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.

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.

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.

Arylsulphatase C and sterol sulphatase activities in microsomes from human placenta.

Microsomes of human placenta were tested with respect to sulphohydrolase activity towards p-nitrophenyl sulphate, oestrone sulphate, dehydroepiandrosterone sulphate and pregnenolone sulphate. Microsomal sulphohydrolases were solubilized by treatment with 0.5% Triton X-100 at pH 9.0. Bio-gel A 1.5 m column chromatography allowed to separate the microsomal arylsulphatase into subfractions differing in molecular mass and substrate specificity. Oestrone sulphate sulphohydrolase appeared to be as specific for microsomal fraction as glucose-6-phosphatase.

Oestrone sulphate sulphohydrolase activity in nuclear envelopes from human placenta cell nuclei.

Procedures for isolation, from human term placenta, of highly purified nuclei and nuclear envelopes with a low content of DNA are described. Both fractions contain oestrone sulphate sulphohydrolase activity. The enzyme from nuclear envelopes can be solubilized with Triton X-100 and, partially, with proteolytic enzymes. It does not require Ca2+ and is insensitive to Ag+ and agents reacting with SH groups. It is strongly inhibited by millimolar concentrations of sulphites and to a much smaller extent by phosphates. Oxidized forms of ascorbic acid, glutathione and NAD+ revealed a pronounced inhibitory effect, whereas reduced forms of these compounds produced a slight activation. It is proposed that oestrone sulphate sulphohydrolase activity in nuclear envelopes from human placenta is not exerted by arylsulphatase but represents a specific enzyme.

Purification of steroid sulphohydrolase from human placenta microsomes.

A procedure for purification of oestrone sulphate sulphohydrolase from human placenta microsomes was elaborated. The use of Concanavalin-A-Sepharose chromatography made it possible to separate, for the first time, oestrone sulphate sulphohydrolase (Mr 36,000, optimum pH 7.0, Km 5.5 X 10(-5) M, specific activity 1563 nmol X min-1 X mg protein-1) from arylsulphatase C (Mr 45,000, optimum pH 7.6, Km 0.96 X 10(-3) M). The observed third subfraction showed both arylsulphate C and oestrone sulphate sulphohydrolase activity. Sigmoidal kinetics of oestrone sulphate sulphohydrolase after DEAE-cellulose chromatography (Mr 130,000) points to the allosteric character of the enzyme.

Studies on oestrone sulphate sulphohydrolase from human placenta nuclear envelopes--solubilization and properties.

Kinetic properties of human placenta nuclear envelope oestrone sulphate sulphohydrolase were determined in envelope suspension, Triton X-100 solubilized enzyme preparations and in partially purified enzyme preparations. Purification of the enzyme(s) extracted with Triton X-100 was performed by three alternative procedures: Bio-gel A 15 m chromatography, DEAE-cellulose chromatography and Con-A-Sepharose chromatography. The nuclear envelope suspension revealed optimal hydrolysis of oestrone sulphate at pH 8.2; the Triton X-100 solubilized enzyme exhibited optimal activities at pH 6.6 and 8.6. Three distinct activity peaks of oestrone sulphate hydrolysis were found for the partially purified enzyme preparations: at pH 6.2 (5.8-6.6), at pH 7.0 and pH 8.4 (8.0-8.8). The influence of the oestrone sulphate concentration on the enzyme activity determined at pH 6.2 exhibited a sigmoid saturation kinetics with all enzyme preparations except that obtained after Con-A Sepharose chromatography. The value of Hill (h) coefficient was 2; S0.5 = 65 microM, the K'm = 4.9 x 10(-9) M. At pH 7.0 and 8.4, the influence of oestrone sulphate concentration on enzyme activity revealed a Michaelis-Menten saturation kinetics; the respective values of Km were 4.2 +/- 1.1 x 10(-6) M and 1.08 +/- 0.11 x 10(-4) M. The Km value of oestrone sulphate hydrolysis of the enzyme preparation after Con-A Sepharose chromatography determined at pH 6.2, 7.0 or 8.1 was 4.0 x 10(-4) M.

A simple procedure for isolation of microsomes from human placenta.

A method for rapid isolation of human placenta microsomes, which does not require facilities for ultracentrifugation was described. Such microsomes were compared with microsomes prepared by conventional ultracentrifugation technique. Both microsomal preparations were tested for protein, RNA and phospholipid content as well as for sulphohydrolase activities and glucose-6-phosphatase activity. The degree of contamination with other subcellular particles were tested as well. The data presented showed that microsomal proteins precipitated at pH 5.3 may be conveniently used for preparative separation of microsomal enzymes.

[Steroid modulation of GABA(A) receptors]. / Steroidowa modulacja receptor-ow GABA(A).

It has been known for many years that steroids influence many processes by genome activation. In 40-th the fast (anesthetic)-effect of steroids on neuronal activity was discovered, and later the molecular mechanism of steroid action as modulators of GABA(A) receptors was documented. Such kind of influence of neuronal activity is characteristic for some glucocorticosteroids and some derivatives of androsterone and progesterone (for instance: THDOC, THP). The endogenous production of several steroids in the brain was proved. Recently modulatory effect (anti-anesthetic properties) of sulphate esters of pregnenolone (P) and dehydroepiandrosterone (DHEA) on GABAA receptors was discovered. The steroid influence on the neuronal activity is still poorly documented and requires further investigations.