Kinetische Untersuchungen und Charakterisierung der solubilisierten Oestradiol-bzw. Testosteron-"sensitiven" 17 β-HSD-Aktivitäten / Microsome-Associated 17β-Hydroxysteroid Dehydrogenases of Human Placenta, II Kinetic Studies and Characterization of the Solubilized Estradioland Testosterone-“Sensitive” 17β-HSD-Activities

Abstract

Abstract Detailed enzyme kinetic parameters of the reactions catalyzed by the two 17β-hydroxysteroid dehydrogenases (17β-HSD), which were solubilized from the microsomes of human placenta by treatment with phospholipase A, followed by enrichment and separation were determined. Both enzymes are strictly substrate specific. The most active substrate of one of the 17β-HSD (fraction A) is estradiol-17β, the other 17β-HSD (fraction B) is sensitive to testosterone. Both NAD and NADP can serve as hydrogen transferring coenzymes, the latter giving about one-third of the initial rate of the former. With respect to the influence of temperature, different buffers and pH values, Michaelis constants [Km] with estradiol-17β and testosterone as substrates, the solubilized and separated microsomal 17β-HSD behave like those isolated from the cytoplasmic fraction. The two 17β-HSD, after solubilization from the microsomal fraction of human placenta, enrichment and separation from each other, show only a little activity for the transfer of hydrogen between C17 of estradiol-17β and C17 of androstenedione. On the other hand, intact microsomes and an integrated system prepared by recombination of the 17β-enzymes by preincubation in phosphate buffer are able to catalyse very actively the transfer of hydrogen between estradiol-17β and androstenedione. The effect of temperature and time on the recombination of the two enriched and separated microsomal enzyme activities and the determination of the pH-optimum of the hydrogen transfer reaction are described. Finally it is proposed that the hydrogen transfer between steroid hormones represents an aspect of the true reaction mechanism of steroid hormones: Steroid hormones function as hydrogen transferring coenzymes by forming part of a chain of hydrogen carriers.