Pregnenolone production by adrenal mitochondria: a new high-performance liquid chromatographic analytical method for cholesterol side-chain cleavage.
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Adrenal Glands↗
Biomedical subjects
J D Lambeth
Publications and source records attributed to J D Lambeth.
Platelet-derived growth factor stimulates phagocytosis and blocks agonist-induced activation of the neutrophil oxidative burst: a possible cellular mechanism to protect against oxygen radical damage.
The effect of platelet-derived growth factor (PDGF) on agonist-induced activation of the superoxide-generating oxidative burst in human neutrophils was tested. PDGF had no effect on the resting level of superoxide generation but inhibited both the rate and the extent of fMet-Leu-Phe-stimulated superoxide production in a dose-dependent manner. The concentration required to inhibit the response by 50% was 95 +/- 26 pM (n = 10). PDGF also blocked activation by other receptor-mediated agonists such as the complement protein C5a and opsonized zymosan, but not by phorbol myristate acetate or arachidonate, both of which may act at postreceptor sites. The growth factor, however, had no effect on the binding of fMet-Leu-Phe to its receptor. PDGF in concentrations that blocked the oxidative burst stimulated phagocytosis of opsonized latex particles. Thus, PDGF functions as a heterologous "down-regulator" of receptor-mediated activation of the neutrophil oxidative burst and an activator of phagocytosis. A model for a feedback regulatory loop between platelets and neutrophils is proposed.
Blood Platelets↗
Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst.
The neutrophil oxidative burst is characterized by increased cellular O2 consumption due to the activation of a membrane-associated superoxide-generating NADPH-oxidase. The response is triggered by a variety of stimuli, including opsonized zymosan, formylmethionylleucinephenylalanine (FMLP), arachidonate, short-chain diacylglycerols, and phorbol myristate acetate (PMA). We herein demonstrate that incubation of cells with sphinganine or sphingosine blocks or reverses activation by these agonists. The inhibition is reversible, does not affect cell viability, and does not affect another complex cell function, phagocytosis. Inhibitory concentrations of sphinganine did not significantly affect cytoplasmic calcium levels or FMLP-generated calcium transients. Structural requirements for inhibition of the oxidative burst include a long aliphatic chain and an amino-containing head-group, and there is modest specificity for the native (erythro) isomer of sphinganine. Inhibition involves stimulus-induced activation mechanisms rather than a direct effect on the NADPH oxidase, since sphinganine did not inhibit NADPH-dependent superoxide generation in isolated membranes containing the active enzyme. Activation by FMLP, diacylglycerol, PMA, opsonized zymosan, and arachidonate was blocked by the same concentrations of sphinganine, indicating that these agonists share a common inhibited step. Three lines of evidence indicate that this step involves protein kinase C. First, in a micelle system and in platelets, long-chain bases are inhibitors of this enzyme (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). Second, sphinganine blocks PMA-stimulated incorporation of 32PO4 into neutrophil proteins. Third, sphinganine inhibits the binding of [3H]phorbol dibutyrate to its cellular receptor, known to be protein kinase C. We suggest that long-chain bases function as physiologic modulators of cellular regulatory pathways involving protein kinase C.
Arachidonic Acid↗
Use of cytochrome P-450scc to measure cholesterol-lipid interactions.
The interaction of cholesterol with phospholipids has been studied with a variety of techniques; however, the possible consequences of such interactions in vivo have not been demonstrated. In this study, the cholesterol-dependent absorbance spectrum of cytochrome P-450scc was used to monitor cholesterol availability in both micellar and vesicular environments. By use of this approach, in conjunction with titration of putative cholesterol binding species, a tight, approximately equimolar complex of cholesterol and digitonin was demonstrated. Sphingomyelin (SM) (both the synthetic N-palmitoyl and bovine brain forms) gave sigmoidal titration curves, suggesting a cooperative interaction between this lipid and cholesterol. The interaction of bovine brain glycerolipids and cholesterol was weaker than that of SM and showed no cooperativity. The importance of the phospholipid head group in these interactions was established by the differences in the ability of synthetic 1-palmitoyl-2-oleoylphosphatidylcholine, -phosphatidylethanolamine, and -phosphatidylserine to affect cholesterol availability. Comparison of these results with those of the bovine brain phospholipids indicates that the acyl chain composition of these molecules is also important to these interactions. Titrations of SM in phospholipid vesicles containing cytochrome P-450scc and different types of phosphatidylcholine established that the SM-cholesterol interactions also occur in a bilayer membrane. This study demonstrates that the association of cholesterol with cytochrome P-450scc is inhibited by concentrations of SM commonly found in biological membranes. Therefore, such cholesterol-lipid interactions can potentially affect the function of membrane enzymes.
