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N Noy

Publications and source records attributed to N Noy.

51 records · Page 3Linked to original sources
PubMed1991

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

The interactions of retinol with rat cellular retinol-binding protein (CRBP) and with rat serum retinol-binding protein (RBP) were studied. The equilibrium dissociation constants of the two retinol-protein complexes (Kd) were found to be 13 x 10(-9) and 20 x 10(-9) M for CRBP and for RBP, respectively. The kinetic parameters governing the interactions of retinol with the two binding proteins were also studied. It was found that although the equilibrium dissociation constants of the two retinol-protein complexes were similar, retinol interacted with CRBP 3-5-fold faster than with RBP; the rate constants for dissociation of retinol from CRBP and from RBP (koff) were 0.57 and 0.18 min-1, respectively. The rate constants for association of retinol with the two proteins (kon) were calculated from the expression: Kd = koff/kon. The kon's for retinol associating with CRBP and with RBP were found to be 4.4 x 10(7) and 0.9 x 10(7) M-1 min-1, respectively. The data suggest that the initial events of uptake of retinol by cells are not rate-limiting for this process and that the rate of uptake is probably determined by the rate of metabolism of this ligand. The data indicate further that the distribution of retinol between RBP in blood and CRBP in cytosol is at equilibrium and that intracellular levels of retinol are regulated by the levels of CRBP.

Animals↗
PubMed1990

Interactions of retinol with binding proteins: implications for the mechanism of uptake by cells.

The kinetic parameters of the interaction of retinol with retinol binding protein (RBP) were studied. The rate constant for association of retinol with the protein (ka) was found to be 1.5 X 10(6) M-1 min-1. The rate constant for dissociation (kd) from the protein was determined by studying the transfer of retinol from RBP to lipid bilayers. It was found that such transfer proceeds via the aqueous phase and its rate-limiting step is the dissociation of retinol from the binding protein. The rate of transfer therefore represents the rate of dissociation. The kd was 0.112 min-1. These values were validated further by the following consideration. The equilibrium dissociation constant of RBP and retinol can be calculated from the expression Kd = kd/ka. The calculated value was 7.5 X 10(-8) M. Kd was also measured directly by fluorometric titration and was found to be 7 X 10(-8) M. The relative avidities of retinol for RBP, the complex RBP-transthyretin (RBP-TTR), and serum albumin were also studied. It was found that binding of RBP to TTR increased its avidity for retinol by about 2-fold. The avidity of albumin for retinol was 30-fold lower than that of RBP. The data imply that retinol spontaneously and rapidly dissociates from sites on binding proteins, which indicates that the vitamin can freely move in vivo between physiologic compartments with avidities for it.

Animals↗
PubMed1990

Kinetic parameters of the interactions of retinol with lipid bilayers.

The process of transfer of vitamin A alcohol (retinol) between unilamellar vesicles of phosphatidylcholine was studied. The transfer was found to proceed spontaneously by hydration from the bilayer and diffusion through the aqueous phase. The rate-limiting step for transfer was the dissociation from the bilayer, a step that was characterized in bilayers of egg phosphatidylcholine (PC) by a rate constant koff = 0.64 s-1. The rate constant for association of retinol with bilayers of egg PC was also determined: kon = 2.9 x 10(6) s-1. The relative avidities for retinol of vesicles comprised of PC lipids with the various fatty acyl chains were measured. It was found that the binding affinity was determined by the composition of the lipids, such that PC with symmetric acyl chains had a lower affinity for retinol vs those with mixed chains. To clarify the mechanism underlying this observation, the rates of dissociation and association of retinol bound to vesicles of dioleoyl-PC were determined. The rate of association of retinol with bilayers strongly depended on the composition of the fatty acyl chains of the lipids. The rate of dissociation of retinol from the bilayers of PC was found to be independent of that composition. The implications of the observations for the interactions of hydrophobic ligands with lipid bilayers are discussed.

Animals↗
PubMed1990

Thermodynamic parameters of the binding of retinol to binding proteins and to membranes.

