Determination of Arylsulfatase a Activity Using Pyrene Derivatives of Cerebroside Sulfate

  • S. Marchesini
  • A. Novati
  • S. Gatt
Conference paper
Part of the NATO ASI Series book series (NSSA, volume 150)

Abstract

Two fluorescent derivatives of cerebroside sulfate have been used as substrates for determining arylsulfatase A activity (AsA.) The respective two compounds were 12-(1- pyrene) dodecanoyl (P12-CS) and 12-(l-pyrenesulfonylamido) dodecanoyl (PSA12-CS) cerebroside sulfates.When incubated at pH 5.0 in the presence of 5 mM of MnCl2 and 5.5 mM of taurodeoxycholate, either substrate was hydrolyzed by arylsulfatase A of human leukocytes.The rate of hydrolysis was proportional to the reaction time and the respective concentrations of enzyme and substrate;in the latter case Michaelis-Menten type kinetics was observed. For following the rate of hydrolysis,the products (P12 and PSA12 galactosyl ceramides) were separated from the unreacted sulfatide on small columns of DEAE Sephadex A-25 and their fluorescence intensities read at 343-378 and 350-380 nm for the excitation and emission wavelengts, respectively

Keywords

Hydrolysis Sedimentation Pyrene Dextran Ceramides 

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Copyright information

© Plenum Press, New York 1988

Authors and Affiliations

  • S. Marchesini
    • 1
  • A. Novati
    • 1
  • S. Gatt
    • 2
  1. 1.Department of Medical Chemistry and BiochemistryUniversity of MilanoItaly
  2. 2.Department of Membrane Biochemistry and NeurochemistryHebrew University - Hadassah School of MedicineJerusalemIsrael

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