Pregnenolone formation from cholesterol

When stimulation of the normal activity of the target tissue requires increased synthesis of key enzymes, intracellular effects of cyclic 3, 5 -AMP have been suggested to take place either at the translation level, as in the stimulation by ACTH of the synthesis of enzymes involved in pregnenolone formation from cholesterol, or at the transcription level, as in the stimulation by TSH of iodide transport in the thyroid. The main evidence for stimulation at the transcription level is the inhibition of the effect by both inhibitors of RNA synthesis (as actinomydn) and of protein synthesis (as puromycin), while stimulation at the translation level is suggested by inhibition with protein-synthesis inhibitors, but not with RNA-syn-tbesis inhibitors. Effects of 3, S -AMP on amino-acid incorporation in acellular systems, and on peptide release by polysomes have been reported, but their relevance to the activation at the translation level of protein synthesis in the intact cell is not known. 

Stoichiometry of C,-Compounds and Pregnenolone Formation from Cholesterol ... 

Pregnenolone Formation from Cholesterol and Some of Its Oxygenated Derivatives by a Soluble Bovine Adrenocortical Mitochondrial Preparation ... 

FIGURE 25.43 The steroid hormones are synthesized from cholesterol, with intermediate formation of pregnenolone and progesterone. Testosterone, the principal male sex hormone steroid, is a precursor to /3-estradiol. Cortisol, a glucocorticoid, and aldosterone, a mineralocorticoid, are also derived from progesterone. 

The main topic of this review will be the summarization of our present knowledge of how pregnenolone is formed from cholesterol in steroid-producing tissues. Since pregnane derivatives are so widely found in nature, some attention will also be paid to the type of C21 steroids isolated, to their distribution, to their precursors and steroids closely related to them, and to the distribution of the enzymes concerned with their formation. Among the major contributions responsible for the advances made in this field, one may list the preparation of appropriate steroid substrate intermediates, both unlabeled and labeled, and the development of analytical techniques for their chromatographic separation and measurement. As in all major fields of science there has been an exponential increase in published material on steroid biosynthesis, not without considerable duplication. However, since various authors look at the subject from dif-... 

Fig. 5. Pattern of product formation during single turnover cycles of anaerobic reduction/oxygenation of the cytochrome P-450,.,. - cholesterol - adrenodoxin complex. The results are expressed as a percentage conversion of the total [14C]cholesterol added to the incubation. Cholesterol, o 22-hydroxy-cholesterol, 20,22-dihydroxycholesterol. a pregnenolone, (from Ref. 38, with permission).

As the SCCE described above (Section 6.3.1.1) maybe part of a protein complex in the mitochondria, more effort was directed to study the possible interaction partners, especially the peripheral-t)q)e benzodiazepine receptor (PBR) (Papadopoulos et al, 1997 Koch, 2002) and the acyl-CoA-binding protein (ACBP Metzner et al, 2000). The ACBPs bind to the peripheral-t)q)e PBR present in the envelope of mitochondria (Gamier et al, 1994). This interaction stimulates the transport of cholesterol into mitochondria (Papadopoulos and Brown, 1995). The cholesterol taken up into the mitochondria is available as a substrate to the side-chain cleavage enzyme which transforms cholesterol into pregnenolone (Papadopoulos et al, 1997). Because of its interaction with PBR, ACBP is also described as diazepam-binding inhibitor or endozepine. Some isoforms of the latter were isolated and characterized from D. lanata (Metzner et al, 2000). Lindemann and Luckner (1997) speculated that cardenolide formation is regulated mainly by the availability of cholesterol and its transport into mitochondria, where the P450scc is assumed to be located. 

When the release of cholesterol from adrenal cytoplasmic lipid inclusion droplets was measured as a function of either SCP2 or FABP concentration, there was a concentration-dependent release of cholesterol from the lipid inclusion droplets with SCPj, but not with FABP [50]. When pregnenolone production was measured there was a concentration-dependent formation of pregnenolone from endogenous mitochondrial cholesterol with SCP2, but not with FABP. In contrast, FABP, but not SCPj, induced a substantial increase in the release of labeled arachidonate from adrenal cytoplasmic lipid inclusion droplets, prelabeled with [l- C]arachidonate [50]. 

Important elements of sterol metabolism can also be used to elucidate where in the cell a particular precursor has moved [7]. The arrival of cholesteryl esters within lysosomes is revealed by cleavage of the fatty acid to yield free cholesterol. The subsequent transport of cholesterol to the ER can be monitored by the action of acyl-CoA cholesterol acyltrans-ferase (Chapter 14) that results in the formation of new molecular species of cholesteryl esters. In addition, sphingomyelinase treatment of the cell surface induces cholesterol movement from the plasma membrane to the ER where its arrival can likewise be monitored by the action of acyl-CoA cholesterol acyltransferase. Import of cholesterol into mitochondria (usually restricted to steroidogenic cells) can be followed by side-chain cleavage reactions that produce pregnenolone [8]. Movement of pregnenolone out of mitochondria can be followed by oxidations at positions 3, 17, and 21, which occur in the ER. 

Activation of the LH receptor results in an increase of intracellular cyclic adenosine monophosphate (cAMP) levels via activation of a G protein and adenylate cyclase. In the presence of elevated concentrations of cAMP, cholesterol esterase activation occurs. This enzyme catalyzes the cleavage of cholesterol esters to free cholesterol, v/hich is then converted in mitochondria to pregnenolone as described previously. The formation of progesterone from pregnenolone is catalyzed by 5-ene-3p-hydroxysteroid dehydrogenase and 3-oxosteroid-4,5-isomerase (steps c and d in Fig. 46.3). 

In the isolation of steroids from natural sources one must, of course, consider their stability prior to enzyme inactivation, which usually occurs at the commencement of the isolation procedure, when the tissue is extracted with organic solvents. Kahnt and Neher (1965) could observe in bovine adrenal homogenates only trace amounts of 20a-hydroxycholesterol and (22R)-20,22-dihydroxy-cholesterol, as well as pregnenolone or progesterone (< figlgm). As stated earlier the absence of certain derivatives may be due to either their lack of formation or their rapid disappearance from the system. Until recently, primarily because of the lack of appropriate substrates of high specific activity, quantitative studies on the elucidation of the mechanism of the desmolase reaction could not be performed. The study of Ichii et al. (1967a) appears to entail the assumption of a steady state, for which no proof seems to have been provided. Also,... 

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