Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5-5 microM) follows an exponential curve described by the general formula y = a.ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 microM cholesterol glucoside, with a Hill coefficient of h = 2.98 +/- 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 microM) or its glucoside (up to 50 microM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 microM) incorporation. Cholesterol (5 microM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 +/- 1.1 degrees C in control SPM, which was elevated to 29.5 +/- 1.4 degrees C in SPM treated with cholesterol glucoside (50 microM) and abolished in SPM treated with cholesterol (5 microM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F- and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 microM) produced an increased packing of the bulk lipids, while cholesterol (5 microM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.     

Time-dependent cadmium-neurotoxicity in rat brain synaptosomal plasma membranes

• 1.1. The modulation of lipid dynamics and lipid protein interactions were studied in rat brain synaptosomal plasma membranes (SPM) up to 24 hr after exposure to cadmium (Cd).
• 2.2. The activity of acetylcholinesterase and adenylate cyclase showed a considerable decrease after 6 hr of Cd exposure, followed by a progressive increase up to 24 hr.
• 3.3. SPM chemiluminescence showed a maximum decrease at 12 hr, demonstrating a considerable increase in lipid peroxidation.
• 4.4. SPM of Cd-exposed animals showed a statistical significant increase in fluorescence anisotropy parameter [(r₀/r) — 1]⁻¹ at 18 and 24 hr compared to SPM of the control, indicating a decrease of membrane fluidity.

     

Phosphatidylserine and calmodulin effects on Ca2+-stimulated ATPase activity of dog brain synaptosomal plasma membranes

Phosphatidylserine (PtdSer)-liposomes when incubated with synaptosomal plasma membranes (SPM) of dog brain, evoked a significant increase (approx 80%) of the Ca2+-stimulated ATPase activity with maximal effect achieved at around 0.7 mumol PtdSer/mg SPM protein. Higher concentrations of PtdSer led to inhibition of the enzyme activity with respect to the maximal percentage of stimulation. Treatment of SPM with EGTA, to minimize the presence of bound cytoplasmic activator calmodulin, resulted in a mixed mechanism of inhibition of the enzyme activity (Vmax was decreased and Km increased) as estimated by Lineweaver-Burk plots. Addition of exogenous calmodulin resulted in an increase of Vmax and in a restoration of Km to control value. Ca2+-stimulated ATPase activity, in EGTA-treated SPM, showed the same figure of changes at different concentrations of PtdSer-liposomes as those of the control, but the turning point was now located at higher PtdSer concentrations. The results suggest that Ca2+-stimulated ATPase activity of SPM is modulated by PtdSer and that calmodulin participates in these interactions, probably, by regulating the contact between the enzyme and Ca2+ ions.     

Anion channels from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Synaptic membranes from rat brain were incorporated into planar lipid bilayers, and the characteristics of two types of anion-selective channels (type I and type II) were investigated. In asymmetric BaCl₂ buffers (cis, 100mm/trans, 25mm), single channel conductances at –40 mV were 70 pS (type I) and 120 pS (type II). Permeability ratios (P _Na:P _Ba:P _Cl) calculated from the Goldman-Hodgkin-Katz current equation for type I and type II channels were 0.230.041 and 0.050.031, respectively. Both channels exhibited characteristic voltage-dependent bursting activities. Open probability for type I channels had a maximum of 0.7 at about 0 mV and decreased to zero at greater transmembrane potentials of either polarity. Type II channels were relatively voltage independent at negative voltages and were inactivated at highly positive voltages. Type I channels showed spontaneous irreversible inactivation often preceded by sudden transition to subconducting states. DIDS blocked type I channels only from thecis side, while it blocked type II channels from either side.     

Temperature effect of cholesterol association with synaptosomal plasma membranes of rabbit brain.

Association of exogenous cholesterol with rabbit brain synaptosomal plasma membranes follows an exponential path described by the general formula y = a X ebx. The co-operative nature of this association was shown when increasing amounts of unlabelled cholesterol glucoside (up to 0.5 mM) were added to a fixed amount (5 microM) of [14C]cholesterol, when a biphasic curve of the binding of [14C]cholesterol into the membranes was obtained. Arrhenius plots of this association revealed two break points which occur at 25 degrees C and 42 degrees C. The first break apparently corresponds to the transition from the crystalline to the gel phase. The second break may be due to the (continuously) increasing entropy of the system which creates at a certain point difficulties in the binding of cholesterol into the lipid bilayer.     

