Calcium-binding of synaptosomes isolated from rat brain cortex

Free ion concentration of some divalent heavy metal ions such as Mn2+, Co2+, Ni2+, Cd2+ and Zn2+ in the synaptosomal suspension was measured to determine binding with synaptosomes isolated from rat brain cortex. A dual wavelength spectrophotometer was utilized to monitor the absorbance changes of murexide raised by stepwise addition of these ions (as chloride salts). Such titration experiments of the synaptosomal suspension revealed that a part of the added divalent cation such as Mn2+, Co2+ or Ni2+ was almost instantaneously bound to synaptosomes in isotonic NaCl media. Our previous study (Kamino, Uyesaka & Inouye, J. Membrane Biol. 17:13, 1974) demonstrated that raised external K+ resulted in a specific noncompetitive inhibition of synaptosomal Ca-binding. Just like the Ca-binding, Mn-, Co- or Ni-binding was almost completely depressed by high external K+ or ruthenium red when the free concentration of the cations was 10 mum or less, while at higher concentrations the binding was not affected. The present results indicate that tested divalent cations bind with both "Ca-binding sites" and "non-Ca-binding sites" of synaptosomal membrane, the nature of the binding sites of both being quite different: the former is sensitive to high external K+ and to ruthenium red but the latter is not.     

Characteristics of thiamine-binding protein from rat brain synaptosomes

The thiamine-binding protein was obtained from rat brain synaptosomes by affinity chromatography and gel-filtration on Sephadex G-200. The protein is homogeneous by the data of SDS gel-electrophoresis, anode electrophoresis and isofocusing between pH 3.5-9.0. The isoelectric point of this protein is near pH 4.8-5.0. The binding nature of the protein with [14C] thiamine was studied. It is shown that metal ions, especially Na+ and Ca2+, increase the thiamine-binding activity. The binding process is of a saturation character at the thiamine concentrations of 10(-7)-10(-5) M. Thiamine possesses two binding sites with KD1 = 3.1 microM and KD2 = 30 microM. Out of the tested thiamine analogues and antagonists of vitamin B1 thiamine-monophosphate and pyrithiamine were the most competitive.     

Calcium-binding of synaptosomes isolated from rat brain cortex

Summary Ruthenium red combines with isolated synaptosomes, resulting in strong inhibition of their Ca²⁺-binding. In isotonic saline media, however, the dye-induced inhibition of Ca²⁺-binding is significantly greater than that expected for the amount of bound dye and Hill's exponent of the Ca²⁺-binding decreases to 1 with an increase in the amount of the dye bound. On the other hand in isotonic mannitol-sucrose solution, inhibition of synaptosomal Ca²⁺-binding brought about by the dye is proportional to the amount of dye bound. Based on these results, the effects of the dye on the co-operative nature of synaptosomal Ca²⁺-binding is discussed.     

Kainate-induced uptake of calcium by synaptosomes from rat brain

Kainic acid induces a rapid increase in 45Ca2+ uptake by crude synaptosomal fractions isolated from rat brain. This enhanced Ca2+ permeability occurs with a half-time of approx. 1 s, similar to the fast phase of depolarization-induced calcium uptake. The depolarization-induced uptake of calcium is inhibited 85% by 3 mM CoCl2, 80% by 100 microM quinacrine and 50% by 15 microM trifluoperazine while these agents had little effect on the kainate-induced uptake. It is proposed that kainate induces receptor-mediated opening of a class of calcium channels with properties different from those of the voltage-dependent channels.     

Characterization of the glucose transporter from rat brain synaptosomes

Our goal was to characterize the glucose transporter in synaptosomes and to compare it to the different forms of transporter already identified. Cross-reactivity with antibodies to the human erythrocyte transporter, Km of glucose uptake, reversibility of NEM inhibition of transport, and insulin sensitivity were all examined. Immunoblotting showed a band at Mr 40,000, and the Km of glucose uptake was determined to be about 4 mM. Treatment with NEM caused irreversible inhibition of glucose uptake, while incubation with insulin failed to stimulate uptake. The results suggest that the transporter in synaptosomes resembles the human erythrocyte transporter.     

Dielectric properties of synaptosomes isolated from rat brain cortex

Dielectric measurements were performed on the suspensions of synaptosomes isolated from rat brain cortex. The synaptosomes in buffered salt media showed typical dielectric dispersions caused by the presence of a thin limiting membrane of sufficiently low conductivity. An analysis of the dielectric data revealed that the electric conductivity of the synaptosome interior was about 37 % of the external medium conductivity under isotonic conditions and that the dielectric constant for the interior phase was about 35. The membrane capacitance (0.7 ΜF cm⁻²) remained constant irrespective of nature and concentration of the univalent salts examined. Significant reduction in both the conductivity and the dielectric constant of the internal phase can be explained theoretically provided that some intra-synaptosomal structure (synaptic vesicles and/or small mitochondria) of non-conducting nature occupies about 50 % of the particulate volume, the remainder being in equilibrium with the external salt medium.     

