Biomedical subjects

N Noy

Publications and source records attributed to N Noy.

At least 37 records · Page 2Linked to original sources

PubMed1996

Purification, cloning, and bacterial expression of retinol dehydratase from Spodoptera frugiperda.

Anhydroretinol and 14-hydroxy-4,14-retro-retinol, retro-retinoids endogenous to both mammals and insects, act as agonist and antagonist, respectively, in controlling proliferation in lymphoblasts and other retinol-dependent cells. We describe here the identification, purification, cloning, and bacterial expression of the enzyme retinol dehydratase, which converts retinol to anhydroretinol in Spodoptera frugiperda. Retinol dehydratase has nanomolar affinity for its substrate and is, therefore, the first enzyme characterized able to utilize free retinol at physiological intracellular concentrations. The enzyme shows sequence homology to the sulfotransferases and requires 3'-phosphoadenosine 5'-phosphosulfate for activity.

Amino Acid Sequence↗

PubMed1996

Individual subunits of heterodimers comprised of retinoic acid and retinoid X receptors interact with their ligands independently.

The retinoid X receptor (RXR) is a member of a family of transcription factors, known as hormone nuclear receptors, that mediate the effects of hydrophobic hormones on gene transcription. RXR, which is activated by 9-cis-retinoic acid (9cRA), can modulate several signaling pathways by virtue of its ability to form heterodimers with other members of the receptor family, as, for example, the retinoic acid receptor (RAR). The roles of the individual receptors within heterodimers are not clear as yet. It was recently reported that heterodimerization inhibits transcriptional activation by RXR, an effect that was attributed to an inability of RXR within heterodimers to bind its ligand. This inhibition was reported to depend on the association of heterodimers with cognate DNA and on the level of saturation of the RAR subunit within the heterodimers. In the present work, the ligand-binding characteristics of RXR and RAR individually and within heterodimers were examined by fluorescence-based methods. The results indicate that heterodimerization with RAR does ot alter the ligand-binding capacity of RXR nor the rate of dissociation of the ligand from this receptor. The ligand-binding capacity of RXR also was not affected by association of heterodimers with cognate DNA nor by the level of saturation of the RAR subunit. The data indicate further that the affinity of RAR for 9cRA is considerably higher as compared to RXR and that this differential affinity is retained within RAR-RXR heterodimers. Thus, binding of ligands by subunits within RAR-RXR heterodimers proceeds independently.

Base Sequence↗

PubMed1995

On the role of ligand in retinoid signaling: positive cooperativity in the interactions of 9-cis retinoic acid with tetramers of the retinoid X receptor.

Previously, we have shown that the retinoid X receptor (RXR) forms tetramers with a high affinity and that interactions of the receptor with its ligand, 9-cis retinoic acid (9cRA), result in dissociation of protein tetramers. Here it is shown by fluorescence anisotropy studies that ligand-induced tetramer dissociation displays a pronounced positive cooperativity. The binding affinity of RXR for 9cRA at low saturation levels of the receptor with ligand was found to be significantly weaker than the affinity observed at higher levels of saturation. In addition, the rate of dissociation of 9cRA from RXR was found to be faster at low vs. high saturation levels of the receptor. These data suggest that the observed positive cooperativity of the ligand-induced dissociation of RXR tetramers stems from positive cooperativity in binding of 9cRA by the receptor. Kinetic studies showed that dissociation of RXR tetramers upon ligand binding is a rapid reaction characterized by a t1/2 of 80 ms, which is about 5 orders of magnitude faster than the rate of dissociation in the absence of ligand. The data indicate that the oligomeric state of RXR is tightly regulated by the precise concentrations of 9cRA and that it rapidly responds to changes in the ligand's concentrations. These findings further substantiate the hypothesis that modulation of the oligomeric state of RXR by 9cRA is an important regulatory step in the pathway by which retinoids affect gene transcription.

Fluorescence Polarization↗

PubMed1995

Role of ligand in retinoid signaling. 9-cis-retinoic acid modulates the oligomeric state of the retinoid X receptor.

