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Biomedical subjects

A J Lawrence

Publications and source records attributed to A J Lawrence.

At least 73 records · Page 4Linked to original sources
PubMed1998

Distribution of GABAA receptors in the limbic system of alcohol-preferring and non-preferring rats: in situ hybridisation histochemistry and receptor autoradiography.

The present study has employed quantitative receptor autoradiography and in situ hybridisation histochemistry to compare the expression of the mRNA encoding the alpha 1 and alpha 2 subunits of the GABAA receptor and the binding density of mature GABAA receptors in the limbic system of alcohol-preferring Fawn-Hooded rats (FH) with Wistar-Kyoto rats (WKY). Quantifiable levels of mRNA encoding the alpha 1 subunit were found in cortical regions, ventral pallidum, substantia nigra, horizontal limb of the diagonal band and the hippocampus of both rat strains. Interestingly, expression of the alpha 1 subunit mRNA was decreased by approximately 30% in the hippocampus of FH compared to WKY rats. Following a 28-day period with free access to 10% ethanol, expression of the alpha 1 subunit transcript, was significantly increased in the piriform cortex and horizontal limb of the diagonal band, unaltered in the hippocampus but decreased in the substantia nigra of FH rats. Quantifiable levels of mRNA encoding the alpha 2 subunit were found in nucleus accumbens, amygdala, cortical regions, lateral septal nucleus, hippocampus, medial habenula and ventral pallidum of both strains. Expression of the alpha 2 subunit mRNA was decreased by approximately 35% in both the hippocampus and occipital cortex of FH compared to WKY rats. However, consumption of 10% ethanol in FH rats had no impact upon expression of the mRNA encoding the alpha 2 subunit in any region examined. Mature GABAA receptors were studied by autoradiography utilising the antagonist radioligand [3H]SR95531 and the agonist radioligand [3H]muscimol. Topographic binding throughout the limbic system of both strains was observed for both radioligands. Specifically, [3H]SR95531 binding was higher in the occipital cortex, hippocampus, lateral septal nucleus, superior colliculus and ventral pallidum of the FH rats compared to WKY rats; however, in the nucleus accumbens [3H]SR95531 binding was lower in FH compared to WKY. Ethanol consumption had no measurable effect on the binding of [3H]SR95531 in FH rats. In the case of [3H]muscimol, binding was higher in the cortex, lateral septum and ventral pallidum of FH compared to WKY. Furthermore, ethanol consumption resulted in a 25-30% increase in [3H]muscimol binding in the lateral septum and striatum of FH rats. These data provide evidence for differential expression of GABAA receptor subunits in FH and WKY rats, and additionally indicate anatomically defined variations in GABAA receptor binding between the two rat strains.

Alcohol Drinking↗
PubMed1998

Nicotinic acetylcholine receptors in the rat and primate nucleus tractus solitarius and on rat and human inferior vagal (nodose) ganglia: evidence from in vivo microdialysis and [125I]alpha-bungarotoxin autoradiography.

