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Biochemical sensing mechansims in olfaction

By Philip Howard Wood

Abstract

The present work, employing biochemical, biophysical and\ud electrophysiological techniques, attempted to identify specific\ud receptor sites in the vertebrate olfactory system for heterocyclic\ud odorants.\ud An in vitro rat preparation was developed and characterised\ud for use in vapour-phase chemical modification experiments; the EOG\ud responses obtained from this preparation were stable for up to\ud 5 hours after the death of the animal. The signals to various\ud compounds were differentially reduced when brominated odorants were\ud employed as vapour-phase labelling reagents; the responses obtained\ud to these derivatives and to their non-reactive analogues were\ud preferentially diminished.\ud The effect of concanavalin A on ECGs obtained from an in vivo\ud frog preparation was examined. This lectin was found to preferentially\ud inhibit the signals elicited by small, sweaty-smelling carboxylic\ud acids; the responses to most of the non-carboxylic acid odorants\ud tested were not significantly inhibited.\ud The failure to identify specific receptor sites by electrophysiological\ud techniques prompted the performance of odorant binding\ud studies. Examinations of the interaction of [3H] 2-isobutyl-3-\ud methoxypyrazine with 13,000 x g supernatant fractions of sheep\ud olfactory epithelium showed that a component of the homogenate\ud fraction exhibited high affinity saturable binding of this odorant (KD-10-8M). However, the presence of large amounts of non-specific\ud binding, substantially decreased the sensitivity and accuracy of the\ud assay. Non-specific binding was observed with tissue fractions of\ud sheep respiratory epithelium, brain and liver. An investigation of binding specificity showed that other bell pepper odorants competed for the 2-iscbutyl-3-methoxypyrazine binding site.\ud The steno requirements for the protein binding of various\ud substituted heterocyclic odorants were examined using nuclear magnetic\ud relaxation techniques. Model studies performed with bovine serum\ud albumin showed that particular side chains of the odorants tested\ud were primarily involved in the binding interaction. The methoxy group\ud of 2-isopropyl-3-methoxypyrazine was found to be responsible for\ud primary recognition by 13,000 x g supernatant fractions of sheep\ud olfactory epithelium

Topics: QH, QP
OAI identifier: oai:wrap.warwick.ac.uk:3943

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