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1.
Polyphosphoinositide activation of cholesterol side chain cleavage with purified cytochrome P-450scc
Highly purified beef adrenal cytochrome P-450 specific for cholesterol side chain cleavage (P-450-scc) has been reconstituted with sonicated vesicles containing cholesterol and either dimyristoyl phosphatidylcholine (DMPC) or dioleoyl phosphatidylcholine (DOPC). When cholesterol was present in DMPC vesicles at 1:15 molar ratio, cardiolipin and L-alpha-phosphatidylinositol 4-monophosphate (DPI) increased side chain cleavage by at least 5-fold (0.7 min-1-3.5 min-1). In DOPC vesicles, a smaller increase was observed (2.8 min-1-5.0 min-1). Activator phospholipids increased the rate of transference of cholesterol both to and from the cytochrome when, respectively, cholesterol-free P-450scc and cholesterol-P-450scc complex are combined with either DMPC or DOPC vesicles. Transfer of cholesterol to and from cytochrome P-450 occurred with similar first order rate constants and was also independent of the concentrations of cholesterol vesicles and P-450. It is suggested that transfer in both directions is limited by the rate of insertion of P-450scc into the membrane. Phospholipid stimulatory effects for both cholesterol transfer and for activation of side chain cleavage occurred with the same ranking, even though cholesterol transfer, following reconstitution, was 5-10 times slower than the turnover of side chain cleavage. DPI increased Vmax for side chain cleavage in both DMPC and DOPC vesicles to the same rate (12 min-1) without effect on the Km for cholesterol, while cardiolipin both produced a similar increase in Vmax and decreased Km (cholesterol). This activation by DPI is attributed to more favorable incorporation of P-450scc in these membranes and is consistent with previously reported effects of acidic phospholipids on other mitochondrial proteins. 相似文献
2.
We examined the effects of glycerol, a stabilizing agent commonly used in cytochrome P450scc purification and analysis, on the spin state, catalytic activity, and molecular volume of the cytochrome. Glycerol induced a sigmoidal low-spin response. The binding of hydroxycholesterol reaction intermediates, but not cholesterol, increased the concentration of glycerol required for the spin transition to be 50% complete (K(1/2)). Glycerol weakened adrenodoxin binding to P450scc but had no effect on CO or 20alpha,22R-dihydroxycholesterol binding. Cytochrome P450scc activity was inhibited by glycerol with the K(1/2) for inhibition being substrate-dependent. The osmotic stress exerted by glycerol on P450scc resulted in decreases in P450scc molecular volume for both the transition to low spin state and the inhibition of activity. From this we determined that two dissociative water molecules are involved in the inhibition of activity with cholesterol as substrate and five or six dissociative waters are involved in the low-spin transition. The dehydration of P450scc by osmotic stress provides an explanation for the effects of glycerol on P450scc spin transition and activity. 相似文献
3.
M C Tobes S J Hays D L Gildersleeve D M Wieland W H Beierwaltes 《Journal of steroid biochemistry》1985,22(1):103-110
The adrenal cortical enzyme systems, 11 beta-hydroxylase, P-450 11 beta, and the side-chain cleavage complex, P-450 scc, differ only in their cytochrome P-450s. Structural modifications of metyrapone, an inhibitor of cytochrome P-450 enzyme systems, have been made to determine the requirement for the A- or B-pyridyl ring for inhibition of P-45011 beta and P-450 scc activities. Three new analogs of metyrapone (A-phenylmetyrapone, B-phenylmetyrapone and diphenylmetyrapone) were synthesized and evaluated as inhibitors using a crude, defatted bovine adrenal cortical mitochondrial preparation. Characterization of the mitochondrial preparation demonstrated: enhancement of both activities by the addition of 15.0 microM adrenodoxin, the addition of 1% ethanol decreased both activities less than 10%, and the apparent Km of deoxycorticosterone for P-45011 beta was 6.8 microM and the apparent Km of cholesterol for P-450 scc was 21.6 microM. Inhibition of P-45011 beta and P-450 scc activities with these compounds demonstrated: the B-pyridyl ring of metyrapone is required for inhibition of both activities whereas requirement for the A-ring is less stringent, and the four metyrapone analogs were more selective inhibitors of P-45011 beta activity. These studies suggest that the A-phenyl metyrapone analog is a good candidate for further development of a selective adrenocortical radiopharmaceutical. 相似文献
4.
