Human liver N-acetylglucosamine-6-sulphate sulphatase. Catalytic properties.

Kinetic parameters (Km and kcat.) of the two major forms (A and B) and a minor form (C) of human liver N-acetylglucosamine-6-sulphate sulphatase [Freeman, Clements & Hopwood (1987) Biochem. J. 246, 347-354] were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin, heparan sulphate and keratan sulphate. Enzyme activity is highly specific towards glucosamine 6-sulphate or glucose 6-sulphate residues. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are hydrolysed with catalytic efficiencies up to 3900 times above that observed for the monosaccharide substrate N-acetylglucosamine 6-sulphate. Forms A and B both desulphate substrates derived from keratan sulphate and heparin. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue 6-carboxy and 2-sulphate ester groups for heparin-derived substrates and penultimate-residue 6-sulphate ester groups for keratan sulphate-derived substrates. The 4-hydroxy group of the N-acetylglucosamine 6-sulphate or the 2-sulphaminoglucosamine 6-sulphate under enzymic attack is involved in the catalytic mechanism. The presence of a 2-amino group in place of a 2-acetamido or a 2-sulphoamino group considerably decreases the catalytic efficiency of the sulphatase, particularly in the absence of a penultimate-aglycone-residue 6-carboxy group. Both forms A and B are exo-enzymes, since activity towards internal sulphate ester bonds was not observed. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone 2-sulphate ester, 6-carboxy group and 6-sulphate ester groups on the glucosamine 6-sulphate residue under attack considerably affects the pH response. Sulphate and phosphate ions are potent inhibitors of enzyme activity.     

Cleavage of the (1 goes to 3)-2-acetamido-2-deoxy-beta-D-glucopyranosyl linkage present in keratan sulfate. The A and B isoenzymes of human liver hexosaminidase (EC 3.2.1.30)

The disaccharide 2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 goes to 3)-D-[1-3H]-galactitol, prepared from keratan sulfate, was rapidly hydrolyzed by the A and B isoenzymes of normal human liver hexosaminidase (EC 3.2.1.30), and by the B isoenzyme prepared from the liver of a patient who had died of Tay-Sachs disease. The disaccharide substrate was also hydrolyzed by extracts of normal, cultured-skin fibroblasts, and fibroblasts of patients with Tay-Sachs disease, whereas it was not hydrolyzed by fibroblast extracts of patients with Sandhoff disease. Thus, effective degradation of keratan sulfate, secondary to a defect of the beta subunits present in the A and B isoenzymes of hexosaminidase, may contribute to the appearance of skeletal lesions in patients affected by Sandhoff disease.     

Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta

1. N-Acetylgalactosamine 6-sulphate sulphatase was purified about 20000-fold from the soluble extract of human placenta with N-acetylgalactosamine 6-sulphate-glucuronic acid-N-acetyl[1-(3)H]galactosaminitol 6-sulphate as substrate in the activity assay. The enzyme appears to be a glycoprotein with a mol.wt. of about 100000 as determined by gel filtration. On gel electrophoresis in the presence of sodium dodecyl sulphate the major protein band had a mol.wt. of 78000. Variable charge heterogeneity was observed in several enzyme preparations. 2. The purified enzyme released up to one sulphate molecule from the disulphated trisaccharide. It was active towards N-acetylgalactosamine 6-sulphate and exhibited no measurable N-acetylglucosamine 6-sulphate sulphatase or any other known lysosomal sulphatase activity. Hydrolysis of [1-(3)H]galactitol 6-sulphate was achieved by incubation neither with a crude nor with a purified enzyme preparation. Chondroitin 6-sulphate and keratan sulphate, as well as heparin and heparan sulphate, served as competitive inhibitors of the enzyme. 3. Purified N-acetylgalactosamine 6-sulphate sulphatase activity was optimal at pH4.9 and 4.4 when assayed in 0.02m-sodium acetate buffer and at pH4.2 and 5.2 in 0.1m-sodium acetate buffer. A single pH-optimum at pH4.8 was observed for the crude enzyme and for the purified enzyme after mild periodate treatment. The sulphatase activity was inhibited by a variety of anions and cations and activated by thiol-specific and thiol reagents.     

Impaired degradation of keratan sulphate by Morquio A fibroblasts.

