Postsynaptic density (PSD) 是一個細緻的圓盤狀結構，它位於後突觸膜的下方，與調控後突觸的訊息傳遞有關，並且經由調控突觸區域的活性，PSD被相信與學習和記憶有所關聯。之前有許多研究，利用不同的方法來研究PSD的組成，發現其中含有多種蛋白，包括glutamate receptors、 scaffold proteins 和 signaling proteins等等，近年來利用高效率及高解析度的質譜儀系統，現已分析出近500種PSD的組成蛋白。我們實驗室利用不同pH值範圍與不同濃度的二維電泳膠片，分做四個區域來研究PSD組成蛋白在二維膠片上的分布，發現到有超過600個具再現性protein spots的表現，相信可能仍有許多未知的PSD組成蛋白尚未被發現。而我們在二維電泳方面的研究，亦大為改善PSD組成蛋白在鹼性pI值與低分子量區域的分析效果。另外，借助MALDI-TOF的分析，我們做了八個major protein spots的鑑定，發現它們表現在2-D gel的pI/MW值與MW的實際值和pI的理論計算值都很接近。α-CaMKⅡ是PSD中的主要蛋白之一，過去的研究沒有明確的找出它在2-D gel上的位置，我們原本想利用二維電泳配合Western Blot做α-CaMKⅡ的定位，卻發現α-CaMKⅡ在不同pH值的溶液中，表現在膠片上的量不同。於是我們做了溶解度測試的實驗，發現pH6-11 IPG buffer是促成α-CaMKⅡ有效溶解的主因。未來我們可以利用pH6-11 IPG buffer配合改良的二維電泳條件，以期正確定位出α-CaMKⅡ在2-D gel的位置，這將有助於了解α-CaMKⅡ不同的磷酸化狀態，並且推測α-CaMKⅡ是如何調控與PSD之間的關係。

Apperson ML, Moon IS, Kennedy MB. (1996) Characterization of densin-180, a new brain-specific synaptic protein of the O-sialoglycoprotein family. J Neurosci. 16, 6839-6852.

Barria A, Muller D, Derkach V, Griffith LC, Soderling TR. (1997) Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation. Science. 276, 2042-2045.

Beesley PW, Mummery R, Tibaldi J. (1995) N-cadherin is a major glycoprotein component of isolated rat forebrain postsynaptic densities. J Neurochem. 64, 2288-2294.

Biederer T, Sudhof TC. (2000) Mints as adaptors. Direct binding to neurexins and recruitment of munc18. J Biol Chem. 275, 39803-39806.

Blitz AL, Fine RE. (1974) Muscle-like contractile proteins and tubulin in synaptosomes. Proc Natl Acad Sci U S A. 71, 4472-4476.

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, 204-225.

Boeckers, T. M., Bockmann, J., Kreutz, M. R., and Gundelfinger, E. D. (2002) ProSAP/Shank proteins ­a family of higher order organizing molecules of the postsynaptic density with an emerging role in human neurological disease. J Neurochem. 81, 903-910

Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248-254.

Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE, Santillano DR, Wu Z, Huang F, Xia H, Peters MF, Froehner SC, Bredt DS. (1996) Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell. 84, 757-767.

Brenman JE, Christopherson KS, Craven SE, McGee AW, Bredt DS. (1996) Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein. J Neurosci. 16, 7407-7415.

Caceres, A., Binder, L.I., Payne, M.R., Bender, P., Rebhun, L. and Steward, O. (1984) Differential subcellular localization of tubulin and the microtubule- associated protein MAP2 in brain tissues as revealed by immunocytochemistry with monoclonal hybridoma antibodies. J. Neurosci. 4, 394-410.

Caceres, A., Payne, M.R., Binder, L.I. and Steward, O. (1983) Immuno- cytochemical localization of actin and microtubule-associated protein MAP2 in dendritic spines. Proc. Natl. Acad. Sci. USA. 80, 1738-1742.

Carlin RK, Bartelt DC, Siekevitz P. (1983) Identification of fodrin as a major calmodulin-binding protein in postsynaptic density preparations. J Cell Biol. 96, 443-448.

Carlin, R.K., Grab, D.J., Cohen, R.S. and Siekevitz, P. (1980) Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities. J. Cell Biol. 86, 831-843.

Carlin, R.K., Grab D.J. and Siekevitz, P. (1982) Postmortem accumulation of tubulin in postsynaptic density preparations. J. Neurochem. 38, 94-100.

