Interaction of 3-alkylpyridinium polymers from the sea sponge Reniera sarai with insect acetylcholinesterase.
3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS-PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS-PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.
Sepcić
K
K
Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia. kristina.sepcic@uni-lj.si
Poklar
N
N
Vesnaver
G
G
Fournier
D
D
Turk
T
T
Macek
P
P
D000110:Acetylcholinesterase / Q000378:metabolism*; D000818:Animals; D019476:Insect Proteins / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D011108:Polymers / Q000378:metabolism*; D011161:Porifera / Q000378:metabolism*; D011726:Pyridinium Compounds / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism
D019476:Insect Proteins; D011108:Polymers; D011726:Pyridinium Compounds; C115254:poly-APS; D000110:Acetylcholinesterase; D014357:Trypsin