PubMed · 4331239
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A N Davison. 1971. Lipids and brain development.. https://pubmed.ncbi.nlm.nih.gov/4331239/
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Replacement of residues Asp74, Trp286, and Tyr72, which are constituents of the peripheral anionic site (PAS) of human acetylcholinesterase (HuAChE), affected similarly both the binding and the inhibition constants of the PAS-specific ligand propidium, demonstrating that changes in the inhibitory activity are a direct consequence of altered binding to the PAS. In contrast, the active center HuAChE mutants W86A and Y133A show respective 350- and 25-fold increased resistance to inhibition by propidium but no change in binding affinities, demonstrating that the allosteric mechanism of PAS-mediated inhibition involves a conformational change of these Trp86 and Tyr133 residues rather than physical obstruction of substrate access by the inhibitor itself. These findings support the recent proposal that the allosteric mechanism operates via transition between active and nonactive conformations of the anionic subsite Trp86 and that replacement of Tyr133 by alanine may stabilize a nonactive Trp86 conformation that occludes the active center [Ordentlich et al. (1995) J. Biol. Chem. 270, 2082]. In further support of this mechanism and the role of Tyr133, we find that (a) the dissociation constants (Kd) for the noncovalent complexes of the irreversible inhibitors diisopropyl phosphorofluoridate or paraoxon with Y133A HuAChE are increased 20-500-fold, relative to either wild-type enzyme or its Y133F or W86A mutants; and (b) access of substrates such as 3,3-dimethylbutyl thioacetate is restored by removal of Trp86 from the Y133A enzyme (i.e., the W86A/Y133A mutant). We suggest that the conformational transition of Trp86 is coupled to the motions of the cysteine loop (Cys69-Cys96) of HuAChE and is inherent to the dynamics of the native enzyme.
Lactacystin (1.3 microM), a metabolite from an actinomycete, induced the formation of bipolar projections at both sides of the cell body of Neuro 2a cells 1 day after treatment and networks at and after 3 days and enhanced acetylcholinesterase activity (a marker of neuronal differentiation). Thus, the neuronal differentiation was characterized both morphologically and functionally. The experiments with various inhibitors of protein kinases and phosphatases revealed that the protein phosphatase inhibitors calyculin A (0.5 nM) and okadaic acid (0.6 nM) inhibit the formation of bipolar projections at 1 day, but does not inhibit the network formation at and after 3 days.
The possible role of post-translational modifications such as subunit oligomerization, protein glycosylation and oligosaccharide processing on the circulatory life-time of proteins was studied using recombinant human acetylcholinesterase (rHuAChE). Different preparations of rHuAChE containing various amounts of tetramers, dimers and monomers are cleared at similar rates from the circulation, suggesting that oligomerization does not play an important role in determining the rate of clearance. An engineered rHuAChE mutant containing only one N-glycosylation site was cleared from the circulation more rapidly than the wild-type triglycosylated enzyme. On the other hand, hyperglycosylated mutants containing either four or five occupied N-glycosylation sites, analagous to those present on the slowly cleared fetal bovine serum acetylcholinesterase (FBS-AChE), were also cleared more rapidly from the bloodstream than the wild-type species. Furthermore, the two different tetraglycosylated mutants were cleared at different rates while the pentaglycosylated mutant exhibited the most rapid clearance profile. These results imply that though the number of N-glycosylation sites plays a role in the circulatory life-time of the enzyme, the number of N-glycan units in itself does not determine the rate of clearance. When saturating amounts of asialofetuin were administered together with rHuAChE, the circulatory half-life of the enzyme was dramatically increased (from 80 min to 19 h) and was found to be similar to that displayed by plasma-derived cholinesterases while desialylation of these enzymes caused a sharp decrease in the circulatory half-life to approximately 3-5 min. Determination of the average number of sialic acid residues per enzyme subunit of the five different N-glycosylation species generated, revealed that the rate of clearance is not a function of the absolute number of appended sialic acid moieties but rather of the number of unoccupied sialic acid attachment sites per enzyme molecule. Specifically, we demonstrate an inverse-linear relationship between the number of vacant sialic acid attachment sites and the values of the enzyme residence time within the bloodstream.