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J D Lambeth

Publications and source records attributed to J D Lambeth.

At least 73 records · Page 4Linked to original sources
PubMed1989

On the biological occurrence and regulation of 1-acyl and 1-O-alkyl-diradylglycerols in human neutrophils. Selective destruction of diacyl species using Rhizopus lipase.

The occurrence and regulation of 1-ether-linked diradylglycerol in human neutrophils were investigated using a sensitive and practical analytical mass method which distinguishes 1-O-alkyl- (EAG) versus 1-acyl (DAG) diglycerides. After phosphorylation of diglycerides to the corresponding [32P]phosphatidic acids using [gamma-32P]ATP and diglyceride kinase (Preiss, J., Loomis, C. R., Bishop, W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986) J. Biol. Chem. 261, 8597-8600), lipase from Rhizopus arrhizus selectively degraded the 1-acyl-containing species (DAG), but the ether lipid (EAG) was resistant and was identified and quantified after thin layer chromatography separation. By using this method, unstimulated neutrophils were demonstrated to contain both DAG and EAG (100-180 and 40-95 pmol/10(7) cells, respectively). The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) caused a rapid (30 s) and transient increase (1.6-fold) in DAG, but no increase in EAG. Opsonized zymosan produced a 6-8-fold sustained increase in DAG peaking at 2 to 3 min, but only a small (1.7-fold) increase in EAG which was not seen until later times (10 min). Thus, under these stimulation conditions, the major diglyceride was DAG. However, in neutrophils "primed" with cytochalasin B or phorbol ester, formyl-methionyl-leucyl-phenylalanine caused a significant increase in EAG. Neutrophils pretreated with cytochalasin B and then stimulated by fMLP showed a rapid (15-60 s) increase (more than 3-fold) in total diglycerides which was sustained beyond 5 min. At the earliest time points (15-30 s), the increase was due almost entirely to DAG (3-fold), but at 1 min and beyond, EAG comprised as much as 40% of the total (up to a 5-fold increase in EAG). Neutrophils pretreated with phorbol ester prior to fMLP stimulation showed a rapid (around 30 s) more than 2-fold increase in both DAG and EAG. Thus, priming conditions (in particular cytochalasin B) may alter either the access of phospholipase(s) C and/or D to membrane phospholipids or may affect their activities, allowing hydrolysis of 1-O-alkyl-containing lipids to generate 1-O-alkyl-containing diglycerides.

Cytochalasin B↗
PubMed1989

Cholesterol sulfate is a naturally occurring inhibitor of steroidogenesis in isolated rat adrenal mitochondria.

We previously reported (Lambeth, J. D., Xu, X. X., and Glover, M. (1987) J. Biol. Chem. 262, 9181-9188) that exogenously added cholesterol sulfate inhibits the conversion of cholesterol to pregnenolone in isolated adrenal mitochondria, and does so by affecting intramitochondrial cholesterol movement but not its subsequent metabolism to pregnenolone by cytochrome P-450scc. We now report that a major kinetic component of the inhibition is noncompetitive with respect to cholesterol, consistent with an allosteric effect at a site other than the substrate binding site of cytochrome P-450scc. We now also report that cholesterol sulfate is present as an endogenous compound in preparations of adrenal mitochondria. Its content varied from 0.05 to 0.8 nmol/mg protein. Cholesterol sulfate level correlated inversely with the mitochondrial cholesterol side-chain cleavage activity. Endogenous cholesterol sulfate thus appeared to account for the variable rates of pregnenolone synthesis which were seen in different mitochondrial preparations. Cholesterol sulfate was metabolized to pregnenolone sulfate by a mitochondrial side-chain cleavage system, but proved to be a relatively poor substrate for an extramitochondrial steroid sulfatase activity present in adrenal cortex. Confirming a role as a naturally occurring inhibitor, removal of endogenous mitochondrial cholesterol sulfate by metabolism to pregnenolone sulfate correlated with a 3-fold activation of cholesterol side-chain cleavage. We suggest that cholesterol sulfate functions in steroidogenic tissues to regulate the magnitude of the steroidogenic response.