The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria were studied by using controlled digestion with trypsin of purified inner mitochondrial membranes. Inhibition of steroid hydroxylase activity by trypsin was only observed in inner mitochondrial membranes which had been disrupted by various techniques. The steroid hydroxylase activity of intact inner membranes was not inhibited by trypsin. The effect of tryptic digestion was monitored by measuring 11 beta-hydroxylase and cholesterol side chain cleavage activities, as well as cytochrome P-450 reduction. The effect of trypsin on the steroid-induced difference spectra using pregnenolone, 20 alpha-hydroxycholesterol, and deoxycorticosterone was also measured. The results were similar regardless of which procedure was utilized and strongly suggest that both cytochrome P-45011 beta and cytochrome P-450scc are located on the matrix side of the mitochondrial inner membrane. 相似文献
5.
Active complex between adrenodoxin reductase and adrenodoxin in the cytochrome P-450scc reduction reaction 总被引:2,自引:0,他引:2
In order to elucidate the mechanism of the electron transfer reaction of mitochondrial steroid hydroxylase, the reduction reaction of cytochrome P-450scc (P-450scc) catalyzed by covalently cross-linked complexes between adrenodoxin reductase (AR) and adrenodoxin (AD) was studied. The reduction rate with the covalent AR-AD complex was very slow (0.030 min-1, as the flavin turnover number) compared with the reduction catalyzed by AR and AD (4.6 min-1). When free AD was added to the reaction mixture containing the AR-AD complex, the rate increased about 30 times. The AD dimer [(AD)2], and a complex between AR and the AD dimer [AR-(AD)2] were then prepared. The Vmax for the P-450scc reduction activity of AR with (AD)2 was 50% of that of AR with AD. The Km value for the total concentration of AD in the P-450scc reduction reaction mixture containing AR and (AD)2 was found to be the same as that in the reaction mixture containing AR and AD. P-450scc reduction by AR-(AD)2 was about 5 times faster than that by AR-AD. The addition of free AD to the AR-(AD)2 complex enhanced the P-450scc reduction about 30 times. AR-AD and AR-(AD)2 were able to reduce external AD, cytochrome c, and acetylated cytochrome c.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Reconstituted human erythrocyte sugar transporter activity is determined by bilayer lipid head groups 总被引:1,自引:0,他引:1
The effects of bilayer lipid head group on human erythrocyte passive sugar transport protein activity were examined by reconstituting the transporter into bilayers of large unilamellar vesicles (LUVs) formed from lipid classes of identical (or similar) acyl chain composition. Two reconstituted transport parameters were measured as a function of temperature. These were Km and turnover number [Tn = Vmax per reconstituted D-glucose-sensitive cytochalasin B binding site (transport molecule)]. Tn for sugar transport was found to be almost entirely a function of the properties of the bulk lipid composition of the reconstituted LUVs. It was found to be independent of both reconstituted transporter density and small amounts (less than or equal to 3%) of endogenous red cell lipids. With the dimyristoylphospholipids, Tn increases at all temperatures in the order phosphatidylcholine less than phosphatidylglycerol less than phosphatidic acid less than phosphatidylserine (at 50 degrees C, Tn for transport in dimyristoylphosphatidylcholine is 100-fold lower than Tn for transport in dimyristoylphosphatidylserine). Similar results are found with egg yolk derived lipids. Only dimyristoyl- and dipalmitoylphosphatidylcholine bilayers are incapable of supporting detectable transport activity at temperatures below the bilayer phase transition, and only the phosphatidylcholines show a clear increase in Tn during the bilayer melt. All other bilayer systems studied (phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and sphingomyelin) support a small or negligible increase in Tn during the bilayer melt, the major change in transport being restricted to altered Km. With the disaturated phosphatidylglycerols (C14-C18), Tn and the activation energy (Ea) for reconstituted transport increase with acyl chain carbon number. Similar results are found with the phosphatidylcholines. Transport in bilayers formed from egg yolk sphingomyelin (a lipid containing a sphingosine rather than a glycerol backbone) is characterized by very high Km and low Tn parameters. Moreover, protein-mediated transport in sphingomyelin bilayers "spikes" during the bilayer phase transition. These and previous findings [Carruthers, A., & Melchior, D.L. (1984) Biochemistry 23, 6901-6911; Connolly, T.J., Carruthers, A., & Melchior, D. L. (1985) Biochemistry 24, 2865-2873] indicate that those bilayer factors influencing reconstituted sugar transporter activity are, in order of importance, lipid head group greater than lipid acyl chain length and saturation/unsaturation greater than lipid backbone greater than bilayer "fluidity". 相似文献
7.