Carlin RK, Siekevitz P. (1984) Characterization of Na+-independent GABA and flunitrazepam binding sites in preparations of synaptic membranes and postsynaptic densities isolated from canine cerebral cortex and cerebellum. J Neurochem. 43, 1011-1007.

Carr DW, Stofko-Hahn RE, Fraser ID, Cone RD, Scott JD. (1992) Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79. J Biol Chem. 267, 16816-19823.

Chen, H. J., RojasSoto, M., Oguni, A., and Kennedy, M. B. (1998) A synaptic RasGTPase activating protein (p135 SynGAP) inhibited by CaM kinase II. Neuron. 20, 895-904.

Cho, K. O., Hunt, C. A., and Kennedy, M. B. (1992) The rat brain postsynaptic density fraction contains a homolog of the Drosophila discslarge tumor suppressor protein. Neuron. 9, 929-942.

Cohen RS, Blomberg F, Berzins K, Siekevitz P. (1977) The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition. J Cell Biol. 74, 181-203.

Cohen RS, Siekevitz P. (1978) Form of the postsynaptic density. A serial section study. J Cell Biol. 78, 36-46.

Colonnier M. (1968) Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study. Brain Res. 9, 268-287.

Condorelli DF, Nicoletti VG, Barresi V, Conticello SG, Caruso A, Tendi EA, Giuffrida Stella AM. (1999) Structural features of the rat GFAP gene and identification of a novel alternative transcript. J Neurosci Res. 56, 219-228.

Cotman CW, Banker G, Churchill L, Taylor D. (1974) Isolation of postsynaptic densities from rat brain. J Cell Biol. 63, 441-455.

Dong H, O'Brien RJ, Fung ET, Lanahan AA, Worley PF, Huganir RL. (1997) GRIP: a synaptic PDZ domain-containing protein that interacts with AMPA receptors. Nature. 386, 279-284.

Dosemeci A, Reese TS. (1995) Effect of calpain on the composition and structure of postsynaptic densities. Synapse. 20, 91-97.

Dosemeci A, Reese TS, Petersen J, Tao-Cheng JH. (2000) A novel particulate form of Ca2+/calmodulin-dependent [correction of Ca2+/CaMKII-dependent] protein kinase II in neurons. J Neurosci. 20, 3076-3084.

Elgersma Y, Fedorov NB, Ikonen S, Choi ES, Elgersma M, Carvalho OM, Giese KP, Silva AJ. (2002) Inhibitory autophosphorylation of CaMKII controls PSD association, plasticity, and learning. Neuron. 36, 493-505.

Faddis BT, Hasbani MJ, Goldberg MP. (1997) Calpain activation contributes to dendritic remodeling after brief excitotoxic injury in vitro. J Neurosci. 17, 951-959.

Fagg, G.E. and Matus, A. (1984) Selective association of N-methyl aspartate and quisqualate types of L-glutamate receptor with brain postsynaptic densities. Proc. Natl. Acad. Sci. USA. 81, 6876-6880.

Fukunaga, K., Goto, S. and Miyamoto, E. (1988) Immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase Ⅱ in rat brain and various tissues. J. Neurochem. 51, 1070-1078.

Furukawa K, Fu W, Li Y, Witke W, Kwiatkowski DJ, Mattson MP. (1997) The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons. J Neurosci. 17, 8178-8186.

Geinisman Y, deToledo-Morrell L, Morrell F. (1991) Induction of long-term potentiation is associated with an increase in the number of axospinous synapses with segmented postsynaptic densities. Brain Res. 566, 77-88.

Geinisman, Y., Morrell, F. and de Toledo-Morrell, L. (1987) Synapses on dendritic shafts exhibit a perforated postsynaptic density. Brain Res. 422, 352-356.

Goelet P, Castellucci VF, Schacher S, Kandel ER. (1986) The long and the short of long-term memory--a molecular framework. Nature. 6, 419-422.

GRAY EG. (1959) Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study. J Anat. 93, 420-433.

Halpain S, Hipolito A, Saffer L. (1998) Regulation of F-actin stability in dendritic spines by glutamate receptors and calcineurin. J Neurosci. 18, 9835-9844.

Hata Y, Butz S, Sudhof TC. (1996) CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins. J Neurosci. 16, 2488-2494.

Hayashi Y, Ishida A, Katagiri H, Mishina M, Fujisawa H, Manabe T, Takahashi T. (1997) Calcium- and calmodulin-dependent phosphorylation of AMPA type glutamate receptor subunits by endogenous protein kinases in the post-synaptic density. Brain Res Mol Brain Res. 46, 338-342.

