Masahiro Kuwada

Neonatal exposure to endocrine disruptors suppresses juvenile testis weight and steroidogenesis but spermatogenesis is considerably restored during puberty.

Neonatal exposure to endocrine disruptors induces developmental abnormalities in the male reproductive system. As to investigate whether neonatal exposure affects spermatogenesis in juvenile and pubertal testes, Sprague-Dawley rat pups were given various endocrine disruptors by a single injection on the day of birth at concentrations ranging between 4 microM and 40 mM and sacrificed on day 21 (juvenile) or 50 (puberty). The testes were weighed and examined histologically at each stage. Further, the metabolites of steroidogenesis were analyzed using normal-phase high performance liquid chromatography. Neonatal exposure significantly reduced testis weights and steroid biosynthesis of juveniles, but they were highly restored at puberty.

Purification and properties of cytochrome P-450 (SCC) from pig testis mitochondria

Cytochrome P-450 was purified from pig testis mitochondria to a specific content of 13.1 n mol/mg of protein. The purified preparation was found to contain a single species of P-450, on sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular weight of about 53000 +/- 2000. The cholesterol side chain-cleavage system could be reconstituted by mixing the purified cytochrome P-450, adrenodoxin reductase, adrenodoxin, cholesterol and NADPH. The rate of conversion of cholesterol to pregnenolone was 6.2 n mol/min/n mol of P-450 under the conditions employed. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 416, 530 and 568 nm. The reduced CO-complex of the cytochrome P-450 exhibited an absorption maximum at 448 nm. The purified P-450 was subjected to microsequence analysis and its NH2-terminal amino acid sequence was found to show considerable homology with that of bovine adrenal P-450 (SCC).

A simplified procedure for purification of cytochrome P-450 by preparative ampholine gel for isoelectric focusing.

ABSTRACT The purification process for cytochrome P-450 is very complicated, involving five or more column chromatography steps for the final preparation. This paper describes a reduction in the number of the steps; it can be easily purified from pig testis microsomes with improved the yield. As the first step, DEAE-Toyopearl column chromatography is performed only once and then, as the second step, the partially purified cytochrome P-450 is completely purified by a preparative Ampholine PAG-plate Gel for Isoelectric Focusing. The combination reduced the purification to a two-step procedure.

Purification of cytochrome P-450 from adult pig testis by hydroxylapatite and deoxycorticosterone affinity column chromatography.

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and deoxycorticosterone affinity column chromatography. Cytochrome P-450 was determined to have an isoelectric point of 6.5 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular mass was estimated to be 52000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A 1000-fold purification was achieved with a yield of 5%.

Purification of cytochrome P-450 from pig testis by aniline-sepharose 4B and isoelectric focusing.

Testicular cytochrome P‐450 was purified by a procedure including preparative isoelectrofocusing. The cytochrome P‐450 was detemained to have an isoelectric point of 6.47 on analytical isoelecfric focusing. The purified cytochrome P‐450 was found to be homogeneous and its molecular weight was estimated to be 52000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited absorption spectrum of a typical cytochrome P‐450. 284‐fold purification was achieved with an yield of 10.6 %. Following preparation of the microsomes, the purification is accomplished by a two‐step procedure utilizing Aniline‐Sepharose 4B column chromatography and preparative isoelectric focusing.

A TWO-STEP PURIFICATION OF CYTOCHROME P-450 FROM ADULT PIG TESTIS BY PREGNENOLONE AFFINITY COLUMN CHROMATOGRAPHY

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and pregnenolone affinity column chromatography. The cytochrome P-450 was determined to have an isoelectric point of 6.43 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A purification of 755x was achieved with a yield of 3.09%.