Enzymes 7- hydroxylase activity cholesterol

Aminoglutethimide (Cytadren) is a competitive inhibitor of desmolase, the enzyme that catalyzes the conversion of cholesterol to pregnenolone it also inhibits 11-hydroxylase activity. This drug also reduces estrogen production by inhibiting the aromatase enzyme complex in peripheral (skin, muscle, fat) and steroid target tissues. 

The properties of 7a-hydroxylase from pigeon liver microsomes305 and from rat liver306,307 have been further described, and new assay methods are available.308,309 Free cholesterol, rather than a cholesteryl ester, was the preferred substrate for the enzyme from rat liver microsomes,310 and the substrate pool for the hydroxylase was about one third of the total amount of cholesterol present in the microsomal preparation.309 Cholesterol 7a-hydroxylase activity is more sensitive to thyroid function than are the activities of the enzymes responsible for cholesterol synthesis,311 and (22f )-22-aminocholesterol, although having no effect on serum or liver cholesterol levels in rats, drastically reduced 7a-hydroxylase activity.312... 

Vitamin D is a fat soluble vitamin derived from cholesterol. In the human epidermis (skin), sunlight spontaneously oxidizes cholesterol to 7-dehydrocholesterol (Fig. 10.10a). The 7-dehydrocholesterol leaks into the blood where it isomerizes to cholecalciferol (vitamin D3, Fig. 10.10b and c). Cholecalciferol is enzymatically hydroxylated at C25 in the liver (25-cholecalciferol) and then passes to the kidney where another enzyme is activated by parathyroid hormone to hydroxylate it at Cl, forming calcitriol (Fig. lO.lOd). The kidney hydroxylase is sensitive to feedback inhibition. As the amount of calcitriol increases, it binds to the hydroxylase and alters the specificity of the kidney enzyme. Additional 25-cholecal-ciferol is hydroxylated to 24,25-dihydroxycholecalciferol (inactive calcitriol) instead of 1,25 dihydroxycholecalciferol (calcitriol). Other vitamin D derivatives that can be converted to calcitriol are obtained enzymatically from cholesterol in other vertebrates. The most common of these are vitamin D3 (lamisterol) and D2 (ergosterol) from cold-water fish such as cod, where their presence keeps membranes fluid at low body temperatures 10-20°C. 

Cholesterol 7a-hydroxyIase has been partially purified from rat and rabbit liver (H5). The enzyme is located in the smooth endoplasmic reticulum and is dependent on cytochrome F-450 and NADPH-cytochrome P-450 reductase for activity (H5). The particular cytochrome P-450 associated with microsomal cholesterol 7a-hydroxylase activity constitutes a small fraction of total liver cytochrome P-450 and, in the rabbit, it appears to be a subfraction of cytochrome P-450lm4 (B28). Measurement of the activity of this enzyme by isotope incorporation is complicated by dilution of added cholesterol by endogenous microsomal cholesterol. A method has now been developed to remove cholesterol fit>m microsomes, so that the mass of 7a-hydroxycholesterol formed during enzyme assay can be accurately calculated (S25). Using this assay, cholic acid feeding was shown to suppress the activity of cholesterol 7a-hydroxylase in rat liver, whereas cholesterol feeding did not (S25). 

A short-term regulation mechanism for cholesterol 7a-hydroxylase activity has been investigated recently in rat liver. The enzyme appears to exist in two forms, which are interconverted by cytosolic fiictors (K12). These foctors may correspond to a protein kinase and a phosphatase, which have been proposed to regulate cholesterol 7a-hydroxylase activity by a phosphorylation (active form)-dephosphorylation (inactive form) mechanism (S9). Another enzyme utilizing cholesterol as substrate, acyl-CoA cholesterol O-acyltransferase (EC 2.3.1.26), may also be regulated in this way, while the biosynthetic enzyme, HMC-CoA reductase, is inhibited in the phosphory-lated form (SIO). Thus, short-term regulation of the concentration of un-esterified cholesterol in the liver may be achieved by coordinate control of these three key enzymes in cholesterol metabolism by reversible phosphorylation (SIO). 