Retinol (vitamin A alcohol) is a hydrophobic compound and distributes in vivo mainly between binding proteins and cellular membranes. To better clarify the nature of the interactions of retinol with these phases which have a high affinity for it, the thermodynamic parameters of these interactions were studied. The temperature-dependence profiles of the binding of retinol to bovine retinol binding protein, bovine serum albumin, unilamellar vesicles of dioleoylphosphatidylcholine, and plasma membranes from rat liver were determined. It was found that binding of retinol to retinol binding protein is characterized by a large increase in entropy (T delta S degrees = +10.32 kcal/mol) and no change in enthalpy. Binding to albumin is driven by enthalpy (delta H degrees = -8.34 kcal/mol) and is accompanied by a decrease in entropy (T delta S degrees = -2.88 kcal/mol). Partitioning of retinal into unilamellar vesicles and into plasma membranes is stabilized both by enthalpic (delta H degrees was -3.3 and -5.5 kcal/mol, respectively) and by entropic (T delta S degrees was +4.44 and +2.91 kcal/mol, respectively) components. The implications of these finding are discussed.

Animals↗
PubMed1989

The physical-chemical basis for sex-related differences in uptake of fatty acids by the liver.

The uptake of fatty acids by the liver was shown previously to be a non-catalyzed process, and rates of uptake were correlated to the affinity of the plasma membranes of liver cells for fatty acids. The experiments in this paper were designed to test whether the known differences in uptake and metabolism of free fatty acids by the livers of male and female rats could be understood based on differences in the affinities of the corresponding plasma membranes for these substrates. The relative affinities for palmitate and oleate of 'male' plasma membranes were found to be lower versus 'female' membranes. Measurements of uptake of palmitate from albumin-palmitate complexes by 'male' and 'female' perfused livers showed higher uptake rates by the latter when correlated with the concentration of the complex. However, the rates of uptake were identical when the concentrations of the fatty acid in the plasma membranes of male and female liver cells were the same.

Albumins↗
PubMed1989

The interactions of bilirubin with model and biological membranes.

The partitioning of bilirubin between albumin and model and biological membranes and the differential partitioning of bilirubin between membranes with different lipid and protein compositions were measured. Partition coefficients were independent of the concentration of bilirubin in membranes up to at least 7 mol of bilirubin/mol of phospholipid. The avidity of albumin for bilirubin was greater than that of membranes, but the avidity of the latter for bilirubin depended on the composition of the membrane. Bilirubin partitioned preferentially into model membranes comprised of microsomal lipids greater than dioleoylphosphatidylcholine = plasma membrane lipids much greater than egg phosphatidylcholine = dimyristoylphosphatidylcholine. Partitioning into membranes was increased if these contained proteins, but the effect of proteins could not be attributed to specific binding to sites on proteins, as reflected by the temperature independence of partition coefficients. Differential partitioning of bilirubin into different membranes of pure lipids also was independent of temperature. Differences in the bulk phase fluidity of membranes does not appear to account for the preferential partitioning of bilirubin into some membranes. It appears that bilirubin partitions into elements of free volume of differing sizes in membranes with variable lipid compositions and that the size of these elements can be increased by adding proteins to membranes.

Animals↗
PubMed1989

Mechanism for binding of fatty acids to hepatocyte plasma membranes.

The purpose of this study was to examine the interaction between fatty acids and plasma membranes from liver cells. We were unable to reproduce the reported effect of heating on the capacity of these membranes to bind [3H]oleate (Stremmel et al. 1985 Proc. Natl. Acad. Sci. USA. 82: 4-8). In fact, the distribution of [3H]oleate between plasma membranes and unilamellar vesicles of lipids extracted from these membranes was in favor of the lipids, indicating the absence of a detectable amount of binding to a putative fatty acid binding protein in plasma membranes. Radius of curvature of vesicles (125 A vs 475 A) had no effect on the partitioning of fatty acid. In addition, the distribution of [3H]oleate between plasma membranes and other phases had the properties of a partition coefficient over a 200-fold range of [3H]oleate. There was no evidence in this experiment for a binding isotherm, i.e., binding of [3H]oleate at a specific site, superimposed on the nonspecific partitioning of [3H]oleate into the lipids of the plasma membrane. There was no competition between [14C]oleate and [3H]palmitate for entry into plasma membranes. Finally, rates of uptake of [14C]oleate and [3H]palmitate by perfused rat liver were not affected by the presence of the other fatty acid in perfusates. These data indicate that the avidity of hepatocyte plasma membranes for [3H]oleate is a simple consequence of the physical chemical properties of oleate, lipids, and water. The data exclude the idea that the uptake of fatty acids into cells is the result of binding proteins and/or catalyzed reactions at the water-membrane interface of the cell or within the plane of the plasma membrane.