Heukeshoven J and Dernick R. (1985)Simplified method for silverstaining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis. 6, 103-112.

Hunt CA, Schenker LJ, Kennedy MB. (1996) PSD-95 is associated with the postsynaptic density and not with the presynaptic membrane at forebrain synapses. J Neurosci. 16, 1380-1388.

Husi, H., Ward, M. A., Choudhary, J. S., Blackstock, W. P., and Grant, S. G. (2000) Proteomic analysis of NMDA receptor adhesion protein signaling complexes. Nat Neurosci. 3, 661-669.

Hsueh YP, Kim E, Sheng M. (1997) Disulfide-linked head-to-head multimerization in the mechanism of ion channel clustering by PSD-95. Neuron. 18, 803-814.

Ichtchenko K, Hata Y, Nguyen T, Ullrich B, Missler M, Moomaw C, Sudhof TC. (1995) Neuroligin 1: a splice site-specific ligand for beta-neurexins. Cell. 81, 435-443.

Irie M, Hata Y, Takeuchi M, Ichtchenko K, Toyoda A, Hirao K, Takai Y, Rosahl TW, Sudhof TC. (1997) Binding of neuroligins to PSD-95. Science. 277, 1511-1515.

Jaffrey SR, Snowman AM, Eliasson MJ, Cohen NA, Snyder SH. (1998) CAPON: a protein associated with neuronal nitric oxide synthase that regulates its interactions with PSD95. Neuron. 20, 115-124.

Jordan BA, Fernholz BD, Boussac M, Xu C, Grigorean G, Ziff EB, Neubert TA. (2004) Identification and verification of novel rodent postsynaptic density proteins. Mol Cell Proteomics. M400045-MCP200.

Kaech, S., Fischer, M., Doll, T., and Matus, A. (1997) Isoform specificity in the relationship of actin to dendritic spines. J Neurosci. 17, 9565-9572.

Kelly PT, Cotman CW. (1978) Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide. J Cell Biol. 79, 173-183.

Kelly, P.T., McGuinness, T.L. and Greengard, P. (1984) Evidence that the major postsynaptic density protein is a component of a Ca2+/calmodulin-dependent protein kinase. Proc. Natl. Acad. Sci. USA. 81, 945-949.

Kennedy M. B. (1993) The postsynaptic density. Curr. Opin. Neurobiol. 3, 732–737.

Kennedy, M.B. (1997) The postsynaptic density at glutamatergic synapses. TINS. 20, 264-268.

Kennedy, M.B. (1998) Signal transduction molecules at the glutamatergic postsynaptic membrane. Brain Res Brain Res Rev. 26, 243-57. Review.

Kennedy, M. B. (2000) Signal-processing machines at the postsynaptic density. Science. 290, 750–754

Kennedy, M.B., Bennett, M.K. and Erondu, N.E. (1983) Biochemical and immunochemical evidence that the “major postsynaptic density protein” is a calmodulin-dependent protein kinase. Proc. Natl. Acad. Sci. USA. 80, 7357-7361.

Kim E, Cho KO, Rothschild A, Sheng M. (1996) Heteromultimerization and NMDA receptor-clustering activity of Chapsyn-110, a member of the PSD-95 family of proteins. Neuron. 17, 103-113.

Kim E, Niethammer M, Rothschild A, Jan YN, Sheng M. (1995) Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature. 378, 85-88.

Kim, J. H., Liao, D., Lau, L. F., and Huganir, R. L. (1998) SynGAP: a synaptic RasGAP that associates with the PSD95/SAP90 protein family. Neuron. 20, 683-691.

Kim TW, Wu K, Xu JL, Black IB. (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, 11642-33644.

Kornau HC, Schenker LT, Kennedy MB, Seeburg PH. (1995) Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science. 269, 1737-1740.

Laemmli UK. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680-685.

Landis DM, Reese TS. (1974) Differences in membrane structure between excitatory and inhibitory synapses in the cerebellar cortex. J Comp Neurol. 1, 93-125.

Landis, D.M.D., Weinstein, L.A. and Reese, T.S. (1987) Substructure in the postsynaptic density of purkinje cell dendritic spines revealed by rapid freezing and etching. Synapse. 1, 552-558.

Lei S, Czerwinska E, Czerwinski W, Walsh MP, MacDonald JF. (2001) Regulation of NMDA receptor activity by F-actin and myosin light chain kinase. J Neurosci. 21, 8464-8472.