The nucleus tractus solitarius is a key brain centre involved in the regulation of numerous autonomic functions. The present study has employed in vitro autoradiography and in vivo microdialysis to investigate the presence and function of nicotinic acetylcholine receptors located in the medial nucleus tractus solitarius of the rat. Autoradiography using [125I]alpha-bungarotoxin (0.5 nM) enabled visualization of binding sites on sections of rat and monkey brainstem. Specific binding was highest in the medial nucleus tractus solitarius. The presence of binding sites was also apparent on sections of rat nodose ganglia/vagus nerve and human inferior vagal ganglia. Subsequent to unilateral ligation of the vagus nerve in the rat, an accumulation of binding sites was visualized adjacent to the ligature. Unilateral nodose ganglionectomy in the rat caused an approximate 97% reduction in [125I]alpha-bungarotoxin binding site density in the medial nucleus tractus solitarius from 814 +/- 183 to 27 +/- 2 d.p.m./mm2. Microdialysis results indicated that local administration of nicotine (1 mM) into the nucleus tractus solitarius of the rat resulted in increases of extracellular L-glutamate of 146 +/- 9% of basal levels. This effect was not reproducible following a second stimulation and was also blocked by prior and co-administration of the nicotinic acetylcholine receptor antagonist mecamylamine (100 microM). Extracellular levels of L-aspartate exhibited a similar pattern although results were not significant. Taken together, these results are supportive of the presence of a population of [125I]alpha-bungarotoxin binding sites located presynaptically with respect to vagal afferent terminals in the medial nucleus tractus solitarius of the rat. It is possible that these binding sites are the site of action of locally administered nicotine on extracellular levels of L-glutamate, the favoured neurotransmitter at primary baroreceptor afferent fibres. These data are discussed in relation to the functional pharmacology of acetylcholine and nicotinic acetylcholine receptors in this region of the brain.

Adult↗
PubMed1998

The distribution of nitric oxide synthase-, adenosine deaminase- and neuropeptide Y-immunoreactivity through the entire rat nucleus tractus solitarius: Effect of unilateral nodose ganglionectomy.

The present study has employed immunocytochemistry on free-floating sections of adult rat medulla oblongata to characterise the distribution of nitric oxide synthase- (NOS), adenosine deaminase- (ADA) and neuropeptide Y- (NPY) immunoreactivity (IR) throughout the entire rostro-caudal axis of the nucleus tractus solitarius (NTS). In addition, unilateral nodose ganglionectomy was performed in a group of rats to determine whether any observed immunoreactivity was associated with central vagal afferent terminals. NOS-IR was found throughout the entire NTS, in cells, and both varicose and non-varicose fibres. Furthermore, unilateral nodose ganglionectomy resulted in a clear reduction in NOS-IR (visualised with diaminobenzidine) in a highly restricted portion of the ipsilateral medial NTS. Similarly, ADA- and NPY-containing cells, fibres and terminals were also found throughout the adult rat NTS. However, following unilateral nodose ganglionectomy, there was no apparent reduction in either ADA-IR or NPY-IR on the denervated side of the NTS. These data indicate a role for nitric oxide, purines and neuropeptide Y as neuromodulators within the rat NTS, although only nitric oxide appears to be primarily associated with vagal afferent input. Adenosine deaminase and neuropeptide Y-containing neurons appear to be predominantly postsynaptic to vagal input, although their possible association with vagal afferents cannot be completely excluded.

Adenosine Deaminase↗
PubMed1998

Effects of the vasopressin V1 agonist [Phe2,Ile3,Orn8]] vasopressin on regional kidney perfusion and renal excretory function in anesthetized rabbits.

To test whether renal V1-receptors selectively influence blood flow in the renal medulla, we compared the effects of infusion of [Phe2,Ile3,Orn8]vasopressin (3-30 ng/kg/min) by the intravenous, renal arterial, and renal medullary interstitial routes in anesthetized rabbits. Intravenous [Phe2,Ile3,Orn8]vasopressin (30 ng/kg/min) reduced renal medullary perfusion (MBF) by 36 +/- 5% but did not significantly affect cortical perfusion (CBF). MBF was also reduced with the renal arterial (35 +/- 5%) and renal medullary interstitial (40 +/- 7%) routes but, in contrast to the intravenous infusion, CBF was also reduced, by 21 +/- 3% and 15 +/- 3%, respectively. Urine flow and sodium excretion were increased by [Phe2,Ile3,Orn8]vasopressin, and with direct intrarenal administration, this effect was similar for both the infused (left) and noninfused (right) kidneys. After a 20-min renal medullary interstitial infusion of [3H]norepinephrine, radiolabel concentration was approximately fivefold greater in the left medulla than in the left cortex. We conclude that [Phe2,Ile3,Orn8]vasopressin acts on V1-receptors to alter regional kidney blood flow and tubular salt and water handling. The V1-receptors involved are almost certainly within the kidney itself, but given the contrasting effects of the different infusion routes on MBF and CBF, we cannot exclude the possibility that some of the observed effects of [Phe2,Ile3,Orn8]vasopressin are mediated by activation of extra-renal V1-receptors.

