Biomedical subjects

S C Myers-Payne

Publications and source records attributed to S C Myers-Payne.

6 recordsLinked to original sources

PubMed1996

Effects of chronic ethanol consumption on sterol transfer proteins in mouse brain.

Although lipids are essential to brain function, almost nothing is known of lipid transfer proteins in the brain. Early reports indicates cross-reactivity of brain proteins with antisera against two native liver sterol transfer proteins, sterol carrier protein-2 (SCP-2) and the liver form of fatty acid-binding protein (L-FABP). Herein, polyclonal antibodies raised against the recombinant liver sterol transfer proteins SCP-2 and L-FABP were used to identify the lipid transfer proteins in the brains of alcohol-treated and control mice. L-FABP was not detectable in brain of either control or chronic ethanol-treated mice. In contrast, SCP-2 not only was present, but its level was significantly (p < 0.05) increased 23 and 50%, respectively, in brain homogenates and synaptosomes of mice exposed to alcohol. To determine whether antibodies against the recombinant liver SCP-2 reflected true levels of SCP-2 in brain, the cDNA sequence for brain SCP-2 was isolated from a brain cDNA library. The mouse brain SCP-2 sequence was 99.99% identical to the mouse liver SCP-2 sequence. The translated sequence differed by only one amino acid, and the replacement was conservative. Thus, unlike the fatty acid binding proteins, the SCP-2 moieties of brain and liver are essentially identical. Polyclonal antibodies against acyl-CoA binding protein, a lipid-binding protein that does not bind or transfer sterol, showed that increased levels of brain SCP-2 with chronic ethanol consumption did not represent a general increase in content of all lipid transfer proteins. Changes in the amount of SCP-2 may contribute to membrane tolerance to ethanol.

Alcoholism↗

PubMed1996

Isolation and characterization of two fatty acid binding proteins from mouse brain.

Two fatty acid binding proteins (FABPs) were isolated from Swiss Webster mouse brains. Neither protein cross-reacted with antisera to recombinant liver L-FABP. One protein, designated brain H-FABP, migrated on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single band at 14.5 kDa with pl 4.9. Brain H-FABP bound NBD-stearic acid and cis-parinaric acid with K D values near 0.02 and 0.5 microM, respectively. Brain H-FABP cross-reacted with affinity-purified antisera to recombinant heart H-FABP. The second protein, mouse brain B-FABP, migrated on tricine SDS-PAGE gels as a doublet at 16.0 and 15.5 kDa with pl values of 4.5 and 4.7, respectively. Brain B-FABP bound NBD-stearic and cis-parinaric acid with K D values near 0.01 and 0.7 microM, respectively. The brain B-FABP doublet was immunoreactive with affinity-purified antibodies against recombinant mouse brain B-FABP, but not with affinity-purified antibodies against heart H-FABP. (3H)Oleate competition binding indicated that the two brain FABPs had distinct ligand binding specificities. Both bound fatty acids, fatty acyl CoA, and lysophosphatidic acid. Although both preferentially bound unsaturated fatty acids, twofold differences in specific saturated fatty acid binding were observed. Brain B-FABP and brain H-FABP represented 0.1% and 0.01% of brain total cytosolic protein, respectively. In summary, mouse brain contains two native fatty acid binding proteins, brain H-FABP and brain B-FABP.

Animals↗

PubMed1995

Probing the ligand binding sites of fatty acid and sterol carrier proteins: effects of ethanol.

Direct effects of ethanol on the interaction of cytosolic lipid transfer proteins with ligands are not known. In this study, recombinant liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2) were used in conjunction with a series of fluorescent fatty acid probe molecules to compare the relative dielectric properties of the ligand binding sites and to examine the effects of ethanol in vitro on ligand interaction with these proteins. L-FABP and SCP-2 exhibited broad but distinct ligand specificities. Although NBD-stearic acid bound with high affinity to both proteins, emission spectra showed that the relative dielectric constant of the ligand binding site in SCP-2 was significantly lower than in L-FABP, 2 vs 24. Furthermore, affinities of L-FABP for NBD-fatty acid probes were NBD-stearic acid > NBD-lauric acid >>> NBD-hexanoic acid, NBD-acetic acid. In contrast, SCP-2 bound only NBD-stearic acid with a Kd of 0.23 microM and Bmax of 0.98 mol/mol. This observation of SCP-2 specifically binding the fluorescent NBD-stearic acid was confirmed with RdB-stearic acid and the naturally fluorescent cis-parinaric acid, both of which had similar affinities and stoichiometries. Ethanol in vitro had no effect on L-FABP-NBD-stearic acid binding. However, ethanol at physiological concentrations (25 mM) dramatically inhibited NBD-stearic acid binding to SCP-2. In conclusion, the data show that both L-FABP and SCP-2 specifically bind fluorescent fatty acids. However, the ligand binding sites of L-FABP and SCP-2 differed dramatically in their dielectric properties and their sensitivity to ethanol.

