Properties of NAD binding by synaptic membranes of rat brain

The binding of [14C]NAD to rat brain synaptic membranes is reversible and depends on incubation time, temperature and protein concentration in the reaction mixture. The value of the rate constant for [14C]NAD binding to the synaptic membranes at 24 degrees C (kl) is 1.1 X 10(-6) M-1 S-1, the rate constant for dissociation of the [14C]NAD-receptor complex (k-1) is 3.3 X 10(-3) S-1. The value of the constant for the ligand dissociation from this complex (Kd) is 3.0 nmole. Treatment of the experimental results in the Scatchard plots for the equilibrium binding of [14C]NAD to the synaptic membranes demonstrated that the receptor sites with high and low affinities for the ligand (Kd1 = 3.3 nmol, Kd2 = 14.4 nmole) and with binding capacities of 44 and 77 pmole of [14C]NAD, respectively. It was found that the synaptosomal membrane components which bind the labelled NAD have a protein nature. Data from [14C]NAD and [nicotinamide-3H]NAD binding suggest that brain synaptic membranes bind NAD at the nicotinamide and adenylic moieties.     

Endogenous inhibitor of [3H]kainate binding to synaptic membrane in rat brain

An understanding of the mechanism of kainic acid toxicity to neurons could provide important clues to pathogenesis of Huntington's chorea. The existence of high-affinity binding sites for kainate, a foreign compound, is suggestive of the existence of kainate-like substances in the brain. In addition to such neurotoxic kainate-like substances, and endogenous inhibitor of kainate binding may also exist in the brain to allow the synaptic function to operate normally. Based on this idea, the existence of molecules which inhibit [3H]kainate binding to synaptic membranes was examined in rat brain. An endogenous inhibitor of [3H]kainate binding to synaptic membranes was found in the supernatant obtained from synaptic membranes of rat brain. The inhibitor is a thermostable, basic protein with a relatively low molecular weight.     

Effect of chronic psychogenic stress on the characteristics of synaptic membrane receptors in the rat brain

It has been shown that the 15-day stressing of rats by means of stochastic footshock combined with light flashes leads to a relatively stable "behavioral depression". Twenty-four hours after the last exposure to stress crude synaptosomes isolated from the whole brain demonstrated an increase in the KD of 3H-dihydroalprenolol specific binding and a lowering of the Bmax of 3H-WB-4101 specific binding sites. Specific binding of 3H-flunitrazepam changes inconclusively: the decrease in the KD is followed by a reduction in the concentration of receptors. Specific binding of 3H-imipramine by brain membranes remains unchanged under the above conditions.     

Effect of benzodiazepines on 5'-nucleotidase activity in rat brain

Experiments on 330 rats were made to study the influence of benzodiazepines (diazepam, dormicum and phenazepam) on 5'-nucleotidase activity in brain homogenates. It was discovered that diazepam and dormicum in doses of 3 and 4 mg, phenazepam in doses of 3.75 and 5 mg per 200 g bw provoked a 16-20% reduction in 5'-nucleotidase activity. The maximal effect of diazepam (3 and 4 mg doses) was attained 1 h after intraperitoneal injection, that of dormicum (3 mg) 30 min and of phenazepam (5 mg) 1 h after intraperitoneal injection. It is assumed that benzodiazepines are involved in AMP metabolism.     

5-Hydroxytryptamine binding to synaptic membranes from rat brain.

The ³H-5HT binding capacity of rat brain synaptic membranes prepared by density gradient centrifugation has been investigated using a rapid ultrafiltration technique. A saturable, high affinity (K_D = 1.10^?9 M), 5HT displaceable binding has been found. It is thermosensitive, temperature dependent and pH dependent. 5HT and related tryptamines are the most effective displacers of bound ³H-5HT, whereas compounds which are not structurally related to 5HT (chlorpromazine, imipramine, cyproheptadine and cinanserine) and other neuro-transmitters (noradrenalin, dopamine) are ineffective. The distribution of 5HT-specific binding sites in the brain is related to serotonergic input. We conclude that these 5HT binding sites might possibly represent 5HT receptor sites.     

