Saturable binding of [3H]phenytoin to rat brain membrane fraction

1981 ◽  
Vol 59 (4) ◽  
pp. 402-407 ◽  
Author(s):  
W. M. Burnham ◽  
L. Spero ◽  
M. M. Okazaki ◽  
B. K. Madras
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Membrane Fraction ◽  
Proteolytic Enzymes ◽  
Subcellular Fractionation ◽  
Brain Membrane ◽  
High Affinity ◽  
Number Of Binding Sites ◽  
Very High ◽  
Maximal Capacity

Preliminary studies indicate that [3H]phenytoin binds in a saturable and reversible fashion to at least two distinct sites in the membrane fraction of whole rat brain. One of these displays a high affinity (Kd = 6 nM) and a low maximal capacity (Bmax = 10 pmol/g protein). The other has a low affinity (Kd = 4.8 μM) and is estimated to have a very high maximal capacity. Phenytoin binding is reduced if the membrane fraction is preincubated with proteolytic enzymes and subcellular fractionation studies indicate that the P2 fraction has the largest number of binding sites. Competition experiments fail to reveal significant binding interactions with putative neurotransmitters or with other drugs except the hydantoins and anticonvulsant barbiturates. Although it is premature to speculate on the clinical significance of these findings, it is encouraging to note that the low affinity site has a Kd very similar to the therapeutic levels of phenytoin found in cerebrospinal fluid and that there seems to be some relationship between binding potency and anticonvulsant potency within the hydantoin series.

MU1: A very high affinity subtype of enkephalin binding sites in rat brain

Life Sciences ◽  
1985 ◽  
Vol 36 (23) ◽  
pp. 2233-2238 ◽  
Author(s):  
R.A. Lutz ◽  
R.A. Cruciani ◽  
P.J. Munson ◽  
D. Rodbard
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
High Affinity ◽  
Very High

scholarly journals [3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

1992 ◽  
Vol 40 (6) ◽  
pp. 771-779 ◽  
Author(s):  
A A Maki ◽  
D G Baskin ◽  
W L Stahl
Keyword(s):  
Nervous System ◽  
Rat Brain ◽  
Cardiac Glycoside ◽  
Binding Sites ◽  
Quantitative Autoradiography ◽  
Affinity Binding ◽  
Ouabain Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Kd Values

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties

Development ◽  
1997 ◽  
Vol 124 (10) ◽  
pp. 1865-1875 ◽  
Author(s):  
A. Joliot ◽  
A. Trembleau ◽  
G. Raposo ◽  
S. Calvet ◽  
M. Volovitch ◽  
...  
Keyword(s):  
Binding Sites ◽  
Cell Adhesion Molecule ◽  
Proteolytic Enzymes ◽  
Subcellular Fractionation ◽  
Low Density ◽  
Toxin Binding ◽  
Neural Cell Adhesion ◽  
Neural Tissues ◽  
Triton X ◽  
Specific Membrane

We report here that the homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electron microscopy, we observed that chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. These data are fully confirmed by subcellular fractionation experiments, which demonstrate that transfected chick-Engrailed-2 is present in low density membrane fractions that are resistant to Triton X-100, enriched in caveolin and solubilized by the addition of a cholesterol-binding detergent, a set of properties highly characteristic of caveolae. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains, i.e. Triton X-100 resistance, saponin solubilization, low density on sucrose gradients, enrichment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprotein F3/F11 and of the acylated growth-associated protein GAP-43. Finally we demonstrate that part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion.

scholarly journals Effect of Phospholipase A2 on Temperature-Induced High-Affinity (3H)Tryptamine Binding Sites in Rat Brain.

1991 ◽  
Vol 56 (4) ◽  
pp. 413-419 ◽  
Author(s):  
Michiko Saito ◽  
Shigefumi Serikyaku ◽  
Ryoichi Ishitani
Keyword(s):  
Phospholipase A2 ◽  
Rat Brain ◽  
Binding Sites ◽  
High Affinity
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