Biomedical subjects

L F Neville

Publications and source records attributed to L F Neville.

23 records · Page 2Linked to original sources

PubMed1992

Excavations into the active-site gorge of cholinesterases.

Acetyl- and butyrylcholinesterase (ACHE, BCHE) from evolutionarily distant species display a high degree of primary sequence homology and have biochemically similar catalytic properties, yet they differ in substrate specificity and affinity for various inhibitors. The biochemical information derived from analyses of ACHE and BCHE from human, Torpedo, mouse, and Drosophila, as well as that from the recombinant forms of their natural variants and site-directed mutants, can currently be re-examined in view of the recent X-ray crystallography data revealing the three-dimensional structure of Torpedo ACHE. The picture that emerges deepens the insight into the biochemical basis for choline ester catalysis and the complex mechanism of interaction between cholinesterases and their numerous ligands.

Acetylcholinesterase↗

PubMed1990

Anionic site interactions in human butyrylcholinesterase disrupted by two single point mutations.

Structure-function relationships of recombinant human butyrylcholinesterase (CHE) variants were investigated by Xenopus oocyte microinjection. A Ser-425 to Pro-425 mutation failed to modify ligand binding properties. In contrast, Asp-70 to Gly-70 substitution significantly reduced CHE binding capacity for succinylcholine and specific inhibitors, demonstrating Asp-70 as a key anionic site component for certain ligands. Furthermore, the presence of both mutations rendered CHE totally resistant to succinylcholine and dibucaine inhibition, while all mutant proteins bound butyrylthiocholine, benzoylcholine, and propionylcholine normally. These findings imply structural interactions between the conserved Asp-70 and Ser-425 regions in cholinesterases and suggest the contribution of additional electronegative amino acids to anionic site binding.

Anions↗

PubMed1990

Aspartate-70 to glycine substitution confers resistance to naturally occurring and synthetic anionic-site ligands on in-ovo produced human butyrylcholinesterase.

The "atypical" allelic variant of human butyrylcholinesterase (BuChE) can be characterized by its failure to bind the local anesthetic dibucaine, the muscle relaxant succinylcholine, and the naturally occurring steroidal alkaloid solanidine, all assumed to bind to the charged anionic site component within the normal BuChE enzyme. A single nucleotide substitution conferring a change of aspartate-70 into glycine was recently reported in the CHE gene encoding BuChE from several individuals having the "atypical" BuChE phenotype, whereas in two other DNA samples, this mutation appeared together with a second alteration conferring a change of serine-425 into proline. To separately assess the contribution of each of these mutations toward anionic site interactions in BuChE, three transcription constructs were engineered with each of these substitutions alone or both of them together. Xenopus oocyte microinjection of normal or mutated synthetic BuChEmRNA transcripts was employed in conjunction with biochemical analyzes of the resultant recombinant BuChE variants. The presence of the Gly-70 mutation alone was found to render the enzyme resistant to 100 microM solanidine and 5 mM succinylcholine; concentrations sufficient to inhibit the "normal," Asp-70 containing BuChE by over 50%. Furthermore, when completely inhibited by the organophosphorous poison diisopropylfluorophosphate (DFP), Gly-70 BuChE failed to be reactivated by 10 mM of the cholinesterase-specific oxime pyridine 2-aldoxime methiodide (2-PAM); a concentration restoring about 50% of activity in the "normal" Asp-70 recombinant enzyme. The Pro-425 mutation alone had no apparent influence on BuChE interactions with any of these ligands. However, it conferred synergistic effects on some of the anionic site changes induced by the Gly-70 mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

2-chloroadenosine attenuates kainic acid-induced toxicity within the rat straitum: relationship to release of glutamate and Ca2+ influx.

1. The mechanism by which 2-chloroadenosine (2-chloroado) exerts a neuroprotective action against the excitotoxic effect of kainic acid (KA) when injected into the rat striatum was investigated. 2. Histological examination two weeks after a single injection of KA (2.2 nmol) into rat striatum revealed widespread neuronal damage. Co-injection of 2-chloroado (6-25 nmol) with the neurotoxin afforded dose-dependent neuroprotection. This effect was reversed by administration of an equimolar concentration of the adenosine receptor antagonist theophylline. 3. Both K+ (30 mM) and KA (1 mM) enhanced the release of endogenous glutamate from guinea-pig purified cerebrocortical synaptosomes in a predominantly (approximately 70%) Ca2+-dependent manner. 2-Chloroado (10 nM-1 microM) inhibited the release of glutamate evoked by both KA and K+. These effects were partially reversed by the selective A1-adenosine receptor antagonist 8-cyclopentyltheophylline (CPT) (1 microM). 4. Crude rat cortical synaptosomes were loaded with the fluorescent calcium indicator quin-2 and Ca2+ influx monitored following two successive depolarising stimuli (30 mM K+; 'S1' and 'S2'). 2-Chloroado (10 nM-1 microM) produced a dose-dependent reduction in the S2:S1 ratio when added before the S2 period of stimulation. This effect was reversed by 1 microM theophylline. However, KA (1 mM) failed to enhance Ca2+ influx in the same preparation. 5. These results suggest that the anti-excitotoxic action of 2-chloroado is mediated primarily through a specific presynaptic receptor mechanism involving reduction of transmitter glutamate release, possibly occurring through an inhibition of Ca2+ influx.

2-Chloroadenosine↗

PubMed1988

2-Chloroadenosine prevents kainic acid-induced toxicity in rat striatum.

The stable adenosine analogue, 2-chloroadenosine was found to afford a dose-dependent protection (effective dose range: 12.5-50 nmol) against the characteristic neurotoxic actions of kainic acid (2.2 nmol) in rat striatum. The two agents were co-injected directly into the striatum and the extent of the excitotoxin-induced lesion assessed two weeks later in 40 microns coronal sections stained with thionin, or by histochemical staining for NADPH-dependent diaphorase. These findings provide further information concerning the possible mechanism(s) underlying kainic acid-induced neurotoxicity and also yield an insight into a possible neuroprotective role for endogenous adenosine-like substances.

2-Chloroadenosine↗