Specific binding of prostaglandin D2 to rat brain synaptic membrane. Occurrence, properties, and distribution

Prostaglandin (PG) D2 bound specifically to a particulate fraction rich in the synaptic membrane of rat brain. The binding was dependent on time and temperature, equilibrium being reached after 5 min at 37 degrees C. The specific binding constituted about 70% of the total binding at 37 degrees C, and 55% at 0 degrees C. The maximal binding was obtained in the presence of 100 mM sodium ion and at pH 8. The equilibrium dissociation constant and the maximal concentration of binding sites as determined by Scatchard analysis were 28 +/- 7 nM and 0.45 pmol/mg of protein (n = 3), respectively. Hill coefficient was 1.15, indicating a single entity of binding sites and no cooperativity. The binding sites were highly specific for PGD2; the Ki values for PGD1 and PGF2 alpha were 523 and 693 nM, respectively. Other PGs including 13,14-dihydro-15-keto-PGD2, an inactive metabolite of PGD2, had 150- to 1000-fold lower affinities than PGD2. The binding was inhibited by boiling or treatment with proteases, phospholipases, or beta-galactosidase. The specific activity of PGD2 binding was highest in the pituitary gland, followed by the hypothalamus and the olfactory bulb od the rat brain, this pattern being almost parallel to that of the cytosolic NADP-linked PGD2 dehydrogenase activity. The results suggest that PGD2 plays a significant role in these regions of the rat brain.     

[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis

Binding of [¹²⁵I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca²⁺-dependent membrane binding of [¹²⁵I]calmodulin was found to have a B_max of 284 pmol/mg protein and an apparent affinity with a K_d of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of [¹²⁵I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10^–1 min^–1 and a half-time (t_1/2) of 1.8 min for the fast component, and a k of 4.8×10^–2 min^–1 and a t_1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10^–2 min^–1 and a t_1/2 of 15.3 min for the fast component, and a k of 5.5×10^–3 min^–1 and a t_1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, [¹²⁵I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (T_d) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [¹²⁵I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10^–7–10^–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - K_d dissociation constant - B_max maximum binding - k first-order rate constant - t_1/2 half-time - T_d transition temperature     

Phosphatidylinositol-attached 5'-nucleotidase from synaptic plasma membrane of rat brain

Synaptic plasma membranes (SPM) of rat brain contained a 5'-nucleotidase that was specifically released by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC). About 30% of the enzyme was readily released and the remainder was less susceptible. Purified 5'-nucleotidase was treated with PIPLC and the resultant enzyme was almost totally partitioned into the detergent-poor phase following phase-separation in Triton X-114 indicating that PIPLC converted the enzyme from an amphipathic to a hydrophilic form. The results suggest that 5'-nucleotidase is anchored into SPM by a covalently attached phosphatidylinositol moiety.     

Sodium-independent taurine binding to brain synaptic membranes

Saturable sodium-independent taurine binding to mouse and rat brain synaptic membranes was exposed after two freezing-thawing cycles combined with Triton X-100 treatments. The amount of saturable taurine binding was fairly low but was enhanced after depletion of brain taurine. Saturable taurine binding was displaceable by some convulsants and anticonvulsants but is specificity still remains to be established.     

The effect of ACTH on rat brain synaptic plasma membrane lipid fluidity

The effect of ACTH on the lipid fluidity was examined in synaptic plasma membranes from rat forebrain. ACTH1-24 increased the fluidity of the synaptic plasma membranes in a dose-dependent way, the lowest effective dose being 10(-5) M. The shorter N-terminal fragment ACTH1-10 was not effective. The significance of this finding is discussed in relation to the known effects of ACTH on synaptic membrane phosphorylation.     

Effects of ATP on calcium binding to synaptic plasma membrane

The release of labeled norepinephrine from preloaded synaptosomes requires the presence of potassium and calcium. ATP-dependent binding of calcium to synaptic plasma membranes (SPM) may provide a means of maintaining the cation in a readily available pool for the triggering of transmitter release. A high Ca-binding capacity was demonstrated in SPM. The Km for calcium is 5.5 X 10(-5) M. The dependence of the system on the gamma phosphate of ATP was demonstrated by an increase in Ca-binding with increasing ATP concentration and by competitive inhibition of binding by ADP and AMP. Magnesium is also required for ATP-dependent Ca-binding. The optimum pH for the Ca binding was 7.0. Pretreatment of SPM with phospholipase A2 lowered the binding capacity. Sulfhydryl groups are also critical for ATP-dependent Ca binding to occur. A model for ATP-dependent Ca-binding was proposed.     