Adrenal Glands↗
PubMed1989

Insulin-stimulated hexose transport and glucose oxidation in rat adipocytes is inhibited by sphingosine at a step after insulin binding.

Spingosine, a naturally occurring inhibitor of protein kinase C, has recently been shown to have potent bioregulatory effects on a variety of cellular processes involving signal transduction mechanisms. In the present studies, we have investigated its effects on activation by insulin of hexose transport and glucose oxidation in isolated rat adipocytes. Preincubation of cells with this long-chain base blocked both the marked activation of these processes by insulin and the smaller activation by phorbol myristate acetate. Inhibition of both insulin and phorbol 12-myristate 13-acetate activation showed the same sphingosine concentration dependence, suggesting a common locus of action. The effectiveness of sphingosine was inversely proportional to the lipid content in the incubation (which was a function of both the age of the animal and the number of cells used) presumably due to dilution of the lipophilic long-chain base into the cellular triglycerides. Sphingosine did not affect either insulin binding to its receptor or the half-maximal concentration of the hormone required to activate hexose transport, but reduced the maximal responses. Thus, the inhibition was at a step distal to the binding of insulin to its receptor. Basal transport activity was not inhibited, suggesting a locus of action prior to the glucose transporter. The inhibitor was also effective when added following activation by insulin of hexose transport and resulted in a rapid reversal of activation (t 1/2 for inhibition was 2-4 min.). Sphingosine and its analogs showed a parallel potency for inhibition both of isolated protein kinase C and of insulin activation in adipocytes, consistent with an essential role for protein kinase C in the activation of hexose transport by insulin.

Adipose Tissue↗
PubMed1989

Structural requirements for long-chain (sphingoid) base inhibition of protein kinase C in vitro and for the cellular effects of these compounds.

Sphingosine, sphinganine, and other long-chain (sphingoid) bases inhibit protein kinase C in vitro and block cellular responses to agonists that are thought to act via this enzyme. To gain further insight into the mechanism of this inhibition, a series of long-chain analogues differing in alkyl chain length (11-20 carbon atoms), stereochemistry, and headgroup were examined for (a) inhibition of protein kinase C activity in vitro, (b) the neutrophil respiratory burst in response to phorbol myristate acetate (PMA), (c) the PMA-induced differentiation of HL-60 cells, and (d) the growth of Chinese hamster ovary cells. In every instance, the effects were maximal with the 18-carbon homologues, which are the same length as the predominant naturally occurring long-chain base (sphingosine). The lower potency of the shorter chain homologues was partially due to decreased uptake by cells. Small differences were obtained with the four stereoisomers of sphingosine (i.e., D and L forms of erythro- and threo-sphingosine), with N-methyl derivatives of the different sphingosine homologues, and with simpler alkylamines (e.g., stearylamine). The potency of the different headgroup analogues may be affected by the degree of protonation at the assay pH. The pKa of sphingosine was measured to be 6.7; the pKa varied among the analogues. These findings establish that the major structural features required for inhibition of protein kinase C and cellular processes dependent on this enzyme are the presence of a free amino group and an aliphatic side chain and that other groups have more subtle effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Diacylglycerol generation and phosphoinositide turnover in human neutrophils: effects of particulate versus soluble stimuli.