Adrenal Cortex↗
Cytochrome P-450scc-substrate interactions. Role of the 3 beta- and side chain hydroxyls in binding to oxidized and reduced forms of the enzyme.
The steroid binding specificity of cytochrome P-450scc has been investigated for different oxidation/reduction and ligand-binding states of the enzyme (oxidized, reduced, oxygen-bound, and carbon monoxide-bound forms). The oxygen of the 3 beta-hydroxyl of cholesterol is important for the initial enzyme-substrate interaction. Significant binding requires the correct stereochemistry and appears to be controlled by the electron density on the 3 beta-oxygen. Interactions at this position (located at least 13 A from the heme iron) can modulate the heme midpoint potential. The binding site in this region contains a cleft which can accommodate up to two carbons joined in an ether linkage to the 3 beta-oxygen. The steroid intermediates of side chain cleavage (22R-hydroxycholesterol and 20 alpha,22R-dihydroxycholesterol) bind more tightly to the ferric enzyme than does cholesterol and utilize specific interactions of these side chain hydroxyls with a grouping(s) on the polypeptide chain (i.e. not with the heme iron). The interaction requires the correct stereochemistry; a 22S-hydroxyl cannot be readily accommodated in the binding site. The specificity of the interaction for hydroxyls at the 22R- versus the 20 alpha-position is altered upon reduction of the enzyme, indicating a reduction-induced conformational change in the active site. The specific interference of binding of 22R-hydroxy steroids by heme-bound carbon monoxide (but not oxygen), together with the known bond angles and distances for Fe-C-O and Fe-O-O, allows localization of the 22R-hydroxyl group on a line perpendicular to the heme plane, between 2 and 3 A from the iron.
Animals↗
NADH dehydrogenase from bovine neutrophil membranes. Purification and properties.
A membrane-associated NADH dehydrogenase from beef neutrophils was purified to homogeneity, using detergent (cholate plus Triton X-100) extraction and chromatography on DEAE-Sepharose CL-6B, agarose-hexane-NAD, and hydroxylapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 17,500, but the enzyme was highly aggregated (Mr greater than 450,000) in nondenaturing gels containing 0.1% Triton X-100. The protein band in nondenaturing gels was also stained for activity using NADH and nitro blue tetrazolium. The enzyme showed greatest electron acceptor activity with ferricyanide (100%), followed by cytochrome c (3.5%), dichloroindophenol (2.7%), and cytochrome b5 (0.34%). No activity was seen with oxygen. The Km values for NADH and ferricyanide were 18 and 9.5 microM, respectively, and NAD+ was a weak competitive inhibitor (Ki = 118 microM). No activity was seen with NADPH. No effects were seen with mitochondrial respiratory inhibitors such as azide, cyanide, or rotenone, but p-chloromercuribenzoate was strongly inhibitory and N-ethylmaleimide was weakly inhibitory. No free flavin was detectable in enzyme preparations. Based upon kinetic, physical, and inhibition properties, this NADH dehydrogenase differs from those previously described in microsomes and erythrocyte plasma membrane.
Animals↗
Cytochrome P-450scc a review of the specificity and properties of the cholesterol binding site.