Evidence for a synaptic plasma membrane associated adenylate kinase in the rat brain

About 5% of the total adenylate kinase activity in the rat forebrain was found in a subcellular fraction enriched in synaptic plasma membrane (SPM). The enzyme remained membrane bound after washing by 1M potassium acetate. It was resistant to trypsin digestion under conditions which destroyed 90% of acetylcholinesterase activity. The SPM enzyme was solubilized by 0.25% Triton X-100 resulting in a 4-fold increase in activity. Similar effects were observed when SPM was treated with phospholipases, melittin and trifluoperazine. These results suggest the occurrence of an adenylate kinase closely associated with SPM the activity of which can only be fully expressed by disturbances to the hydrophobic lipid bilayer. The enzyme can be seen as strategically located to play a role in regenerating ATP required for the manifold activities of the synaptic membrane.     

High-affinity glutamic acid binding to brain synaptic membranes

Rat brain homogenate preparations exhibited two types of glutamine binding, one a high-affinity (K₁ = 0.2 μM) and the other a low-affinity type (K₂ = 4.4 μM). The high-affinity binding was primarily associated with the plasma membrane subcellular fractions and in particular with the synaptic membrane subfraction. This l-glutamate binding was found to be strongly stereospecific for the l-form and was almost totally reversible. The synaptic membrane glutamate binding was partialy inhibited by neuro-excitatory and neuro-inhibitory amino acids but was not affected by amino acids lacking in neuropharmacologic activity. The membrane-associated l-glutamate binding system could be solubilized by Triton X-100 without loss of its high-affinity binding activity. The chemical nature of this glutamate binding component was found to be that of a glycolipoprotein. It is proposed that this glutamate binding system represents the physiologic receptor on neuronal membranes of this amino acid.     

Modulation of serotonin binding in rat brain by membrane fluidity

Preincubation of rat brain homogenates with increasing concentrations of n-hexanol reduced specific serotonin (5-HT) binding and increased membrane fluidity as measured by fluorescence depolarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. At 5 mM ascorbate maximal reductions of both membrane fluidity and specific 5-HT binding were observed. Both effects were enhanced in the presence of ferrous sulphate and oxygen. In the presence of ascorbate (5.7 mM) only one 5-HT binding site was observed in contrast with high and low affinity binding sites (K_D1 = 0.08 ± 0.04 nM, K_D2 = 28.8 ± 1.3 nM) found in the absence of ascorbate. The ascorbate induced decrease of specific 5-HT binding may be explained by lipid peroxidation, which decreases membrane fluidity, and by ascorbate's reducing properties. Since different correlations were found between membrane fluidity and specific 5-HT binding depending upon the presence of ascorbate or n-hexanol, the results suggest that membrane fluidity is a critical factor, however, just one of several determinants in 5-HT binding studies.     

The effect of ionizing radiation on the binding of muscimol by synaptic membranes in the rat brain

A study was made of the effect of gamma-radiation on binding of muscimol, a GABA agonist, by synaptic membranes of rat brain cortex. Exposure to 2 Gy radiation was shown to reduce [3H]-muscimol binding to membranes.     

High-affinity binding of proline to mouse brain synaptic membranes

There is evidence suggestive of the possible neuromodulatory role forl-proline in the mammalian brain. The binding of proline to whole mouse brain synaptic membranes has been partially characterized. Several binding sites for this imino acid have been identified; one in the nanomolar range and at least two in the submicromolar range. The binding of proline is inhibited by NaCl. Pipecolic acid (40 M), ornithine, aminooxyacetic acid (AOAA), glycine, GABA, and glutamate were capable of significantly inhibiting proline binding. Although detailed pharmacological and functional studies are needed, these results are consistent with a brain-specific function for this imino acid, as well as, with the presence of specific binding site(s) for proline.     

The effect of hypothermia on kinetic characteristics of the rat brain synaptic membrane Na,K-ATPase

The effect of profound hypothermia (acute or prolonged) on Km for ATP, Vm and strophanthine K affinity to Na,K-ATPase in the rat brain synaptosomal membranes was investigated. The temperature dependence of Na,K-ATPase activity in temperature range 5-40 degrees C was also studied. Hypothermia decreases Km and Vm, and increases affinity of strophanthine K to the enzyme. There are two linear sections in Arrhenius plots ofNa,K-ATPase activity. Hypothermia does not change position of the break point in Arrhenius plots. The mechanisms and biological significance of the changes revealed are discussed.