Angiotensin II↗
PubMed1998

Duodenal volvulus in free-living green turtles from coastal United Arab Emirates.

Post-mortem examinations performed during May and August of 1997 on three free-living green turtles (Chelonia mydas) in the United Arab Emirates revealed that all had stomachs full of fresh seagrass (approximately 99% of the total ingesta) and presented with a duodenal volvulus involving a length of approximately 100 cm. Duodeni appeared empty and necrotic with diffuse purple-black mucosa. No apparent signs of obstruction by foreign objects, acute endoparasitism, or other disorders were observed. In all cases, duodenal volvulus was diagnosed, the cause of which may have been dietary in origin. The rise in water temperature, with an associated rise in the temperature of seagrass, thus enhancing the over-fermentation of ingesta with the subsequent liberation of excessive amounts of gas was the probable cause of volvulus formation. Ingestion of foreign bodies and physical movement also are discussed as causes of digestive disorders. It is recommended that handlers should avoid 360 degrees rotation when overturning turtles onto their carapaces.

Animals↗
PubMed1997

[3H]zolpidem binding in alcohol-preferring and non-preferring rat brain.

The present study has employed in vitro autoradiography to study the distribution and density of [3H]zolpidem binding sites, which are regarded as an index of ethanol-sensitive gamma-aminobutyric acid (GABA)A receptors, in the brains of alcohol-preferring Fawn-Hooded (FH) rats compared to non-alcohol preferring Wistar-Kyoto (WKY) rats. Binding of [3H]zolpidem showed a similar distribution profile in both rat strains examined and included cerebellum, globus pallidus, nucleus of the solitary tract and a number of midbrain/hindbrain nuclei. Densitometric quantitation of binding revealed that FH rats possessed a significantly higher density of [3H]zolpidem binding compared to WKY rats in cortical regions, substantia nigra pars reticulata and the ventral pallidum. These data indicate that FH rats may have an increased number of ethanol-sensitive GABA(A) receptors in regions intimately involved in reward processes, and may partially explain the alcohol-seeking nature of the FH rat.

Animals↗
PubMed1997

Prepro-neuropeptide Y mRNA and NPY binding sites in human inferior vagal ganglia.

The inferior vagal ganglia contain the cell bodies of centrally projecting vagal afferent neurones. Using in situ hybridization-histochemistry with a combination of two antisense neuropeptide Y (NPY) oligonucleotides, we have demonstrated that a population of human inferior vagal perikarya express mRNA encoding prepro-NPY, the precursor of NPY. In vitro receptor autoradiography, using both [125I]Bolton Hunter-NPY ([125I]BH-NPY, 15 pM) and [125I]peptide YY ([125I]PYY, 25 pM), enabled visualization of NPY binding sites. Competition binding with NPY (1 microM), PYY (1 microM) and [Leu31,Pro34]NPY (100 nM), suggest that both Y1 and Y2 receptor subtypes are present on human vagal afferent neurones. These observations suggest a potential role for NPY in neuromodulation of vagal transmission in humans.

Autoradiography↗
PubMed1997

Complex interactions between nitric oxide and adenosine receptors in the rat isolated nodose ganglion.