In contrast to other P450 enzymes purified from rat liver microsomes, purified P450 IIIA1 (P450p) is catalytically inactive when reconstituted with NADPH-cytochrome P450 reductase and the synthetic lipid, dilauroylphosphatidylcholine. However, purified P450 IIIA1 catalyzes the oxidation of testosterone when reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, an extract of microsomal lipid, and detergent (Emulgen 911). The present study demonstrates that the microsomal lipid extract can be replaced with one of several naturally occurring phospholipids, but not with cholesterol, sphingosine, sphingomyelin, ceramide, cerebroside, or cardiolipin. The ratio of the testosterone metabolites formed by purified P450 IIIA1 (i.e., 2 beta-, 6 beta-, and 15 beta-hydroxytestosterone) was influenced by the type of phospholipid added to the reconstitution system. The ability to replace microsomal lipid extract with several different phospholipids suggests that the nature of the polar group (i.e., choline, serine, ethanolamine, or inositol) is not critical for P450 IIIA1 activity, which implies that P450 IIIA1 activity is highly dependent on the fatty acid component of these lipids. To test this possibility, P450 IIIA1 was reconstituted with a series of synthetic phosphatidylcholines. Those phosphatidylcholines containing saturated fatty acids were unable to support testosterone oxidation by purified P450 IIIA1, regardless of the acyl chain length (C6 to C18). In contrast, several unsaturated phosphatidylcholines supported testosterone oxidation by purified P450 IIIA1, and in this regard dioleoylphosphatidylcholine (PC(18:1)2) was as effective as microsomal lipid extract and naturally occurring phosphatidylcholine or phosphatidylserine. These results confirmed that P450 IIIA1 activity is highly dependent on the fatty acid component of phospholipids. A second series of experiments was undertaken to determine whether microsomal P450 IIIA1, like the purified enzyme, is dependent on cytochrome b5. A polyclonal antibody against purified cytochrome b5 was raised in rabbits and was purified by affinity chromatography. Anti-cytochrome b5 caused a approximately 60% inhibition of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation by purified P450 IIIA1 and inhibited these same reactions by approximately 70% when added to liver microsomes from dexamethasone-induced female rats. Overall, these results suggest that testosterone oxidation by microsomal cytochrome P450 IIIA1 requires cytochrome b5 and phospholipid containing unsaturated fatty acids. 相似文献
8.