Animals↗
PubMed1987

A physical-chemical model for cellular uptake of fatty acids: prediction of intracellular pool sizes.

If the uptake of fatty acids by liver is a physical, not a biological, process, then the size and location of the intrahepatic pool of fatty acids can be predicted from uptake rates and thermodynamic data. The purpose of the experiments in this paper was to test the accuracy of this idea. Rat livers were perfused with palmitate bound to albumin, and the total amounts of palmitate removed from the perfusate were measured at 3-s intervals. The intrahepatic pools of palmitate calculated from these data were 13.8 and 23.0 nmol/g of liver at ratios of palmitate/albumin (mol/mol) (afferent side) of 2/1 and 4/1, respectively, in the steady state. The intrahepatic pools of palmitate calculated from the distributions of palmitate between membranes, H2O, albumin, and fatty acid binding protein and the measured first-order rate constants for acyl-CoA ligases in mitochondria and microsomes were 12.1 and 34.6 nmol/g for perfusate ratios of palmitate/albumin of 2/1 and 4/1, in the steady state. Intrahepatic pools of palmitate measured after establishment of a steady-state rate of uptake were 15.0 and 31.8 nmol/g for these ratios of palmitate/albumin of 2/1 and 4/1.

Animals↗
PubMed1986

Physical-chemical model for the entry of water-insoluble compounds into cells. Studies of fatty acid uptake by the liver.

The spontaneous transfer of water-insoluble substances from plasma to the interior of cells would involve a series of steps in which the substance of interest dissociates from albumin in plasma, enters the outer half of the plasma membrane of a cell, crosses the bilayer, and then dissociates from the inner half of the plasma membrane to enter cell cytosol and diffuses to sites of its metabolism. We have examined the behavior of long-chain fatty acids in the uptake process, assuming that none of these steps is facilitated by the cell during the entry of fatty acids into the liver. Comparison of the spontaneous rates for each individual step with rates of uptake of fatty acid by perfused liver leads to the conclusion that the uptake of fatty acids is not limited by kinetic factors but is determined instead by the equilibrium distribution (Keq) of fatty acids between albumin in plasma and the phospholipids of the plasma membrane. This idea was examined further by determining whether there was a relationship between the value for Keq and rates of uptake of a fatty acid and the pattern of kinetics for uptake. The data indicate that there is a linear relationship between Keq and the rate of uptake, that uptake rates can be predicted with a high degree of accuracy from thermodynamic data, and that the pattern of kinetics of uptake is compatible with the idea that the uptake rate is determined by the relative affinity of a fatty acid for albumin and membranes.

Animals↗
PubMed1985

Fatty acids bound to unilamellar lipid vesicles as substrates for microsomal acyl-CoA ligase.

Palmitate incorporated into single-layered vesicles of phosphatidylcholine was used as a substrate for palmitoyl coenzyme A ligase (palmitoyl-CoA ligase) in microsomes from rat liver. This was done in order to avoid the use of detergents for dispersal of the water-insoluble palmitate and the possibility of precipitating palmitate added to the aqueous assay as a salt suspension. The activity of the ligase measured when palmitate was added to assays as a component of phospholipid vesicles was 10-40-fold greater vs. activities reported in the literature using other methods for adding fatty acids to the assay system. Phospholipids, however, had no direct effect on the activity of palmitoyl-CoA ligase. The data indicate, therefore, that the activity of this enzyme has been underestimated because of the manner in which fatty acid was added to the assay, which has a significant effect on the activity of the ligase. It is shown too that the rate of spontaneous transfer of palmitate from unilamellar vesicles of phosphatidylcholine to microsomes via a hydrated intermediate is far more rapid than the inherent catalytic activity of the fatty acyl-CoA ligase. The data also suggest that the membrane-associated pool of fatty acid and not fatty acid in the aqueous phase of the assay is the pool of substrate interacting with the ligase.