Upon incubation of keratan [35S]sulphate with normal fibroblasts both [35S]sulphate and N-acetylglucosamine 6-[35S]sulphate are liberated. From the products obtained after digestion with various mutant fibroblasts and with purified N-acetylgalactosamine 6-sulphate sulphatase we suggest that (i) [35S]sulphate is released almost exclusively from galactose 6-sulphate residues; (ii) N-acetylgalactosamine 6-sulphate sulphatase exhibits galactose 6-sulphate sulphatase activity; (iii) both sulphatase activities are deficient in Morquio disease type A.     

Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties.

Human iduronate-2-sulphatase (EC 3.1.6.13), which is involved in the lysosomal degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate, was purified more than 500,000-fold in 5% yield from liver with a six-step column procedure, which consisted of a concanavalin A-Sepharose-Blue A-agarose coupled step, chromatofocusing, gel filtration on TSK HW 50S-Fractogel, hydrophobic separation on phenyl-Sepharose CL-4B and size separation on TSK G3000SW Ultrapac. Two major forms were identified. Form A and form B, with pI values of 4.5 and less than 4.0 respectively, separated at the chromatofocusing step in approximately equal amounts of recovered enzyme activity. By gel-filtration methods form A had a native molecular mass in the range 42-65 kDa. When analysed by SDS/PAGE, dithioerythritol-reduced and non-reduced form A and form B consistently contained polypeptides of molecular masses 42 kDa and 14 kDa. Iduronate-2-sulphatase was purified from human kidney, placenta and lung, and form A was shown to have similar native molecular mass and subunit components to those observed for liver enzyme. Both forms of liver iduronate-2-sulphatase were active towards a variety of substrates derived from heparin and dermatan sulphate. Kinetic parameters (Km and Kcat) of form A were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparan sulphate, heparin and dermatan sulphate. Substrate with 6-sulphate esters on the aglycone residue adjacent to the iduronic acid 2-sulphate residue being attack were hydrolysed with catalytic efficiencies up to 200 times above that observed for the simplest disaccharide substrate without a 6-sulphated aglycone residue. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure, substrate concentration, buffer type and the presence of other proteins. Sulphate and phosphate ions and a number of substrate and product analogues were potent inhibitor of form A and form B enzyme activities.     

Multiple forms of 2-deoxy-D-glucoside-2-sulphamate sulphohydrolase from human placenta.

2-Deoxyglucoside-2-sulphamate sulphohydrolase was purified about 10 000-fold from the soluble extract of human placenta by using as substrate [N-sulpho-35S]heparin. Differently charged enzyme forms were observed on chromatography on DEAE-cellulose, all of which had an apparent mol.wt. of 110 000 as determined by gel filtration. By using immobilized heparan sulphate as affinity matrix the sulphamate sulphohydrolase could be separated into two forms, a minor one with low and a major one with high affinity for the adsorbent. When tested with [N-sulpho-35S]heparan sulphate the low-affinity form had a Km of 0.2 mM, and the high-affinity form a Km of 0.03 mM. Both forms exhibited the same Km of 10 microM towards [N-sulpho-35S]heparin and were equally well adsorbed to immobilized heparin. The two forms could be distinguished by their pH-optima and by the influence of KCl on heparan sulphate sulphohydrolase activity.     

The effect of benzyl beta-D-xyloside on keratan sulphate synthesis in ox articular cartilage

Cartilage from adult bovine hock joints was incubated with [3H]galactose or [35S]sulphate in the presence of benzyl beta-D-xyloside. Radioisotope incorporation into proteoglycan was inhibited by the xyloside; the magnitude of this inhibition depended on the concentration of xyloside used. With 0.2mM xyloside radioisotope incorporation into keratan sulphate was not altered but inhibition was observed at xyloside concentrations of 1.0mM or higher. The decrease in radioisotope incorporation into keratan sulphate in the presence of 1.0mM benzyl beta-xyloside was directly related to a reduction in the average length of the keratan sulphate chains. This effect of beta-xyloside on keratan sulphate biosynthesis was markedly different from its effect on chondroitin sulphate biosynthesis.     

A [3H]lysine-containing synthetic peptide substrate for human protocollagen lysyl hydroxylase

A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the alpha chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to N alpha-t-butyloxycarbonyl-N epsilon-o-chlorobenzyloxycarbonyl [3H]lysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.     

Absence of keratan sulphate from skeletal tissues of mouse and rat.

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.     

Dimeric nature and amino acid compositions of homogeneous canine prostatic human liver and rat liver acid phosphatase isoenzymes. Specificity and pH-dependence of the canine enzyme.

