Synaptosomal plasma membranes

The modulation of synaptosomal plasma membranes (SPMs) by adriamycin and the resultant effects on the activity of membrane-bound enzymes have been reported [58]. Again DPH was used as fluorescence probe. Adriamycin increased the lipid fluidity of the membrane labeled with DPH, as indicated by the steady-state fluorescence anisotropy. The lipid-phase separation of the membrane at 23.3 °C was perturbed by adriamycin so that the transition temperature was reduced to 16.2 °C. At the same time it was found that the Na+,K+-stimulated ATPase activity exhibits a break point at 22.8 °C in control SPMs. This was reduced to 15.8 °C in adriamydn-treated SPMs. It was proposed that adriamycin achieves this effect through asymmetric perturbation of the lipid membrane structure and that this change in the membrane fluidity may be an early key event in adriamycin-induced neurotoxicity. 

An asymmetric lipid distribution between the exoplasmic and cytoplasmic leaflets of plasma membranes is typical (Devaux, 1991). Por example, it is well documented that highly unsaturated species of PE and PS are found primarily on the inner leaflet of many membranes. This has been reported for human erythrocytes (Knapp et al., 1994), murine synaptosomal plasma membranes (Pontaine et al., 1980), and human lymphocytes (Bougnoux et al., 1985), among others. It has also been shown that polyunsaturated fatty acids, including DHA, are found in higher concentrations in the aminophospholipids in the inner, cytoplasmic leaflet (Crinier et al., 1990 Hullin et al., 1991). As a result, the cytoplasmic leaflet of erythrocytes is more fluid than the exoplasmic leaflet (Morrot et al., 1986). The addition of polyunsaturated fatty acids to membranes have been shown to translocate cholesterol to the outer leaflet, where its efflux from membranes is enhanced (Dusserre et al., 1995). 

Fontaine RN, Harris RA, 8chroeder F. Aminophospholipid asymmetry in murine synaptosomal plasma membrane. J Neurochem 1980 34 269-277. 

Heemskerk, F.M.J., Dontenwill, M., Greney, H., Vonthron, C., Bousquet, P., 1998. Evidence for the existence of imidazoline specific binding sites in synaptosomal plasma membranes of the bovine brainstem. J. Neurochem. 71, 2193-2202. 

Preparation of Synaptosomal Plasma Membranes by Subcellular Fractionation... 

It appeared, furthermore, that synaptosomal plasma membranes (SPMs) could easily be obtained after lysis and ftuther subcellular fractionation of such synaptosomal preparations. Essentially, this procedure permits isolation of synaptosomal plasma membranes with minimal contamination by glial cell elements. Throughout the years, this method has been modified and improved in many labs to permit detailed studies of neuronal signal transduction using isolated synaptosomal plasma membranes in vitro. Recently, synaptosomal plasma membranes have been used to study protein phosphorylation and dephosphorylation events (6-9), polyphosphomositide metabolism (10), and protein-protein interactions using chemical crosslinkers (9,11) and unmuno-precipitation techniques (6,12). Synaptosomal plasma membranes have also been used as source material for isolation of membrane proteins (13). 

In this chapter, the preparation of synaptosomal plasma membranes using centrifugation techniques will be described in detail. The method here is based on that described previously by Kristjansson et al. (14) with only slight modifications. Section 3.1. outlines protocols for dissection and homogenization of the brain, indicating the parameters most important to obtain synaptosomal plasma membrane preparations of reproducibly high quality. Section 3.2. describes the subcellular fractionation procedure itself, and Section 3.3. outlines a protocol for assessment of yield of the synaptosomal plasma membranes employing a protein determination assay described by Bradford (15). 

To prepare a total synaptosomal plasma membrane fraction (T-SPM), proceed to step 8 (see Note 12). 

To prepare a light synaptosomal plasma membrane fraction (L-SPM), centrifuge the P2 lysate at 10,000g for 20 min using a fixed-angle rotor Collect the supernatant approx 7 mL will be recoverable (see Note 12)... 

Fig. 1. Preparation of synaptosomal plasma membranes. See Section 3.2. for complete details.

Make dilutions of the synaptosomal plasma membrane preparation (20 pL total vol per tube, in triplicate) such that the protein concentrations will fall within the range of the BSA standard senes (see Section 3.2., step 12 and Note 14). 

The protein yield of the synaptosomal plasma membrane preparation is interpolated from a plot of absorbance at 595 nm against protein concentration for the BSA standards. 

Decapitation without using anesthetics is preferable, because anesthetics may affect the biological properties of the synaptosomal plasma membrane preparation The brain should be dissected on ice immediately after decapitation. Brains from species other than rat can also be used to prepare synaptosomal plasma membranes using this protocol... 

For further dissection, prepare a chilled dissection surface by placing a circular Whatman filter paper, soaked with isotonic sucrose solution, on top of an inverted, ice-filled Petn dish (see Note 3). Dissect the brain, as required, on this ice-cold surface (e g., if preparing synaptosomal plasma membranes from forebrain only, set the brain on the filter paper and remove the brainstem and cerebellum). 

At these low speeds, the yield of synaptosomes—and thus of synaptosomal plasma membranes—will be decreased by a loss of smaller-sized particles into the so-called microsomal fraction (S2). On the other hand, to use these speeds minimizes microsomal contamination of the crude mitochondrial/synaptosomal P2 ft action. Forces as high as 17,000g for 1 h have been used to maximize the yield of synaptosomes in the P2 flection, at the expense of increasing the microsomal contamination. 

The addition of water to a crude P2 fraction changes the osmotic pressure of the medium. The synaptosomes will easily lyse as a result of this osmotic shock, whereas the mitochondria and vesicles are largely unaffected. The lysis efficiency of the synaptosomes determines the yield of the synaptosomal plasma membrane preparation. 

Step 7 in the procedure of synaptosomal plasma membranes is optional and only required if L-SPM is desired. To produce T-SPM, this centrifugation step is omitted. Besides a difference in protein yield, T-SPM and L-SPM show a difference in the sizes of the membrane fragments Membrane sheets of L-SPM are smaller than those of T-SPM. In general, L-SPM is more homogenous and slightly more accessible for exogenous treatments (in particular after heat-inactivation of SPM) than T-SPM. 

The synaptosomal plasma membranes layer at the 0.4/1.OAf sucrose interface. The mitochondria are foimd in the pellet and the synaptic vesicles are found on top of the 0.4A/sucrose fraction. Use a clean, disposable Pasteur pipet to harvest the SPMs, after first removing the top fractions (removing about one-half of the 0.4Af sucrose to prevent contamination of the SPM fraction). 

To remove glycerol or to replace the buffer medium of the synaptosomal plasma membrane preparation, wash the preparation by centrifugation at 15,000g for at least 5 min at 4°C in SPM buffer. 

Papaphihs A, Dehconstantinos G. Modulation of serotonergic receptors by exogenous cholesterol in the dog synaptosomal plasma membrane. Biochem Pharmacol. 1980 29(24) 3325-3327. 

Morgan, I. G., Reith, M., Marinari, U., Breckenridge, W. C., and Gombos, G., 1972, The isolation and characterization of synaptosomal plasma membranes, in Glycoli-pids, Glycoproteins, and Mucopolysaccharides of the Nervous System (V. Zambotti, G. Tettamanti, and M. Arrigoni, eds.), pp. 209-228, Plenum Press, New York. 

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