Serum-treated, or "opsonized" zymosan (OZ), a particulate material which can be phagocytized by polymorphonuclear leukocytes, activates the superoxide-generating respiratory burst in these cells. The use of dual wavelength spectroscopy in the present studies has allowed accurate continuous monitoring of superoxide generation (cytochrome c reduction) upon cellular activation by this turbid material; activation occurs after a short lag period (about 20 s) which is similar to the lag seen after activation with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Unlike the fMLP response which terminates after about 90 s, superoxide generation in response to OZ continues beyond 10 min, and is similar in this regard to the response seen with the protein kinase C activator phorbol myristate acetate (PMA). OZ and fMLP, but not PMA, also activate receptor-linked phospholipase C mechanisms as judged by the appearance of inositol trisphosphate (IP3) (as well as other inositol phosphates) and diacylglycerol (DAG), with the latter measured by a mass assay. The appearance of these potential mediators corresponded to the loss of phosphoinositides, in particular phosphatidylinositol 4,5-bisphosphate (PIP2). The magnitude of DAG and inositol sugar generation as well as the breakdown of PIP2 was considerably greater using OZ than with fMLP. In addition, while fMLP resulted in a transient increase in IP3 and DAG, OZ resulted in a sustained elevation of these molecules. With both agonists, the onset and duration of generation of putative mediators corresponded to the period of generation of O2-, consistent with a role for DAG and/or IP3 in the activation of the respiratory burst.

Diglycerides↗
PubMed1989

Cholesterol sulfate: a naturally-occurring inhibitor of cholesterol side-chain cleavage which functions at the level of intramitochondrial cholesterol translocation.

Cholesterol sulfate is a naturally occurring compound which is widely distributed among various tissues including the adrenal cortex. When added to adrenal mitochondria from ether-stressed rats, it inhibits pregnenolone synthesis from exogenous but not endogenous cholesterol. Evidence supports a locus of action at the level of an intramitochondrial cholesterol translocation system with no effect on the side-chain cleavage enzymatic system (cytochrome P-450scc). Levels of endogenous cholesterol sulfate in the adrenal are similar to the Ki for inhibition by this compound, suggesting a possible physiological role. Moreover, quantities of cholesterol sulfate present in isolated mitochondria from ether-stressed animals are variable, and levels correlate inversely with rates of pregnenolone production by these preparations. In the presence of malate, mitochondrial cholesterol sulfate is metabolized slowly to pregnenolone sulfate, and its removal correlates with activation of cholesterol side-chain cleavage. Cholesterol sulfate levels are not regulated acutely by stress, but can be decreased significantly after several weeks of daily injection of ACTH (a model of chronic stress). Such treatments result in an increased capacity of isolated adrenal mitochondria to synthesize pregnenolone, and increased activity correlates with increased levels of circulating corticosterone. Thus, we propose that cholesterol sulfate is a new physiological regulator of steroid hormone biosynthesis which may function to regulate the magnitude of the steroidogenic response of the adrenal cortex to ACTH.

Adrenal Cortex↗
PubMed1988

The superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane. Phosphatidylserine as an effector of the activated enzyme.

The superoxide-generating respiratory burst oxidase is an integral membrane enzyme found in the plasma membrane of polymorphonuclear leukocytes (neutrophils). NADPH-dependent superoxide generation is seen in isolated plasma membranes and in their detergent extracts following activation of the intact cells with phorbol myristate acetate. We have herein examined the effects of phospholipids on the activity of the solubilized oxidase. Solubilization of plasma membranes with 0.5% each of Tween 20 plus deoxycholate resulted in an approximately 2-fold enhancement of activity. Inclusion of phospholipids in the extraction medium resulted in further activation. At 1.0 mg/ml the order of effectiveness was phosphatidylserine (PS) greater than cardiolipin greater than phosphatidylethanolamine greater than phosphatidylinositol; phosphatidylcholine and phosphorylated inositol lipids were not effective. The concentrations required for half-maximal activation by PS and phosphatidylethanolamine were 85 and 200 micrograms/ml, respectively. When PS was used at a maximally activating concentration (0.5 mg/ml), the activity was enhanced 3-5-fold. Detergent solubilization alone elevated the Km of the oxidase for NADPH from 68 microM in intact plasma membranes to 123 microM, but inclusion of PS with detergent restored the Km to near or below that seen in intact membranes. PS also increased the Vmax by a factor of 2-3, but had no effect on the pH optimum. A plot of the activity versus enzyme concentration was linear when membranes were used, but activity showed a quadratic dependence on concentration in solubilized membrane, with lower than expected activity at lower enzyme concentration. PS restored linearity of the concentration-activity plot. The activation by PS was not influenced by the addition of Ca2+, EGTA, or dioctanoylglycerol, indicating that activation was not dependent on protein kinase C. These results implicate phosphatidylserine as a direct effector of the NADPH-oxidase.