The present study has employed in vitro electrophysiology, utilising the isolated rat nodose ganglion preparation, to determine whether nitric oxide (NO) and adenosine interact with each other in vagal afferent neurons. The nucleophile NO donor, diethylamine-NO, caused reproducible, concentration-related depolarisations of the isolated rat nodose ganglia. Pre-incubation of the isolated rat nodose ganglia with the adenosine A2A receptor agonists CGS 21680 (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride) and DPMA (N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine) (both 1 microM) resulted in a functional antagonism of the ability of diethylamine-NO to depolarise the preparation. A similar effect was observed with adenosine (10 microM) only in the presence of the adenosine A1 receptor antagonist PACPX (1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine, 100 nM). Conversely, the adenosine A1 receptor agonists ENBA (N6-[2-endo-norbomyl]adenosine, 1 microM) and cyclohexyladenosine (100 nM) potentiated the effect of diethylamine-NO on isolated rat nodose ganglia. Inclusion of either adenosine A3 agonists or ATP had no effect on the diethylamine-NO concentration-response curve. These data suggest an ability of NO to interact, in opposing manner, with adenosine A2A and A1 receptors in rat vagal afferent neurons. On the other hand, neither A3 receptors nor ATP appear capable of interacting with NO.

Adenosine↗
PubMed1997

Adenosine-dopamine receptor interactions in the isolated rat nodose ganglion but not in membranes of dorsal vagal complex.

The present study has employed in vitro electrophysiology and radioligand binding assays to determine whether dopamine and adenosine receptors interact with each other on rat vagal afferent neurons. Preincubation of the isolated rat nodose ganglion with the adenosine A2a agonists CGS 21 680 or DPMA (Both 1 microM) resulted in a functional antagonism of the ability of dopamine to depolarise the preparation. Specifically, the concentration-response curve to dopamine was significantly shifted to the right in the presence of CGS 21 680 and DPMA. On the other hand, adenosine itself, A1 and A3 receptor agonists and ATP were all incapable of modulating the electrophysiological response to dopamine. In contrast to the nodose ganglion, CGS 21 680 did not significantly affect the ability of the dopamine D2 ligands quinpirole or raclopride to displace [125I]NCQ298 binding to dopamine D2 receptors in membranes prepared from rat dorsal brain stem. These data indicate the presence of an interaction between high affinity adenosine A2 receptors and dopamine D2 receptors on the soma of rat vagal afferent neurons, whereas the situation in the brain stem remains less clear.

Adenosine↗
PubMed1997

Functional GABAA receptors on rat vagal afferent neurones.

1. In the present study, in vitro electrophysiology and receptor autoradiography were used to determine whether rat vagal afferent neurones possess gamma-aminobutyric acid (GABA)A receptors. 2. GABA (1-100 microM) and isoguvacine (3-100 microM) caused a concentration-dependent depolarization of the rat isolated nodose ganglion preparation at room temperature. When applied to the tissue 20 min before the agonist, SR95531 (3 microM) and bicuculline (3 microM) caused a parallel shift to the right of the GABA and isoguvacine concentration-response curves, yielding shifts of 81 fold and 117 fold for SR95531 and 4 fold and 12 fold for bicuculline, respectively. 3. Baclofen (10 nM-100 microM) was unable to elicit a depolarization of the rat isolated nodose ganglion preparation at either room temperature or at 36 degrees C, whilst 5-aminovaleric acid (10 microM), a GABAB receptor antagonist, was unable to antagonize significantly the GABA-induced depolarization at either room temperature or at 36 degrees C. 4. [3H]-SR95531 (7.2 nM), a GABAA receptor-selective antagonist, bound topographically to sections of rat brainstem. Specific binding was highest in the medial nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMVN). Binding was also observed in certain medullary reticular nuclei, in particular the parvocellular reticular nucleus. 5. Unilateral nodose ganglionectomy caused a reduction in GABAA binding site density in the medial NTS from 93 +/- 7 to 68 +/- 6 d.p.m./mm2. This procedure also caused a reduction in GABAA binding site density in the side of the NTS contralateral to the lesion, from 151 +/- 12 to 93 +/- 7 d.p.m./mm2. Sham surgery had no effect on the binding of [3H]-SR95531 in rat brainstem. 6. The present data provide evidence for the presence of GABAA receptors located on the soma and central terminals of rat vagal afferent neurones. Additionally, a population of GABAA receptors is evidenced postsynaptically in the rat NTS with respect to vagal afferent terminals. These data are discussed in relation to the functional pharmacology of GABA in this region of the NTS.

