To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase (BtChoEase; EC 3.1
Cholinesterases are serine esterases that rapidly hydrolyze the neurotransmitter acetylcholine. In humans, cholinesterases exhibit extensive polymorphism in terms of their substrate specificity, sensitivity to selective inhibitors, hydrophobicity, and cellular as well as subcellular localization. It is not yet known whether the various cholinesterase forms originate from different genes or are products of posttranscriptional and posttranslational processing. The extent to which these enzyme forms are homologous in their amino acid sequence is also not known. However, a consensus organophosphate-binding hexapeptide sequence Phe-Gly-Glu-Ser-Ala-Gly was found both in "true" acetylcholinesterase from the electric organ of Torpedo [McPhee-Quigley et al: J Biol Chem 260:12185-12189, 1985] and in "pseudocholinesterase" (butyrylcholinesterase) from human serum [Lockridge: "Cholinesterases--Fundamental and Applied Aspects." New York: de Gruyter pp 5-12, 1984], suggesting that this region in the protein is conserved in all cholinesterases. Based on this common sequence, we prepared synthetic oligodeoxynucleotides and used them as labeled probes to screen a cDNA library from fetal human brain mRNA, cloned in lambda gt10 phages. A cDNA clone of 770 nucleotides in length was isolated. It contains an open reading frame terminating with the sequence Ser-Val-Thr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala, which includes the consensus hexapeptide used for designing the DNA probe. Furthermore, the sequence of this 12-amino acid peptide is identical to the sequence reported for the organophosphate binding site of human serum pseudocholinesterase [Lockridge: "Cholinesterases--Fundamental and Applied Aspects." New York: de Gruyter, pp 5-12, 1984]. These findings confirm that the isolated clone is indeed part of a human cholinesterase cDNA.
The bicyclic [2S-(2 alpha,3 beta,4 beta)]-2-carboxy-4-(1-hydroxy-1-methylethyl)-3- pyrrolidineacetic acid delta-lactone (4), as well as its 4-[1-hydroxy-1-(iodomethyl)ethyl], 4-[1-hydroxy-1-(hydroxymethyl)ethyl], and 4-[1-hydroxy-1-[(phenyl-thio)methyl]ethyl] analogues, 6, 7, and 9, respectively, were designed and synthesized as potential selective antagonists of neuroexcitatory amino acids. When applied to rat brain slices, these lactones, which are chemically derived from kainic acid, inhibit the stimulation of Na+ fluxes induced by the neuroexcitants kainic acid and N-methyl-D-aspartic acid. Lactone 4 and the hydroxy lactone 7 block preferentially the response to N-methyl-D-aspartic acid, while the iodo lactone 6 and the phenylthio lactone 9 are mainly kainic acid antagonists. Total inhibitions can be obtained, half of the maximal effect being observed at lactone concentrations in the range of 0.2-3 mM.
Specific 22Na' efflux rates from preloaded rat striatal slices are increased in a dose-dependent manner by L-glutamate and other excitatory amino acids displaying the following order of efficiency:
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