Cell Membrane↗
PubMed1988

Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils but inhibits phosphoinositide hydrolysis. Implications for the mechanism of PMA priming of the respiratory burst.

Pretreatment ("priming") of neutrophils with a non-activating concentration (2 nM) of phorbol myristate acetate (PMA) augments superoxide (O2-) production in response to the chemoattractant formylmethionylleucylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), on activation of primed neutrophils. In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supporting a common inhibited site. PMA also augmented (about 2-fold) fMLP-induced generation of sn-1,2-diglyceride (DG), the level of which correlated with O2- generation. In contrast to its effects on DG, PMA diminished by about 50% the magnitude of the fMLP-stimulated rise in cytosolic Ca2+. Thus, PMA priming dissociates the fMLP-stimulated Ca2+ increase from DG and O2- generation. The effect of PMA on Ca2+ levels appeared to be due in part to lowered levels of inositol trisphosphate. Lowering of inositol phosphate levels correlated with inhibition of fMLP-induced hydrolysis of inositol-containing phospholipids, particularly phosphatidylinositol 4,5-bisphosphate. PMA did not inhibit (and in fact augmented at early time points) formation of [32P] phosphatidic acid in response to fMLP, indicating that the increase in DG was not due to inhibition of cellular diglyceride kinase. Thus, the data suggest that PMA enhances fMLP-stimulated DG generation concomitant with switching the source of DG from phosphatidylinositol 4,5-bisphosphate to an alternative lipid(s). Increased DG and inhibition of activation by sphinganine are consistent with a role for protein kinase C in activation of the respiratory burst in PMA-primed neutrophils.

Diglycerides↗
PubMed1988

A menadione-stimulated pyridine nucleotide oxidase from resting bovine neutrophil membranes. Purification, properties, and immunochemical cross-reactivity with the human neutrophil NADPH oxidase.

A menadione-stimulated, superoxide-generating enzyme was purified 127-fold from resting bovine polymorphonuclear leukocyte (neutrophil) membranes with a yield of 34%. The enzyme was extracted with Triton X-100 and purified by chromatography on DEAE-Sepharose CL-6B, NAD-agarose, and Sephacryl S-200. The purified enzyme contained FAD and had an apparent molecular mass of 93 kDa by sodium dodecyl sulfate gel electrophoresis. In a nondenaturing gel electrophoresis system, the enzyme was multimeric (Mr greater than 400,000). The oxidase showed 3-4-fold higher activity (Vm) with NADH compared with NADPH, but the Km for both pyridine nucleotides was similar (39 and 47 microM, respectively). The enzyme transferred electrons to cytochrome c, dichlorophenolindophenol, and nitro blue tetrazolium. Cytochrome c reduction was stimulated 4-fold by menadione and was inhibited 70% by superoxide dismutase. Cytochrome c reduction was not inhibited by several mitochondrial respiratory chain inhibitors (azide, cyanide, and rotenone) but was sensitive to thiol-reactive agents (p-chloromercuribenzoate and monoiodo acetate). The catalytic properties of this enzyme distinguish it from the NADPH-dependent superoxide-generating respiratory burst oxidase (NADPH-oxidase) of human neutrophils. Nevertheless, antibodies to this enzyme inhibited not only the purified menadione-stimulated oxidase, but also the respiratory burst oxidase in membranes isolated from activated human neutrophils, indicating similar antigenic determinants are shared by these enzymes. Western blots of human neutrophil membranes visualized a plasma membrane protein of molecular mass 67 kDa, corresponding in size to a protein previously reported in preparations of the human respiratory burst oxidase.