Cortisol effect on (Na++K+)-stimulated ATPase activity and on bilayer fluidity of dog brain synaptosomal plasma membranes

The binding of [¹⁴C]cortisol into dog brain synaptosomal plasma membranes (SPM) follows an exponential path described by the general formula y=a.e^bx. The specific activity of the SPM-bound (Na⁺+K⁺)-stimulated ATPase was linearly increased at different concentrations of cortisol. Changes in the allosteric properties of (Na⁺+K⁺)-stimulated ATPase by fluoride (F^–) (i. e. changes of Hill coefficients) indicate that cortisol increases the membrane fluidity. The fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene-labeled SPM decreased in cortisol treated SPM compared to untreated (control) SPM, which is consistent with a general increase in membrane fluidity. This increase of fluidity by cortisol may play a role in the physiological effects of this hormone in the brain.     

Interaction of bilirubin with the synaptosomal plasma membrane

The interaction of the neurotoxic pigment bilirubin with synaptosomal plasma membrane vesicles (SPMV) isolated from rat brain was investigated. The interaction seems to involve three steps: (a) a rapid formation of an electrostatic complex between bilirubin and polar lipid head groups; (b) a slow inclusion of the pigment into the hydrophobic core of the membrane; and (c) a SPMV-induced bilirubin aggregation, observed when membrane capacity for bilirubin is exceeded. The association constant of the initial complex increased markedly when pH was lowered below 7.4, particularly in SPMV isolated from newborn rats. A preferential binding of bilirubin to pure gangliosides and sphingomyelin was observed, thus suggesting a role for these lipids as first targets of the pigment in the synaptic membrane. The inclusion of bilirubin into the membranes was gradually enhanced when decreasing the pH or the age of the rats from which SPMV were isolated. In addition, membranes from 2-day-old rats have a higher capacity for bilirubin incorporation compared to those from adult rats. Experiments with reconstituted liposomes of varying protein and cholesterol contents suggest that the effect of age may be related to changes in synaptosomal membrane fluidity during development. Our results support the hypothesis that the interaction of bilirubin with the synaptic membrane plays an important role in the molecular mechanisms of bilirubin neurotoxicity.     

Structural rearrangements in synaptosomal membranes of the brain]

Studies were carried out on structural state of synaptosomes and synaptic plasma membranes from brain of rats and guinea pigs at 5--50C. In membranes from guinea pig brain there were two breaks at 8 and at 18--20C in the curves of temperature dependence of light scattering. At the same temperatures adhesive properties of membranes had changed in jump-like manner when evaluating the degree of aggregation at pH4. The degree of aggregation in membranes from rat brain as well as rate of solubilization by 0.2% SDS (stoped-flow measurements) are characterized by single break point at 27C in Arrhenius curves. The breaks were interpreted as phase transitions with membrane surface involvement. Chlorpromazine and tetraciane (10(4)--10(3) M) abolished or modified these transitions. Because the number of negative charges and tryptophanyl and ANS fluorescence parameters were unchanged it was suggested that redistribution of ionogenic and hydrophobic goups took place at membrane surface during phase transitions.     

Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes

Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain.     

Endogenous phosphorylation of rat brain synaptosomal plasma membranes in vitro: Some methodological aspects

The time course of endogenous phosphorylation in vitro of total or separted synaptic plasma membrane proteins (SPM) has been correlated with that of hydrolysis of the phosphate donor (ATP) in the incubation medium. The ATP/SPM ratio in the medium was varied. In a low-ratio medium (7.5 M ATP; 2.2 g SPM/l) a complete hydrolysis of ATP occurred almost instantaneously as was measured by the release of free phosphate in and the disappearance of ATP from the medium. As a consequence, only a very short peak of phosphorylation, followed by dephosphorylation was observed. However, when higher ATP/SPM ratios were used (200 M ATP; 0.4 g SPM/l and 500 M ATP; 0.4 g SPM/l), the incorporation of phosphate into SPM proteins was linear for 20 sec, and the maximum level of phosphate incorporation was increased. Similar results were obtained after separation of³²P-labeled phosphoproteins by slab gel electrophoresis. However, analysis of the autoradiographs obtained fromone SPM preparation under different ATP/SPM ratios revealed dependence of phosphorylation of individual protein bands on the conditions used.     