Cytochrome P-450scc is unusual among members of this class of enzymes in showing a high degree of substrate specificity. Features of the cholesterol structure which are particularly important for binding include the 3 beta-hydroxyl, the delta 5-ring configuration, and the side-chain organization in the 20-22 region. Regarding the ring system, binding appears to require planarity and limited size at the 4-5-6 carbons (the A-B ring juncture). In the region of the 3 beta-hydroxyl, a "cleft" in the binding site extends about 4 A beyond the hydroxyl and can accommodate two additional ether-linked carbons. Evidence indicates that an enzyme residue hydrogen-bonds to the oxygen of the 3 beta hydroxyl, providing much of the energy for the initial enzyme-substrate interaction. The cytochrome shows less specificity for the side-chain structure, except in the region of carbons 20-22 where hydroxylation/side-chain cleavage takes place. The binding cleft for the side-chain is limited to approximately the length of the isocaproic group but can accommodate structural variations beyond the 22-position. Evidence indicates that the region near the 20-22 bond is more limited in size, and that an amino acid residue near the heme iron binds strongly and stereospecifically to the 22R-hydroxyl of the cleavage intermediates, 22R-hydroxycholesterol and 20 alpha, 22R-dihydroxycholesterol. The 22R-hydrogen of cholesterol is very close to the heme iron (approximately 3 A), while the 22S-hydrogen is slightly further (about 4 A). The size and bonding properties of the steroid binding/active site suggest a mechanism which accounts for the stereospecificity and sequence of reactions catalyzed by cytochrome P-450scc.
Adrenal Cortex↗
Cytochrome P-450scc-adrenodoxin interactions. Ionic effects on binding, and regulation of cytochrome reduction by bound steroid substrates.
The binding of adrenodoxin to cytochrome P-450scc and the intracomplex electron transfer from the iron-sulfur center to the heme have been studied. Salt sensitivity of the protein complex suggests the participation of electrostatic forces, as is also seen for the complex of adrenodoxin with NADPH-adrenodoxin reductase. Differences in ion specificities for the complexes of adrenodoxin with the other two proteins suggest some differences in binding requirements. Insensitivity of the heme reduction to solution conditions (salt, detergent) and kinetic analysis indicate that the protein complex is formed rapidly and that intracomplex electron transfer then occurs more slowly. Factors governing the rate of this electron transfer were investigated; binding of a series of cholesterol derivatives was used to perturb the spin state, midpoint potential, and reduction rate of the heme, and thus to test for relationships among these parameters. A linear free energy relationship between the substrate-induced midpoint potential and reduction rate is seen, but none of the other parameters (including the strength of substrate binding) are correlated. Data indicate that factors other than spin state (i.e. steric requirements and bonding groups within the steroid-binding site) regulate the strength of steroid binding. The bound steroid then modulates both midpoint potential/reduction rate and spin state but by independent mechanisms.
Adrenodoxin↗
Regulation of mitochondrial compartment volumes in rat adrenal cortex by ether stress.
In vivo ether stress of rats causes release of pituitary adrenocorticotropin (ACTH) leading to activation of steroidogenesis in adrenal cortex mitochondria. The present studies show that this treatment also induces a decrease in the volume of the intermembrane space in isolated adrenal mitochondria. This decrease is accompanied by an increase in the volume of the matrix, thus leaving the total mitochondrial volume approximately constant. These effects are prevented by the protein synthesis inhibitor, cycloheximide, and are specific to the adrenal gland. The decrease in the intermembrane space (or increase in the matrix volume) is correlated with activation of the cholesterol side chain cleavage reaction (the regulated step in steroidogenesis). We propose as a working hypothesis that these changes reflect a hormonally regulated alteration in the relationship between the outer and inner mitochondrial membranes, which may facilitate the rate-limiting movement of cholesterol from the outer to the inner membrane where the side chain cleavage enzyme is located.
Adrenal Cortex↗
NADPH-cytochrome P-450 reductase-cytochrome b5 interactions: crosslinking of the phospholipid vesicle-associated proteins by a water-soluble carbodiimide.
Detergent-solubilized and purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome b5 were coreconstituted into phospholipid vesicles. When the proteoliposomes were incubated with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a new higher-molecular-weight band was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The band was purified by chromatography on DEAE-Sepharose CL-6B, 2'5'-ADP-Sepharose 4B, and Sephadex G-100. The heme absorption spectrum and fluorophotometric assay of flavin of the purified material demonstrate that this product is a 1:1 crosslinked complex containing one molecule each of the flavoprotein and cytochrome. Proteolysis of the crosslinked form indicates that the hydrophilic catalytic domains participate in the covalent attachment, and that the hydrophobic membrane-attachment peptide is necessary for the protein interaction. The purified crosslinked derivative showed no activities for reduction of either cytochrome c or ferricyanide. About half of the enzyme-associated flavin was reduced rapidly by NADPH, as was 20-30% of the crosslinked cytochrome, indicating that, in at least some of the complexes, the flavin-mediated pathway for reduction of cytochrome by pyridine nucleotide was intact. These data suggest that the output- rather than the input-electron transfer site(s) in the flavoprotein was (were) blocked by the covalently attached cytochrome.