Lidov HG, Byers TJ, Watkins SC, Kunkel LM. (1990) Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons. Nature. 348, 725-728.

Li KW, Hornshaw MP, Van Der Schors RC, Watson R, Tate S, Casetta B, Jimenez CR, Gouwenberg Y, Gundelfinger ED, Smalla KH, Smit AB. (2004) Proteomics analysis of rat brain postsynaptic density. Implications of the diverse protein functional groups for the integration of synaptic physiology. J Biol Chem. 279, 987-1002.

Lujan, R., Nusser, Z., Roberts, J. D., Shigemoto, R., and Somogyi, P. (1996) Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus. Eur J Neurosci. 8, 1488-1500.

Luscher C, Nicoll RA, Malenka RC, Muller D. (2000) Synaptic plasticity and dynamic modulation of the postsynaptic membrane. Nat Neurosci. 3, 545-550.

Matus A, Ackermann M, Pehling G. (1981) Regularity and differentiation within the structure of brain postsynaptic densities. J Neurocytol. 10, 889-896.

Matus A, Pehling G, Ackermann M, Maeder J. (1980) Brain postsynaptic densities: the relationship to glial and neuronal filaments. J Cell Biol. 87, 346-359.

Matus AI, Taff-Jones DH. (1978) Morphology and molecular composition of isolated postsynaptic junctional structures. Proc R Soc Lond B Biol Sci. 203, 135-151.

Matus AI, Walters BB. (1975) Ultrastructure of the synaptic junctional lattice isolated from mammalian brain. J Neurocytol. 4, 369-375.

Monahan JB, Michel J. (1987) Identification and characterization of an N-methyl-D-aspartate-specific L-[3H]glutamate recognition site in synaptic plasma membranes. J Neurochem. 48, 1699-1708.

Moon, I.S., Apperson, M.L. & Kennedy, M.B. (1994) The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B. Proc. Natl. Acad. Sci. USA. 91, 3954–3958.

Muller BM, Kistner U, Kindler S, Chung WJ, Kuhlendahl S, Fenster SD, Lau LF, Veh RW, Huganir RL, Gundelfinger ED, Garner CC. (1996) SAP102, a novel postsynaptic protein that interacts with NMDA receptor complexes in vivo. Neuron. 17, 255-265.

Mummery R, Sessay A, Lai FA, Beesley PW. (1996) Beta-dystroglycan: subcellular localisation in rat brain and detection of a novel immunologically related, postsynaptic density-enriched protein. J Neurochem. 66, 2455-2459.

Nguyen T, Sudhof TC. (1997) Binding properties of neuroligin 1 and neurexin 1beta reveal function as heterophilic cell adhesion molecules. J Biol Chem. 272, 26032-26039.

Niethammer, M., Kim, E., and Sheng, M. (1996) Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD95 family of membraneassociated guanylate kinases. J Neurosci. 16, 2157-2163.

Ouimet, C.C., McGuinness, T.L. and Greengard P. (1984) Immunocytochemical localization of calcium/calmodulin-dependent protein kinase Ⅱ in rat brain. Proc. Natl. Acad. Sci. USA. 81, 5604-5608.

Peng J, Kim MJ, Cheng D, Duong DM, Gygi SP, Sheng M. (2004) Semiquantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry. J Biol Chem. 279, 21003-21011.

Sabatini, B. L., and Svoboda, K. (2000) Analysis of calcium channels in single spines using optical fluctuation analysis. Nature 408, 589-593.

Satoh, K., Takeuchi, M., Oda, Y., DeguchiTawarada, M., Sakamoto, Y., Matsubara, K., Nagasu, T., and Takai, Y. (2002) Identification of activityregulated proteins in the postsynaptic density fraction. Genes Cells. 7, 187-197.

Sheng M. (2001) Molecular organization of the postsynaptic specialization. Proc. Natl. Acad. Sci. U.S.A. 98, 7058–7061.

Sheng M. and Sala C. (2001) PDZ domains and the organization of supramolecular complexes. Annu. Rev. Neurosci. 24, 1–29.

Shevchenko A, Wilm M, Vorm O, Mann M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 68, 850-858.

Srivastava S, Osten P, Vilim FS, Khatri L, Inman G, States B, Daly C, DeSouza S, Abagyan R, Valtschanoff JG, Weinberg RJ, Ziff EB. (1998) Novel anchorage of GluR2/3 to the postsynaptic density by the AMPA receptor-binding protein ABP. Neuron. 21, 581-591.

