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N-Acetylgalactosamine-6-Sulfate Sulfatase in Man. Absence of the Enzyme in Morquio Disease

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N-Acetylgalactosamine-6-Sulfate Sulfatase in Man. Absence of the Enzyme in Morquio Disease N-acetylgalactosamine-6-sulfate sulfatase in man. Absence of the enzyme in Morquio disease. J Singh, … , P Niebes, D Tavella J Clin Invest. 1976;57(4):1036-1040. https://doi.org/10.1172/JCI108345. Research Article Human N-acetylgalactosamine-6-sulfate sulfatase (6-sulfatase) activity is measured by using as a substrate a sulfated tetrasaccharide obtained by digesting purified chondroitin-6-sulfate (C-6-S) with testicular hyaluronidase. The amount of inorganic sulfate released is measured turbidimetrically. The enzyme from human kidney has a pH optimum of 4.8; its activity is augmented by low levels of NaCl and inhibited by phosphate and high levels of NaCl. Free glucuronate, acetylgalactosamine, inorganic sulfate, polymeric C-6-S, or tetrasaccharide obtained from chondroitin-4-sulfate do not affect the enzyme activity. The method may be used for the diagnosis of Morquio disease since extracts of Morquio fibroblasts are devoid of 6-sulfatase activity. Find the latest version: https://jci.me/108345/pdf N-Acetylgalactosamine-6-Sulfate Sulfatase in Man ABSENCE OF THE ENZYME IN MORQUIO DISEASE JAGAT SINGH, NICOLA Di FERRANTE, PAUL NIEBES, and DANIELA TAVELLA From the Departments of Biochemistry and Medicine, and the Division of Orthopedic Surgery of the Department of Surgery, Baylor College of Medicine, Houston, Texas 77025 and Zyma, S.A., Nyon, Switzerland A B S T R A C T Human N-acetylgalactosamine-6-sulfate measurement, and the need to ascertain whether the sulfatase (6-sulfatase) activity is measured by using as sulfate released was in position 4 or 6 of the galactos- a substrate a sulfated tetrasaccharide obtained by di- amine moieties make the method (1) rather laborious gesting purified chondroitin-6-sulfate (C-6-S) with tes- and not ideal for the routine assay of the enzyme ac- ticular hyaluronidase. The amount of inorganic sulfate tivity. Using a 6-sulfated tetrasaccharide as a substrate, released is measured turbidimetrically. The enzyme we have developed a simple method for the measurement from human kidney has a pH optimum of 4.8; its of 6-sulfatase activity. This report describes the prepara- activity is augmented by low levels of NaCl and in- tion of the substrate, the optimal conditions of the en- hibited by phosphate and high levels of NaCl. Free zyme assay as established with a crude enzyme prepara- glucuronate, acetylgalactosamine, inorganic sulfate, tion from human kidney, and the applicability of the polymeric C-6-S, or tetrasaccharide obtained from chon- method for the diagnosis of Morquio disease. droitin-4-sulfate do not affect the enzyme activity. The method may be used for the diagnosis of Morquio dis- METHODS ease since extracts of Morquio fibroblasts are devoid of Unsaturated 6-sulfated (A-Di-6S) and nonsulfated (A-Di- 6-sulfatase activity. OS) disaccharides, shark cartilage chondroitin-6-sulfate (C- 6-S), and whale cartilage chondroitin-4-sulfate were from Miles Laboratories, Inc., Miles Research Div., Elkhart, INTRODUCTION Ind.; bovine testicular hyaluronidase (3,000 USP U/mg) Matalon et al. (1) demonstrated the presence of chon- from Worthington Biochemical Corp., Freehold, N. J.; droitin sulfate N-acetylgalactosamine-6-sulfate sulfatase gelation from ICN Nutritional Biochemicals Div., Interna- ' tional Chemical & Nuclear Corp., Cleveland, Ohio; AG (6-sulfatase) in extracts of cultured human skin fibro- 1x2 (200400 mesh, Cl- form) and Bio-Gel A-0.5 m (100- blasts and have provided evidence that its deficiency is 200 mesh) from Bio-Rad Laboratories, Richmond, Calif. responsible for the pathogenesis of Morquio disease Dermatan sulfate from pig intestinal mucosa was a gift of (mucopolysaccharidosis IV). For the assay, Matalon et Dr. J. A. Cifonelli of University of Chicago. Tritiated hepa- rin fractions were prepared as described (2). al. (1) used biologically labeled polymeric chondroitin- Hexuronate (3), hexosanine (4), chloride (5), and re- 4/6-sulfate extracted from femural and tibial epiphyses ducing groups (6) were measured as previously published. of chick embryos or oligosaccharides derived from it. Inorganic sulfate was assayed as described under Assay of The required purification of the substrate, the short kidney enzyme by a modification of Dodgson's method (7). The sulfate content of the mucopolysaccharides was assayed half-life of the 35SO4 label, the need for electrophoretic by Dodgson and Price's method (8) after hydrolysis in 1 N separation of the released inorganic "5SO4 before its HCl for 5 h at 110'C. When polymeric C-6-S was tested as a possible substrate or inhibitor of the enzyme activity, Dr. Tavella was on leave of absence from the Pediatric or when enzyme assays were performed with urine con- Institute, Universita Cattolica, Roma, Italy. Dr. Niebes' centrates or extracts of leukocytes or fibroblasts, sulfate address is: Zyma, S. A., Nyon, Switzerland. was determined according to Saito et al. (9) (see Assay of Received for publication 20 October 1975 and in revised fibroblast enzyme); in this method (9), polymeric glycos- form 11 December 1975. aminoglycans are precipitated with cetylpyridinium chloride ' Abbreviations used in this paper: C-6-S, chondroitin-6- before addition of the gelatin-BaCl2 reagent. sulfate; 6-sulfatase, N-acetylgalactosamine-6-sulfate sulfa- Substrate preparation. 