D W Seybert J R Lancaster J D Lambeth H Kamin 《The Journal of biological chemistry》1979,254(23):12088-12098
Cytochrome P-450scc can be reconstituted into a phospholipid bilayer in the absence of added detergent by incubation of purified hemoprotein with preformed phosphatidylcholine vesicles. Salt effects demonstrate that the primary interaction between the cytochrome and phospholipid vesicles is hydrophobic rather than ionic; in contrast, neither adrenodoxin reductase nor adrenodoxin will bind to phosphatidylcholine vesicles by hydrophobic interactions. Insertion of cytochrome P-450scc into a phospholipid bilayer results in conversion of the optical spectrum to a low spin type, but this transition is markedly diminished if cholesterol is incorporated within the bilayer. Vesicle-reconstituted cytochrome P-450scc metabolizes cholesterol within the bilayer (turnover = 13 nmol/min/nmol of cytochrome P-450scc); virtually all (greater than 94%) of the cholesterol within the vesicle is accessible to the enzyme. "Dilution" of cholesterol within the bilayer by increasing the phospholipid/cholesterol ratio at a constant amount of cholesterol and cytochrome P-450scc results in a decreased rate of side chain cleavage, and cytochrome P-450scc incorporated into a cholesterol-free vesicle cannot metabolize cholesterol within a separate vesicle. In addition, activity of the reconstituted hemoprotein is sensitive to the fatty acid composition of the phospholipid. These results indicate that the cholesterol binding site on vesicle-reconstituted cytochrome P-450scc is in communication with the hydrophobic bilayer of the membrane. The reducibility of vesicle-reconstituted cytochrome P-450scc as well as spectrophotometric and activity titration experiments show that all of the reconstituted cytochrome P-450scc molecules possess an adrenodoxin binding site which is accessible from the exterior of the vesicle. Activity titrations with adrenodoxin reductase also demonstrate that a ternary or quaternary complex among adrenodoxin reductase, adrenodoxin, and cytochrome P-450scc is not required for catalysis, a finding consistent with our proposed mechanism of steroidogenic electron transport in which adrenodoxin acts as a mobile electron shuttle between adrenodoxin reductase and cytochrome P-450 (Lambeth, J.D., Seybert, D.W., and Kamin, H. (1979) J. Biol. Chem. 254, 7255-7264. 相似文献
9.
The conversion of cholesterol to pregnenolone by cytochrome P450scc is the rate-determining step in placental progesterone synthesis. The limiting component for placental cytochrome P450scc activity is the concentration of adrenodoxin reductase in the mitochondria, where it permits cytochrome P450scc to work at only 16% of maximum velocity. Adrenodoxin reductase serves to reduce adrenodoxin as part of the electron transfer from NADPH to cytochrome P450scc. We therefore measured the proportion of adrenodoxin in the reduced form in intact mitochondria from the human placenta during active pregnenolone synthesis, using EPR. We found that the adrenodoxin pool was only 30% reduced, indicating that the adrenodoxin reductase concentration was insufficient to maintain the adrenodoxin in the fully reduced state. As both oxidized and reduced adrenodoxin can bind to cytochrome P450scc we tested the ability of oxidized adrenodoxin to act as a competitive inhibitor of pregnenolone synthesis. This was done in a fully reconstituted system comprising 0.3% Tween 20 and purified proteins, and in a partially reconstituted system comprising submitochondrial particles, purified adrenodoxin and adrenodoxin reductase. We found that oxidized adrenodoxin is an effective competitive inhibitor of placental cytochrome P450scc with a Ki value half that of the Km for reduced adrenodoxin. We conclude that the limiting concentration of adrenodoxin reductase present in placental mitochondria has a two-fold effect on cytochrome P450scc activity. It limits the amount of reduced adrenodoxin that is available to donate electrons to cytochrome P450scc and the oxidized adrenodoxin that remains, competitively inhibits the cytochrome. 相似文献
10.
E C Müller A Lapko A Otto J J Müller K Ruckpaul U Heinemann 《European journal of biochemistry》2001,268(6):1837-1843
NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin -P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin-P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system. 相似文献
11.
