Toshiaki Setoguchi

Purification and characterization of 25-hydroxyvitamin D3 1α-hydroxylase from rat kidney mitochondria

We purified extensively 25‐hydroxyvitamin D3 1α‐hydroxylase (calcidiol, NADPH: oxygen oxidoreductase (1‐hydroxylating), EC 1.14.13.13) from kidney mitochondria of rachitic rats and disclosed its peculiar properties as a P450. The final preparation was identified as a 55 kDa protein having an intense absorption at 417 nm characteristic of P450. The specific activity was 4.8 nmol/min/mg of protein indicating a 350‐fold purification. Specific content of P450 was 1.1 nmol/mg of protein and turnover number was 4.4 min−1.

Sex differences in serum 7α-hydroxycholesterol levels in the rat reflect hepatic activity of 3β-hydroxy-Δ5-C27-steroid dehydrogenase and cholesterol 7α-hydroxylase

Factors that affect serum levels of 7α-hydroxycholesterol were studied in the rat. Serum levels of 7α-hydroxycholesterol differed in male and female rats fed regular chow (male; 0.2±0.1 nmol/ml (mean ±SD)n=8; female; 0.4±0.1 nmol/ml;n=8). When rats were fed with chow to which 3% cholestyramine had been added, the level increased significantly, particularly in female rats (male: 0.6±0.3 nmol/ml;n=8; female; 2.4±1.5 nmol/ml;n=8). The liver activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme for degradation of cholesterol, did not show any sex differences, irrespective of whether the animals were fed with regular chow (male; 51±15 pmol/min per mg protein;n=8; female; 58±21 pmol/min per mg protein;n=8), or the cholestyramine-supplemented chow (male; 162±33pmol/min per mg protein;n=8; female; 172±33 pmol/min per mg protein;n=8). In contrast, the activity of 3β-hydroxy-Δ5-C27-steroid dehydrogenase, which acts after cholesterol 7α-hydroxylase in the catabolism of cholesterol, showed a marked difference between the sexes. In both sexes this enzyme activity was higher in cholestyramine-treated rats (male; 963±78 pmol/min per mg protein;n=8; female; 708±106 pmol/min per mg protein,n=8) compared to that in that rats received regular chow (male; 622±83pmol/min per mg protein;n=8). If the serum level of 7α-hydroxycholesterol depended solely on the enzyme activity of cholesterol 7α-hydroxylase, it would be difficult to explain these sex differences, since there were no sex differences in levels of cholesterol, 7α-hydroxylase. These results clearly indicate that, in the rat, the serum level of 7α-hydroxycholesterol depends not only on cholesterol 7α-hydroxylase activity but also on 3β-hydroxy-Δ5-C27-steroid dehydrogenase activity.

Immune Complex Transfer Enzyme Immunoassay for Anti-Thyroglobulin IgG Using 2,4-Dinitrophenyl-thyroglobulin, Biotinyl-thyroglobulin and Streptavidin-β-D-galactosidase Conjugate

Abstract A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG in serum is described. Anti-thyroglobulin IgG in serum was reacted simultaneously with 2,4-dinitrophenyl-thyroglobulin and biotinyl-thyroglobulin. The immune complex formed of the three components was trapped onto polystyrene balls coated with (anti-2,4-dinitrophenyl group) IgG and, after washing, reacted with streptavidin-β-D-galactosidase conjugate. After washing, the immune complex was eluted from the polystyrene balls with eN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with (anti-human IgG γ-chain) IgG. β-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. Biotinylthyroglobulin and streptavidin-β-D-galactosidase conjugate could be prepared more easily than thyroglobulin-β-D-galactosidase conjugate used in the previous immunoassay. Inactive β-D-galactosidase, used to eliminate interference by anti-β-D-galactosidase antibodies in ...