Affinity purification of a rat‐brain junctional protein, connexin 43

Abstract

Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affnity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A -Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDSjPAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5 -17, 55-56 and 314-322 of rat heart CX43. The NH,-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to al connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product.

Phosphorylated Forms of Connexin43 Predominate in Rat Brain: Demonstration by Rapid Inactivation of Brain Metabolism

“…In a n earlier study involving microdissection of discrete brain regions, we reported that both phosphorylated and unphosphorylated forms of Cx43 were present in all areas examined and that the ratio of these forms was fairly uniform among regions . In more rapidly excised CNS tissues, we have recently found considerable variability in the relative quantities of the 43-and 4 1-kDa forms (authors' unpublished data), an observation consistent with similar findings in the literature (Dupont et al, 1991;Kadle et al, 1991;Miragall et al, 1992). These disparate results suggested that Cx43 in brain may be subject t o postmortem metabolism, especially dephosphorylation.…”

Identification of synaptic plasma membrane proteins co‐precipitated with fibrillar β‐amyloid peptide

“…1995), opioid‐binding cell adhesion molecule (Miyata et al . 2003), gap junction α‐1 protein (Dupont et al . 1991), contactin 2 precursor (Furley et al .…”

“…(3) Neuronal cell adhesion molecules: contactin 1 (Hosoya et al 1995), contactin-associated protein 1 precursor (Peles et al 1997), flotillin-1 (Lang et al 1998), neurotrimin (Struyk et al 1995), opioid-binding cell adhesion molecule (Miyata et al 2003), gap junction a-1 protein (Dupont et al 1991), contactin 2 precursor (Furley et al 1990) and ELKS-Rab6-interacting protein-Cytomatrix at the active zone associated structural protein (ERC) protein 2 (Ohtsuka et al 2002). It has been suggested that cell adhesion molecules such as integrins bind to Ab and play an important role in AD (for review, see ).…”

Distribution pattern of connexin 43, a gap junctional protein, during the differentiation of mouse heart myocytes

“…These immunogold labeling experiments were carried out in vitro, on isolated rat heart gap junctions [15], and in situ, on sections of adult rat myocardium [ 151 and cultured mouse astrocytes [20]. These same antibodies were also used to immunopurify CX 43 from rat brain [14]. We have also shown by immunoblotting that these antibodies recognize only CX 43 in total extracts prepared from myocardium of mouse embryos and newborns as well as adult mouse [19].…”

“…In later stages, the accumulation of junctions in the intercalated discs makes it difficult to distinguish the immunoreactive sites individually. Comparison of observations (Table 1) made at different embryonic development stages (11,14,16 days post coitum and newborn) shows that the abun- 1 wpp, a and b) and two-weekold (2 wpp, c and d) mouse myocardium. Double immunofluorescence experiments.…”