Metabolism of the potent hepatocarcinogen N-nitrosodimethylamine (NDMA) was evaluated in reconstituted monooxygenase systems containing each of 11 purified rat hepatic cytochrome P-450 isozymes. The reaction has an absolute requirement for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH, as well as a partial dependence on dilauroylphosphatidylcholine. Of the cytochrome P-450 isozymes evaluated, only cytochrome P-450j, purified from livers of ethanol- or isoniazid-treated rats, had high catalytic activity for the N-demethylation of NDMA. At substrate concentrations of 0.5 and 5 mM, rates of NDMA metabolism to formaldehyde catalyzed by cytochrome P-450j were at least 15-fold greater than the rates obtained with any of the other purified isozymes. At the pH optimum (approximately 6.7) for the reaction, the Km,app and Vmax were 3.5 mM and 23.9 nmol/min/nmol cytochrome P-450j, respectively. With hepatic microsomes from ethanol-treated rats, which contain induced levels of cytochrome P-450j, the Km,app and Vmax were 0.35 mM and 3.9 nmol/min/nmol cytochrome P-450, respectively. Inclusion of purified cytochrome b5 in the reconstituted system containing cytochrome P-450j caused a six-fold decrease in Km,app (0.56 mM) of NDMA demethylation with little or no change in Vmax (29.9 nmol/min/nmol cytochrome P-450j). Trypsin-solubilized cytochrome b5, bovine serum albumin, or hemoglobin had no effect on the kinetic parameters of the reconstituted system, indicating a specific effect of intact cytochrome b5 on the Km,app of the reaction. These results demonstrate high isozyme specificity in the metabolism of NDMA to an ultimate carcinogen and further suggest an important role for cytochrome b5 in this biotransformation process. 相似文献
12.
CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro. 总被引:2,自引:0,他引:2
In mammals, hydrocortisone synthesis from cholesterol is catalyzed by a set of five specialized enzymes, four of them belonging to the superfamily of cytochrome P-450 monooxygenases. A recombinant yeast expression system was recently developed for the CYP11B1 (P45011beta) enzyme, which performs the 11beta hydroxylation of steroids such as 11-deoxycortisol into hydrocortisone, one of the three mitochondrial cytochrome P-450 proteins involved in steroidogenesis in mammals. This heterologous system was used to test the potential interaction between CYP11B1 and CYP11A1 (P450scc), the mitochondrial cytochrome P-450 enzyme responsible for the side chain cleaving of cholesterol. Recombinant CYP11B1 and CYP11A1 were targeted to Saccharomyces cerevisiae mitochondria using the yeast cytochrome oxidase subunit 6 mitochondrial presequence fused to the mature form of the two proteins. In yeast, the presence of CYP11A1 appears to improve 11beta hydroxylase activity of CYP11B1 in vivo and in vitro. Fractionation experiments indicate the presence of the two proteins in the same membrane fractions, i.e. inner membrane and contact sites of mitochondria. Thus, yeast mitochondria provide interesting insights to study some molecular and cellular aspects of mammalian steroid synthesis. In particular, recombinant yeast should permit a better understanding of the mechanism permitting the synthesis of steroids (sex steroids, mineralocorticoids and glucocorticoids) with a minimal set of enzymes at physiological level, thus avoiding disease states. 相似文献
13.
Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin 总被引:1,自引:0,他引:1
Tuckey RC Nguyen MN Slominski A 《The international journal of biochemistry & cell biology》2008,40(11):2619-2626
Vitamin D3 can be hydroxylated sequentially by cytochrome P450scc (CYP11A1) producing 20-hydroxyvitamin D3, 20,23-dihydroxyvitamin D3 and 17,20,23-trihydroxyvitamin D3. The aim of this study was to characterize the ability of vitamin D3 to associate with phospholipid vesicles and to determine the kinetics of metabolism of vitamin D3 by P450scc in vesicles and in 2-hydroxypropyl-beta-cyclodextrin (cyclodextrin). Gel filtration of phospholipid vesicles showed that the vitamin D3 remained quantitatively associated with the phospholipid membrane. Vitamin D3 exchanged between vesicles at a rate 3.8-fold higher than for cholesterol exchange and was stimulated by N-62 StAR protein. The Km of P450scc for vitamin D3 in vesicles was 3.3 mol vitamin D3/mol phospholipid and the rate of conversion of vitamin D3 to 20-hydroxyvitamin D3 was first order with respect to the vitamin D3 concentration for the range of concentrations of vitamin D3 that could be incorporated into the vesicle membrane. 20-Hydroxyvitamin D3 was further hydroxylated by P450scc in vesicles, producing primarily 20,23-dihydroxyvitamin D3, with Km and kcat values 22- and 6-fold lower than those for vitamin D3, respectively. 20,23-dihydroxyvitamin D3 was converted to 17,20,23-trihydroxyvitamin D3 with even lower Km and kcat values. Vitamin D3 and cholesterol were metabolized with comparable efficiencies in cyclodextrin, but the Km for both showed a strong dependence on the cyclodextrin concentration, decreasing with decreasing cyclodextrin. This study shows that vitamin D3 quantitatively associates with phospholipid vesicles, can exchange between membranes, and can be hydroxylated by membrane-associated P450scc but with lower efficiency than for cholesterol hydroxylation. The kcat values for metabolism of vitamin D3 in vesicles and 0.45% cyclodextrin are similar, but the ability to solubilize vitamin D3 at a concentration higher than its Km makes the cyclodextrin system more efficient for producing the hydroxyvitamin D3 metabolites for further characterization. 相似文献
14.