2-Amino-5-phosphonovalerate↗
PubMed1996

Actions of nitric oxide and expression of the mRNA encoding nitric oxide synthase in rat vagal afferent neurons.

The present study has investigated whether nitric oxide (NO) is involved in neurotransmission of rat vagal afferent neurons. The diethylamine-NO complex (diethylamine-NO, 10-100 microM) and S-nitroso-N-acetylpenicillamine (3-100 microM) both elicited a concentration-dependent depolarisation of the isolated rat nodose ganglion preparation. Pre-treatment with 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 300 nM), 6-(phenylamino)-5,8-quinolinedione (LY83,583, 30 microM) and Methylene blue (100 microM) all caused a significant shift to the right in the concentration-response curve to diethylamine-NO. Incubation of rat nodose ganglion sections with a 35S-labeled antisense oligonucleotide to neuronal NO synthase resulted in visualisation of the mRNA encoding NO synthase over vagal afferent perikarya. The anatomical findings, therefore, suggest that a number of rat vagal afferent perikarya possess the ability to produce the enzyme required for the biosynthesis of NO. Collectively, these data suggest that NO may be functionally important as a neuromodulator of rat vagal afferent neurons.

Aminoquinolines↗
PubMed1996

Loss of [125I]-pindolol binding to beta-adrenoceptors on rat nodose ganglion after chronic isoprenaline treatment.

The nodose ganglion contains the cell bodies of afferent nerves which convey predominantly sensory information from the viscera to the central nervous system (CNS). Autoradiographic studies show binding sites for beta-adrenoceptor ligands are present on sections of the rat nodose ganglion and also on the corresponding inferior vagal ganglion in humans, indicating the presence of beta-adrenoceptors in these ganglia. Since prolonged stimulation of beta-adrenoceptors in rats with the nonselective beta-adrenoceptor agonist isoprenaline (400 micrograms kg-1 day-1 s.c.) for 14 days results in desensitisation and/or down-regulation of receptors in peripheral tissues, such as heart, kidney and blood vessels, the effects of this treatment on the beta-adrenoceptor population on the nodose ganglion have been examined. Using [125I]-pindolol as a radioligand, autoradiographic studies revealed that specific binding was reduced by 74% in ganglia from isoprenaline-pretreated rats compared to that in ganglia from vehicle-pretreated rats, demonstrating down-regulation of receptors by isoprenaline. [125I]-Pindolol binding was sensitive to inhibition by ICI 118.551 (selective beta 2-adrenoceptor antagonist) but not to atenolol (selective beta 1-adrenoceptor antagonist), indicating receptors are predominantly of the beta 2-adrenoceptor subtype. No change in binding was apparent over the vagus nerve. The nodose ganglion appears to be an additional site at which beta 2-adrenoceptors may be down-regulated in vivo, possibly interfering with normal baro-, chemo- and sensory reflexes.

Animals↗
PubMed1996

Radioligand binding and autoradiographic visualization of adenosine transport sites in human inferior vagal ganglia and their axonal transport along rat vagal afferent neurons.

