buffer (.2 mol dm-3, ph 7.5) over the range mol dm-3 NaC1.

4 5 Glyosulphatase from Pseudomonas arrageenovora Ativity of the purified sulphatase against ph was measured over the range ph using Tris/ imidazole buffer (.5 mol dm-3 Tris,.5 mol dm-3 imidazole,.25 mol dm-3 sodium hloride). Rates of desulphation of neoarrabiose 4--sulphate were determined over a range of substrate onentration ( mmol dm-3) in Tris buffer (.2 rnol dmp3 Tris,.25 mol dmp3 sodium hloride, ph 7.5). The K, value was alulated by regression analysis of the reiproal rate and reiproal substrate onentration. Phosphate-free neoarrabiose 4--sulphate (25 mg galatose ontent) was further prepared for this experiment by gel filtration on Sephadex G-1 (18 x 1.65 m) equilibrated with the assay buffer. The effet of inorgani phosphate (.5 mol dm-3) was examined on the enzymi desulphation of neoarrabiose 4--sulphate (.15 mmol dm-3) under assay buffer onditions. Glyosulphatase Desulphation of Neoarrabiose 4--(3 SJSulphate and Neoarratetraose 4--(35S]Sulphate Disaharide (4.7 pmol, 8.8 x 1 ounts min- ) or tetrasaharide (2.3 pmol, 7.2 x 1 ounts min- ) in Tris buffer (8. m,.1 rnol dmp3 Tris,.25 mol dm-3 sodium hloride, ph 7.5) was inubated (25 C with purified glyosulphatase (3 pg). Samples (.2 m3) were taken at intervals, mixed with aqueous trihloroaeti aid (1 % w/v,.5 m3) and the released inorgani [3 S]sulphate estimated by the sulphatase assay. At 6 min the remaining inubation mixtures were frozen and lyophilized. Frationation of the reation produts from the disaharide inubation was by gel filtration on a olumn of Sephadex G-15 (98 x 1.45 m) equilibrated with ammonium aetate buffer (.1 mol dm-3, ph 8.8). Frations were assayed for arbohydrate and radioativity. In the ase of the tetrasaharide gel filtration on Sephadex G-15 was followed by paper eletrophoresis. Eletrophoretograms were examined for sugars (silver nitrate reagent) and radioativity. Preparation of Reation Produts for I3C-NMR Spetrosopy Neoarrabiose 4--sulphate (.51 mmol, 185 mg galatose ontent, phosphate-free) in imidazole buffer (2m3,.5 mol dmp3, ph 7.5) was inubated (25 C) with purified glyosulphatase (3 pg). At zero time an aliquot of the reation mixture was removed and mixed with [3 S]disaharide (2.9 pmol, 4 x 1 ounts min-i). The progress of desulphation was followed by assaying inorgani [3 S]sulphate. At 9 h, when the reation was estimated to be 8 % omplete, the inubation mixture was frozen, lyophilized and applied in deionized water to a olumn of Sephadex G-1 (18 x 1.65 m) equilibrated with aetate buffer (.1 mol dmp3 sodium hloride,.25 mol dm-3 SOdium aetate, ph 5.). Frations ontaining the disaharide were pooled, lyophilized and examined by 13C-NMR spetrosopy. Further purifiation of the sample to remove remaining sulphated neoarrabiose was by passage through a olumn of AG 1x8 hloride (14 x 1. m). Spetra were ompared with one of neoarrabiose 4--sulphate. Neoarratetraose 4--sulphate (.57 mmol, 49 mg galatose ontent) was treated with glyosulphatase as desribed for the disaharide. At 5 h the reation was estimated to be 95 % omplete, the reation mixture frozen, lyophilized and applied to a olumn of Sephadex G-15 (116 x 2.4 m) equilibrated with aetate buffer as desribed for the disaharide. Frations ontaining the tetrasaharide were pooled, lyophilized and examined by I3CNMR spetrosopy. In both experiments, samples were taken at zero time and upon ompletion for 3,6-anhydro sugar determination. RESULTS Purifiation ~f(~ S]Oligosaharides Elution profiles for the purifiation steps of 3 Slabelled substrates (Fig. 1) indiate that after two gel filtrations radiohemially pure oligosaharides were obtained. Signifiant quantities of impurities whih