Adrenodoxin, purified from bovine adrenal cortex, was subjected to trypsin cleavage to yield a trypsin-resistant form, designated TT-adrenodoxin. Sequencing with carboxypeptidase Y identified the trypsin cleavage site as Arg-115, while Edman degradation indicated no NH2-terminal cleavage. Native adrenodoxin and TT-adrenodoxin exhibited similar affinity for adrenodoxin reductase as determined in cytochrome c reductase assays. In side chain cleavage assays using cytochrome P-450scc, however, TT-adrenodoxin demonstrated greater activity than adrenodoxin with cholesterol, (22R)-22-hydroxycholesterol, or (20R,22R)-20,22-dihydroxycholesterol as substrate. This enhanced activity is due to increased affinity of TT-adrenodoxin for cytochrome P-450scc; TT-adrenodoxin exhibits a 3.8-fold lower apparent Km for the conversion of cholesterol to pregnenolone. TT-Adrenodoxin was also more effective in coupling with cytochrome P-450(11) beta, exhibiting a 3.5-fold lower apparent Km for the 11 beta-hydroxylation of deoxycorticosterone. In the presence of partially saturating cholesterol, TT-adrenodoxin elicited a type I spectral shift with cytochrome P-450scc similar to that induced by adrenodoxin, and spectral titrations showed that oxidized TT-adrenodoxin exhibited a 1.5-fold higher affinity for cytochrome P-450scc. These results establish that COOH-terminal residues 116-128 are not essential for the electron transfer activity of bovine adrenodoxin, and the differential effects of truncation at Arg-115 on interactions with adrenodoxin reductase and cytochromes P-450 suggest that the residues involved in the interactions are not identical. 相似文献
15.
Cholesterol side-chain cleavage (CSCC) catalyzed by purified bovine adrenal mitochondrial cytochrome P-450scc is highly dependent on the vesicles that supply cholesterol. Six-fold higher rates are achieved with large unilamellar dioleoylphosphatidylcholine vesicles (diameter 150 nm) prepared by octyl glucoside (OG) dialysis (DOPC-LUV) than with small sonicated vesicles (diameter 30 nm) (DOPC-SUV) (Vmax = 25 and 4 min-1, respectively. Extensive dialysis that may remove OG decreased Vmax rates for DOPC-LUV almost to rates seen with DOPC-SUV. These dialyzed DOPC-LUV were, however, very sensitive to addition of OG (EC50 = 2.5 microM, 4.3-fold stimulation) while DOPC-SUV were only weakly affected (EC50 = 100 microM, 1.6-fold stimulation). This enhancement of CSCC in LUV by OG only occurred when the cholesterol:DOPC exceeded 0.1 and was associated with a 15-fold increase in the Km for cholesterol. Structural changes in both SUV and LUV at high cholesterol:DOPC ratios (0.1-1) were indicated by decreases in internal volume that were insensitive to OG and did not affect the external diameters. Stearic acid produced a similar stimulation of CSCC in LUV (EC50 = 50 microM) and had no effect on SUV. The Vmax for CSCC, produced by OG activation of DOPC-LUV, is comparable to the highest attained for cytochrome P-450scc (Tween 20/cholesterol). In LUV, a minor proportion of OG (1-5% of cholesterol) is thus sufficient to generate a domain of reactive cholesterol that maintains a near-optimum turnover. This increased CSCC was paralleled by increased binding of cholesterol to P-450scc, suggesting that this cholesterol is more readily donated by the membrane to the cytochrome. 相似文献
16.
