Solubilization and various properties of NAD-binding receptor protein from rat brain synaptic membranes

The NAD-binding receptor protein has been solubilized from the synaptic membranes of the rat brain by different detergents. Digitanin proved to be the most effective detergent which exerted no action on the specific binding of [14C]NAD with the solubilized receptor protein. Kinetic parameters of the soluble ligand-receptor complex were studied. The affinity of the solubilized receptor protein to NAD did not change as compared to the protein of native membranes. The specific binding of [14C]NAD was saturated at Kd = 0.53 microM, Bmax = 0.011 nmol per 1 mg of protein. The molecular weight of the soluble NAD-receptor complex determined under native conditions was equal to 115 kDa.     

Solubilization and physical characterization of acceptors for dendrotoxin and beta-bungarotoxin from synaptic membranes of rat brain

Dendrotoxin (DTX), an Mr 7000 convulsant polypeptide from the venom of Dendroaspis angusticeps, or its facilitatory homologues act through blockade of certain voltage-sensitive K+ currents in a variety of neurons. High-affinity acceptors for DTX have been demonstrated in synaptic plasma membranes of rat or chick brain, and a fraction of these avidly bind beta-bungarotoxin (beta-BuTX), a presynaptically active protein whose lighter B polypeptide is homologous to this toxin. Extraction of rat synaptic plasma membranes using Triton X-100 in K+-containing buffer yielded binding sites with KD values of approximately 0.5 and 0.7 nM for 125I-labeled DTX and beta-BuTX, respectively. The content of high-affinity sites obtained for beta-BuTX, including the contribution of a lower affinity component, approximates to the Bmax (approximately 1.3 pmol/mg of protein) obtained for the apparent single set of DTX acceptors. On solubilization, the pharmacological specificity of the acceptor for neurotoxic DTX congeners was retained. 125I-beta-BuTX binding (2.1 nM) was blocked efficaciously by DTX (IC50 = 1.6 nM) while the binding of 2.1 nM 125I-DTX was inhibited completely by beta-BuTX (IC50 = 25 nM); the lower potency of the latter could relate to the noncompetitive nature of the mutual competition and to the presence of high- and low-affinity sites for beta-BuTX. On gel filtration, or sedimentation analysis in H2O/sucrose and 2H2O/sucrose gradients, one peak of DTX binding activity was observed, and this was inhibitable by beta-BuTX. From the hydrodynamic properties of the acceptor/detergent/lipid complex (s20,w = 13.2 S; Stokes radius = 8.6 nm), a molecular weight of 405,000-465,000 was estimated.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Dalargin-binding proteins of synaptic membranes from the rat brain: isolation and comparison of their properties with opiate receptor properties]

The proteins isolated from rat brain synaptic membranes were studied by affinity chromatography on dalargin-omega-aminohexyl-Sepharose 4B and specific elution with DAGO (Tyr-D-Ala-Gly-N-Me-Gly-ol). These proteins were shown to bind specifically 3H-naloxone (Kd = 6.6 nM; Bmax = 690 pmol/mg of protein). SDS electrophoresis of the dalargin-binding proteins termed as DBPDAGO revealed one major protein band with M(r) of 42 kDa and two minor bands with M(r) of 29 and 67 kDa. The glycoprotein component was found in DBPDAGO; their isoelectric properties were established (pI 5.4). The close similarity of DBP properties with those of isolated brain opiate receptors suggest them to be opiate receptor components.     

Comparative study of beta-adrenoreceptors of rat brain synaptic membranes

Affinity of beta-adrenoreceptors in the rat brain synaptic membranes to agonists isoproterenol and norepinephrine, as well as to antagonist 125I-hydroxybenzylpindolol is lower in young (1 month) and old (24--26 months) than in mature (8--12 months) rats. Desensitization toward isoproterenol is expressed in the young ones only. In the old but not in other groups simultaneous action of isoproterenol and N-ethylmaleimide decreases the following binding of the antagonist while the same agents added separately produced no effect. It is suggested that beta-adrenoreceptors undergo age-related changes in their conformational state due to modification of the membrane environment.     

Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes

A peptidase that cleaved neurotensin at the Pro10-Tyr11 peptide bond, leading to the formation of neurotensin-(1-10) and neurotensin-(11-13), was purified nearly to homogeneity from rat brain synaptic membranes. The enzyme appeared to be monomeric with a molecular weight of about 70,000-75,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography filtration. Isoelectrofocusing indicated a pI of 5.9-6. The purified peptidase could be classified as a neutral metallopeptidase with respect to its sensitivity to pH and metal chelators. Thiol-blocking agents and acidic and serine protease inhibitors had no effect. Studies with specific peptidase inhibitors clearly indicated that the purified enzyme was distinct from enzymes capable of cleaving neurotensin at the Pro10-Tyr11 bond such as proline endopeptidase and endopeptidase 24-11. The enzyme was also distinct from other neurotensin-degrading peptidases such as angiotensin-converting enzyme and a recently purified rat brain soluble metalloendopeptidase. The peptidase displayed a high affinity for neurotensin (Km = 2.6 microM). Studies on its specificity revealed that neurotensin-(9-13) was the shortest neurotensin partial sequence that was able to fully inhibit [3H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule as well as substitutions in positions 8, 9, and 11 by D-amino acids strongly decreased the inhibitory potency of neurotensin. Among 20 natural peptides, only angiotensin I and the neurotensin-related peptides (xenopsin and neuromedin N) were found as potent as unlabeled neurotensin.     