o-elute from the Sephadex G-25 olumn were resolved from the sugars when re-run on Sephadex G-15 equilibrated with the buffer speified. Thus in the ase of the disaharide, a trae of inorgani [3 S]sulphate, whih eluted within the disaharide peak in the first gel filtration, was ompletely resolved on Sephadex G-15 along with any remaining inorgani phosphate. Similarly, another labelled impurity was separated from the tetrasaharide (frations 31-35, Fig. 1 C). These steps also preluded the red algal produt, holine -sulphate [2] from ontaminating the purified oligosaharides. In the partiular preparation desribed the yields of disaharide and tetrasaharide were respetively 21 mg and 21 mg (galatose ontent) at a speifi ativity of 3.5 x 1 ounts min- mol- galatose. Purifiation of Glyosulphatase Chromatography on Sepharyl S-2 of soluble material from disrupted Pseudomonas arrageenovora resolved the sulphatase ativity from any x-arrageenase (Fig.2A). In the six preparations so far arried out in our laboratory, the sulphatase onsistently eluted as a disrete peak whereas the x-arrageenase peak tended to spread from the leading edge. Under idential buffer onditions the purified x-arrageenase

5 M. W. MLean and F. B. Williamson o.8 E 4.6 T.4.4 E 5 m a, W m L " n - Fig. 1, Purifiation oj [35S]oligosi~~l~uri/i~.~ froi~ x-.url.igreiiri.se digestion of utzfia,tionated [35S],urr.ageenan(.54 g) )om Chondrus rispus. (A) Initial hromatography of oligosaharides on Sephadex G-25 (77 x 1.45 m). (B) Further purifiation of disaharide on Sephadex G-15 (98 x 1.45 m). (C) Further purifiation of tetrasaharide on Sephadex G-15 (98 x 1.45 m). Total arbohydrate (@), radioativity (O), and inorgani phosphate (----) ran as a disrete peak although retarded for its moleular size. During hromatography of the rude extrat the ion-exhange property of Sepharyl S-2 [21] may have ompeted with exluded material and resulted in the observed leading-edge spreading. Ion-exhange hromatography with DEAE-Sepharose CL-6B of the desalted sulphatase peak from the Sepharyl S-2 olumn resolved the ativity into a peak eluting at the beginning of the salt gradient (Fig. 2 B). Dodeylsulphate/polyarylamide gel ele pool Fig. 2. Purifiation of glyosulphatase. (A) Gel filtration of soluble extrat of Pseudomonas arrageenovora on Sepharyl S-2 (1 15 x 2.4 m). (B) Ion-exhange hromatography on DEAE-Sepharose CL-6B (7 x 1.1 m) of desalted glyosulphatase peak from Sepharyl S-2 elution. Glyosulphatase ativity (e), il-arrageenase ativity () and,428 ", (-- ). (C) Dodeylsulphate/polyarylamide gel eletrophoresis of frations of DEAE-Sepharose CL-6B elution, and pooled frations as quoted in Table 1, step 3

6 ~ 8 52 Glyosulphatase from Pseudomonas arrageenovoru Table 1. Purgiation of'glyosulphatase Step Volume Protein x Glyosulphatase Purifiation Yield ativity m3 mg units 1, Supernatant, disrupted ells Sepharyl S DEAE-Sepharose fold % trophoresis of frations ontaining the sulphatase peak indiated that one major protein speies was present and orresponds exatly to the peak observed in the ativity profile and A28 nm (Fig. 2C). This purifiation has been repeated with no signifiant differenes with another bath eah of Sepharyl S-2 and DEAE- Sepharose CL-6B. Thus in routine preparations assays are onveniently made over the first A28 nm peak eluting from the salt gradient. Quantifiation for the purifiation desribed is summarized in Table 1. x 2.s z._ > Properties of Purified Glyosulphatase Moleular weight as determined by dodeylsulphate/polyarylamide gel eletrophoresis was 55. This estimation would be that of a subunit if the native enzyme has quarternary struture. Profiles of ativity against NaCl and ph are depited in Fig.3. Maximal ativity