400 mg of shark cartilage C-6-S tase. dissolved in 8 ml of a pH 4.8, 0.125 M Tris-acetate buffer 1036 The Journal of Clinical Investigation Volume 57 April 1976* 1036-1040 containing 1.6 ;Lmol of NaN3 were incubated at 370C with 12,000 USP units of testicular hyaluronidase for 48 h, and its depolymerization was monitored turbidimetrically (10). Upon completion of incubation, the digest was boiled 12 A for 20 min to inactivate hyaluronidase and was clarified A by centrifugation. The supernate, which is the substrate, was 8 diluted with a pH 4.8, 0.125 M Tris-acetate buffer to give 32 ttmol hexuronate/ml, and stored frozen in portions until used. A portion of the degraded C-6-S was chromato- graphed on an AG 1 X 2 column (20 X 0.9 cm) packed with 0 water and eluted with a linear gradient of 0-3.0 M NaCt in water (Fig. 1). The molecular weight of the degraded C- 6-S was determined by gel filtration (Fig. 2) on a Bio- Gel A-0.5 m column (98 X 1 cm), packed and eluted with 0.025 M NaCl, as previously described (11-13). The column 0 40 80 was calibrated with various carbohydrates of known molecu- Elution Volume, mil lar weight (11) (Fig. 3) and its void volume was assessed FIGURE 2 Elution patterns of polymeric C-6-S (A) and with blue dextran 2000. degraded C-6-S (B) from a calibrated Bio-Gel A-0.5 m Enzyme preparation. Whole normal human kidney was column. Vo, void volume. homogenized at 50C in a Waring blendor (Waring Products Div., Dynamics Corp. of America, New Hartford, Conn.) with 1.2 ml of cold (50C) 0.15 M NaCl/g of wet tissue. minus substrate. After incubation at 37°C for 8 h, the re- After centrifugation at 600 g for 30 min at 0C, the super- leased inorganic sulfate was measured as follows. To each nate (50 ml) was dialyzed for 24 h at 50C against four tube (except for the one containing substrate alone) was changes of 0.15 M NaCl (1 liter each). The retentate was added 1.6 ml of 4% trichloroacetic acid to precipitate pro- centrifuged at 20,200 g for 30 min at 0C and the supernate teins. At this time, the substrate control was mixed with (which had 16.5 mg protein/ml [14]) was stored frozen the reaction components of the other control tube. The until used. precipitate in each tube was removed by centrifugation at Human skin fibroblasts from normal individuals and from 685 g for 25 min at 5°C and the supernates were recovered. Morquio patients were cultured (11) and homogenized (15) To a 1.4-ml portion of each supernate, 0.5 ml of gelatin- as described previously. Urinary concentrates were prepared BaCI2 reagent was added. After 15 min at room tempera- by precipitation of fresh urine with (NH)2SO4 at 70% ture, the turbidity produced was measured at 360 nm saturation (11). Leukocytes, harvested from whole blood against water. Net absorbance values were obtained by sub- according to Fallon et al. (16), were homogenized by the tracting the value of the control from that of corresponding same method used for fibroblasts. Before enzyme assay, all complete reaction mixture. 1 unit of enzyme activity is extracts and homogenates were dialyzed against 0.15 M defined as that amount releasing 1 nmol inorganic sulfate/ NaCI, as described for the preparation of kidney extract. h at 37°C. Specific activity is units per milligram of protein. Assay of kidney enzyme. Assays were performed in glass Optimal conditions for the kidney enzyme (pH, length of tubes (100 X 12 mm) prewashed in concentrated HNO3. incubation, amount of enzyme needed) and the effect upon it The standard reaction mixture (total volume, 0.6 ml) con- of various ions were determined with minor modifications tained: 0.3 ml of water containing 0.31 ,umol of NaN3, 0.15 of the described technique (see Figs. 4-7 and their legends). ml of pH 4.8, 0.6 M Tris-acetate buffer, 0.05 ml of sub- Assay of fibroblast enzyme was performed under condi- strate solution (containing 1.6 ,umol of hexuronate), and 0.1 tions given in the first paragraph of the preceding section ml (1.65 mg protein) of kidney extract. Control tubes con- but with an incubation period of 16 h. 0.1 ml of fibroblast tained only the substrate or all of the other components extract usually had 0.4 mg protein.
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