Many of the effects of retinoids on cells are mediated by the transcription factors known as retinoid nuclear receptors, but the mechanisms by which retinoids regulate the activity of the receptors are not known. It was previously shown that the retinoid X receptor (RXR) forms tetramers with a high affinity. In the present work it is demonstrated that binding of 9-cis-retinoic acid to RXR leads to rapid dissociation of receptor tetramers. In addition, fluorescence anisotropy studies indicate that ligand-binding results in a significant conformational change such that holo-RXR is more compactly folded as compared to the apo-protein. These findings suggest that the initial event in signaling by 9-cis-retinoic acid is a change in the oligomeric state of RXR. The data also imply that tetramer formation is a regulatory feature of the pathway by which RXR mediates the effects of retinoids on gene transcription.

Binding Sites↗

PubMed1995

Fatty acid flip-flop in phospholipid bilayers is extremely fast.

The rate of movement of fatty acids (FA) across phospholipid bilayers is an important consideration for their mechanism of transport across cell membranes but has not yet been measured. When FA move undirectionally across phospholipid bilayers, the rapid movement of un-ionized FA compared to ionized FA results in transport of protons. We have previously used this property to show that FA move spontaneously ("flip-flop") across the bilayer of small unilamellar vesicles within approximately 1 s (Kamp & Hamilton, 1992, 1993). This work extends the time resolution of this assay into the millisecond time range by use of stopped flow fluorometry. In small unilamellar vesicles (diameter, approximately 25 nm) at neutral pH, flip-flop of all fatty acids studied (lauric, myristic, palmitic, oleic, and stearic) was > or = 80% complete within 5-10 ms. In large unilamellar vesicles (diameter, approximately 100 nm), the same fatty acids exhibited fast flip-flop but with a measureable rate (t 1/2 = 23 +/- 12 ms). The calculated pseudounimolecular rate constant of the un-ionized FA (kFAH) approximately 15 s-1. There was no dependence of the flip-flop rate on the fatty acid chain length or structure. We also monitored the rate of desorption and transbilayer movement of (anthroyloxy)stearic acid in small unilamellar vesicles. Whereas previous studies suggested slow flip-flop of this FA analogue, the present studies suggest that (anthroyloxy)stearic acid flip-flops rapidly and that earlier studies did not truly measure the transbilayer movement step. These findings further support the view that proteins are not required for translocation of FA across cell membranes.

Arylsulfonates↗

PubMed1995

Retinoid X receptor alpha forms tetramers in solution.

Protein-protein interactions allow the retinoid X receptor (RXR) to bind to cognate DNA as a homo- or a heterodimer and to participate in mediating the effects of a variety of hormones on gene transcription. Here we report a systematic study of the oligomeric state of RXR in the absence of a DNA template. We have used electrophoresis under nondenaturing conditions and chemical crosslinking to show that in solution, RXR alpha forms homodimers as well as homotetramers. The dissociation constants governing dimer and tetramer formation were estimated by fluorescence anisotropy studies. The results indicate that RXR tetramers are formed with a high affinity and that at protein concentrations higher than about 70 nM, tetramers will constitute the predominant species. Tetramer formation may provide an additional level of the regulation of gene transcription mediated by RXRs.

Base Sequence↗

PubMed1995

Interactions of retinol with lipid bilayers: studies with vesicles of different radii.

The interactions of retinol with vesicles of dioleoylphosphatidylcholine of varying radii were studied. The rate constants of dissociation of retinol from bilayers (k(off)) and the equilibrium partition constants (Keq) of retinol into bilayers of different sized vesicles were measured. The rate constants for association of retinol with vesicles were calculated from the expression Keq = k(on)/k(off). k(off) was 10-fold faster in the smallest versus the largest vesicles tested. K(on) was also somewhat faster in vesicles with small radii, but the effect on k(off) was more pronounced, leading to an overall higher affinity for retinol of bilayers in large vesicles. The thermodynamic parameters of the dissociation reaction were studied in vesicles with 0.025, 0.1, and 0.4 microns diameter. The enthalpy of activation decreased while the entropy of activation of the dissociation of retinol from bilayers increased as the vesicles become larger. It is suggested that restructuring of lipid-lipid interactions within the bilayer play a role in determining the rate by which retinol is solvated off bilayers. Overall, the data indicate that the rates by which retinol moves between different cell types in vivo may depend on the geometry of cellular surfaces.

Lipid Bilayers↗

PubMed1994

Pure and functionally homogeneous recombinant retinoid X receptor.