Animals↗
PubMed1988

Modulation of the free sphingosine levels in human neutrophils by phorbol esters and other factors.

Because free long-chain bases have been recently found to have potent pharmacological effects when added to neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623) and other cell types, the levels in human neutrophils were measured by high-performance liquid chromatography. Sphingosine was the major free long-chain base in freshly isolated cells and ranged from 13 to 101 pmol/10(7) cells for different donors (mean +/- S.E. of 50 +/- 5, n = 17). Upon incubation at 37 degrees C, there was a time-dependent increase in free sphingosine (57 +/- 8% in 1 h, n = 17), but no change was seen at 4 or 25 degrees C. The sphingosine was apparently derived from more complex sphingolipids because little (less than 1%) could be accounted for by new synthesis from [14C]serine. Greater increases in free sphingosine were obtained when neutrophils were incubated with serum, plasma, or serum lipoproteins (about 2-fold higher than for cells incubated alone). In contrast, agonists such as phorbol 12-myristate 13-acetate, A23187, arachidonic acid, low concentrations (10 nM) of N-formyl-methionyl-leucyl-phenylalanine, and opsonized zymosan either decreased the amount of free sphingosine or blunted the time-dependent increase. This may be due to enhanced removal of free sphingosine because phorbol 12-myristate 13-acetate-treated cells exhibited an increased conversion of exogenously added [3H]sphinganine to ceramides. Endogenous sphingosine was approximately one-tenth the level found in neutrophils when exogenous long-chain bases were added to inhibit protein kinase C. Hence, depending on the subcellular localization of the endogenous versus exogenous long-chain bases, the amounts of free sphingosine in neutrophils might be sufficient to affect the function of these cells.

Calcimycin↗
PubMed1988

Sphinganine effects on chemoattractant-induced diacylglycerol generation, calcium fluxes, superoxide production, and on cell viability in the human neutrophil. Delivery of sphinganine with bovine serum albumin minimizes cytotoxicity without affecting inhibition of the respiratory burst.

Long chain bases (sphinganine and sphingosine) are potent inhibitors of protein kinase C in an in vitro mixed micelle-reconstituted system (Hannun, Y. A., Loomis, C. R., Merrill, A. H. J., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609) and block activation of the superoxide-generating respiratory burst in human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). In the present studies, we have investigated the effects of sphinganine on cellular levels of the second messengers related to phosphoinositide turnover: diacylglycerol and calcium. We find that sphinganine added from a stock solution containing equimolar or greater bovine serum albumin had no effect on either formyl-methionyl-leucyl-phenylalanine-stimulated calcium fluxes or diacylglycerol generation, at levels which completely blocked activation of superoxide generation. In addition, there was no effect of sphinganine on cell viability in this concentration range. These data indicate an inhibitory effect subsequent to the generation of second messengers and are consistent with protein kinase C as the locus of action. When sphinganine was added from a stock in dimethyl sulfoxide, significant cytotoxic effects (assayed by trypan blue exclusion, release of cellular lactate dehydrogenase, and leakage of Quin2) were seen at concentrations nearer those which inhibited the respiratory burst. Cytotoxicity was inversely proportional to cell concentration and was probably due to detergent micelle formation which occurs in the absence of albumin. These studies emphasize the importance of the method of delivery and the consideration of cytotoxic effects, but indicate that long-chain bases possess potent inhibitory properties which make them useful probes of signal transduction mechanisms.

Calcium↗
PubMed1988

Activation of the respiratory burst oxidase in neutrophils: on the role of membrane-derived second messengers, Ca++, and protein kinase C.

