Regional and laminar specificity of kainate-stimulated cobalt uptake in the rat hippocampal formation.

Kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors were initially found to be impermeable to calcium, but cloning and expression studies subsequently revealed that certain kainate and AMPA receptor subunit combinations display substantial divalent cation permeability. The regional and cellular distribution of calcium-permeable kainate/AMPA receptors has not been extensively investigated, however. In this study, we used a histochemical technique, the kainate-stimulated cobalt uptake assay, to localize calcium-permeable kainate responsive receptors in the rat hippocampal formation. In the presence of cobalt, kainate produced a highly localized, dark granular precipitate in dendrites, perikarya, or both, of hippocampal neurons. Kainate-stimulated cobalt uptake was time- and concentration-dependent (1 microM-1 mM) and was blocked by the glutamate receptor antagonist, kynurenate. The specific cellular location of cobalt labeling varied regionally within the hippocampal formation, switching from selective labeling of only apical dendrites in Ammon's horn subregion la (CA1a) to a diffuse band of punctate labeling in CA1c to labeling of cell bodies along with their proximal dendrites in CA3. Furthermore, increasing the kainate concentration not only enhanced the intensity of cobalt labeling, but also changed the pattern of cellular staining from exclusively dendritic labeling to extensive staining of both cell bodies and dendrites in CA1a pyramidal neurons. High kainate concentrations or prolonged incubation times produced a diffuse cellular labeling suggestive of neurotoxicity. These data are consistent with evidence that specific kainate and AMPA receptor subunit combinations are targeted to specific synapses in hippocampal pyramidal neurons.

Characterization of antisera to glutamate and aspartate.

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.

Thiol and aspartyl proteolytic activities in secretory vesicles of bovine pituitary.

Thiol and aspartyl proteolytic activities in isolated secretory vesicles of neural (NL) and intermediate (IL) lobes of bovine pituitary were characterized with heterologous enkephalin and tachykinin precursor substrates, 35S-(Met)-preproenkephalin and 35S-(Met)-beta-preprotachykinin. IL and NL secretory vesicles contained thiol-dependent proteolytic activity that cleaved the enkephalin precursor with a pH optimum of 4.5; this activity resembled a novel "prohormone thiol protease' previously purified and characterized from adrenal medulla chromaffin granules. IL and NL vesicles also demonstrated aspartyl proteolytic activity with acidic pH optimum, as shown by pepstatin A inhibition of tachykinin and enkephalin precursor cleaving activity. This activity may be related to a previously characterized chromaffin granule aspartyl protease (CGAP) related to cathepsin D (2), as indicated by the presence of immunoreactive CGAP in NL secretory vesicles by anti-CGAP immunoblots. These results show that pituitary secretory vesicles, like chromaffin granules, may contain similar thiol-dependent and aspartyl proteolytic activities.

Two-step purification of tryptophan-accepting tRNA from Bacillus stearothermophilus.

Tryptophan-accepting tRNA has been purified essentially to homogeneity from Bacillus stearothermophilus. Crude tRNA was chromatographed first on benzoylated DEAE-cellulose and then on Sepharose 4B with reverse salt gradient elution. The product has tryptophan acceptor activity in excess of 2 nmol [14C]tryptophan per A260 unit. This procedure avoids costly aminoacylation, a step characteristic of other one- and two-step procedures. In two separate purifications 7 and 11 mg of tRNAtrp were prepared from 750 and 1000 g of frozen cells, respectively. This yield compares favorably with that from other procedures. The pure tRNAtrp has been crystallized under several different conditions.