was observed at NaCl onentration mol dmp3 and ph 7.. The K,,, value alulated for the disaharide is.21 mmol dmp3 (Fig.3C). At.15 mmol dm-3 neoarrabiose 4-O-sulphate,.5 mmol dmp3 phosphate inhibited the rate of desulphation by 5 %. n o.1 a a5 Sodium hloride (mol dri3) I B E 25 1 Ation of Purfied Glyosulphatase on [ 35 S]Oligosaharides Release of inorgani [35S]mlphate with respet to time (Fig. 4A) tended towards ompletion in the ase of the disaharide and 5 % for the tetrasaharide. Sephadex G-15 hromatography of neoarrabiose 4-O-[35S]sulphate reation produts (Fig. 4B) resolved inorgani [35S]sulphate from the resulting disaharide. The elution profile indiates that desulphation is quantitative, no radioativity being deteted in the arbohydrate peak. Sephadex G-I 5 hromatography of the [35S]tetrasaharide desulphation produts (Fig. 4C) resulted in o-elution of arbohydrate and radioativity. Highvoltage paper eletrophoresis resolved the omponents into a single, radioative arbohydrate speies and inorgani [35S]sulphate (Fig. 4D). These results indiate the quantitative onversion of the tetrasaharide to a monosulphated derivative t PH r 3. - C I I I I I I I l/[s] (rnrnol-'drn3) Fig. 3. (A) Avtivity qf purified glyosulphatase 1vitli respet to NuC'I onentration over the range.-1. mol dm-3 hujfered with Tris (.2 niol dm-3, ph 7.5). (B) Ativity ofpurifird glyosulphatase with respet toph over the rangeph in Tris/imidazole huffer ontaining sodium hloride (.25 mol dm-'). (C) Detevnination of K, jilr neoarrabiose 4--sulphate. K, =.21 mnzol dm-3,/iom regression analysis

7 M. W. MLean and F. B. Williamson Time (rnin) D i ii iii IV I II 111 IV I A -.2 " Ln h E -.lo.s L r al w Fig. 4. Ation ofpurified glyosulphatase on [35S]oligosaharides.(A) Time ourses of loss of [35S]sulpl~ate from labelled substrates, neoarrabiose 4--[35S]sulphate () neoarratetraose 4--[35S]sulphate ().(B) Chromatography on Sephadex (3-15 (98 x 1.45 m) of the reation produts of the disaharide time ourse. Total arbohydrate (O), radioativity (O), area under urves normalized. (C) Sephadex G-I 5 hromatography of produts of tetrasaharide time ourse. Total arbohydrate (O), radioativity (O), area under urves normalized. (D) Paper eletrophoretogram of lyophilized frations 35-4 of Sephadex G-I 5 hromatography of tetrasaharide produts. Left: detetion by silver nitrate reagent, Right: X-ray film autoradiograph. (i) Neoarratetraose 4--[35S]sulphate, (ii) neoarrabiose 4--[35SJsulphate, (iii) lyophilized frations 35-4 of Sephadex (3-15 hromatography of [35S]tetrasaharide,(iv) (i) + (iii) l3 C-NMR Spetrosopy of Reation Produts Spetra of neoarrabiose 4--sulphate and neoarratetraose 4--sulphate together with spetra of the orresponding glyosulphatase reation produts are seen in Fig. 5. Differenes between the disaharide spetra inlude on desulphation the upfield shift (.3 ppm) of the anomeri resonane of the anhydro sugar from 95.6ppm and the appearane or a new peak at 68. ppm (arrowed in Fig. 5 A) orresponding in area to a resonane in the a form of the reduing sugar. An inspetion of the tetrasaharide spetra reveals the resolution of the oinident anhydro ano- meri resonanes into a doublet, the new peak being.3 ppm upfield of the resonane orresponding to to the original peak (Fig.5B). In the ase of the tetrasaharide, a peak analogous to that arrowed in the spetrum of the desulphated disaharide is not observed. In addition, the resonane arrowed in Fig. 5B (attributable to a arbon in the reduing sugar) persists after glyosulphatase digestion. These observations are onsistent with the reduing sugar of the tetrasaharide remaining unhanged and therefore with the sulphate loss being from the residue proximal to the non-reduing end.