Two isoenzymes of rat liver acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) have been purified to homogeneity, at least one of these for the first time. Both of the rat liver isoenzymes have identical specific activities towards p-nitrophenyl phosphate. Molecular weights of the native enzymes are 92 000 for rat liver isoenzyme I and 93 000 for isoenzyme II, while the subunit molecular weights are 51 000 and 52 000 respectively. Data on substrate specificity and pH dependence are presented for the homogeneous canine prostatic enzyme, which is also isolated as a dimeric enzyme of (native) molecular weight 89 000. Carbohydrate analysis data are presented for canine prostatic acid phosphatase and it is further noted that both isoenzymes of rat liver acid phosphatase are also glycoproteins. The amino acid compositions of the two rat liver isoenzymes are presented together with those of the similar dimeric acid phosphatase of human liver and of canine prostate. Comparison of these results with published data for the amino acid composition of human prostatic acid phosphatase shows substantial similarities. However, significant differences are seen in the amino acid composition of rat liver acid phosphatase isoenzyme I as compared to a previous literature report. Most notably, 17 histidine residues are found per mol of isoenzyme I and 18 for isoenzyme II.     

Purification of carboxypeptidase B from human pancreas.

Carboxypeptidase B of the human pancreas was purified by chromatography on DEAE-cellulose and CM-cellulose columns. Two forms of the enzyme, named carboxypeptidase B1 and B2, were separated. They have similar mol.wts. (34250 +/- 590) as established by polyacrylamide-gel disc electrophoresis and by gel filtration. Carboxypeptidase B2 migrates further towards the anode in disc electrophoresis. When the amino acid content of the enzymes was analysed, carboxypeptidase B2 had four more glycine and three more aspartic acid residues than had form B1. The amino acid sequence of the human carboxypeptidase B1 differs from that of the bovine enzyme only in two places in the N-terminal 20-amino-acid sequence. The N-terminal amino acid in carboxypeptidase B1 and B2 is alanine. The peptide 'map' of the tryptic digest of carboxypeptidase B1 contained more peptides than did that of form B2. The Km, the Vmax. and the pH optimum of the cleavage of the peptide substrate hippurylarginine and the ester substrate hippurylargininic acid were similar for both enzymes. CoCl2 accelerated the peptidase activity, and cadmium acetate enhanced the esterase activity, of human carboxypeptidases B1 and B2. Urea and sodium dodecyl sulphate inhibited the enzymes.     

Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue.

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].     

The enzymatic sulphation of heparan sulphate by hen's uterus

A sulphotransferase preparation from hen's uterus catalysed the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to N-desulphated heparan sulphate, heparan sulphate, N-desulphated heparin and dermatan sulphate. Heparin, chondroitin sulphate and hyaluronic acid were inactive as substrates for the enzyme. N-desulphated heparin was a much poorer substrate for the enzyme than N-desulphated heparan sulphate suggesting that properties of the substrate other than available glucosaminyl residues influenced enzyme activity. N-acetylation of N-desulphated heparin and N-desulphated heparan sulphate reduced their sulphate acceptor properties so it was unlikely that the N-acetyl groups of heparan sulphate facilitated its sulphatiion. Direct evidence for the transfer of [³⁵S]sulphate to amino groups of N-desulphated haparan sulphate was obtained by subsequent isolation of glucosamine $\text{N-[}{}^{35}\text{S}\text{]}\text{sulphate}$ from heparan [³⁵S]sulphate product. This was made possible through the use of a flavobacterial enzyme preparation which contained “heparitinase” activity but had been essentially freed of sulphatases. Attempts to transfer [³⁵S]sulphate to glucosamine or N-acetylglucosamine were unsuccessfull.     

Biosynthesis of dermatan sulphate. Loss of C-5 hydrogen during conversion of D-glucuronate to L-iduronate.

The formation of L-iduronic acid during biosynthesis of dermatan sulphate has been studied in culture human fibroblasts and in microsomes from the same cells. The cells were incubated with D-[14C]glucose and D-[5-3H]glucose for 72 h. The [14C,3H]dermatan sulphate was hydrolysed and the disaccharides obtained were acetylated and separated by ion-exchange chromatography. The ratio of 3H/14C was 0.36 for N-acetyldermosine and 1.36 N-acetylchondrosine. A microsomal preparation from the fibroblasts was incubated with UDP-D-[5-3H]glucuronic acid, UDP-D-[14C]glucuronic acid, UDP-N-acetyl-D-galactosamine and 3'-phospho-5'-adenylyl sulphate. The polymeric products were separated into nonsulphated and sulphated components which had 3H/14C ratios of 0.51 and 0.20 and contained 9% and 70% of their uronosyl residues in the L-ido-configuration, respectively. Chondroitinase-AC digestion of these polymers liberated all of the remaining 3H activity. Hydrolysis and N-acetylation followed by paper chromatography showed that the L-iduronic acid-containing products were devoid of 3H. The data obtained indicate that the epimerization of D-glucuronosyl to L-iduronosyl residues during biosynthesis of dermatan sulphate involves an abstraction of the C-5 hydrogen of the uronosyl residue.     

