생리활성소재연구실

Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the elderly, characterised by widespread loss of central cholinergic function. The only symptomatic treatment proven effective to date is the use of cholinesterase (ChE) inhibitors to augment surviving cholinergic activity. ChE inhibitors act on the enzymes that hydrolyse acetylcholine (ACh) following synaptic release. In the healthy brain, acetylcholinesterase (AChE) predominates (80%) and butyrylcholinesterase (BuChE) is considered to play a minor role in regulating brain ACh levels. In the AD brain, BuChE activity rises while AChE activity remains unchanged or declines. Therefore both enzymes are likely to have involvement in regulating ACh levels and represent legitimate therapeutic targets to ameliorate the cholinergic deficit. The two enzymes differ in location, substrate specificity and kinetics. Recent evidence suggests that BuChE may also have a role in the aetiology and progression of AD beyond regulation of synaptic ACh levels. Experimental evidence from the use of agents with enhanced selectivity for BuChE (cymserine, MF-8622) and ChE inhibitors such as rivastigmine, which have a dual inhibitory action on both AChE and BuChE, indicate potential therapeutic benefits of inhibiting both AChE and BuChE in AD and related dementias. The development of specific BuChE inhibitors and the continued use of ChE inhibitors with the ability to inhibit BuChE in addition to AChE should lead to improved clinical outcomes.

During the search for anticholinesterase compounds from marine organisms, two known plastoquinones, sargaquinoic acid (1) and sargachromenol (2), were isolated from Sargassum sagamianum. Both compounds showed moderate acetylcholinesterase (AChE) inhibitory activity in a micromole range (IC50 23.2 and 32.7 µM, respectively). However, for butyrylcholinesterase (BuChE), a new target for the treatment of Alzheimer’s disease (AD), compound 1 showed particularly potent inhibitory activity (IC50 26 nM), which is 1000-fold greater than for AChE. Hence, sargaquinoic acid represents an effective and selective inhibitor of BuChE with a potency similar to or greater than the anticholinesterases in current clinical use, making it an interesting potential drug candidate for AD. Copyright © 2007 John Wiley & Sons, Ltd.

The plastoquinones sargaquinoic acid and sargachromenol are major components of brown alga Sargassum sagamianum and are known to be involved in neurite growth and survival. In this study, we describe their novel pro-apoptotic activities in vitro and in vivo. In vitro, treatment with sargaquinoic acid or sargachromenol promoted cell death and activation of caspase-3, caspase-8, caspase-9 and poly (ADP-ribose) polymerase (PARP) in a concentration-dependent manner. Sargaquinoic acid- or sargachromenol-induced apoptosis was enhanced by co-treatment with UVB irradiation. It showed much higher levels of cleaved caspase-3, caspase-8, caspase-9, and PARP than with sargaquinoic acid and sargachromenol alone, while it had no effect on Bcl-2 and Bax expression level. Examination by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) and immunohistochemistry showed that topical application of sargaquinoic acid (1 mg/ml) before UVB irradiation (2.5 kJ/m(2)) of hairless mice also enhanced apoptosis including activation of caspase-3. Since a combination of phototherapy using UVB with topical reagents has been clinically applied to treat hyperproliferative skin disease, these results suggest that sargaquinoic acid and sargachromenol could be effective therapeutic agents.

The closure of the two domains of the catalytic chains of Escherichia coli aspartate transcarbamoylase, which is critical for completion of the T → R transition, is stabilized by salt-bridges between Glu-50 and both Arg-167 and Arg-234. Mutation of Glu-50 to Ala shifts the enzyme toward a low activity, low affinity state (Newton, C. J., and Kantrowitz, E. R.(1990) Biochemistry, 29, 1444-1451). Kinetic isotope effects (KIE) and equilibrium isotope exchange kinetics (EIEK) have been used to probe the dynamic properties of the Glu-50 → Ala enzyme. Unlike the behavior of the wild-type enzyme, the observed kinetic isotope effect for 13C versus12C at the carbonyl group of carbamoyl phosphate (CP) increased upon the binding of ligands which promote the formation of the R-state (Asp, N-phosphonacetyl-L-aspartate (PALA), or ATP). The maximum rate for the [14C]Asp ⇋ Carbamoyl aspartate (CAsp) exchange with the Glu-50 → Ala enzyme was 500-fold slower than for the wild-type enzyme; however, the rate for the [14C]CP ⇋ CAsp exchange was only 50-fold slower, reversing the relative rates observed with the wild-type enzyme. In addition, upon variation of substrate pairs involving Asp or CAsp, loss of inhibition effects in the CP ⇋ CAsp exchange indicated that the Glu-50 → Ala substitution caused the kinetic mechanism for the mutant enzyme to shift from ordered to random. Computer simulations of the EIEK data indicate that the Glu-50 → Ala mutation specifically causes strong decreases in the rates of catalysis and association-dissociation for Asp and CAsp, with minimal effects on the CP and Pi on-off rates. With substrates bound, the Glu-50 → Ala enzyme apparently does not attain a full R-state conformation. The PALA-activated Glu-50 → Ala enzyme, however, exhibits substrate affinities comparable to those for the wild-type enzyme, but fails to restore the preferred order substrate binding. Unlike the wild-type enzyme, both the T and R-states of the Glu-50 → Ala enzyme contribute to catalysis. A third state, I, is proposed for the Glu-50 → Ala enzyme, in which random order substrate binding is exhibited, and the catalytic step contributes significantly to overall rate limitation.

Acetylcholinesterase activity has been detected in extracts of guard cell protoplasts from Vicia faba L. and Nicotiana glauca Graham. Guard cell protoplast homogenates from V. faba exhibited 16.4 and 6.7-fold greater specific activities for acetylthiocholine hydrolysis compared to homogenates of mesophyll cell protoplasts or whole leaves, respectively. Extracts of N. glauca guard cell protoplasts also displayed highest specific activity for acetylthiocholine hydrolysis. Guard cell protoplast extracts from both species displayed a distinct substrate preference for acetylthiocholine. In contrast, no substrate specificity for choline ester hydrolysis was observed in extracts of mesophyll cell protoplasts or whole leaves. In both species, specific reversible inhibitors of mammalian acetylcholinesterase, BW284c51 and neostigmine, inhibited 40–90% of guard cell protoplast acetylcholinesterase activity. Exogenously applied acetylcholine (1 mM) induced an 80% closure of stomata in abaxial epidermal peels of V. faba leaves within 5 min, while only a 10–15% stomatal closure was induced by either butyrylcholine or propionylcholine. BW284c51, neostigmine and eserine also induced varying degrees of stomatal closure in epidermal peels of V. faba. Results from these studies demonstrate that guard cells have acetycholinesterase activity and suggest that acetylcholine might have a physiological role in stomatal movement.