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.     

Isolation and partial characterization of rat brain synaptic plasma membranes

Abstract— Synaptic plasma membranes from the cortices of adult rat brain were isolated from synaptosomes prepared by flotation of a washed mitochondrial pellet (P2) in a discontinuous Ficoll-sucrose gradient. Contamination of the synaptosome fraction by microsomes was estimated by enzymic and chemical analysis to be less than 15 per cent. (2) The purified synaptosome fraction was subjected to osmotic shock, subfractionated on a discontinuous sucrose gradient and the distribution of enzymic and chemical markers for synaptic plasma membranes, microsomal membranes and mitochondria was determined. (3) Comparison of synaptosome subfractions prepared in the presence and absence of 1 mM NaH₂ PO₄/0.1 mM EDTA buffer pH 7.5, indicated that the ionic composition of the isolation medium markedly affected the distribution and enzymic composition of the subfractions. (4) Synaptic plasma membranes prepared in the presence of PO₄/EDTA exhibited a 10-fold enrichment in [Na⁺+ K⁺] ATPase and were characterized by less than 15 and 10 per cent contamination by microsomes and mitochondria respectively. (5) The polypeptide composition of the purified synaptic plasma membranes was compared with the microsomes and mitochondria by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. No differences between the protein and glycoprotein composition of the synaptic plasma membranes and microsomes were detected. The mitochondria, in contrast, possessed a unique protein composition.     

Similar physical and kinetic properties of rat brain synaptic membrane and cytosol phosphoinositide phospholipases C

Phosphoinositide phospholipase C (PLC) was extracted from the synaptic membrane fraction of rat brain by 1% sodium deoxycholate. The molecular weight and sedimentation coefficient of the membrane PLC were about 160,000 and 6.7 as estimated by gel filtration and sucrose density gradient centrifugation, respectively. These values of the membrane PLC were identical with those of the cytosol PLC of the same tissue. Moreover, the membrane PLC showed the substrate specificity for phosphatidylinositol, phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate and the sensitivity to Ca2+, sodium deoxycholate and N-ethylmaleimide similar to those of the cytosol PLC. These results indicate that the rat brain synaptic membrane PLC is indistinguishable from the cytosol PLC in physical and kinetic properties.     

Investigation of oxidation in freeze-dried membranes using the fluorescent probe C11-BODIPY

Incorporation of the fluorescent probe C11-BODIPY^581/591 in two dried membrane systems, soy bean phosphatidylcholine liposomes freeze-dried in a carbohydrate/protein matrix and Lactobacillus acidophilus (La-5) freeze-dried in a carbohydrate matrix, was successful and could be visualised by Confocal Laser Scanning Microscopy (CLSM). The C11-BODIPY^581/591 probe is a lipid oxidation reporter molecule, which is known to associate with the lipids of biological membranes and exhibit a fluorescence shift from the red range to the green range of the visible spectrum when it is oxidised together with the lipids. The present study is the first to demonstrate that the C11-BODIPY^581/591 probe can be used in dried membrane systems, and that a detection of oxidation is possible by CLSM analysis directly on the dried samples.     

Lipid peroxidation-induced changes in physical properties of annular lipids in rat brain synaptosomal membranes

The effects of lipid peroxidation (LPO) on the physical state (fluidity) of the rat brain synaptosomal lipid bilayer matrix and the annular lipid domains were investigated using the fluorescent probe pyrene. The parameters of pyrene fluorescence intensity alpha = IE/IM were measured at excitation wavelengths 280 nm and 340 nm (alpha 280 and alpha 340), reflecting fluidity of lipid bilayer matrix and annular lipids, respectively. LPO induction was shown to result in changes of fluidity of both the bilayer and annular lipids. Upon reducing formation of LPO products by carnosine, fluidity changes of both the lipid bilayer matrix and annular lipids were diminished. Conformational changes of the annular lipid domain by LPO may therefore be considered as a possible cause of the functional changes in the receptor mediated responses and of the inactivation of membrane-bound enzymes by oxidative stress.     