A major bactericidal mechanism of neutrophils involves activation of the respiratory burst oxidase to generate superoxide (O2-). The oxidase is activated rapidly, often within a minute, in response to extracellular signals such as chemoattractants, inflammatory mediators, and invading microorganisms. Increasing evidence indicates that lipases also respond rapidly, releasing potent regulatory molecules from progenitor lipids. Released molecules include potential regulators of protein kinase C--diacylglycerol (DAG), arachidonate, and sphingosine--and levels of one of these, DAG, frequently correlate with O2- production. In this author's view, the available data implicate DAG and protein kinase C as key factors in the regulation of the respiratory burst. Herein, the array of activating agonists, the generation and function of some lipid-derived mediators, and evidence pertaining to the participation of protein kinase C are reviewed.

Animals↗
PubMed1988

Photoreactive cardiolipin analogues.

New photoreactive analogues of cardiolipin have been chemically synthesized. Photoreactive aryl azido acyl groups were placed at two different locations within the cardiolipin molecule: at the 2-sn position of the 2-sn glycerol of cardiolipin; at the 2-sn position of the 3-sn-phosphatidyl group; or at both locations to provide a dual labeled analogue. Thus three different cardiolipin analogues distinguished by the positions of the aryl azido acyl groups were prepared. Two different aryl azido acyl groups were employed in the above syntheses and the site of acylation was stereospecifically identified using several phospholipids of known specificity for cardiolipin. Acylation of cardiolipin with the symmetrical anhydride of either acyl aryl azido fatty acid analogue, 2-(N-4-azido-2-nitrophenyl)beta-alanine or 12-(N-4-azido-2-nitrophenyl)aminododecanoic acid provided 1-(3-sn-phosphatidyl)-2-(acyl aryl azido)-3-(3-sn-phosphatidyl)-sn-glycerol. Acylation of monolysocardiolipin (1-(3-sn-phosphatidyl)-3-(1-acyl-2-lyso-glycero(3)phospho)-sn-glyce++ + rol provided two products. 1-(3-sn-phosphatidyl)-3-(1-acyl-2-(acyl aryl azido)-glycero(3)phospho)-sn-glycerol and the doubly labeled 1-(3-sn-phosphatidyl)-2-(acyl aryl azido)-3-(1-acyl-2-(acyl aryl azido)glycero(3)phospho)-sn-glycerol. These are the first reported photoreactive analogues for cardiolipin. The analogues were positive effectors for cytochrome P-450sec, and as shown by SDS-PAGE, they labeled the single subunit of cytochrome P-450sec and the smallest subunits of cytochrome c oxidase from beef heart.

Azides↗
PubMed1987

Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils.

Sphingoid long-chain bases (sphinganine and sphingosine) have recently been shown to inhibit protein kinase C both in vitro [Y. Hannun et al. (1986) J. Biol. Chem. 261, 12604-12609] and in intact human neutrophils, in which they block activation of the superoxide-generating respiratory burst [E. Wilson et al. (1986) J. Biol. Chem. 261, 12616-12623]. In the present study we have used sphingosine to investigate the pathways for agonist-induced secretion of neutrophil granule contents. Induction of secretion of the specific granule component lactoferrin by a variety of agonists [phorbol 12-myristate-13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187] was completely inhibited by sphingosine with an ED50 of 6 to 10 microM. PMA-induced secretion of lysozyme (present in both the azurophilic and specific granules) was completely blocked with an ED50 of 10 microM, whereas fMLP-induced secretion was only about 50% inhibited. Secretion of the azurophilic granule proteins beta-glucuronidase and myeloperoxidase was activated by fMLP and A23187, but not by PMA, and was not affected by sphingosine. The use of A23187 in the presence of sphingosine allowed differentiation between calcium activation of protein kinase C-dependent versus-independent pathways. The effect of sphingosine was not mediated by neutralizing intracellular acidic compartments, since treatment of neutrophils with inhibitory concentrations of sphingosine did not significantly alter the uptake of labeled methylamine. We conclude that at least two mechanisms participate in the regulation of specific and azurophilic granule secretion, respectively: a protein kinase C-dependent pathway and a calcium-dependent pathway which does not involve protein kinase C.