Mouse retinoid X receptor alpha (RXR alpha) lacking the amino-terminal region A/B (RXR alpha delta AB) has been purified to more than 98% purity and functional homogeneity from bacterial and baculovirus-based recombinant expression systems with yields of 2-8 mg/liter of culture. The purified protein is soluble, and fluorescence quenching analysis demonstrated that it binds its cognate ligand 9-cis-retinoic acid (9-cis-RA) stoichiometrically, and with high affinity. Compared with RXR delta AB expressed in COS-1 cells, bacterially and baculovirus-expressed proteins bind approximately 10 and 5 times less efficiently to direct repeat 1 (DR1) DNA elements, respectively, suggesting that animal cell-specific modification of RXR or interaction with other animal cell-specific factors may modulate DNA binding. 9-cis-RA did not stimulate DR1 binding of functional RXR delta AB expressed in Escherichia coli, Sf9 or COS-1 cells. The previously reported ligand effect that can be observed with in vitro made receptor may therefore be a consequence of a conformational stabilization of improperly folded in vitro synthesized protein.

Amino Acid Sequence↗

PubMed1994

Studies on the efflux of heme from biological membranes.

It is unknown how heme is distributed intracellularly from its site of synthesis in the mitochondria to other organelles. In previous work (Biochemistry 23, 3715, 1984) the transfer of heme from lipid bilayers to soluble proteins had been found to be independent of the recipient proteins' affinity for heme. Here, we investigated whether proteins are involved in the transfer of heme from biological membranes into aqueous media. We followed the release of 14C-labeled heme, from mitochondria preloaded with the heme, to BSA and found that only about 28%, of the heme was extracted on the first wash. After the third wash 35-50% of the heme that had been partitioned into the membranes was extracted. Fourth and fifth washes with BSA or a cytosolic heme-binding protein (HBP, also known as liver fatty acid binding protein) removed only insignificant amounts of 14C-labeled heme. Similarly, a large portion of the preloaded 14C-labeled heme could not be extracted from a variety of isolated membranes (inner and outer mitochondrial membranes, plasma membranes of liver cells, kidney cortex cells and erythrocyte membranes). By contrast, essentially all [14C]palmitate preloaded in biological membranes and all 14C-labeled heme preloaded in synthetic membranes was released to albumin (Biochemistry 23, 3715, 1984). These observations suggest that, in general, heme associates with membrane components which can be distinguished into two compartments. One compartment releases its heme spontaneously, while another compartment binds heme so tightly that a specific process has to be evoked for its release.

Animals↗

PubMed1994

Retinoid specificity of interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP), the predominant protein in the interphotoreceptor matrix of retina, has been implicated in transfer of retinoids between retinal pigment epithelium and photoreceptor cells. In this work, the interactions of several retinoids with IRBP were studied in order to clarify whether the protein displays specificity toward particular forms of these ligands. The equilibrium dissociation constants of complexes of 11-cis- and all-trans-retinols and retinaldehydes with IRBP were measured. It was found that IRBP contains two binding sites for 11-cis-retinaldehyde and for all-trans-retinaldehyde and retinol. Binding affinities followed the order: 11-cis-retinaldehyde > all-trans-retinol > all-trans-retinaldehyde > 11-cis-retinol. The kinetic parameters of the dissociation of these retinoids from binding sites on IRBP were measured by monitoring the rate of transfer of the retinoids from IRBP to synthetic unilamellar vesicles. 11-cis-Retinaldehyde and all-trans-retinol were found to dissociate from the strong binding site of IRBP 3-4-fold slower than all-trans-retinaldehyde and 11-cis-retinol. The higher binding affinities and the slower rates of dissociation from IRBP displayed by 11-cis-retinaldehyde and by all-trans-retinol correspond to the physiological need to shuttle these particular retinoids between pigment epithelium and photoreceptor cells across the interphotoreceptor matrix as part of the visual cycle.

Animals↗

PubMed1994

Control of substrate flow at a branch in the visual cycle.

