Site-directed Antisera Research Articles

Identification and cellular localisation of NPW 1 (GPR7) receptors for the novel neuropeptide W-23 by [ 125I]-NPW radioligand binding and immunocytochemistry

Using a novel radioligand, we have identified high-affinity binding sites ( K D=0.44±0.13 nM) for the recently discovered peptide, neuropeptide W (NPW), in rat brain. Binding density was highest in the amygdala ( B max=149.9±13.8 fmol/mg protein), thalamic, and hypothalamic nuclei. A similar distribution was observed in mouse brain. We have confirmed the identity of these binding sites as the G-protein-coupled receptor, NPW 1 (previously designated orphan receptor GPR7), using site-directed antisera that revealed receptors were expressed by neuronal cell bodies and processes. Additionally, we have demonstrated the presence of NPW-like immunoreactivity in neuronal cell bodies in areas projecting to the amygdala, such as the dorsal raphe nucleus and ventral tegmental area, providing evidence for an emerging new transmitter system.

Characterization of site-directed antisera against endothelin-converting enzymes.

Site-directed antisera have been developed against the two endothelin-converting enzyme-1 (ECE-1) isoforms cloned to date in humans, ECE-1 alpha and ECE-1 beta. Antisera were raised in rabbits against synthetic peptides corresponding to the deduced amino acid sequences that differ between ECE-1 alpha and ECE-1 beta. Antisera were highly selective for their corresponding antigen (titer 1 x 10(4)) and did not detect ET-1 or big ET-1. Furthermore, no detectable crossreactivity was observed between the different site-specific antisera and the other immunizing peptides, suggesting that the antisera would be selective for ECE-1 alpha and ECE-1 beta. Standard displacement curves have been developed to determine the levels of immunoreactive ECE-1 alpha and ECE-1 beta in solubilized microsomal fractions of human tissue. In conclusion, we have described the first production and characterization of site-directed antisera raised against ECE-1 alpha and ECE-1 beta capable of discriminating between the two ECE-1 isoforms. Furthermore, using these antisera, we have found that ECE-1 alpha appears to be the predominant isoform in human tissue.

Primary sequence and immunological characterization of beta-subunit of high conductance Ca(2+)-activated K+ channel from smooth muscle.

The charybdotoxin receptor, purified from bovine tracheal smooth muscle, consists of two subunits (alpha and beta) and, when reconstituted into planar lipid bilayers, forms functional high conductance Ca(2+)-activated K+ channels. Amino acid sequence, obtained from proteolytic fragments of the beta-subunit, was used to design oligonucleotide probes with which cDNAs encoding this protein were isolated. The cDNAs encode a protein of 191 amino acids that contains two hydrophobic (putative transmembrane) domains and bears little sequence homology to subunits of other known ion channels. Site-directed antisera, raised against putative extracellular epitopes of this protein, specifically immunoprecipitated 125I-labeled Bolton-Hunter beta-subunit as well as [125I]charybdotoxin-cross-linked beta-subunit. Under nondenaturing conditions, however, these anti-beta sera immunoprecipitated a complex consisting of both the alpha- and beta-subunits. The data demonstrate that, in vivo, the high conductance Ca(2+)-activated K+ channel exists as a multimer containing both alpha- and beta-subunits, and this cDNA represents the first beta-subunit of a potassium channel cloned to date. Furthermore, we demonstrate that the cloned protein is the subunit to which charybdotoxin is specifically and covalently incorporated when cross-linked to the channel.

Enhancement of bovine growth hormone activity by antibodies against growth hormone peptides

The biological activity of growth hormones can be enhanced by complexing with monoclonal antibodies of appropriate specificity. In order to define the regions associated with the phenomenon, site-directed antisera to ovine GH (oGH) were prepared by vaccination of sheep with synthetic peptides. Peptides from six distinct regions of the oGH molecule raised antibodies which recognized the hormone in solid-phase radioimmunoassay; however, only one peptide elicited high-affinity antibody as determined by liquid-phase assay. This peptide, corresponding to amino acid sequence 35-53, resulted in circulating hormone antibody in the majority of vaccinated sheep. Immunoglobulin prepared from the serum of immunized animals produced an enhancement of the somatotrophic activity of exogenously administered GH in dwarf mice as determined by the incorporation of [35S]sulphate into costal cartilage. The identification of an antigenic peptide sequence from oGH/bovine GH which elicits enhancing antisera, raises the possibility of a growth-promotion vaccine.

Chronic opiate receptor activation in vivo alters the level of g-protein subunits in guinea-pig myenteric plexus

Studies with the longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum were undertaken to investigate the relationship between guanine nucleotide-binding proteins (G-proteins) and chronic opioid receptor activation in vivo. Treatment with the narcotic agonist fentanyl, at doses which render the preparation tolerant and dependent, led to an increase of pertussis toxin-catalysed incorporation of ADP-ribose in a protein of approximately 40,000 mol. wt. Quantitative immunoblotting, using site-directed antisera, demonstrated an upregulation of G αi/G αo and, to an even greater degree, of G β. However, the level of G βs was decreased by the same treatment. All alterations observed were abolished by the concomitant presence of the antagonist naloxone. The implications of this differential regulation of G-protein subunits for opiate-induced tolerance and dependence are discussed.

Site of covalent attachment of alpha-scorpion toxin derivatives in domain I of the sodium channel alpha subunit.

Purified and reconstituted sodium channels from rat brain have been photoaffinity labeled with a photoactivable derivative of the alpha-scorpion toxin V from Leiurus quinquestriatus (LqTx). A battery of sequence-specific antibodies has been used to determine which of the peptides produced by chemical and enzymatic cleavage of the photolabeled sodium-channel alpha subunit contain covalently attached LqTx. Nearly all of the covalently attached LqTx is found within homologous domain I. Two site-directed antisera, which recognize residues 317 to 335 and residues 382 to 400, respectively, specifically immunoprecipitate a 14-kDa peptide produced by CNBr digestion to which LqTx is covalently attached. It is proposed that a portion of the receptor site for alpha-scorpion toxins is formed by peptide segment(s) between amino acid residues 335 and 378 which is located in an extracellular loop between transmembrane helices S5 and S6 of homologous domain I of the sodium channel alpha subunit.

Immunological studies on the D-1 and D-2 proteins of photosystem II preparations from the thermophilic cyanobacterium, Synechococcus sp.

Immunoblotting of Synechococcus photosystem II preparations with site-directed antisera against the spinach D-2 protein and an antiserum against a large polypeptide segment of the spinach D-1 protein showed (i) that the 28 and 31 kDa polypeptides of the cyanobacterial photosystem II core complex are equivalent to the D-1 and D-2 proteins of higher plants and algae and (ii) that the β-octylglucoside treatment of the photosystem II preparation diminishes band intensities of the 28 and 31 kDa proteins in Coomassie blue-stained gels mainly by increasing the amounts of two proteins which migrate as a dimer of D 1 or D 2 or its aggregate, and of proteolytic digestion products.