The regional distribution, age dependent variation and species differences of brain arylsulphatases.

The relative proportions of arylsulphatase A and B were determined by the method of BAUM, DODGSON and SPENCER (1959) in brains of various animal species and it was found that there was a considerable variation in the concentration of these two enzymes. Arylsulphatase A and B of various animal species including rat, man, monkey, sheep and chicken were partially separated using zinc acetate fractionation procedure and gel electrophoresis. The chicken brain arylsulphatase A had a similar electrophoretic mobility to that of arylsulphatase B of other species. Further, chicken brain arylsulphatase A p r e cipitated at a zinc acetate concentration of 0.005 M, a condition under which arylsulphatase B from the brain of other species precipitated. Kinetic properties such as K, value and inhibitory effect of sulphite and phosphate ions indicated that chicken brain arylsulphatase A was similar to arylsulphatase A of other species. The results on regional distribution of arylsulphatase A and B activities in monkey brain and in developing rat brain suggest a relationship between arylsulphatase A and sulphatides and arylsulphatase B and mucopolysaccharides. THERE are several lines of evidence indicating a relationship between arylsulphatases and mucopolysaccharides and sulphatides. The occurrence and importance of mucopolysaccharides and sulphatides in brain have already been established (AUSTIN, 1960; BRANTE, 1957; GUHA, NORTHOVER and BACHHAWAT, 1960; YOUNG and ABOOD, 1960). A significant decrease in arylsulphatase A activity and accumulation of sulphatides was observed in various tissues of metachromatic leucodystrophy patients (AUSTIN, 1960; AUSTIN et al., 1963). MEHL and JATZKEWITZ (1968) have recently demonstrated that cerebroside-3-sulphate is a naturally occurring substrate for arylsulphatase A. BALASUBRAMANIAN and BACHHAWAT (19634 have reported a relationship between high arylsulphatase activity and the sulphatide content of some regions of brain. Collective evidence thus indicates that arylsulphatase A may have a role in regulating the sulphatide content in brain. A marked increase in arylsulphatase B activity compared to A was observed in Hurler’s disease (AUSTIN et al., 1964; ABRAHAM et al., 1969). Furthermore, ABRAHAM et al. (1969) found that although there was an increase in heparan sulphate and dermatan sulphate fraction in Hurler’s disease compared to normal, the total mucopolysaccharide content in Hurler’s and normal brain was the same. The present communication describes (1) the determination of arylsulphatase A and B in brains of various animal species, (2) the partial separation of arylsulphatases A and B of brain by fractionation with zinc acetate and comparison of properties of arylsulphatase A from avian and mammalian species (3) the determination of arylsulphatases A and B in different regions of monkey brain and (4) the variation of arylsulphatase A and B activities in developing rat brain. 635 636 A. A. FAROOQUI and B. K. BACHHAWAT MATERIALS A N D METHODS Dipotassium salt of nitrocatechol sulphate was purchased from Sigma Chemical Co., U.S.A. Anylamide and N-N'-methylene bis acrylamide were obtained from Eastman Organic Chemicals, Distillation Products Industries, U.S.A., Triton X-100 was purchased from Rohm and Haas, Philadelphia, Pa. Coomassie Brilliant Blue was obtained from Sigma Chemical Co., U S A . and all other chemicals used were of analytical grade. The brains of various animal species studied were taken out after decapitation, immediately chilled in ice and were homogenized with 2 ml/g of 0.05 M-tris-HCl, pH 7.4 in a Potter-Elvehjem homogenizer for 2 min at 0°C. Homogenates were centrifuged at 800 g for 30 d n and the supernatant fluid suitably diluted, was used for the assay of enzyme activity. Since the original supernatant was diluted 40 times, the possibility of inhibition by phosphate ions present in the supernatant fluid was negligible. Arylsurphntrrre assay. Arylsulphatases A and B were assayed by the method of BAUM, DODGSON