Steroid sulphohydrolase (EC 3.1.6.2) has received in recent years the considerable attention due to its involvement in pathogenesis of diseases like X-linked ichthyosis or breast cancer. Steroid sulphohydrolase from human placenta lysosomal membranes is highly specific to cholesterol sulphate as a substrate and acts optimally at pH 3.4. The cholesterol sulphate sulphohydrolase (CHS-ase) is capable of hydrolysing the exogenous cholesterol sulphate as well as the cholesterol sulphate present in lysosomal membranes. Free cholesterol and sulphate ion are products of the enzymatic reaction catalyzed by this enzyme.

Up to now the determination of CHS-ase activity was based on the loss of cholesterol sulphate assayed by the method of Roy [1]. We adapted the enzymatic CHOD/PAP method [2] of cholesterol quantification to the determination of CHS-ase activity. Application of this method in crude lysosomal fraction required the addition of 30 mM sodium cholate for the effective solubilization of lysosomal membranes and free cholesterol.

The amount of cholesterol released in the enzymatic reaction catalyzed by CHS-ase in the lysosomal membranes rised during 60 minutes of incubation to 8.95 ± 1.2 μmoles/ml and unexpectedly, after next 60 minutes decreased to 4.05 ± 0.03 μmoles/ml. Addition of exogenous cholesterol (5 μmoles/ml) to the lysosomal membranes and incubation under the above conditions also effected in the linear decrease of cholesterol concentration depending on the amount of membranes used.

The above changes suggest the conversion of cholesterol to other derivatives. Our current studies are focused on the identification of these derivatives.

Conclusions: 1/ The application of enzymatic CHOD/PAP method of cholesterol quantification allowed us to find that cholesterol produced by CHS-ase in the lysosomal membranes is subsequently converted to other derivatives. 2/ The concentration of membranous cholesterol must be precisely controlled since it influences the stability of the membranes. Cholesterol sulphate is then the reserve pool of the inactive steroid for the production of cholesterol as well as its derivatives. 3/ We presume that CHS-ase present in lysosomal membranes from human placenta may be a part of the multienzymatic complex involved in regulation of cholesterol and its derivatives content in these membranes.

1. A.B. Roy, The enzymic synthesis of steroid sulphates, Biochem. J. 63 (1956) 294-300.
2. W. Richmond, Preparation and properties of a bacterial cholesterol oxidase from Nocardia sp. and its application to enzyme assay of total cholesterol in serum, Clin. Chem. 19 (1973) 1350-1356.

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