Do-Hyun Jo

The human cytochrome P4507B1: catalytic activity studies

The cytochrome P4507B1 (P4507B1) in the human hippocampus is responsible for the production of 7alpha-hydroxylated derivatives of dehydroepiandrosterone (DHEA) and other 3beta-hydroxylated neurosteroids. Minor quantities of the 7beta-hydroxylated derivatives are also produced. Neuroprotective action of these 7-hydroxysteroids was reported. Recombinant human P4507B1 was prepared from yeast coexpressing the human hippocampal P450 cDNA and the human P450 reductase genes. Microsomal P4507B1 activity was tested in the presence of NADPH and (14)C-labeled steroid substrates to deduce kinetic parameters and to study inhibitor responses. The K(M) values obtained for DHEA, pregnenolone, epiandrosterone, 5alpha-androstane-3beta,17beta-diol and estrone were 1.90 +/- 0.06, 1.45 +/- 0.03, 1.05 +/- 0.12, 0.8 +/- 0.04 and 1.20 +/- 0.26 microM, respectively. Production of limited amounts of 7beta-hydroxylated derivatives was also observed, but only with DHEA, 5alpha-androstane-3beta,17beta-diol and epiandrosterone. K(M) values determined for 7beta-hydroxylation were identical to those for 7alpha-hydroxylation. The DHEA 7alpha-hydroxylation was inhibited by estrone and estradiol (mixed type inhibition) and by the [25-35] beta-amyloid peptide (non-competitive inhibition). These results indicate that in human, the 7-hydroxylation catalysed by P4507B1 preferentially takes place on DHEA, 5alpha-androstane-3beta,17beta-diol and epiandrosterone with major and minor formation of 7alpha- and 7beta-hydroxylated derivatives, respectively. Both estrogens and a beta-amyloid component inhibit the P4507B1-mediated production of the 7-hydroxysteroid metabolites.

Distribution and characterization of neurosteroid sulfatase from the bovine brain.

We investigated the regional and subcellular distribution of neurosteroid sulfatase (NSS) in the bovine brain and its enzymatic properties by using dehydroepiandrosterone sulfate (DHEA-S) as a substrate. Bovine NSS was highly concentrated in the region of the midbrain and in the hypothalamus. The enzyme was found to be a microsomal enzyme. The optimal temperature of the enzyme was 50 degrees C, which was slightly lower than that of other steroid sulfatases. The optimal pH of bovine NSS was 7.4 with a second optimum at pH 4.0. The second optimal pH of 4.0 was the most characteristic property of bovine NSS. Employing DHEA-S as the substrate, apparent Km and Vmax values were 113 +/- 21 microM and 4.1 +/- 0.4 nmol/mg protein/h, respectively, whereas Km and Vmax values were found to be 1.6 +/- 0.2 M and 1.9 +/- 0.3 micromol/mg protein/h with p-nitrophenyl sulfate (NP-S) as the substrate. NSS has thus been shown to have a higher affinity for the steroid sulfate than the phenolic compound. When DHEA-S was used as the substrate, pregnenolone sulfate (Preg-S) was a competitive inhibitor with an apparent Ki value of 46 microM, and NP-S was a non-competitive inhibitor (apparent Ki=12 mM).

Chitinolytic enzymes from the gizzard and the chyme of the broiler (Gallus gallus L.)

Crude chitinolytic enzymes were prepared from the chyme, the mucous lining and the remaining tissue of the gizzard. Freezing and thawing of the sample did not affect their activity. The thermostability was variable, depending upon the source of the enzyme and the type of enzyme action. Under experimental conditions 165 mg N-acetylchitooligomers with high degree of polymerization could be obtained from a gizzard over 18 h.

Subunits of neurosteroid sulfatase from bovine brain

We have purified the neurosteroid sulfatase (NSS) from Triton X-100 solubilized microsomes of bovine brain about 100-fold. The purified enzyme is composed of two catalytic units (MW: 57 kDa) and two regulatory units (MW: 38 kDa), making it an alpha(2)beta(2) heterotetramer, whose apparent molecular weight was 180 kDa by gel filtration in the presence of Triton X-100.

The length of the polyoxyethylene chain in the Triton X detergents modulates the apparent activation of neurosteroid sulfatase in bovine brain

The effect of the Triton X series on the solubilization and enzyme activity of neurosteroid sulfatase (NSS) in the bovine midbrain was investigated. Triton X-100 and X165 stimulated NSS activity in the bovine midbrain, while Triton X-305 did not. This apparent activation was attributed to the action of the detergents, and not to the latency of the enzyme or the removal of some inhibitory substance from the microsomes. The maximum stimulation was obtained when the length of the polyoxyethylene chain of the detergent was 16.