and SPENCER (1959) with slight modification. Assay mixture for arylsulphatase A consisted of 0.1 ml of Reagent A (0.01 M-nitrocatecho1 sulphate in 0.5 M-sodium acetate-acetic acid buffer containing 5 x M-sodium pyrophosphate and 10% (w/v) NaCl, pH 5.0), 0.1 ml of Triton x-100 and enzyme in a total volume of 0.3 ml. After incubation at 37"C, for 1 h the reaction was stopped by the addition of 2.7 ml of 0.11 M-NaOH and the red colour developed was measured in a Klett Summerson Colorimeter using filter 50. Assay mixture for arylsulphatase B consisted of 0.1 ml of reagent B (0.05 M-nitrocatecho1 sulphate in 0-5 M-sodium acetate buffer containing M-barium acetate, pH 6). The tubes were incubated at 37°C for 0.5 and 1-5 hand thereaction stopped as indicated above. The amount of nitrocatechol formed was calculated for 1 h as described by BAUM, DODGSON and SPENCER (1959). The specific activity was expressed as pmol nitrocatechol formed/mg protein/h. Partial separation of aryisulphatases A and B. Frozen brain was homogenized with 2 vol. of 0.03 M-tris-HC1 pH 7.4 for 2 min in a Waring blender. The homogenate was cooled at 5°C in a bath of ethylene glycol and then subjected to ethanol fractionation with constant mechanical stirring. Ethanol 20% (v/v) was added dropwise and after 10 min the mixture was centrifuged at 12,000 g for 1 h at 5°C. A small portion (approx. 5 ml) of supernatant was dialysed against 100 vol. of 0.001 M-acetate buffer, pH 5, containing O.OOO1 M-MgCI, for 8 h. The rest of the supernatant was subjected to zinc acetate fractionation. Zinc acetate solution (0.2 M) was added to a final concentration of 0405 M at 5°C with constant mechanical stirring. After 15 min the mixture was centrifuged at 12,000 g for 1 h at 5°C. The reddish precipitate was dissolved in 0.1 Mcitrate buffer pH 7 and dialysed for 4 h against 500 vol. of 0.001 hi-acetate buffer, pH 5, containing 0.0001 M-MgCI,. To the supernatant, zinc acetate was added to a final concentration of 0.02 M. After 15 min it was centrifuged at 12,OOO g for 1 h at 5°C. The supernatant was discarded and the white precipitate was dissolved in 0.1 Mcitrate buffer pH 7 and dialysed as above. Gel electrophoresis. Polyacrylamide gel electrophoresis was carried out by the method of O R N S ~ E M and DAVJS (1962) in 0.03 M-barbitone sodium buffer pH 8.0 for 10 h using 8 mA/tUbe using a Buchler instrument. The proteins were stained by Coomassie brilliant blue (CHRAMBACH, REISFELD, WYCKOFF and Znccm, 1967). For elution of protein, the whole gel was cut into 1 cm segments and from each segment the protein was eluted by homogenizing the gel in a Potter-Elvehjem homogenizer with 0-03 M-tns-Ha, pH 7.4. The homogenate was centrifuged at 0°C. The supernatant after dialysis against 0-001 M-actate buffer pH 5.0 containing OW01 M-MgC12 was analysed for arylsulphatase A and B activities. Prorein determinations. Protein was determined by the method of LOWRY, ROSEBROUGH, FARR and RANDALL (1951) with crystalline bovine serum albumin as a standard. RESULTS The proportion of arylsulphatase A and B in brains of various animal species. Table 1 shows that in a lower vertebrate like frog the ratio of arylsulphatase A : B is 1 : 1. In birds, like pigeon and chicken arylsulphatase A accounts for greater part of the total arylsulphatase activity. In fact in chicken the activity of arylsulphatase A is exceptionally high compared to B. In mammals like rabbit and sheep arylsulphatase A is high while in rat, monkey and man arylsulphatase B predominates. Zinc acetate fractionation results in partial separation of arylsulphatase A and B in all species studied (Table 2). It should be noted here that arylsulphatase B (0.005 M-zinc acetate precipitate) was contaminated with 6 to 18 per cent of arylsulphatase A, and arylsulphatase A (0.02 M-ZinC acetate precipitate was contaminated with 1-8 to 12 per cent Arylsulphatases of the brain 637 Species Specific activity A specific Ratio of activity specific B activities A:B Rat Man (adult Child (2 years) Hurler's syndrome patient (7 Years) Sanfilippo syndrome patient (8 years) Monkey Sheep