Jadwiga Gniot-Szulżycka

A “soluble” form of sterol sulphate sulphohydrolase from cell nuclei of human placenta tissue—examinations with oestrone sulphate as substrate

DN-ase digestion of the nuclear envelope-chromatin complex of the cell nuclei preparations from human placenta, released a soluble form of sterolsulphohydrolase. The enzyme revealed three pH optima, at 4.0, 6.2 and 7.4. The Km value was 4.16 +/- 1.44 x 10(-5) M. The molecular mass determined by gel filtration on Bio-gel A 15 m was 406 kDa. The enzyme is sensitive to -SH group reacting reagents such as cysteine, p-chloromercuribenzoate and iodoacetamide. Oxidized and reduced forms of NAD, FAD, dithiothreitol and glutathione moderately inhibited enzyme activity. Ascorbic acid (reduced and oxidized) exerted slight activation. The enzyme was insensitive to phosphate ions.

Sterolsulphate sulphohydrolase from human placenta microsomes—30 kDa molecular weight form of cholesterol sulphate sulphohydrolase

The cholesterol sulphate sulphohydrolase (CHS-ase) exhibiting molecular weight of 30 kDa was purified from human placenta microsomes. The microsomal proteins were extracted with 0.5% Triton X-100. The DEAE-cellulose chromatography of the solubilized microsomal proteins, performed at pH 7.6 allowed to separate two enzymatically active fractions. One of them was associated with the protein fraction unbound by DEAE-cellulose, the other was tightly bound by ion exchanger. The 30 kDa cholesterol sulphate sulphohydrolase was purified to homogenity from the protein fraction tightly bound by DEAE-cellulose. The highly purified enzyme preparation (specific activity 385 nmol min(-1)mg(-1) of protein) exhibited optimal activity at pH 6.4, the K(m) was established to be 6.7 x 10(-6)M, the pI value was 7.4. The 30 kDa cholesterol sulphate sulphohydrolase, in contrast to the CHS-ase form originated from the protein fraction unbound by DEAE-cellulose, was not sensitive to alkaline phosphatase treatment and phosphohydrolase inhibitors. The effects of steroids, -SH reacting agents and sulphohydrolase inhibitors on the enzyme activity were tested.

Cholesterol sulphate sulphohydrolase from human placenta microsomes--purification and properties of the dephosphorylated form of enzyme.

The procedure for purification of cholesterol sulphate sulphohydrolase (ChS-ase) from human placenta microsomes was elaborated. The highly purified enzyme preparation (specific activity 2000 nmol x min(-1) x mg protein(-1)) exhibited optimal activity at pH 9.0. The K(m) value was established to be 1.5+/-0.85 x 10(-5) M. The high molecular weight form (200 kDa) and the low molecular weight form (20 kDa) of the enzyme were separated. The interconversion of the high molecular weight variant into the low one occurs under the influence of dephosphorylation. Both forms exhibited typical Michaelis-Menten saturation kinetics. The effect of different compounds on the enzyme activity was tested.

Subunits of sterol sulphate sulphohydrolase from human placenta microsomes

A procedure for separation of the catalytic and regulatory subunits of sterol sulphate sulphohydrolase from human placenta microsomes with the use of Concanavalin A-Sepharose chromatography is presented. The Km value for the catalytic subunit with oestrone sulphate is 1.2 x 10(-5) M. The Hill coefficient value h, for the reconstituted enzyme complex is 3, the S0.5 = 0.68 x 10(-3) M and the value of Km is 0.31 x 10(-12) M. The regulatory subunit is trypsin sensitive, while the catalytic one is resistant to trypsin digestion.

Cholesterol sulphate sulphohydrolase of human placenta lysosomal membrane

In this paper we report that the activity of cholesterol sulphate sulphohydrolase (CHS-ase) is associated with the lysosomal membranes. The procedure of purification of CHS-ase from human placenta lysosomes was elaborated. The purified enzyme is highly specific to cholesterol sulphate (specific activity 2126.60+/-940.90 nmol min(-1) mg protein(-1)) and acts optimally at pH 3.4. The K(M) value for the hydrolysis of cholesterol sulphate is 3.6+/-0.95 x 10(-5)mol/l. The isoelectric point (pI) has the value 5.7, molecular weight estimated by SDS-PAGE electrophoresis is 38 kDa. The described enzyme may be involved in a regulation of cholesterol and cholesterol sulphate levels in the lysosomal membrane.