Photoisomerization of rhodopsin's chromophore, 11-cis-retinaldehyde, and subsequent regeneration of the 11-cis configuration are accomplished in vertebrates by a series of reactions known as the visual cycle. At one point in the cycle, 11-cis-retinol can either be enzymatically oxidized to 11-cis-retinaldehyde and exported for visual pigment regeneration or be enzymatically esterified and stored. Partition of substrate at this branch was examined in this study and found to be influenced by cellular retinaldehyde-binding protein (CRALBP), a retinoid-binding protein found in retina. Esterification was reduced to about 10% and oxidation stimulated 2-3-fold in the presence of this protein. Other experiments confirmed that "free" 11-cis-retinol was esterified more rapidly than 11-cis-retinol complexed with CRALBP and that CRALBP.11-cis-retinol was not an inhibitor of the esterification. Following oxidation of CRALBP.11-cis-retinol, the reaction product, 11-cis-retinaldehyde, was found associated with the binding protein. 11-cis-Retinaldehyde is not available for reaction with carbonyl reagents when the retinoid is bound to CRALBP. However, enzymatic oxidation of CRALBP.11-cis-retinol in the presence of O-ethylhydroxylamine produced ca. 30% retinaldehyde O-ethyloxime and 70% free 11-cis-retinaldehyde, suggesting that about one-third of the retinol oxidized had dissociated from the binding protein. Neither oxidation nor esterification of CRALBP.11-cis-retinol was inhibited by including CRALBP.11-cis-retinaldehyde in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Interactions of all-trans-retinol and long-chain fatty acids with interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP), a predominant protein in the interphotoreceptor matrix of the retina, has been implicated in transfer of retinoids between retinal pigment epithelium and photoreceptor cells. The interactions of IRBP with all-trans-retinol have been studied by three fluorescence-based methods and by measurements of binding of 3[H]-labeled all-trans-retinol to this protein. It was found that IRBP contains two sites with similar but not identical affinities for all-trans-retinol. The dissociation constant of the all-trans-retinol-IRBP complex at the first site was 0.1 microM, which is about 10-fold lower than previously reported values. The second site had about 2.5-fold lower affinity for all-trans-retinol as compared to the first site. Long-chain fatty acids were found in this study to displace all-trans-retinol from the stronger retinol-binding site on IRBP. Displacement of all-trans-retinol was used to study the interactions of fatty acids with this protein. It was found that docosahexaenoic acid (DHA C22:6n-3), an essential fatty acid which plays an important role in vision, had the highest apparent affinity for the site probed on IRBP of all the fatty acids studied.

Animals↗

PubMed1993

Anhydroretinol: a naturally occurring inhibitor of lymphocyte physiology.

Vitamin A (retinol) is an essential cofactor for growth of B lymphocytes in culture and for activation of T lymphocytes by antigen receptor-mediated signals. 14-hydroxy-4,14-retro-retinol (14-HRR) a metabolite of retinol, has been implicated as the intracellular mediator of this effect. Anhydroretinol (AR) is a retinol derivative with retro structure produced in activated human B lymphocytes and the insect cell lines SF 21 and Schneider S2. AR reversibly inhibits retinol- and 14-HRR-dependent effects and blocks B lymphocyte proliferation as well as activation of resting T lymphocytes. The intracellular signaling pathway blocked by AR in T cell activation is distinct from the calcineurin/interleukin 2 pathway inhibitable by cyclosporine A or FK-506.

B-Lymphocytes↗

PubMed1993

The interactions of retinoids with retinol-binding protein. A resonance Raman spectroscopic study.

We have measured the pre-resonance Raman spectrum of retinal, retinoic acid and retinol in dilute CCl4 solutions and when bound to the bovine-serum retinol-binding protein. The comparison reveals that the binding interaction does not involve any specific interactions of the head group and/or the polyene chain with a particular protein residue. The data indicate hydrogen bonding of bound retinal's head-group oxygen to water, as well as some torsional angle change of its polyene chain upon binding.

Ethylenes↗

PubMed1992

Interactions of retinol with binding proteins: studies with retinol-binding protein and with transthyretin.

The interactions within the molecular complex in which retinol circulates in blood were studied. To monitor binding between retinol-binding protein (RBP) and transthyretin (TTR), TTR was labeled with a long-lived fluorescence probe (pyrene). Changes in the rotational volume of TTR following its association with RBP were monitored by fluorescence anisotropy of the probe. Titration of TTR with holo-RBP revealed the presence of 1.5 binding sites characterized by a dissociation constant Kd = 0.07 microM. At 0.15 M NaCl, binding of RBP to TTR showed an absolute requirement for the native ligand, retinol. At higher ionic strength (0.5 M NaCl), RBP complexed with retinal also bound to TTR with high affinity (Kd = 0.134 microM). RBP containing retinoic acid did not bind to TTR even at the high salt concentration. The data suggest that the TTR binding site on RBP is in close proximity to the retinoid binding site and that the head group of retinoic acid, when bound to RBP, presents steric hindrance for the interactions with TTR. The implications of the data for selectivity in retinoid transport in the circulation are discussed. The kinetics of the steps leading to complete dissociation of the retinol-RBP-TTR complex was also studied. The first step of this process was dissociation of retinol, which had a rate constant of 0.06/min. Following loss of retinol, the two proteins dissociate. The rate of dissociation is slow (k = 0.055/h), however, indicating that the complex apo-RBP-TTR will be an important factor in regulating serum levels of retinol.

