Secondary 15N isotope effects at the N-1 position of 3-acetylpyridine adenine dinucleotide have been determined, by using the internal competition technique, for horse liver alcohol dehydrogenase (LADH) with cyclohexanol as a substrate and yeast formate dehydrogenase (FDH) with formate as a substrate. On the basis of less precise previous measurements of these 15N isotope effects, the nicotinamide ring of NAD has been suggested to adopt a boat conformation with carbonium ion character at C-4 during hydride transfer [Cook, P. F., Oppenheimer, N. J. & Cleland, W. W. (1981) Biochemistry 20, 1817]. If this mechanism were valid, as N-1 becomes pyramidal an 15N isotope effect of up to 2-3% would be observed. In the present study the equilibrium 15N isotope effect for the reaction catalyzed by LADH was measured as 1.0042 +/- 0.0007. The kinetic 15N isotope effect for LADH catalysis was 0.9989 +/- 0.0006 for cyclohexanol oxidation and 0.997 +/- 0.002 for cyclohexanone reduction. The kinetic 15N isotope effect for FDH catalysis was 1.004 +/- 0.001. These values suggest that a significant 15N kinetic isotope effect is not associated with hydride transfer for LADH and FDH. Thus, in contrast with the deformation mechanism previously postulated, the pyridine ring of the nucleotide apparently remains planar during these dehydrogenase reactions.

Gastrin releasing peptide (GRP) is a 27 amino acid peptide hormone which is homologous to the amphibian peptide bombesin. Two series of novel GRP antagonists were developed by C-terminal modification of N-acetyl-GRP-20-27 amide. Peptide derivatives within each series resist enzymatic degradation in serum and exhibit strong affinity for the GRP receptor. The first series of compounds replaces the Leu26-Met27 region of GRP with an alkyl ether N-acetyl-GRP-20-25-NH-[(S)-1-ethoxy-4-methyl-2-pentane], specifically blocked radiolabeled GRP binding with an IC50 of 6 nM. In the second series of antagonists the oxygen of the ether moiety is replaced with a methylene group, resulting in GRP antagonists which are equipotent to native GRP in receptor binding assays (IC50 = 2 nM) and are also resistant to proteolytic degradation in vitro. All of the C-terminally modified peptides tested blocked GRP-stimulated mitogenesis in Swiss 3T3 mouse fibroblasts. Representative compounds also blocked GRP-induced elevation of [Ca2+]i in human SCLC cells, and inhibited GRP-independent release of gastrin in vivo.

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