Steroid binding to synaptic plasma membrane: differential binding of glucocorticoids and gonadal steroids

The specific binding of [3H]-corticosterone, [3H]-17 beta-estradiol, [3H]-testosterone, and [3H]-progesterone to synaptic plasma membrane (SPM) prepared from rat brain has been characterized. The dissociation constant is estimated as on the order of 1 x 10(-7) M for corticosterone and 1 x 10(-8) M for the other three steroids. In a competition experiment, none of the 3H-steroids was displaced by the other steroids at 500-fold excess, indicating the presence of specific binding sites on the membrane for each type of steroid. Moreover, pre-incubation of the SPM with phospholipase A2 or phospholipase C totally destroys the membrane binding of corticosterone and testosterone, but the binding of estradiol and progesterone remains intact. Since the SPM prepared from brain tissue is derived from many different neuronal cell types, it is possible that the membrane binding sites for glucocorticoids and for gonadal steroids are present in different neurons.     

Topography of glycoproteins in the chick synaptosomal plasma membrane

Chick brain synaptosomes or synaptic subfractions were treated with neuraminidase (EC 3.2.1.18) and/or galactose oxidase (EC 1.1.3.9) preparations in which proteolytic activity was inhibited with phenylmethanesulfonyl fluoride followed, after washing, by reductive incorporation of sodium boro[³H]hydride to identify galactose residues exposed on the synaptosomal external surface. Control experiments to demonstrate restriction of labeling to the external surface involved comparing the radioactivity in synaptoplasmic, soluble polypeptides isolated after labeling with labeled, isolated synaptoplasm and examining incorporation into fractions incubated without enzymes. Intactness of the synaptic plasma membrane after labeling was shown by trypsin digestion studies. Polypeptides were separated on sodium dodecyl sulfate polyacrylamide gels and were detected by a liquid scintillation counting procedure. Eleven major radioactive peaks were found after galactose oxidase treatment and reduction of isolated synaptic membranes. When intact synaptosomes were labeled, the same components were detected. When isolated synaptic membranes or intact synaptosomes were treated with neuraminidase before galactose oxidase treatment, three additional components were labeled. These results suggest that (a) chick synaptic membranes have a complex mixture of glycoproteins, (b) all major chick synaptic membrane glycoproteins labeled by galactose oxidase have most or all carbohydrate groups exposed at the exterior surface of the synaptosome, (c) all major, externally-disposed polypeptides of these synaptic membranes are glycoproteins.     

Determination of electrical characteristics of synaptosomal membranes of the brain

A correct method for evaluating the potential differences across plasma membranes of synaptosomes from the brain is presented. It takes into consideration the multicompartment synaptosome organization and is based on the accumulation of the radioactive permeant cation [3H]tetraphenylphosphonium. It is shown that upon potassium depolarization of the synaptosomes to about -5 mv there is a sharp decrease in the ion selectivity of the synaptic membranes.     

Characterization of an 11,000-dalton beta-bungarotoxin: binding and enzyme activity on rat brain synaptosomal membranes

The binding and phospholipase A2 activity of an 11,000-dalton beta-bungarotoxin, isolated from Bungarus multicincutus venom, have been characterized using rat brain subcellular fractions as substrates. 125I-labeled beta-bungarotoxin binds rapidly (k = 0.14 min-1 and 0.11 min-1), saturably (Vmax = 130.1 +/- 5.0 fmoles/mg and 128.2 +/- 7.1) fmoles/mg), and with high affinity (apparent Kd = 0.8 +/- 0.1 nM and 0.7 +/- 0.1 nM) to rat brain mitochondria and synaptosomal membranes, respectively, but not to myelin. The binding to synaptosomal membranes is inhibited by divalent cations and by pretreatment with trypsin. The binding results suggest that the toxin binds to specific protein receptor sites on presynpatic membranes. The 11,000-dalton toxin rapidly hydrolyzes synaptosomal membrane phospholipids to lysophosphatides and manifests relative substrate specificity in the order phosphatidyl ethanolamine greater than phosphatidyl choline greater than phosphatidyl serine. These results indicate that the 11,000-dalton beta-bungarotoxin is a phospholipase A2 and can use presynaptic membrane phospholipids as substrates. The binding, phospholipase activity and other biological properties of the 11,000-dalton toxin are contrasted with those of the beta-bungarotoxin found in highest concentration in the venom (the 22,000-dalton beta-bungarotoxin), and the two toxins are shown to have qualitatively similar properties. Finally the results are shown to support the hypothesis that beta-bungarotoxins act in a two-step fashion to inhibit transmitter release: first, by binding to a protein receptor site on the presynatic membrane associated with Ca2+ entry, and second, by perturbing through enzymatic hydrolyses the phospholipid matrix of the membrane and thereby causing an increase in passive Ca2+ permeability.