Cerebroside 3-sulfate as a physiological substrate of arylsulfatase A

Abstract

1. 1.A method was developed for isolating a pure enzyme with cerebroside-sulfatase activity from the pig kidney. It was found that arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was confined to the same isolated enzyme preparation. As disc electrophoresis revealed, this enzyme is strikingly different from the arylsulfatase A from ox live which was isolated in Roy's laboratory by an essentially different procedure. Nevertheless, Roy's enzyme also exhibited, like ours, a slight degree of cerebroside sulfatase activity which in both preparations was strongly enhanced by addition of the same heat-stable ‘complementary fraction’ obtained from pig kidney. Analytical disc electrophoresis under various conditions indicated purity of the two arylsulfatases A. The quotient of the sulfatase activities with aryl and cerebroside sulfate as substrate remained constant after further disc-electrophoresis separation. 2. 2.As sulfatase activity with 35S-labelled synthetic galactose 6-sulfate and cerebroside 6-sulfate could not be detected, the enzyme is highly specific for its naturally occurring substrate, cerebroside 3-sulfate ( K m = 0.105 mM) (see ref. 5). 3. 3.Galactose 3-sulfate, galactose 6-sulfate and cerebroside 6-sulfate were found to inhibit the activity with cerebroside 3-sulfate as substrate as well as with arylsulfate (2-hydroxy-5-nitrophenyl sulfate). 4. 4.Cerebroside 3-sulfate was found to inhibit sulfatase activity with 2-hydroxy-5-nitrophenyl sulfate as substrate and vice versa; a result which indicates a common enzyme for both substrates. 5. 5.Arylsulfatase B is only slightly active towards cerebroside sulfate, provided the heat-stable complementary fraction is added. Thus it is very evident that cerebroside 3-sulfate is a naturally occurring substrate of arylsulfatase A.