Tuckey RC Headlam MJ Bose HS Miller WL 《The Journal of biological chemistry》2002,277(49):47123-47128
The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in steroidogenic cells. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P-450scc converts this cholesterol into pregnenolone. We tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P-450scc to acceptor vesicles containing P-450scc but no cholesterol, using P-450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P-450scc activity in acceptor vesicles 5-10-fold following the addition of donor vesicles. Transfer of cholesterol to acceptor vesicles was rapid and sufficient to maintain a linear rate of pregnenolone synthesis for 10 min. The effect of N-62 StAR in stimulating P-450scc activity was specific for cholesterol transfer and was not due to vesicle fusion or P-450scc exchange between vesicles. Maximum stimulation of P-450scc activity in acceptor vesicles required preincubation of N-62 StAR with phospholipid vesicles prior to adding donor vesicles. The amount of N-62 StAR causing half-maximum stimulation of P-450scc activity in acceptor vesicles was 1.9 microm. Half-maximum stimulation required more than a 10-fold higher concentration of R182L N-62 StAR, a mutant associated with congenital lipoid adrenal hyperplasia. N-62 StAR-mediated transfer of cholesterol between vesicles showed low dependence on the cholesterol concentration in the donor vesicles. Thus StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria. 相似文献
17.
H Mochizuki K Suhara M Katagiri 《The Journal of steroid biochemistry and molecular biology》1992,42(1):95-101
The minor steroid hydroxylase activity of purified bovine adrenocortical mitochondrial P-450 is described. The results indicate that both P-450scc and P-450(11 beta) act on deoxycorticosterone and androstenedione to form 6 beta-hydroxydeoxycorticosterone and 6 beta-hydroxyandrostenedione (6 beta-hydroxylase), respectively. Both forms of P-450 also catalyze 6-desaturation of androstenedione to form 4,6-androstadiene-3,17-dione (6-desaturase). 相似文献
18.
Slominski A Semak I Zjawiony J Wortsman J Li W Szczesniewski A Tuckey RC 《The FEBS journal》2005,272(16):4080-4090
We show that cytochrome P450scc (CYP11A1) in either a reconstituted system or in isolated adrenal mitochondria can metabolize vitamin D3. The major products of the reaction with reconstituted enzyme were 20-hydroxycholecalciferol and 20,22-dihydroxycholecalciferol, with yields of 16 and 4%, respectively, of the original vitamin D3 substrate. Trihydroxycholecalciferol was a minor product, likely arising from further metabolism of dihydroxycholecalciferol. Based on NMR analysis and known properties of P450scc we propose that hydroxylation of vitamin D3 by P450scc occurs sequentially and stereospecifically with initial formation of 20(S)-hydroxyvitamin D3. P450scc did not metabolize 25-hydroxyvitamin D3, indicating that modification of C25 protected it against P450scc action. Adrenal mitochondria also metabolized vitamin D3 yielding 10 hydroxyderivatives, with UV spectra typical of vitamin D triene chromophores. Aminogluthimide inhibition showed that the three major metabolites, but not the others, resulted from P450scc action. It therefore appears that non-P450scc enzymes present in the adrenal cortex to some extent contribute to metabolism of vitamin D3. We conclude that purified P450scc in a reconstituted system or P450scc in adrenal mitochondria can add one hydroxyl group to vitamin D3 with subsequent hydroxylation being observed for reconstituted enzyme but not for adrenal mitochondria. Additional vitamin D3 metabolites arise from the action of other enzymes in adrenal mitochondria. These findings appear to define novel metabolic pathways involving vitamin D3 that remain to be characterized. 相似文献
19.
