Harry Martin

Subcellular Localisation of 14‐3‐3 Isoforms in Rat Brain Using Specific Antibodies

Abstract: The 14‐3‐3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14‐3‐3 proteins are cytosolic, a small but significant proportion of 14‐3‐3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform‐specific antisera we find that the ε, η, γ, β, and ζ isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the η, ε, and γ isoforms but not the β and ζ isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the ε and γ isoforms were present and the β and ζ isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.

Antibodies against the major brain isofbrms of 14-3-3 protein: An antibody specific for the N-acetylated ammo-terminus of a protein

14‐3‐3 proteins are apparently ubiquitous eukaryotic proteins that comprise a large number of isoforms. They have been implicated in the regulation of a wide range of biological processes [reviewed in Aitken et al. (1992) Trends Biochem. Sci. 17,498‐501]. We have raised specific antibodies against each mammalian brain isoform of 14‐3‐3 employing peptides synthesised from the amino‐tenninal regions. The peptides were, like the proteins from mammalian brain, N‐acetylated. The antiserum specific for the epsilon isoform did not recognise the recombinant form of this protein (lacking the N‐acetyl co‐translational modification) expressed in E. coli until it was chemically acetylated.14-3-3 proteins are apparently ubiquitous eukaryotic proteins that comprise a large number of isoforms. They have been implicated in the regulation of a wide range of biological processes [reviewed in Aitken et al. (1992) Trends Biochem. Sci. 17, 498-501]. We have raised specific antibodies against each mammalian brain isoform of 14-3-3 employing peptides synthesised from the amino-terminal regions. The peptides were, like the proteins from mammalian brain, N-acetylated. The antiserum specific for the epsilon isoform did not recognise the recombinant form of this protein (lacking the N-acetyl co-translational modification) expressed in E. coli until it was chemically acetylated.

Research lettersAntibodies against the major brain isofbrms of 14-3-3 protein: An antibody specific for the N-acetylated ammo-terminus of a protein

14‐3‐3 proteins are apparently ubiquitous eukaryotic proteins that comprise a large number of isoforms. They have been implicated in the regulation of a wide range of biological processes [reviewed in Aitken et al. (1992) Trends Biochem. Sci. 17,498‐501]. We have raised specific antibodies against each mammalian brain isoform of 14‐3‐3 employing peptides synthesised from the amino‐tenninal regions. The peptides were, like the proteins from mammalian brain, N‐acetylated. The antiserum specific for the epsilon isoform did not recognise the recombinant form of this protein (lacking the N‐acetyl co‐translational modification) expressed in E. coli until it was chemically acetylated.14-3-3 proteins are apparently ubiquitous eukaryotic proteins that comprise a large number of isoforms. They have been implicated in the regulation of a wide range of biological processes [reviewed in Aitken et al. (1992) Trends Biochem. Sci. 17, 498-501]. We have raised specific antibodies against each mammalian brain isoform of 14-3-3 employing peptides synthesised from the amino-terminal regions. The peptides were, like the proteins from mammalian brain, N-acetylated. The antiserum specific for the epsilon isoform did not recognise the recombinant form of this protein (lacking the N-acetyl co-translational modification) expressed in E. coli until it was chemically acetylated.

Identification of a thioredoxin — related protein associated with plasma membranes

A low molecular weight membrane associated sulphydryl protein was detected on a wide range of nucleated cells when [14C]-iodoacetamide was used as a probe. This protein was extracted from THP-1 monocytes, purified to homogeneity and its isoelectric point, Mr and N-terminal amino acid sequence determined. These were shown to be almost identical to the corresponding values for both human thioredoxin and a Tac interleukin-2 receptor activator, indicating that the protein may be a member of this family and function as an essential growth factor.

PURIFICATION OF 14-3-3 PROTEIN AND ANALYSIS OF ISOFORMS IN CHICKEN BRAIN

14-3-3 proteins are apparently ubiquitous eukaryotic proteins that comprise a large number of isoforms. We have used specific antibodies raised against each mammalian isoform to probe for 14-3-3 isoforms in adult hen brains. The results suggest that there is a remarkable degree of similarity in primary structure (at least in the regions containing the epitopes). Reverse-phase HPLC of the purified avian 14-3-3 proteins indicates a high overall degree of similarity in sequence and levels of expression of each isoform that are remarkably similar to their mammalian counterparts.