Binding of latrotoxin to synaptosomes and synaptic membranes of the rat brain

The binding of [125I] alpha-latrotoxin to synaptosomes from the rat brain is studied. It is shown that the constant rate of toxin association with the synaptosome receptor at 37 degrees C is equal to 8.2 +/- 1.3 x 10(7) M-1.s-1, while that of synaptosomal membrane -7.6 +/- 2.7 x 10(6) M-1 s-1. Depolarization of the synaptosome membrane induced by 55 mM KCl decreases the binding rate of toxin to the receptor, the rate constant being equal to 3.9 +/- 1.5 x 10(7) m-1 s-1. The pattern of the dissociation process of the toxin-receptor complex of synaptosomes and of synaptosomal membrane is different. In the first case dissociation follows two stages with the rate constants 3.6 x 10(-3) s-1 and 1.2/10(-4) s-1, in the second case it follows one stage with the constant equalled 2.0 x 10(-5) s-1. The quantity of the toxin binding sites on synaptosomes may vary under the action of agents modifying the activity of calcium fluxes which are induced by alpha-latrotoxin. It is supposed that a decrease in the ATP level in synaptosomes as well as deenergy of the surface membrane leads to a change in the state of the alpha-latrotoxin receptor.     

Free radical lipid oxidation and physical properties of lipid layer of biological membranes

The results obtained mainly by the author and coworkers are summarized. One efficient method to detect free radicals in biological samples is chemiluminescence (CL). In the absence of activators CL of membraneous systems is due to lipid peroxide free radicals, whereas in the presence of luminol it is initiated by oxygen radicals. Low levels of free radicals in the cells and blood plasma are maintained by antioxidants, enzymes included. Ferrous ions increase free radical concentrations in the cells and tissues. Deleterious action of hydroxyl radicals is the result of the breakage of DNA strains and of lipid peroxidation (LPO). The latter reaction brings about the damage of the membrane barriers due to a decrease of the electrical stability of the membrane lipid bilayer and "self-breakdown" of the membranes by potential differences produced in the living cells.     

Inactivation of muscarinic acetylcholine receptors in brain synaptic membranes by free fatty acids. Evaluation of the role of lipid phase

Arachidonic, linolenic and linoleic acids decreased the binding of the m-cholinergic antagonist [3H] QNB and did not affect the ratio of high to low affinity binding sites to the agonist carbamoylcholine in rat brain synaptic membranes. In the presence of arachidonic acid, SH-reagent N-ethylmaleimide acquired the ability to block QNB binding to receptor. Lipids in the bilayer and annular regions were probed by fluorescence of 1,6-diphenyl-1, 3, 5-hexatriene and pyrene. A microviscosity drop induced by increasing temperature from 10 to 37 degrees C did not affect the level of QNB equilibrium binding, whereas arachidonic acid strongly inhibited the binding at concentrations inducing the same drop in microviscosity as that induced by heating. For various unsaturated fatty acids an equal extent of receptor blocking was reached at quite different degrees of bilayer fluidization, the state of annular lipid being not changed under these conditions. It is suggested that the effect of unsaturated acids is reached through their direct interaction with the receptor, which undergoes a conformational change, rather than by an alteration of the physical state of the lipid phase of the membrane.     

Degradation of bradykinin and its metabolites by rat brain synaptic membranes

Bradykinin (BK) (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) was degraded by rat brain synaptic membranes at a rate comparable to that found for Met-enkephalin, but approximately 40 times the rate for vasopressin and oxytocin. The catabolic pathway for BK and its metabolites was elucidated through the use of high performance liquid chromatography for metabolite identification and peptidase inhibitors for blocking specific cleavage sites. BK was hydrolyzed at three sites: at the -Phe5-Ser6- bond by metalloendopeptidase 24.15, at the -Pro7-Phe8- bond by an apparently novel peptidyl dipeptidase, and at the -Phe8-Arg9 bond by a carboxypeptidase B-like enzyme. Each enzyme contributed about equally to BK degradation under the assay conditions used. Some of the resulting metabolites were further hydrolyzed: BK(1-8) to BK(1-7) + Phe by a DFP inhibitable prolyl carboxypeptidase-like enzyme, BK(1-8) to BK(1-5) + BK(6-8) by metalloendopeptidase 24.15, BK(1-7) slowly to BK(1-5) by a second peptidyl dipeptidase which was captopril inhibited, and Phe-Arg to Phe + Arg by a bestatin-inhibited dipeptidase. A number of properties of the individual enzymes were determined including sensitivity to a variety of peptidase inhibitors. These results provide a starting point for investigating the potential physiological role of each enzyme in BK function in the brain.     

Age-related characteristics of the Na+,K+-ATPase activity of synaptic membranes from the rat brain

The study of albino rats aged 6-7 months and 25-27 months revealed the age-related increase of maximal activity (V) of Na+, K+-ATPase of synaptosomal plasma membranes, separated from the cerebral cortex, while the level of Km remained stable. It is shown that in old rats as compared to the adult ones the affinity of Na+, K+-ATPase to sodium ions increases and the character of the ATP hydrolysis schedule changes in the presence of different ration of ions-activators. There are no significant changes in the inhibiting effect of strophantidin K on Na+, K+-ATPase activity of synaptosomal plasma membranes.