Animals↗
Paramagnetic probes of multicomponent electron-transfer systems.
Electron-transfer systems associated with steroid hormone production in the adrenals and with photosynthetic NADP+ reduction in the chloroplast share some important features. Complexes between components in both systems affect the exposure of prosthetic groups to water-soluble paramagnetic groups. The sensitivity of the components to probes of different charges suggests that electrostatic interactions are important in complex formation. Haem plane orientation in cytochrome P-450 of the adrenal system is parallel to the membrane plane in both native and reconstituted systems.
Adrenal Glands↗
Adrenodoxin interaction with adrenodoxin reductase and cytochrome P-450scc. Cross-linking of protein complexes and effects of adrenodoxin modification by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
Modification of the three carboxyl groups on adrenodoxin using a water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) caused a weakening of the binding of this iron-sulfur protein to both its electron donor protein, adrenodoxin reductase, and its electron acceptor protein, cytochrome P-450scc. Based upon the proximity of the modified groups, the site on adrenodoxin for interaction with the other two proteins is likely to be either identical or highly overlapping, and formation of a ternary complex among the proteins is precluded. Upon incubation of adrenodoxin and either adrenodoxin reductase or cytochrome P-450 plus the carbodiimide (1:1), covalently cross-linked species were formed. When all three proteins were incubated with the cross-linker, only the binary complexes were formed, and no higher order (e.g. 1:1:1 or 1:2:1) complexes were seen. These studies indicate that adrenodoxin forms exclusive binary complexes with its electron transfer partner proteins, and thus provide a physical explanation for the proposed role of adrenodoxin as a mobile electron shuttle between NADPH-adrenodoxin reductase and cytochrome P-450scc.
Adrenodoxin↗
Cytochrome b558 from (bovine) granulocytes. Partial purification from Triton X-114 extracts and properties of the isolated cytochrome.
A membrane-associated b-type cytochrome (a proposed component in the neutrophil microbicidal superoxide generating system) has been partially purified from nonactivated beef granulocytes to a specific heme content of 20 nmol of heme/mg of protein, a value about 10-fold higher than those previously reported. The hemoprotein was solubilized at low temperature (4 degrees C) from mixed granule (30,000 X g) cell fractions using Triton X-114 detergent. Warming the extract to 25 degrees C allowed separation into detergent and aqueous phases; cytochrome b558 partitioned exclusively into the detergent phase, allowing separation from other visible-absorbing species (e.g. myeloperoxidase) and indicated an intrinsic membrane localization (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). The partitioned cytochrome was chromatographed on hydroxylapatite and a hydrophobic affinity matrix, allowing a 185-fold (heme content) purification from the granule extract. The cytochrome preparation revealed three equal-staining protein bands by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis; apparent molecular weights were 14,000, 12,000, and 11,000. The question of heterogeneity of the preparation versus subunit structure is not resolved at present. The hemoprotein binds carbon monoxide, consistent with a proposed role as a terminal oxidase, and has an unusually negative oxidation-reduction potential (-225 mV) similar to that observed in granulocyte membranes. The preparation is devoid of NAD(P)H-diaphorase and cytochrome c reductase activities.
Animals↗
Oxygen dependence of adrenal cortex cholesterol side chain cleavage. Implications in the rate-limiting steps in steroidogenesis.