C R Jefcoate W H Orme-Johnson H Beinhert 《The Journal of biological chemistry》1976,251(12):3706-3715
The binding of cholest-5-ene-3beta,20alpha-diol (20alpha-hydroxycholesterol), 11-deoxycorticosterone, and aminoglutethimide to cytochrome P-450 in bovine adrenal mitochondria was measured by changes in optical spectra at room temperature and by EPR spectra at 14 K. The two methods provided nearly identical quantitation of these interactions with cytochrome P-450. Two distinct high spin forms of cytochrome P-450 were revealed by EPR spectra. The predominant high spin species (g = 8.2) was decreased by addition of 20alpha-hydroxycholesterol and elevated pH but was increased by addition of cholesterol. The minor high spin species (g = 8.1) was incrreased by addition of deoxycorticosterone but decreased by low concentrations of metyrapone. The two forms were evidently not in equilibrium and have been assigned to distinct forms of cytochrome P-450 involved in, respectively, cholesterol side chain cleavage (P-450scc) and steroid 11beta hydroxylation (P-450(11)beta). The high spin states are derived from complexes of these P-450 cytochromes with endogenous substrates, which are, respectively, cholesterol and deoxycorticoids. A high to low spin transition was observed when these complexes were turned over by initiating hydroxylation with malate. The contributions of cytochromes P-450(11)beta and P-450scc to the low spin spectrum were also resolved by similar means. At least 20% of P-450scc is in the low spin state while about 90% of P-450(11)beta is low spin in isolated beef adrenal mitochondria. Low spin complexes of cytochrome P-450scc with 20alpha-hydroxycholesterol and 3beta-hydroxypregn-5-ene-20-one (pregnenolone) gave distinct EPR spectra. Aminoglutethimide interacted with the total cytochrome P-450 content of the bovine adrenal mitochondria forming low spin complexes. Both optical and EPR data indicated binding to two forms of cytochrome P-450. These results suggest a detailed correlation between the spin state and absorbance changes seen at room temperature, illustrate that EPR allows the distinction of two principal forms of P-450, and suggest that there is no appreciable change in the spin state of either cytochrome between 14 K and 300 K. 相似文献
20.
The present study was to investigate the effects and action mechanisms of digoxin and ouabain on steroidogenesis in human adrenocortical NCI-H295 cells. Administration of digoxin or ouabain for 24 h decreased the basal and angiotensin II (Ang II)-stimulated release of aldosterone by NCI-H295 cells. The conversions of corticosterone (substrate of cytochrome P450 aldosterone synthase, P450c11AS) to aldosterone or deoxycortisol (substrate of cytochrome P450 11beta-hydroxylase, P450c11beta) to cortisol were reduced by digoxin or ouabain. The basal and 22-hydroxy-cholesterol (a membrane-permeable cholesterol, substrate of cytochrome P450 side-chain cleavage enzyme, P450scc)-stimulated pregnenolone release in mitochondria was inhibited by digoxin or ouabain. Digoxin or ouabain suppressed the basal and Ang II-stimulated protein expression of steroidogenic acute regulatory (StAR) protein and P450scc. Incubation of digoxin or ouabain for 24 h reduced P450c11AS mRNA expression in NCI-H295 cells. Digoxin or ouabain (10(-6) M, 24 h)-treated cells showed a lower resting intracellular Ca2+ concentration ([Ca2+]i) and an attenuated response of [Ca2+]i to Ang II. Since no significant cytotoxicity was observed at 10(-6) M digoxin or ouabain, the digoxin- or ouabain-induced decrease of aldosterone or cortisol release was not associated with cytotoxicity. These results demonstrate that digoxin or ouabain inhibits the aldosterone or cortisol release via reduction of P450c11AS or P450c11beta and P450scc activities, inhibition of StAR and P450scc protein expression, suppression of P450c11AS mRNA expression, and attenuation of Ca2+ mobilization in NCI-H295 cells. 相似文献