Abstract
A protocol presents a purification of postsynaptic density (PSD), from rat brain by subcellular fractionation using solubilization of membrane with Triton X-100 and sucrose density centrifugation. The protocol also includes purification of other synapse sub-domains such as synaptosome, synaptic plasma membrane (SPM), synaptic membrane raft, PSD lattice, P1 (nuclei and cell debris), P2 (crude mitochondria fraction), S3 (soluble fraction), and P3 (microsomal fraction). The PSD purification method presented in this text is the one established by Siekevitz group. The PSDs obtained by this method are mainly excitatory type I PSDs. These methods are useful for biochemical analyses such as identification of proteins associated with these sub-domains by proteomics methods and western blotting, and morphological analyses at the electron microscopic level. The purification protocol for the synaptic membrane raft using sucrose gradient ultracentrifugation is a useful means by which to analyze the relationship between the PSD and synaptic membrane raft by isolating both preparations simultaneously.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Somerville RA, Merz PA, Carp RI (1984) The effects of detergents on the composition of postsynaptic densities. J Neurochem 43(1):184–191
Cohen RS et al (1977) The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition. J Cell Biol 74(1):181–203
Wu K, Carlin R, Siekevitz P (1986) Binding of L-[3H]glutamate to fresh or frozen synaptic membrane and postsynaptic density fractions isolated from cerebral cortex and cerebellum of fresh or frozen canine brain. J Neurochem 46(3):831–841
Carlin RK et al (1980) Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities. J Cell Biol 86(3):831–845
Kim TW, Wu K, Black IB (1995) Deficiency of brain synaptic dystrophin in human Duchenne muscular dystrophy. Ann Neurol 38(3):446–449
Hahn CG et al (2009) The post-synaptic density of human postmortem brain tissues: an experimental study paradigm for neuropsychiatric illnesses. PLoS One 4(4):e5251
Suzuki T et al (1993) Characterization of protein kinase C activities in postsynaptic density fractions prepared from cerebral cortex, hippocampus, and cerebellum. Brain Res 619(1–2):69–75
Kim TW et al (1992) Detection of dystrophin in the postsynaptic density of rat brain and deficiency in a mouse model of Duchenne muscular dystrophy. Proc Natl Acad Sci U S A 89(23):11642–11644
Wu K, Black IB (1987) Regulation of molecular components of the synapse in the developing and adult rat superior cervical ganglion. Proc Natl Acad Sci U S A 84(23):8687–8691
Wu K, Siekevitz P (1988) Neurochemical characteristics of a postsynaptic density fraction isolated from adult canine hippocampus. Brain Res 457(1):98–112
Suzuki T et al (1997) Excitable membranes and synaptic transmission: postsynaptic mechanisms. Localization of alpha-internexin in the postsynaptic density of the rat brain. Brain Res 765(1):74–80
Matus A et al (1980) Brain postsynaptic densities: the relationship to glial and neuronal filaments. J Cell Biol 87(2 Pt 1):346–359
Suzuki T et al (2007) Characterization of mRNA species that are associated with postsynaptic density fraction by gene chip microarray analysis. Neurosci Res 57(1):61–85
Cotman CW, Taylor D (1972) Isolation and structural studies on synaptic complexes from rat brain. J Cell Biol 55(3):696–711
Nieto-Sampedro M, Bussineau CM, Cotman CW (1981) Optimal concentration of iodonitrotetrazolium for the isolation of junctional fractions from rat brain. Neurochem Res 6(3):307–320
Cotman CW et al (1974) Isolation of postsynaptic densities from rat brain. J Cell Biol 63(2 Pt 1):441–455
Kelly PT, Montgomery PR (1982) Subcellular localization of the 52,000 molecular weight major postsynaptic density protein. Brain Res 233(2):265–286
Kelly PT, Cotman CW (1976) Intermolecular disulfide bonds at central nervous system synaptic junctions. Biochem Biophys Res Commun 73(4):858–864
Kelly PT, Cotman CW (1981) Developmental changes in morphology and molecular composition of isolated synaptic junctional structures. Brain Res 206(2):251–257
Lai SL et al (1999) Interprotein disulfide bonds formed during isolation process tighten the structure of the postsynaptic density. J Neurochem 73(5):2130–2138
Sui CW, Chow WY, Chang YC (2000) Effects of disulfide bonds formed during isolation process on the structure of the postsynaptic density. Brain Res 873(2):268–273
Suzuki T et al (1994) Rapid translocation of cytosolic Ca2+/calmodulin-dependent protein kinase II into postsynaptic density after decapitation. J Neurochem 63(4):1529–1537
Carlin RK, Grab DJ, Siekevitz P (1982) Postmortem accumulation of tubulin in postsynaptic density preparations. J Neurochem 38(1):94–100
Cheng HH et al (2009) Cold-induced exodus of postsynaptic proteins from dendritic spines. J Neurosci Res 87(2):460–469