Most studies on substrate specificity of cholesterol 7a-hydroxylase have been performed with intact microsomes. Results of such studies may be difficult to interpret since the enzyme system is embedded in a lipoprotein membrane, and may not be directly accessible to potential substrates [59]. Thus, differences in the rate of 7a-hydroxylation of various steroids could be due to differences in the rate at which the substrate reaches the active site of. the enzyme rather than to differences in the intrinsic ability of the enzyme to interact catalytically with the substrate [59], Further, occurrence of 7a-hydroxylation of a certain steroid may not reflect the substrate specificity of cholesterol 7a-hydroxylase activity since different species of cytochrome P-450 are present in the microsomes. 

The mitochondrial enzyme has a broad substrate specificity and catalyses 26-hydroxylation of a number of C27-steroids. The most important substrates in vivo are believed to be 5)8-cholestane-3a,7a-diol, 7a-hydroxy-4-cholesten-3-one and 5j8-cholestane-3a,7a,12a-triol. Bjorkhem and Gustafsson found that 5j8-cholestane-3a,7a,12a-triol and 7a-hydroxy-4-cholesten-3-one were the best substrates in rat liver mitochondria and that the least efficient 26-hydroxylation occurred with cholesterol as substrate [126,130]. There was also a small extent of 25-hydroxylation of cholesterol in the mitochondrial fraction [130]. The major part of the 26-hydroxylase is bound to the inner mitochondrial membranes [130,131]. Thus the hydroxylase activity is low with intact mitochondria and NADPH as cofactor. Under such conditions citric acid and isodtric acid, which are able to penetrate the inner mitochondrial membrane, stimulate 26-hydroxylation much more efficiently than NADPH [130,131]. It is evident that citric acid and isocitric acid generate NADPH inside the mitochondrial membrane. When using leaking mitochondria, NADPH stimulates the reaction about as efficiently as isocitrate [130,131]. 

Conclusive evidence that a species of cytochrome P-450 was involved in the hydroxylation was presented by Okuda et al., who showed that the photochemical action spectrum for reversal of the carbon monoxide inhibition of 26-hydroxylation of 5)8-cholestane-3a,7a,12a-triol in rat liver exhibited a maximum at 450 nm [134]. Pedersen et al. [135] and Sato et al. [136] reported simultaneously that small amounts of cytochrome P-450 could be solubilized from the inner membranes of rat liver mitochondria that was active towards cholesterol as well as 5)8-cholestane-3a,7a,12a-triol in the presence of ferredoxin, ferredoxin reductase and NADPH. The mechanism of hydroxylation is thus the same as that operative in the biosynthesis of steroid hormones in the adrenals and in the la-hydroxylation of 25-hydroxyvitamin D in the kidney (Fig. 8). The liver mitochondrial cytochrome P-450 was not active in the presence of microsomal NADPH-cytochrome P-450 reductase [135,136]. Ferredoxin reductase as well as ferredoxin were active regardless of whether they were isolated from rat liver mitochondria or bovine adrenal mitochondria [133]. The partially purified cytochrome P-450 had a carbon monoxide difference spectrum similar to that of microsomal cytochrome P-450 from liver microsomes and adrenal mitochondria. In the work by Pedersen et al. [133], the concentration of mitochondrial cytochrome P-450 in rat liver mitochondria from untreated rats was calculated to be only about 0.1 nmole/mg protein. Treatment of rats with phenobarbital increased the specific content of cytochrome P-450 in the mitochondria more than 2-fold, without significant increase in the 26-hydroxylase activity. The carbon monoxide spectrum of the reduced cytochrome P-450 solubilized from liver mitochondria of phenobarbital-treated rats exhibited a spectral shift of about 2 nm as compared to the corresponding spectrum obtained in analysis of preparations from untreated rats. This was taken as evidence that more than one species of cytochrome P-450 was present in the preparation. It was later shown by Pedersen et al. [137] and Bjbrkhem et al. [138] that the preparation was also able to catalyse 25-hydroxylation of vitamin D3 and that different enzymes are involved in... 

K. Okuda, and T. Setoguchi (2002). Half-life of cholesterol 7a-hydroxylase activity and enzyme mass differ in animals and humans when determined by a monoclonal antibody against human cholesterol 7a-hydroxylase. J. Steroid Biochem. Mol. Biol. 81, 377-380. 