The oxygen dependence of cholesterol side chain cleavage to form pregnenolone was measured, using both purified phospholipid vesicle-reconstituted cytochrome P-450scc and rat adrenal mitochondria. At saturating cholesterol and nonlimiting electron supply (via NADPH-adrenodoxin reductase and adrenodoxin) the Km(O2) is low (4 microM). Limitations in the availability of both cholesterol and reductant caused elevations in the observed Km(O2). Pregnenolone synthesis was measured in mitochondria from variously pretreated rats, using a phospholipid-cholesterol dispersion as the source of exogenous substrate. In mitochondria obtained from ether-stressed rats (which elevates adrenocorticotropic hormone) two phases of malate-supported pregnenolone production are seen, a rapid (first 2 min) highly oxygen-dependent phase (Km = 150 microM) and a slow (2-10 min) relatively oxygen-independent phase (Km less than 10 microM). Comparison of side chain cleavage rates with mitochondrial 11 beta-hydroxylation rates at various oxygen concentrations suggests that the rapid phase is limited by the availability of reducing equivalents. In cycloheximide-pretreated ether-stressed rats, only a linear slow rate of pregnenolone production was seen (about 25% of the rate of the slow phase in the ether-stressed group), while in mitoplasts from both groups only a linear rapid rate was seen. Data are consistent with the proposal (Privalle, C. T., Crivello, J. F., and Jefcoate, C. R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 702-706) that the adrenocorticotropic hormone-regulated cycloheximide-inhibitable rate of cholesterol side chain cleavage is limited by the rate of cholesterol transfer from outer to inner mitochondrial membranes.
Adrenal Cortex↗
Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization.
Addition of homogeneous rat liver sterol carrier protein2 (SCP2) or an adrenal cytosolic fraction enhanced pregnenolone production by adrenal mitochondria. Pretreatment of SCP2 or adrenal cytosol with anti-SCP2 IgG abolished the stimulatory effect of both preparations on mitochondrial pregnenolone output. Incubation of mitochondria with aminoglutethimide, which blocks interaction of cholesterol with inner membrane cytochrome P-450scc, resulted in decreased pregnenolone production and a decreased level of mitoplast cholesterol. Addition of SCP2 to the incubation media caused an almost 2-fold increase in cholesterol associated with the mitoplast, but did not enhance mitochondrial pregnenolone production. Studies with reconstituted cytochrome P-450scc in phospholipid vesicles also suggested that SCP2 did not affect interaction of cholesterol with the hemoprotein. Treatment of rats with cycloheximide alone or with adrenocorticotropic hormone resulted in a dramatic increase in mitochondrial cholesterol. However, these mitochondria did not exhibit increased levels of pregnenolone output under control incubation conditions. When SCP2 was included in the mitochondrial incubation media, pregnenolone production was significantly increased over that observed with adrenal mitochondria from untreated or adrenocorticotropic hormone-treated rats. The results imply that SCP2 enhances mitochondrial pregnenolone production by improving transfer of mitochondrial cholesterol to cytochrome P-450scc on the inner membrane, but does not directly influence the interaction of substrate with the hemoprotein.
Adrenal Glands↗
Cytochrome P-450scc-adrenodoxin complex. Reduction properties of the substrate-associated cytochrome and relation of the reduction states of heme and iron-sulfur centers to association of the proteins.
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Adrenodoxin↗
Cytochrome P-450scc-phospholipid interactions. Evidence for a cardiolipin binding site and thermodynamics of enzyme interactions with cardiolipin, cholesterol, and adrenodoxin.
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Adrenal Glands↗
22-Ketocholesterol. A potent competitive inhibitor of cytochrome P-450scc-dependent side-chain cleavage of cholesterol.
22-Ketocholesterol binds with high affinity to purified, phospholipid vesicle-reconstituted cytochrome P-450scc. Binding, quantitated using reversal of cholesterol-induced absorbance changes in the Soret region of the enzyme, indicates an affinity 3-5 times greater than that for the normal substrate cholesterol. The ketosteroid cannot be hydroxylated at position 22 and thus acts as a potent inhibitor of cholesterol side-chain cleavage. Steady-state kinetics demonstrate competitive inhibition by this steroid and provide a KI value several-fold lower than the cholesterol Km. On the basis of recently proposed mechanisms for hydroxylation by cytochromes P-450, 22-ketocholesterol may exert its inhibitory effect by acting as a tightly bound analogue that resembles the enzyme-bound cholesterol from which a hydrogen has been abstracted from position 22.
Adrenal Cortex↗