Binding Sites↗

PubMed1992

The ionization behavior of retinoic acid in aqueous environments and bound to serum albumin.

The ionization behavior of retinoic acid (RA) in an aqueous phase and when bound to bovine serum albumin was studied. Titrations of RA in the various phases were followed by monitoring the red shift in the absorption maximum of RA that occurred upon deprotonation. The apparent pK of RA was dependent on the concentration of this compound. At the concentration range 6-20 microM, the pK of RA in water had a value of approximately 8.0. As the concentration was decreased in the range 1-6 microM, the value of the pK decreased continuously. The lowest pK observed was approximately 6.0. It was concluded that RA in an aqueous phase at concentrations in the microM range, forms micelles, and that the values of the pK of RA monomers and micelles in water are less than 6.0 and 8.0, respectively. The presence of 0.15 M NaCl caused a decrease in the pK of RA micelles and lowered the value of the CMC. Titration of RA in the presence of bovine serum albumin revealed the presence of a heterogeneous population comprised of three distinct microenvironments for RA associated with this protein. Two populations of RA were found to undergo complete titration in the pH range 4-8. A third population became apparent at pH greater than 9.5.

Absorption↗

PubMed1992

The ionization behavior of retinoic acid in lipid bilayers and in membranes.

The ionization behavior of retinoic acid (RA) incorporated in unilamellar vesicles of different lipid compositions and in biological membranes was studied. Titration of RA in the various membranes was followed by monitoring the red shift in the absorption maximum of RA that occurred upon deprotonation. It was found that, similar to other hydrophobic carboxylic acids, the protonated form of RA is stabilized by incorporation into bilayers vs. RA monomers in an aqueous phase. The pK of RA in bilayers comprised of neutral phospholipids was approximately 7 regardless of the composition of the fatty acyl chains. Incorporation of RA in bilayers comprised of negatively charged phospholipids stabilized the protonated form to a larger extent vs. neutral lipids, resulting in pK's that were about 1 pH unit higher. The ionization behavior of RA in plasma membranes from rat liver and in erythrocyte membranes was similar to its behavior in negatively charged bilayers. The data indicate that RA incorporated in membranes is predominantly protonated at physiologic pH.

Animals↗

PubMed1992

The kinetics of interactions of bilirubin with lipid bilayers and with serum albumin.

Rate constants for the hydration of bilirubin bound to unilamellar bilayers of dioleoylphosphatidylcholine and albumin were measured by stopped-flow methods. Rate constants for association of bilirubin with these vesicles and albumin were calculated from measured rate constants for dissociation and the equilibrium binding constants of bilirubin and lipids or albumin. Rate constants for hydration (dissociation) for bilirubin bound to dioleoylphosphatidylcholine and albumin were 71 s-1 and 1.8 s-1 respectively. Rate constants for association were 4.0 10(7) s-1 and 1.1 10(9) M-1 s-1, respectively. Both rates for interactions of bilirubin with bilayers were essentially independent of temperature in the range 0-40 degrees C, indicating that barriers to entry and exit of bilirubin from bilayers were entropic. Rates of transbilayer movement of bilirubin in dioleoylphosphatidylcholine were too fast to resolve by measuring rates of hydration of bilirubin. Rate constants for hydration of bilirubin bound to bilayers with less avidity for bilirubin as compared with dioleoylphosphatidylcholine also were too fast to measure with stopped-flow methods. In addition to providing details of the energetic basis for interactions between bilirubin and membranes, the data allow for calculating the maximal rates at which bilirubin could transfer spontaneously from sites on albumin in blood to the interior of cells. The data show, in this regard, that this rate is 10-50 fold faster than measured rates of uptake of bilirubin by intact liver.

Bilirubin↗