4-Chloro-7-nitrobenzofurazan↗

PubMed1995

Structure and polarity of mouse brain synaptic plasma membrane: effects of ethanol in vitro and in vivo.

Structural and dielectric alteration by ethanol in vitro and chronic ethanol consumption were examined in synaptic plasma membranes (SPM) using diphenylhexatriene and charged diphenylhexatriene derivatives. These fluorophores, in combination with multifrequency phase and modulation fluorometry, allowed the examination of the surface and interior core of SPM. Limiting anisotropy and rotational relaxation time demonstrated that the synaptosomal plasma membrane surface domain was more rigid than the interior core domain. Ethanol in vitro fluidized the interior core and surface domains in SPM of the control, but not chronic ethanol-treated mice. Although the latter membranes were more rigid than control membranes, the intrinsic rigidity of the interior core of the synaptosomal plasma membrane did not strictly correlate with effects of ethanol in vitro. SPM of irradiated membranes were more rigid, but ethanol fluidized those membranes. Diphenylhexatriene lifetime and photoreactivity were sensitive to the range of dielectric constants in the SPM interior core. Ethanol in vitro increased both the surface and interior core range of dielectric constants of SPM from control but not chronic ethanol-treated animals. Thus, ethanol in vitro altered not only the fluidity but also the range of dielectric constants in both the surface and interior core domains in SPM of control but not chronic ethanol-treated mice.

Animals↗

PubMed1995

Cholesterol esterase: a cholesterol transfer protein.

Rat pancreatic cholesterol esterase was examined for its ability to effect sterol transfer between small unilamellar vesicle (SUV) preparations. Sterol exchange was determined using SUV composed of palmitoyloleoylphosphatidylcholine/sterol (65:35) with or without 10 mol % phosphatidylserine or phosphatidic acid. This recently developed assay does not require separation of donor and acceptor vesicles (Butko et al., 1992). Cholesterol esterase stimulated cholesterol exchange when SUV contained phosphatidylserine and even more so in the presence of phosphatidic acid. Cholesterol esterase increased the initial rate of sterol transfer between phosphatidic acid-containing SUV by approximately 80%. The enzyme increased sterol exchange by significantly decreasing the half-times of sterol transfer and by significantly increasing the initial rates of sterol exchange. In the absence of negatively charged phospholipids, cholesterol esterase was ineffective at increasing sterol transfer. Monolayer studies showed that negatively charged phospholipids seem to play a key role in cholesterol esterase adsorption to lipid interfaces. Finally, a mutant cholesterol esterase lacking a histidine (435) residue essential for esterasic catalysis was found to be equally capable of increasing sterol transfer and binding to charged monolayers. In summary, cholesterol esterase enhances sterol transfer in SUV containing negatively charged phospholipids, independent of esterasic activity.

Animals↗

PubMed1995

Cholesterol interaction with recombinant human sterol carrier protein-2.

The interaction of human recombinant sterol carrier protein-2 (SCP-2) with sterols was examined. Two independent ligand binding methods, Lipidex 1000 binding of [3H]cholesterol and a fluorescent dehydroergosterol binding assay, were used to determine the affinity of SCP-2 for sterols. Binding analysis indicated SCP-2 bound [3H]cholesterol and dehydroergosterol with a Kd of 0.3 and 1.7 microM, respectively, and suggested the presence of a single binding site. Phase fluorometry and circular dichroism were used to characterize the SCP-2 sterol binding site. Alterations in dehydroergosterol lifetime, SCP-2 tryptophan lifetime, and SCP-2 tryptophan quenching by acrylamide upon cholesterol binding demonstrated a shielding of the SCP-2 tryptophan from the aqueous solvent by bound sterol. Differential polarized phase fluorometry revealed decreased SCP-2 tryptophan rotational correlation time upon cholesterol binding. Circular dichroism of SCP-2 indicated that cholesterol elicited a small decrease in SCP-2 alpha helical content. The data suggest that SCP-2 binds sterols with affinity consistent with a lipid transfer protein that may act either as an aqueous carrier or at a membrane surface to enhance sterol desorption.

Acrylamide↗