The binding of neurotensin to synaptic membranes from rat brain was studied at 24°with the use of [3HJ neurotensin. The binding was found to be highly specific, saturable, and reversible. Values for KD of 2 nM and 0.9 nM were derived from equilibrium and kinetic experiments, respectively. Virtually no degradation of neurotensin was observed in the incubation medium after exposure to synaptic membranes under the conditions of the binding studies. Competitive inhibition of [3Hlneurotensin binding by partial sequences of neurotensin revealed that the addition of the residue arginine-8 to the neurotensin(9-13)pentapeptide increases about 500-fold the relative binding potency, whereas the remaining portion of the NH2-terminal region is mainly responsible for full pharmacological potency; the COOH-terminal leucyl residue is essential for bin ing.

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Bordetella pertussis and Bacillus anthracis, two taxonomically distinct bacteria, secrete adenylate cyclase toxins that are activated by the eukaryotic protein calmodulin. The two enzymes contain a well-conserved stretch of 24 amino acid residues [Escuyer et al. (1988) Gene 71, 293-298]. Antibodies have been obtained against two synthetic heptadecapeptides, covering part of the conserved sequences. The anti-peptide antibodies specifically reacted in Western blots with the rat brain adenylate cyclase as well as with the two bacterial enzymes. Anti-rat brain adenylate cyclase serum contained antibodies that were retained by the immobilized peptides, and the affinity-purified antibodies yielded the same recognition pattern of the eukaryotic enzyme as did the unfractionated serum. These results indicate that the eukaryotic adenylate cyclase contains an epitope closely related to that specified by the conserved bacterial sequence. The synthetic peptides and the bacterial adenylate cyclases appeared to compete for ATP (KD of the ATP-peptide complex ca. 0.2 mM), suggesting that the conserved sequence may be part of the substrate binding site in these two enzymes.

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