Arylsulphatase activity of corn (Zea mays L.) seedling roots

The crude lysosomal fraction of corn seedling root tips contains an arylsulphatase (E.C. 3.1.6.1) which hydrolysed p-nitrophenyl sulphate at pH 8.0 but had no activity towards p-nitrocatechol sulphate. The Km value for p-nitrophenyl sulphate was 1.24 mM. The hydrolysis of p-nitrophenyl sulphate was linear up to 2 h and the rate was proportional to the amount of enzyme added. The enzyme was strongly inhibited by cyanide, fluoride and phosphate ions and did not resemble the arylsulphatases of bacterial and animal origin.     

Purification and some properties of the D-lactate-2-sulphatase of Pseudomonas syringae GG.

A soil bacterium grown on propan-2-yl sulphate as sole source of carbon and sulphur yielded extracts containing an enzyme capable of liberating sulphate from racemic lactate-2-sulphate. The enzyme was purified to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, batch treatment with DEAE-cellulose, and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was monomeric with an Mr of 55 000-60 000. The enzyme activity was specific for D-lactate-2-sulphate (Km 6.6 nM; maximal specific activity 14.3 mumol/min per mg of protein) and showed no activity towards the L-isomer. The products of the enzyme's action were inorganic sulphate and D-lactate which were released in equimolar amounts and stoicheiometrically with the amount of ester hydrolysed. No L-lactate was formed. Retention of configuration implied cleavage of the O-S bond of the C-O-S ester link and this was confirmed by 18O-incorporation experiments in which 18O from 18O-enriched water in the incubation medium was incorporated exclusively and quantitatively into inorganic sulphate. Only two other esters (serine-O-sulphate and p-nitrophenyl sulphate) of a total of 29 compounds tested were substrates for the enzyme. D-Lactate, L-lactate-2-sulphate and the substrate analogues glycollate-2-sulphate and butyrate-2-sulphate were significantly inhibitory.     

The structure of keratan sulphates from various sources.

Quantitative structural comparisons were made between keratan sulphates isolated from various sources, namely pig nucleus pulposus, bovine cornea, and the costal cartilages of children, a young adult with Marfan syndrome and of old human autopsies. In human costal cartilage the amount of keratan sulphate increases markedly with age, although total mucopolysaccharide decreases to some extent, concomitant with a decrease in chondroitin 4-sulphate and an increase in chondroitin 6-sulphate. Comparison of molecular weights estimated by gel chromatography with those calculated from the molar ratio of galactose to mannose indicates that keratan sulphates of human costal cartilages of children and of a young adult with Marfan syndrome, and of pig nucleus pulposus, contain one mannose residue per chain, whereas keratan sulphates of old human costal cartilage and of bovine cornea contain one to two, and two, per chain respectively. After mild acid-catalysed desulphation of pig nucleus pulposus keratan sulphate, approx. 12% of the mucopolysaccharide aggregates irreversibly once the water is removed from the polysaccharide. The following conclusions have been drawn from a methylation analysis of keratan sulphates of various sources, aided by g.l.c.-mass spectrometry. (1) Fucose and N-acetylneuraminic acid are non-reducing terminal residues and the sialic acid is linked to the 3-position of galactose residues. (2) Pig nucleus pulposus keratan sulphate has approximately 4 non-reducing terminal groups per molecule and appears to be slightly less branched than the costal-cartilage keratan sulphate of children. The branching in human costal-cartilage keratan sulphates decreases with age. Bovine corneal keratan sulphate appears to be unbranched. (3) Mannose residues are linked by 3 different substituents in human costal-cartilage and bovine corneal keratan sulphates, and by two different substituents in pig nucleus pulposus keratan sulphate. (4) The sulphate ester groups are all on the 6-position of N-acetyl-glucosamine and galactose residues. The degree of sulphation increases with age in costal keratan sulphates with the increase mainly of the galactose 6-sulphate residues.     

Characterization of the keratan sulphate proteoglycans from bovine corneal stroma.