Strader CD, Pickel VM, Joh TH, Strohsacker MW, Shorr RG, Lefkowitz RJ, Caron MG. (1983) Antibodies to the beta-adrenergic receptor: attenuation of catecholamine-sensitive adenylate cyclase and demonstration of postsynaptic receptor localization in brain. Proc Natl Acad Sci U S A. 80, 1840-1844.

Strack S, Colbran RJ. (1998) Autophosphorylation-dependent targeting of calcium/ calmodulin-dependent protein kinase II by the NR2B subunit of the N-methyl- D-aspartate receptor. J Biol Chem. 273, 20689-20692.

Suzuki, T., Okumura-Noji, K., Tanaka, R., Ogura, A., Kyoko, N., Kudo, Y. and Tada, T. (1993) Characterization of protein kinase C activities in postsynaptic density fractions prepared from cerebral cortex, hippocampus, and cerebellum. Brain Res. 619, 69-75.

Togashi, H., Abe, K., Mizoguchi, A., Takaoka, K., Chisaka, O., and Takeichi, M. (2002) Cadherin regulates dendritic spine morphogenesis. Neuron. 35, 77-89.

Toni N, Buchs PA, Nikonenko I, Povilaitite P, Parisi L, Muller D. (2001) Remodeling of synaptic membranes after induction of long-term potentiation. J Neurosci. 21, 6245-6251.

Vinade L, Petersen JD, Do K, Dosemeci A, Reese TS. (2001) Activation of calpain may alter the postsynaptic density structure and modulate anchoring of NMDA receptors. Synapse. 40, 302-309.

Walikonis, R. S., Jensen, O. N., Mann, M., Provance, D. W., Jr., Mercer, J. A., and Kennedy, M. B. (2000) Identification of proteins in the postsynaptic density fraction by mass spectrometry. J Neurosci. 20, 4069-4080.

Walsh, M. J., and Kuruc, N. (1992) The postsynaptic density: constituent and associated proteins characterized by electrophoresis, immunoblotting, and peptide sequencing. J Neurochem. 59, 667-678.

Walters BB, Matus AI. (1975) Tubulin in postynaptic junctional lattice. Nature. 257, 496-498.

Westenbroek RE, Ahlijanian MK, Catterall WA. (1990) Clustering of L-type Ca2+ channels at the base of major dendrites in hippocampal pyramidal neurons. Nature. 347, 281-284.

Wolf M, Burgess S, Misra UK, Sahyoun N. (1986) Postsynaptic densities contain a subtype of protein kinase C. Biochem Biophys Res Commun. 140, 691-698.

Wu K, Carlin R, Sachs L, Siekevitz P. (1985) Existence of a Ca2+-dependent K+ channel in synaptic membrane and postsynaptic density fractions isolated from canine cerebral cortex and cerebellum, as determined by apamin binding. Brain Res. 360, 183-194.

Wyszynski M, Lin J, Rao A, Nigh E, Beggs AH, Craig AM, Sheng M. (1997) Competitive binding of alpha-actinin and calmodulin to the NMDA receptor. Nature. 385, 439-442.

Xia J, Zhang X, Staudinger J, Huganir RL. (1999) Clustering of AMPA receptors by the synaptic PDZ domain-containing protein PICK1. Neuron. 22, 179-187.

Yamauchi T. (2002) Molecular constituents and phosphorylation dependent regulation of the post-synaptic density. Mass Spectrometry Rev. 21, 266–286.

Yoshimura Y, Sogawa Y, Yamauchi T. (1999) Protein phosphatase 1 is involved in the dissociation of Ca2+/calmodulin-dependent protein kinase II from postsynaptic densities. FEBS Lett. 446, 239-242.

Yoshimura Y, Yamauchi Y, Shinkawa T, Taoka M, Donai H, Takahashi N, Isobe T, Yamauchi T. (2004) Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography-tandem mass spectrometry. J Neurochem. 88, 759-768.

Zagon IS, Higbee R, Riederer BM, Goodman SR. (1986) Spectrin subtypes in mammalian brain: an immunoelectron microscopic study. J Neurosci. 6, 2977-2986.

Ziff, E.B. (1997) Enlightening the postsynaptic density. Neuron. 19, 1163-1174.

賴森林 (1998) 後突觸質密區結構之研究-蛋白質間雙硫鍵的形成與影響 國立清華大學生命科學所碩士論文