Li X et al (2007) Two pools of Triton X-100-insoluble GABA(A) receptors are present in the brain, one associated to lipid rafts and another one to the post-synaptic GABAergic complex. J Neurochem 102(4):1329–1345
Ratner N, Mahler H (1983) Isolation of postsynaptic densities retaining their membrane attachment. Neuroscience 9(3):631–644
Cho KO, Hunt CA, Kennedy MB (1992) The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron 9(5):929–942
Walikonis RS et al (2000) Identification of proteins in the postsynaptic density fraction by mass spectrometry. J Neurosci 20(11):4069–4080
Murphy JA, Jensen ON, Walikonis RS (2006) BRAG1, a Sec7 domain-containing protein, is a component of the postsynaptic density of excitatory synapses. Brain Res 1120(1):35–45
Suzuki T (2002) Lipid rafts at postsynaptic sites: distribution, function and linkage to postsynaptic density. Neurosci Res 44(1):1–9
Blomberg F, Cohen RS, Siekevitz P (1977) The structure of postsynaptic densities isolated from dog cerebral cortex. II. Characterization and arrangement of some of the major proteins within the structure. J Cell Biol 74(1):204–225
Matus AI, Taff-Jones DH (1978) Morphology and molecular composition of isolated postsynaptic junctional structures. Proc R Soc Lond B Biol Sci 203(1151):135–151
Gurd JW, Gordon-Weeks P, Evans WH (1982) Biochemical and morphological comparison of postsynaptic densities prepared from rat, hamster, and monkey brains by phase partitioning. J Neurochem 39(4):1117–1124
Matus A (1981) The postsynaptic density. Trends Neurosci 4:51–53
Garner AE, Smith DA, Hooper NM (2008) Visualization of detergent solubilization of membranes: implications for the isolation of rafts. Biophys J 94(4):1326–1340
Shogomori H, Brown DA (2003) Use of detergents to study membrane rafts: the good, the bad, and the ugly. Biol Chem 384(9):1259–1263
Phillips GR et al (2001) The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution. Neuron 32(1):63–77
Suzuki T, Yao W-D (2014) Molecular and structural bases for postsynaptic signal processing: interaction between postsynaptic density and postsynaptic membrane rafts. J Neuro-Oncol 2:1–14
Pike LJ (2006) Rafts defined: a report on the Keystone Symposium on lipid rafts and cell function. J Lipid Res 47(7):1597–1598
Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387(6633):569–572
Suzuki T et al (2011) Association of membrane rafts and postsynaptic density: proteomics, biochemical, and ultrastructural analyses. J Neurochem 119(1):64–77
Liu Q, Yao W-D, Suzuki T (2013) Specific interaction of postsynaptic densities with membrane rafts isolated from synaptic plasma membranes. J Neurogenet 27(1–2):43–58
Zhao L, Sakagami H, Suzuki T (2014) Detergent-dependent separation of postsynaptic density, membrane rafts and other subsynaptic structures from the synaptic plasma membrane of rat forebrain. J Neurochem 131(2):147–162
Dosemeci A et al (2016) The postsynaptic density: there is more than meets the eye. Front Synaptic Neurosci 8:23
Matus AI, Walters BB (1975) Ultrastructure of the synaptic junctional lattice isolated from mammalian brain. J Neurocytol 4(3):369–375
Suzuki T et al (2017) Protein components of postsynaptic density lattice, a backbone structure for type I excitatory synapses. J Neurochem 144(4):390–407. in press
Suzuki T et al (2018) Protein components of post-synaptic density lattice, a backbone structure for type I excitatory synapses. J Neurochem 144(4):390–407
Chang HW, Bock E (1980) Pitfalls in the use of commercial nonionic detergents for the solubilization of integral membrane proteins: sulfhydryl oxidizing contaminants and their elimination. Anal Biochem 104(1):112–117
Adam RM et al (2008) Rapid preparation of nuclei-depleted detergent-resistant membrane fractions suitable for proteomics analysis. BMC Cell Biol 9:30
Fried RC, Blaustein MP (1978) Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes). J Cell Biol 78(3):685–700
Warburg O, Christian W (1942) Isolierung and Kristallisation des Garungsferment. Biochem Z 310:384–421
Acknowledgments
The author learned the method of PSD purification in the Philip Siekevitz laboratory, Rockefeller University, New York. The author heartily thanks Dr. Philip Siekevitz and Marie LeDoux for their instruction.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Suzuki, T., Shirai, Y., Li, W. (2019). Isolation of Synapse Sub-Domains by Subcellular Fractionation Using Sucrose Density Gradient Centrifugation: Purification of the Synaptosome, Synaptic Plasma Membrane, Postsynaptic Density, Synaptic Membrane Raft, and Postsynaptic Density Lattice. In: Li, K. (eds) Neuroproteomics. Neuromethods, vol 146. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9662-9_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9662-9_3
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9661-2
Online ISBN: 978-1-4939-9662-9
eBook Packages: Springer Protocols