The present study has employed membrane-binding studies and in vitro autoradiography to demonstrate the presence of adenosine transport sites in human inferior vagal ganglia using [3H]nitrobenzylthioinosine ([3H]NBMPR), a potent inhibitor of adenosine transport. In addition, [3H]NBMPR was used to determine whether adenosine transport sites are subject to axonal transport along the rat vagus nerve. Binding of [3H]NBMPR to human inferior vagal ganglia membranes was saturable and reversible. Saturation experiments revealed a single class of high affinity-binding sites with a Kd of 93.73 +/- 23.13 pM and Bmax of 413.50 +/- 50.40 fmol/mg protein. In displacement experiments, the adenosine transport inhibitor dipyridamole was the most potent displacer of [3H]NBMPR binding (Ki = 42.7 +/- 28.0 nM). Adenosine itself was able to fully displace [3H]NBMPR binding with a Ki of 115.0 +/- 34.0 microM. The A1/A2a adenosine receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA) was able to fully displace [3H]NBMPR binding in only one experiment at a concentration of 100 microM, yielding an affinity 1000-fold higher than its affinity for adenosine receptors. All competition curves obtained from displacement experiments displayed monophasic profiles, indicating the presence of a single class of [3H]NBMPR binding sites. Incubation of human inferior vagal ganglia sections with [3H]NBMPR (0.7 nM) revealed dense binding which appeared to be consistent with the distribution of neuronal cell bodies in this tissue. Following unilateral ligation of the vagus nerve in the rat, accumulation of [3H]NBMPR binding sites occurred both proximal and distal to the vagal ligatures. These results suggest that [3H]NBMPR binds with high affinity to a single class of adenosine transport sites, and that these sites are present on vagal afferent neurons in the human and undergo bidirectional axonal transport along the rat vagus nerve.

Adenosine↗
PubMed1996

Localisation and properties of AMPA-insensitive kainate sites: receptor autoradiography and gene expression in rat brain.

Kainic acid (KA)-sensitive glutamate sites have been investigated by receptor autoradiography and in situ hybridisation histochemistry (ISHH) to evaluate their relationship to specific high-affinity KA receptors identified in molecular biological studies. Autoradiography with [3H]KA in the presence of the AMPA-selective antagonist NBQX (1 microM) revealed a widespread distribution of receptors through brain, especially in neocortex, hippocampal CA3, corpus striatum and granule cell layer of cerebellum. Specific binding was insensitive to the AMPA-selective agonist, S-5-fluorowillardiine, but inhibited by kainoids in a manner suggestive of receptor heterogeneity. Expression of the KA-2 receptor subunit mRNA by ISHH was also localised in hippocampal CA3 and cerebellar granule cells, suggesting some high-affinity native KA receptors labelled by [3H]KA were likely to include the KA-2 subunit in their heteromeric assembly.

Alanine↗
PubMed1996

Neuropeptide Y gene expression and receptor autoradiography in hypertensive and normotensive rat brain.

Neurones containing neuropeptide Y (NPY) may participate in central cardiovascular control by tonically influencing barosensitive neurones within the nucleus tractus solitarius. The present study has employed both in situ hybridisation histochemistry and receptor autoradiography, to visualise the expression of prepro-NPY mRNA in the forebrain and to determine the NPY receptor subtype(s) in the brainstem, respectively. Prepro-NPY gene expression was visualised in the hypothalamus, cortex, dentate gyrus and lateral reticular thalamus from age-matched spontaneously hypertensive rats (SHR) and normotensive Don Ryu rats (DRY) and Wistar Kyoto rats (WKY). Quantitative densitometry revealed an increase in the NPY transcript in the arcuate nucleus of SHR rats compared to their normotensive counterparts. Autoradiography using [125I]Bolton-Hunter-NPY (BH-NPY, 15 pM) demonstrated NPY binding sites in the area postrema, the commissural nucleus tractus solitarius (cNTS) and the inferior olivary complex. NPY (1 microM) and peptide YY (1 microM), but not [Leu31,Pro34]NPY (10-100 nM), fully inhibited the binding of [125I]BH-NPY. These results indicate that NPY receptors of the Y2 subtype predominate in the dorsal vagal complex. Unilateral nodose ganglionectomy resulted in a partial loss of NPY binding sites in the commissural NTS, but not the area postrema, suggesting that a proportion of binding sites (Y2 subtype) are present on central vagal terminals. While all three rat strains appear to have the same relative proportions of NPY receptor subtypes in the brainstem, the relevance of the differential NPY gene expression in the arcuate nucleus regarding central cardiovascular control mechanisms and/or the pathogenesis of hypertension remains to be elucidated.