Microwave induced stimulation of32Pi incorporation into phosphoinositides of rat brain synaptosomes

Summary Exposure of synaptosomes to microwave radiation at a power density of 10 mW/sq cm or more produced stimulation of the³²Pi-incorporation into phosphoinositides. The extent of³²Pi incorporation was found to be much more pronounced in phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP₂) as compared to phosphatidylinositol (PI) and phosphatidic acid (PA). Other lipids were also found to incorporate³²Pi but no significant changes in their labeling were seen after exposure to microwave radiation. Inclusion of 10 mM lithium in the medium reduced the basal labeling of PIP₂, PIP and PI and increased PA labeling. Li⁺ also inhibited the microwave stimulated PIP₂, PIP and PI labeling but had no effect on PA labeling. Calcium ionophore, A₂₃₁₈₇, inhibited the basal and microwave stimulated³²Pi labeling of PIP and PIP₂, stimulated basal labeling of PA and PI and had no effect on microwave stimulated PA and PI labeling. Calcium chelator, EGTA, on the other hand, had no effect on basal labeling of PA and PI, stimulated basal PIP and PIP₂ labeling but did not alter microwave stimulated labeling of these lipids. Exposure of synaptosomes to microwave radiation did not alter the chemical concentration of phosphoinositides indicating that the turnover of these lipids was altered. These results suggest that low frequency microwave radiation alter the metabolism of inositol phospholipids by enhancing their turnover and thus may affect the transmembrane signalling in the nerve endings.     

Metabolism of oleoyl-CoA in rat brain synaptosomes

The hydrolysis of acyl-CoA by acyl-CoA hydrolase (EC 3.1.2.2.) in brain synaptosomes was inhibited by calcium. This inhibition was partly due to interaction of Ca²⁺ with the acyl-CoA, which was present in the soluble form, and partly due to complex formation among acyl-CoA, Ca²⁺ and membrane phospholipids. The inhibition of acyl-CoA hydrolase activity, as well as the complex formation. could be reversed if incubation was carried out in the presence of Ca²⁺ chelating agents. Synaptosomes isolated from brain samples after 1 min of postdecapitative treatment showed a decrease in oleoyl-CoA hydrolase activity. The physiological implication of acyl-CoA metabolism in relation to synaptic function is discussed.Abbreviations FFA Free fatty acids - GPC glycerophosphocholines - GPE glycerophosphoethanolamines - GPI glycerophosphoinositols - GPS glycerophosphoserines     

Dielectric properties of synaptosomes isolated from rat brain cortex.

Dielectric measurements were performed on the suspensions of synaptosomes isolated from rat brain cortex. The synaptosomes in buffered salt media showed typical dielectric dispersions caused by the presence of a thin limiting membrane of sufficiently low conductivity. An analysis of the dielectric data revealed that the electric conductivity of the synaptosome interior was about 37% of the external medium conductivity under isotonic conditions and that the dielectric constant for the interior phase was about 35. The membrane capacitance (0.7 muF cm-2) remained constant irrespective of nature and concentration of the univalents salts examined. Significant reduction in both the conductivity and the dielectric constant of the internal phase can be explained theoretically proveded that some intra-synaptosomal structural (synaptic vesicles and/or small mitochondria) of non-conducting nature occupies about 50% of the particulate volume, the remainder being in equilibrium with the external salt medium.     

d-glucosamine uptake by rat brain synaptosomes

Uptake of d-glucosamine by rat brain synaptosomes occurs via a saturable transport process (K_m 2.1 mM, V 3.0 nmol/mg per min) which was clearly distinguishable from simple diffusion. This transport process is highly sensitive to cytochalasin (K_i = 7 · 10^?5 mM. d-Glucose competitively inhibits d-glucosamine uptake with a K_i value of 8 · 10^?1 mM.     

Topographic studies of gangliosides of intact synaptosomes from rat brain cortex

Gangliosides in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups with galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components fractionated on diatrizoate to give four synaptosomal membrane fractions (A-D) and a mitochondrial pellet (E). Fractions A and B represent synaptosomal plasma membranes. When intact synaptosomes were labeled, the major portion of the total radioactivity incorporated into ganglioside fraction was found to be in G _M1 ³ species. With isolated membrane fractions little selectivity was seen: (1) more label was present compared to intact synaptosomes, and (2) zones corresponding to G_M2, G_M1, G_D1a, G_D1b were the major gangliosides labeled. The results confirm the conclusion that membrane fractions A and B are derived from the exposed synaptosome surface and also show that G_M1 is the major ganglioside species available for enzyme oxidation at the surface.     

Topographic studies of glycoproteins of intact synaptosomes from rat brain cortex

Glycoproteins in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups by galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components were fractionated on diatrizoate to give four synaptosomal membrane fractions (A to D) and a mitochondrial pellet (E). Fractions A and B represent highly purified synaptosomal plasma membranes. After separation of their polypeptides by electrophoresis, $\text{4}\text{5}$ of the label was present in two bands: one about 72 000 and the other between 7800 and 3200 daltons. Seven other bands were labeled to various degrees: 160 000, 96 000, 53 000, 39 000, 34 000, 23 000 and 16 000 daltons. With isolated membranes (which incorporate 5–6 times more label) $\text{4}\text{5}$ of label was present in polypeptides in three ranges: 160 000–96 000, 70 000–40 000 and 7800-3200. The number of polypeptides that can be labeled by treatment of isolated membranes is very large. In comparison, glycoproteins whose topographical distribution permits interaction with large molecules at the synaptic surface are very limited. It is further suggested that the external synaptosome membrane involves a relatively tight network of interacting molecules that cannot be readily penetrated by large molecules.