Arylsulfatase B Acetylgalactosamine 4Sulfatase Fluharty et al 1982

Arylsulfatase B catalyzes the hydrolysis of the sulfate from UDP-Gal-NAc-4-sulfate to form UDP-GalNAc and sulfate. This activity is found in normal fibroblasts but not in Maroteaux-Lamy fibroblasts.

The substrate was separated from the product using ion-paired reversed-phase HPLC (Ultrasphere-ODS). The samples were eluted isocratically at room temperature using a mobile phase composed of methanol and 20 mM potassium phosphate (pH 2.5), (40:60, v/v) containing 15 mM tetrabutylam-monium phosphate. The eluant was monitored at 262 nm.

The reaction was carried out in a solution containing UDP-GalNAc-4-S, acetate buffer at pH 3.5, and the arylsulfatase B. Reactions were incubated at 37°C for 30 minutes and terminated by heating in a boiling water bath for 1 minute. Longer heating resulted in the formation of a new component, which overlapped the product peak on the HPLC. The heated material was centrifuged at 8000g for 2 minutes, and a sample was used directly for analysis. Figure 9.65 shows chromatograms of samples taken at zero time and after 30 minutes of incubation. The appearance of the new peak at about 4.5 minutes indicates the formation of the reaction product. The time course of sulfatase activity is shown in Figure 9.66.

The enzyme was prepared from human liver. This substrate is not hydro-lyzed by arylsulfatase A.

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Time, min

Figure 9.65 HPLC analysis of arylsulfatase B reaction with UDP-GalNAc-4-S The reaction mixture contained 5 mU of enzyme. (From Fluharty et al., 1982).

Time, min

Figure 9.65 HPLC analysis of arylsulfatase B reaction with UDP-GalNAc-4-S The reaction mixture contained 5 mU of enzyme. (From Fluharty et al., 1982).

Figure 9.66 Time course of arylsulfatase B reactions with UDP-GalNAc-4-S. The reaction mixture contained 3 mU of enzyme. (From Fluharty et al., 1982.)

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