L-Tryptophan benzyl ester hydrochloride

Ghrelin increases electrically evoked, neuronally mediated contractions of rat isolated forestomach, a prokinetic-like activity. Since the nerve type sensitive to ghrelin is unclear, we examined the activity of ghrelin in the presence of antagonists at receptors for the main gastric motor neurotransmitters. Electrical field stimulation (EFS; 5 Hz, 0.5 ms, +/-50 V, 30 s every 3 min) of circular muscle preparations evoked tetrodotoxin 1 microM-sensitive responses, consisting of a small initial contraction followed by a further contraction or more usually, by muscle relaxation. Termination of EFS evoked a large rapidly developing after-contraction. Atropine 1 microM prevented contractions during EFS, increased any relaxations and prolonged the after-contractions. Nomega-Nitro-L-arginine-methyl-ester-hydrochloride (L-NAME) 0.3 mM prevented relaxations during EFS, changing the triphasic response into a monophasic contraction. The tachykinin NK1 and tachykinin NK2 receptor antagonists N-acetyl-L-tryptophan-3,5-bistrifluoromethyl-benzyl-ester (L-732,138 1 microM) and Cyclo[Gln-Trp-Phe-Gly-Leu-CH2N(CH3)-Leu] (MDL-29,913 1 microM) each reduced EFS-evoked relaxations; the latter also reduced the after-contractions. The tachykinin NK3 receptor antagonist (-)-(S)-N-(alpha-ethylbenzyl)-3-(carboxymethoxy)-2-phenylquinoline-4-carboxamide (SB-235375, 0.1 microM) had no effects. The combination of tachykinin NK(1,2,3) receptor antagonists reduced the after-contractions and abolished relaxations during EFS, replacing this with a contraction. In control tissues, ghrelin 1 microM increased EFS-induced contractions and tended to reduce any relaxations. In the presence of atropine 1 microM, L-NAME 0.3 mM or the tachykinin receptor antagonists (as above), ghrelin 1 microM increased any EFS-induced contraction but in the presence of atropine had no effects on EFS-evoked relaxations. We conclude that EFS evokes responses mediated by acetylcholine, nitric oxide and tachykinins. Ghrelin facilitates both cholinergic and tachykininergic excitatory pathways, consistent with activity within the enteric nervous system and possibly the vagus nerve.

The reactions of tryptophanase, tyrosine phenol-lyase, and tryptophan synthase with a new class of substrates, the O-acyl-L-serines, have been examined. A method for preparation of O-benzoyl-L-serine in high yield from tert.-butyloxycarbonyl (tBoc)-L-serine has been developed. Reaction of the cesium salt of tBoc-L-serine with benzyl bromide in dimethylformamide gives tBoc-L-serine benzyl ester in excellent yield. Acylation with benzoyl chloride and triethylamine in acetonitrile followed by hydrogenolysis with 10% palladium on carbon in trifluoroacetic acid gives O-benzoyl-L-serine, isolated as the hydrochloride salt. O-Benzoyl-L-serine is a good substrate for beta-elimination or beta-substitution reactions catalyzed by both tryptophanase and tyrosine phenol-lyase, with Vmax values 5- to 6-fold those of the physiological substrates and comparable to that of S-(o-nitrophenyl)-L-cysteine. Unexpectedly, O-acetyl-L-serine is a very poor substrate for these enzymes, with Vmax values about 5% of those of the physiological substrates. Both O-acyl-L-serines are poor substrates for tryptophan synthase, measured either by the synthesis of 5-fluoro-L-tryptophan from 5-fluoroindole and L-serine catalyzed by the intact alpha 2 beta 2 subunit or by the beta-elimination reaction catalyzed by the isolated beta 2 subunit. With all three enzymes, the elimination of benzoate appears to be irreversible. These results suggest that the binding energy from the aromatic ring of O-benzoyl-L-serine is used to lower the transition-state barrier for the elimination reactions catalyzed by tryptophanase and tyrosine phenol-lyase. Our findings support the suggestion (M. N. Kazarinoff and E. E. Snell (1980) J. Biol. Chem. 255, 6228-6233) that tryptophanase undergoes a conformational change during catalysis and suggest that tyrosine phenol-lyase also may undergo a conformational change during catalysis.