Calcimycin↗
PubMed1987

Immunocytochemical localization of protein kinase C in resting and activated human neutrophils.

An immunocytochemical method was used to determine possible changes in the subcellular distribution of protein kinase C (PKC) in human neutrophils in response to opsonized latex beads and zymosan. While in resting cells most of the PKC immunoreactivity was localized in the cytoplasm, a redistribution of PKC to the plasma and phagosomal membranes was observed in cells treated with latex beads or zymosan for 5-20 min, suggesting a participation of PKC in endocytosis.

Adult↗
PubMed1987

The use of a specific fluorescence probe to study the interaction of adrenodoxin with adrenodoxin reductase and cytochrome P-450scc.

The single free cysteine at residue 95 of bovine adrenodoxin was labeled with the fluorescent reagent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS). The modification had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc, suggesting that the AEDANS group at Cys-95 was not located at the binding site for these molecules. Addition of adrenodoxin reductase, cytochrome P-450scc, or cytochrome c to AEDANS-adrenodoxin was found to quench the fluorescence of the AEDANS in a manner consistent with the formation of 1:1 binary complexes. Förster energy transfer calculations indicated that the AEDANS label on adrenodoxin was 42 A from the heme group in cytochrome c, 36 A from the FAD group in adrenodoxin reductase, and 58 A from the heme group in cytochrome P-450scc in the respective binary complexes. These studies suggest that the FAD group in adrenodoxin reductase is located close to the binding domain for adrenodoxin but that the heme group in cytochrome P-450scc is deeply buried at least 26 A from the binding domain for adrenodoxin. Modification of all the lysines on adrenodoxin with maleic anhydride had no effect on the interaction with either adrenodoxin reductase or cytochrome P-450scc, suggesting that the lysines are not located at the binding site for either protein. Modification of all the arginine residues with p-hydroxyphenylglyoxal also had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc. These studies are consistent with the proposal that the binding sites on adrenodoxin for adrenodoxin reductase and cytochrome P-450scc overlap, and that adrenodoxin functions as a mobile electron carrier.

Adrenodoxin↗
PubMed1987

Cholesterol sulfate inhibits adrenal mitochondrial cholesterol side chain cleavage at a site distinct from cytochrome P-450scc. Evidence for an intramitochondrial cholesterol translocator.

Cholesterol sulfate inhibits (K1/2, 6 microM) the side chain cleavage of exogenous cholesterol in intact rat adrenal mitochondria. Inhibition is at a site other than cytochrome P-450scc: the spin state of the hemoprotein is not perturbed, and its activity is unaffected as judged by the failure to inhibit the metabolism both of 25-hydroxycholesterol and of endogenous cholesterol in a mitochondrial "steroidogenic pool." In contrast, 25-hydroxycholesterol, known to interact with the cytochrome, prevented the cleavage of both endogenous and exogenous cholesterol and produced the expected optical changes in the hemoprotein. Inhibition was specific, since a variety of related compounds including pregnenolone sulfate were not effective. Metabolic conversion to other species was insufficient to account for inhibition, indicating that cholesterol sulfate is the effective molecule. A hallmark of an inhibitor of a transport system is that disruption of the barrier to transport eliminates inhibition. Sonic disruption of mitochondria abated by 70% the effect of cholesterol sulfate, but did not affect inhibition by 25-hydroxycholesterol. Thus, the cholesterol sulfate appears to inhibit an intramitochondrial cholesterol translocation system that functions to move cholesterol into a steroidogenic pool. The high content of cholesterol sulfate in adrenal cortex (Drayer, N.M., Roberts, K.D., Bandi, L., and Lieberman, S. (1964) J. Biol. Chem. 239, 3112-3114) suggests a possible regulatory role for this molecule.

Adrenal Glands↗