Animals↗
PubMed1985

Rates of hydration of fatty acids bound to unilamellar vesicles of phosphatidylcholine or to albumin.

The rates of hydration of naturally occurring fatty acids bound to unilamellar vesicles of dimyristoylphosphatidylcholine were measured by following the rate of quenching of the inherent fluorescence of albumin. Rates of hydration of fatty acids bound to albumin could be estimated from the same data. The data show that these rates depend on the chain length and unsaturation of the fatty acid. Increasing chain length diminishes the rate of hydration whereas increasing unsaturation increases this rate. Rates of hydration of fatty acids bound to lipid vesicles appear to be rapid enough to account for intracellular movement between compartments in the absence of carrier proteins. It is uncertain whether this is true for hydration of fatty acids bound to albumin. Rates for this process are about 100-300 times slower vs. rates of hydration of fatty acids bound to lipid vesicles.

Animals↗
PubMed1985

Substrate specificity of fatty-acyl-CoA ligase in liver microsomes.

The substrate specificity of fatty-acyl-CoA ligase in liver microsomes has been studied in a system in which fatty acids are present initially as complexes with unilamellar vesicles of phosphatidylcholine. The latter were prepared by cosonication of phospholipids and different fatty acids. As compared with previous studies of the enzyme the activity of acyl-CoA ligase is several-fold higher for assays carried out with fatty acid substrates added as components of a bilayer. This was true for all fatty acids studied. Also as compared with data reported previously in the literature there was a systematic relationship between the structure of fatty acids, activity at Vmax for synthesis of acyl-CoA and avidity of binding to the ligase. Activity at Vmax was greatest for lauric acid and decreased with increasing chain length. The apparent avidity of enzyme for fatty acids was greatest for octanoic acid and decreased as chain length increased.

Adenosine Triphosphate↗
PubMed1985

Age-related changes in the redox status of rat muscle cells and their role in enzyme-aging.

The redox status of three biological components capable of undergoing oxidation-reduction reactions, glutathione, NAD and NADP, were determined in muscle tissues of young and old rats. A considerable increase in the relative concentration of the oxidized form, at the expense of the reduced one was found in the old tissue reflecting a significantly less reducing environment than in young cells. The effects of varying the ratio of reduced/oxidized glutathione in vitro on the activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase extracted from young and old animals were compared. It was found that concentrations of GSSG as found in old muscle tissue do not affect enzyme samples extracted from young muscle. The accumulation of oxidized glutathione observed in old cells does not, therefore, directly cause the age-related activity loss of this enzyme.

Aging↗
PubMed1984

Age-related effects in enzyme catalysis.

Rat muscle glyceraldehyde-3-phosphate dehydrogenase is one of several enzymes which have been found to undergo age-related modifications. While the amount of this enzyme in muscle tissue does not change with age, both its specific activity and affinity towards its co-enzyme are significantly reduced in the old tissue. Age-related structural changes were found to exist in the nicotinamide binding site of the enzyme and the reactions leading to the activity loss in 'old' glyceraldehyde-3-phosphate dehydrogenase were shown to involve a reversible modification of the essential cysteine-149 residue at the active site of the enzyme. The aging effects were simulated by a controlled oxidation of cys-149 in samples of 'young' glyceraldehyde-3-phosphate dehydrogenase and subsequent reduction of this residue by 2-mercaptoethanol. The enzyme modified in this way closely resembles native 'old' glyceraldehyde-3-phosphate dehydrogenase, indicating that the structural modifications in the latter enzyme are indeed introduced by a post-translational process. The mechanism for aging of glyceraldehyde-3-phosphate dehydrogenase which is proposed, based on these observations, thus assumes an oxidation of cys-149 as its first step followed by irreversible conformational changes in the enzyme molecule. The aging of glyceraldehyde-3-phosphate dehydrogenase may thus be triggered by the reduced ability of old muscle tissue to protect its constituents against oxidation.

Adenine Nucleotides↗