Animals↗
PubMed1996

Neurochemical modulation of cardiovascular control in the nucleus tractus solitarius.

The central control of cardiovascular function has been keenly studied for a number of decades. Of particular interest are the homeostatic control mechanisms, such as the baroreceptor heart-rate reflex, the chemoreceptor reflex, the Bezold-Jarisch reflex and the Breuer-Hering reflex. These neurally-mediated reflexes share a common termination point for their respective centrally-projecting sensory afferents, namely the nucleus tractus solitarius (NTS). Thus, the NTS clearly plays a critical role in the integration of peripherally initiated sensory information regarding the status of blood pressure, heart rate and respiratory function. Many endogenous neurochemicals, from simple amino acids through biogenic amines to complex peptides have the ability to modulate blood pressure and heart rate at the level of the NTS. This review will attempt to collate the current knowledge regarding the roles of neuromodulators in the NTS, the receptor types involved in mediating observed responses and the degree of importance of such neurochemicals in the tonic regulation of the cardiovascular system. The neural pathway that controls the baroreceptor heart-rate reflex will be the main focus of attention, including discussion of the identity of the neurotransmitter(s) thought to act at baroafferent terminals within the NTS. In addition, this review will provide a timely update on the use of recently developed molecular biological techniques that have been employed in the study of the NTS, complementing more classical research.

Animals↗
PubMed1996

Conformational changes associated with activation of bee venom phospholipase A2.

Bee venom PLA2 possesses a binding site for long-chain fatty acids that can be acylated by long-chain fatty acid imidazolides [Drainas, D. and Lawrence, A.J. (1978) Eur. J. Biochem. 91, 131-138]. Occupation of the site either by oleic acid or the oleoyl residue enhances the catalytic activity by 45.7-fold in the presence of 20% 1-propanol and occupation of the site by the oleoyl residue increases the lytic activity against rabbit erythrocytes by 60-fold. Treatment of the enzyme with oleic acid and glutaraldehyde is known to produce irreversible activation [Lawrence, A.J. and Moores, G.R. (1975) FEBS Lett. 49, 287-291]. Here we show that reduction of the glutaraldehyde-treated enzyme with borohydride stabilizes the activated state and enables the fatty acid to be removed, revealing that a large proportion of the induced activation does not require the presence of oleic acid and indicating that activation is due to a change in the conformation rather than the hydrophobicity of the protein. A kinetic study of enzyme activated by oleoyl imidazolide showed that this modification stabilizes the protein against reversible inactivation by 1-propanol. Comparison of the CD spectra of native and oleoyl imidazolide-activated enzyme shows a change in secondary structure with apparent increase in both alpha-helix and beta-sheet content. During reaction of the enzyme with oleoyl imidazolide, the protein fluorescence shows a biphasic response with an initial fall attributed to occupation of the binding site followed by a progressive decrease with a shift of the emission maximum from 341 to 348 nm. The rate of the second phase closely matched the rate of increase in catalytic activity of the enzyme. Free oleic acid caused a rapid fall in fluorescence emission without the subsequent slow change. These results support the proposal that oleic acid or the oleoyl residue occupy a very similar site on the protein and that occupation of this site increases the exposure of one or both of the Trp residues to the aqueous environment. Binding studies show that activation by oleoyl imidazolide does not increase the affinity of the enzyme for the erythrocyte membrane. It is proposed that occupation of a long-chain fatty acid binding site on the enzyme enhances catalytic activity by changing the conformation of the protein rather than acting as a hydrophobic anchor to the substrate surface.

1-Propanol↗