8 54 Glyosulphatase from Pseudomonas rarrageenovora A J L Fig. 6. Struture of partially desulphated produt of glyosulphaias ation on neoarrutetraose 4--sulphate as dedued from the I3C- NMR data L I I Chemial shift (ppm) Chemial shift (ppm) Fig. 5. "C-NMR spetra of' oligosaharides and reation produts of desulphated oligosaharides. (A) Neoarrabiose 4--sulphate (top) and desulphated produt (bottom). (B) Neoarratetraose 4--sulphate (top) and partially desulphated produt (bottom). Chemial shift, 6, relative to tetramethylsilane DISCUSSION A glyosulphatase has been purified from Pseudomonas arrageenovora and partially haraterized. Two substrates have been investigated, namely neoarrabiose 4--sulphate and neoarratetraose 4-- sulphate, the two simplest degradation produts of x-arrageenase digestion of x-arrageenan. In a previous study of this enzyme by Weigl and Yaphe [5], neoarrabiose 4--sulphate as well as galatose 4-- sulphate and galatose 6--sulphate were tested as potential substrates for a partially purified glyosulphatase. These authors reported ativity with neoarrabiose 4--sulphate and galatose 6--sulphate but not with galatose 4--sulphate., Although desulphation of neoarrabiose 4--sulphate yields neoarrabiose, an analogous produt is not observed in the ase of neoarratetraose 4--sulphate, whih is only partially desulphated. Interpretation of the I3C-NMR data suggests that the ester hydrolysed is the one proximal to the non-reduing end (Fig. 6). Aumulation of disaharide and tetrasaharide on exhaustive digestion of x-arrageenan (.f. Fig. 1 A) indiates a slow onversion of the tetrasaharide to disaharide by x-arrageenase. In ontrast, the partial desulphation of neoarratetraose 4--sulphate proeeds rapidly (Fig. 4A). These onsiderations suggest that the baterium might assimilate the disulphated tetrasaharide before degradation by the x-arrageenase. A P-galatosidase would at some stage leave the unique linkage in the tetrasaharide whih is absent in the disaharide. Both CI and P-galatosidase ativities are easily detetible in the soluble fration from disrupted Pseudomonas urrageenovora by a hallenge with the respetive 4-nitrophenyl galatopyranoside. Partially desulphated neoarratetraose 4--sulphate (Fig.6) may be regarded as a model struture for the desulphated x-arrageenan previously prepared by the method of Usov et al. [22] and studied with 13C-NMR by Bhattaharjee and Yaphe [23]. The 13C-NMR spetrum (Fig.5) is in good agreement with that desribed in the latter referene. A hemial shift of 95.3 ppm reorded for the a-anomeri arbon adjaent to a desulphated galatose in a %-arrageenan oligosaharide is still about 4ppm upfield of the standard, methyl 3,6-anhydro-a-~-galatopyranoside (99.1 ppm). In the analogous agar derivative, neoagarobiose, in whih the anhydro sugar is of L-galato onfiguration, the orresponding arbon atom resonates at 99.3 ppm [24], a shift in good agreement with that of the methyl 3,6-anhydro-a-~-galatopyranoside. A tentative interpretation would be that the D-galatose moiety of the arrageenan derivative interats with the 3,6-anhydro-~-galatopyranoside to perturb the latter's freely assumed onformation. The authors aknowledge Mr Rihard Thomas for tehnial assistane and Mr David Sim for photographi assistane. We thank the Siene Researh Counil in assoiation with the Philip Lyle Memorial Laboratory, Reading, for the maintenane of one of us (M. ML.).

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