In the apparently major pathway for the conversion of cholesterol into 5 -cholestane-3a,7a,12a-triol, the step following the formation of 7a-hydroxy-4-cholesten-3-one is a 12a-hydroxylation yielding 7a,12a-dihydroxy-4-cholesten-3-one (Fig. 1). The reaction is catalyzed by the microsomal fraction fortified with NADPH (15,37). The conversion of 5-cholestene-3, 7a-diol into 5-cholestene-3/5,7a,12a-triol, which is a reaction in another pathway for the formation of 5/5-cholestane-3a,7a,12a-triol, is also catalyzed by the microsomal fraction fortified with NADPH (30,37), as is the 12a-hydroxylation of 5/5-cholestane-3a,7a-diol and 7a-hydroxy-5)5-cholestan-3-one (37). The rates of 12a-hydroxylation of these C27-steroids differ considerably the rate with 5-cholestene-3/5,7a-diol is about one-tenth and with 5 -cholestane-3a,7a-diol about half of that with 7a-hydroxy-4-cholesten-3-one (37). Einarsson (37) and Suzuki et al. (38) have studied some properties of the 12a-hydroxylase system with special reference to the possible participation of electron carriers such as NADPH-cytochrome c reductase and cytochrome P-450. The 12a-hydroxylation of 7a-hydroxy-4-cholesten-3-one was inhibited by cytochrome c, indicating that NADPH-cytochrome c reductase might be involved. However, no direct evidence for the participation of flavins was obtained. If NADPH-cytochrome c reductase participates, it is not rate-limiting, since the activity of this enzyme increases upon treatment with thyroxine whereas the activity of the 12a-hydroxylase decreases (39). Suzuki et al. (38) found no inhibition of 12a-hydroxylation by carbon monoxide, whereas Einarsson (37) obtained some inhibition. The 12a-hydroxylase activity was unaffected by methylcholanthrene treatment (40) and lowered by phenobarbital treatment (37,38). These observations indicate that the cytochrome(s) P-450 induced by methylcholanthrene and... 

Previously published evidence from our laboratory (12, 13) concludes that cholesterol 7a -hydroxylase activity is similarly regulated that is, the phosphorylated form of the enzyme is active, whereas dephosphorylated form of the enzyme is inactive. Here I will briefly show some of this evidence. 

The data discussed here so far provides a basis for proposing a mechanism (1 ) that may regulate the amount of unesterified cholesterol present in the cell (Fig. 8). In the liver cell, the utilization of cholesterol is regulated by ACAT and cholesterol 7a-hydroxylase. Both of these enzymes appear to be active in the phosphorylated state. The enzyme which regulates cholesterol synthesis, that is, HMG-CoA reductase, is active when dephosphorylated. Therefore, synthesis and utilization are oppositely regulated by phosphorylation/dephosphorylation. 

The regulatory implication of such a proposal is shown in Fig. 9. In the event of cholesterol excess, such as dietary cholesterol entering the cell, the regulatory adjustment would be as follows HMG-CoA reductase activity would decline, as a consequence of phosphorylation, whereas the activities of AC AT and 7 a-hydroxylase enzymes would be stimulated. In the instance of cholesterol deprivation, for example a cholesterol-free diet or a cultured liver cell grown in lipid-deficient medium, the regulatory adjustment would be HMG-CoA reductase activity would increase as a consequence of dephosphorylation, but AC AT and 7a-hydroxylase activities would decline under these conditions. 

The lowered concentration of bile acids returning to the liver by the enterohepatic circulation results in derepression of 7-a-hydroxylase, the rate-limiting enzyme for conversion of cholesterol to bile acids. This results in increased use of cholesterol to replace the excreted bile acids and lowering of hepatic cholesterol (mechanism VI in Fig. 23.2). Thus, similar to the statins, the ultimate actions of the bile acid-sequestering resins are up-regulation of transcription of the LDL receptor gene, increased hepatic receptor activity, and lowering of plasma LDL cholesterol (mechanism VII in Fig. 23.2). 

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