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

Abstract

Synaptic membrane (SPM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brain, either fresh or frozen and isolated from either fresh or frozen tissue, were found to contain L-[3H]glutamate binding sites. It was found that there was a concentration of L-glutamate binding sites in CTX-PSD and CL-PSD over the respective membrane fractions, and the Bmax value of CL-PSD (92.0 pmol/mg protein) was about three times that of CTX-PSD (28.9 pmol/mg). The results, together with those of others, suggest that the thin CL-PSD are probably derived from the excitatory synapses in the molecular layer. The ion dependency of L-glutamate binding to canine CTX-SPM fraction was found to be similar to that reported for a rat brain SPM fraction: (a) Cl- increased the number of L-glutamate binding sites and the effect was enhanced by Ca2+; Ca2+ alone had no significant effect; (b) the Cl-/Ca2+-sensitive binding sites were abolished by 2-amino-4-phosphonobutyrate (APB) or freezing and thawing; (c) the effect of Na+ ion was biphasic; low concentration of Na+ (less than 5 mM) decreased Cl-/Ca2+-dependent L-glutamate binding sites, whereas at higher concentrations of Na+ the binding of glutamate was found to increase either in the presence or absence of Ca2+ and Cl-. In addition, the K+ ion (50 mM) was found to decrease the Na+-independent and Cl-/Ca2+-independent binding of L-glutamate to fresh CTX-SPM by 18%, but it decreased the Na+-dependent and Cl-/Ca2+-independent L-glutamate binding by 93%; in the presence of Cl-/Ca2+, the K+ ion decreased the Na+-dependent binding by 78%. Freezing and thawing of CTX-SPM resulted in a 50% loss of the Na+-dependent L-glutamate binding sites assayed in the absence of Ca2+ and Cl-. The CL-SPM fraction showed similar ion dependency of L-glutamate binding except for the absence of Na+-dependent glutamate binding sites. The CTX-PSD fraction contained neither Na+-dependent nor APB (or Cl-/Ca2+)-sensitive L-glutamate binding sites and its L-glutamate binding was unaffected by freezing and thawing, in agreement with the reported findings using rat brain PSD preparation. L-Glutamate binding to CTX-SPM or CTX-PSD fraction was not affected by pretreatment with 10 mM L-glutamate, nor by simultaneous incubations with calmodulin.(ABSTRACT TRUNCATED AT 400 WORDS)

Proteomics Analysis of Rat Brain Postsynaptic Density

“…For two decades the 2ϫ Triton extraction method to yield the PSD fraction from rat brain as developed by Carlin and coworkers (19,24) has been the standard protocol for the purification of PSD proteins and has been used in previous studies as the source for the characterization of novel PSD protein components. In the present study two independent proteome approaches were used, namely two-dimensional gel electrophoresis in conjunction with MALDI tandem mass spectrometry, and the ICAT peptide derivatization technique with nano-liquid chromatography coupled online to electrospray tandem mass spectrometry.…”

“…Purification of the PSD-The PSD fraction was isolated either as described (23) based on the original method of Carlin et al (19), or based largely on a variant of the original method (24). In brief, in the original method, forebrains of 30 days old untreated rats were homogenized in homogenization buffer (5 mM Hepes, pH 7.4; 320 mM sucrose) containing a protease inhibitor mixture (Roche Applied Science).…”

Early Presynaptic and Late Postsynaptic Components Contribute Independently to Brain-Derived Neurotrophic Factor-Induced Synaptic Plasticity

“…Adult wild-type and Rab3A knock-out (two animals each) mice were used to prepare synaptosomes according to the first part of the protocol for postsynaptic density preparations (Wu et al, 1986). Fresh cortices were gently homogenized (five strokes at 800 rpm) with a Teflon/glass homogenizer in 5 vol (w/v) in buffer containing protease inhibitors (0.32 M sucrose, 0.5 mM CaCl 2 , 1 mM MgCl 2 , and 1 mM NaHCO 3 ).…”

On the identity of the major postsynaptic density protein.

“…However, CKII has been localized to the cytosol immunohistochemically (22). In direct contrast, the mPSDp, as part of PSD, is insoluble in high concentrations of salt, various detergents, and the denaturant guanidine hydrochloride (23)(24)(25)(26)(27)(28). Moreover, the mPSDp is relatively nonantigenic (K.W.…”

“…Cerebral cortices from 70 adult rat brains were used for preparation of the mPSDp. Highly purified PSDs were obtained as described (27). The mPSDp was eluted from an SDS gel using 10 tTo whom reprint requests should be addressed.…”