The keratan sulphate proteoglycans that can be prepared from bovine corneal stroma [Axelsson & Heineg?rd (1975) Biochem. J. 145, 491-500] were characterized by gel chromatography, gel electrophoresis and analytical ultracentrifugation in associative (0.6 M-NaCl) and dissociative (6M-guanidinum chloride) solvents. The proteoglycans aggreagated at low salt concentrations and pH. The weight-average molecular weight of the monomer proteoglycans was established. Keratan sulphate peptides and oligosaccharide peptides were isolated after proteolysis. Their composition indicated that both are linked to protein via asparagine residues. A tentative model for corneal keratan sulphate proteoglycans is suggested.     

Studies of a monoclonal antibody to skeletal keratan sulphate. Importance of antibody valency.

A mouse monoclonal antibody (AN9P1) to keratan sulphate is described. In a competitive-inhibition solution-phase radioimmunoassay employing 125I-labelled intact proteoglycan, it reacts preferentially with keratan sulphate bound to the core protein of adult human articular-cartilage proteoglycan and to a much lesser degree with keratan sulphate purified from this proteoglycan. Proteolytic cleavage of the proteoglycan by pepsin and trypsin has little effect on antibody binding, but treatment with papain decreases binding considerably and more than does treatment with keratanase. An even greater decrease in binding is observed after treatment with alkaline borohydride. A comparison of binding of antibody AN9P1 with that of another previously described monoclonal antibody, 1/20/5-D-4, to keratan sulphate [Caterson, Christner & Baker (1983) J. Biol. Chem. 258, 8848-8854] revealed similar binding characteristics, both showing much diminished binding after papain digestion of proteoglycan and even less with purified skeletal keratan sulphate. Removal of the Fc piece of antibody AN9P1 had no significant effect on the differential binding of divalent F(ab')2 fragment to proteoglycan, to papain-digested proteoglycan and to keratan sulphate, although there was a small decrease in binding to papain-digested proteoglycan. Conversion of the antibody into univalent Fab fragment with removal of the Fc piece resulted in diminished binding to proteoglycan, compared with that observed with IgG, and in enhanced binding to free keratan sulphate and to papain-digested proteoglycan. These results suggest that close proximity of keratan sulphate chains on the core protein of proteoglycans favours preferential reactivity of bivalent antibody with these species through cross-bridging of chains by antibody. Conversely, much decreased binding to keratan sulphate on proteoglycan core-protein fragments and to free keratan sulphate results from a lack of close proximity of keratan sulphate. By using univalent Fab fragment in these assays these differences in binding are minimized by preventing cross-bridging and thereby enhancing detection of smaller fragments without sacrificing too much sensitivity of detection of larger proteoglycan species. The persistent preferential binding of Fab fragment to proteoglycan is probably in part the result of the increased epitope density in the intact molecule compared with keratan sulphate in a more disperse form.     

The active site of Sulfolobus solfataricus aspartate aminotransferase

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus binds pyridoxal 5' phosphate, via an aldimine bond, with Lys-241. This residue has been identified by reducing the enzyme in the pyridoxal form with sodium cyanoboro[3H]hydride and sequencing the specifically labeled peptic peptides. The amino acid sequence centered around the coenzyme binding site is highly conserved between thermophilic aspartate aminotransferases and differs from that found in mesophilic isoenzymes. An alignment of aspartate aminotransferase from Sulfolobus solfataricus with mesophilic isoenzymes, attempted in spite of the low degree of similarity, was confirmed by the correspondence between pyridoxal 5' phosphate binding residues. Using this alignment it was possible to insert the archaebacterial aspartate aminotransferase into a subclass, subclass I, of pyridoxal 5' phosphate binding enzymes comprising mesophilic aspartate aminotransferases, tyrosine aminotransferases and histidinol phosphate aminotransferases. These enzymes share 12 invariant amino acids most of which interact with the coenzyme or with the substrates. Some enzymes of subclass I and in particular aspartate aminotransferase from Sulfolobus solfataricus, lack a positively charged residue, corresponding to Arg-292, which in pig cytosolic aspartate aminotransferase interacts with the distal carboxylate of the substrates (and determines the specificity towards dicarboxylic acids). It was confirmed that aspartate aminotransferase from Sulfolobus solfataricus does not possess any arginine residue exposed to chemical modifications responsible for the binding of omega-carboxylate of the substrates. Furthermore, it has been found that aspartate aminotransferase from Sulfolobus solfataricus is fairly active when alanine is used as substrate and that this activity is not affected by the presence of formate. The KM value of the thermophilic aspartate aminotransferase towards alanine is at least one order of magnitude lower than that of the mesophilic analogue enzymes.