Fluorescent organophosphates: novel probes for studying aging-induced conformational changes in inhibited acetylcholinesterase and for localization of cholinesterase in nervous tissue.
Aging of acetylcholinesterase (AChE) inhibited by certain organophosphates such as diisopropylfluorophosphate apparently involves dealkylation of the bound organophosphoryl moiety; this renders the inactive enzyme resistant to reactivation by quaternary oximes such as 2-pyridinealdoxime methiodide (2-PAM) which are used in therapy of organophosphate intoxication. The fluorescent pyrenyl organophosphates synthesized in this study were designed to detect putative conformational changes which might explain this resistance. The following inhibitors: 1-pyrenebutyl phosphorodichloride (PBPDC), 1-pyrenebutyl ethylphosphorochloridate (PBEPC), and 1-pyrenebutyl ethylphosphorofluoridate (PBEPF), react specifically with purified electric eel AChE (ki = 10(6)-10(7) M-1 min-1). AChE inhibited by PBEPC and PBEPF was readily reactivated by 2-PAM, while enzyme inhibited PBPDC could not be reactivated. Conjugates were prepared of both PBEPC and PBPDC with AChE, each containing one molecule of florophore per catalytic subunit. Thus two stoichiometric conjugates, PBEP-AChE (non-aged) and POBP-AChE (aged), were obtained. The two complexes exhibited identical absorption spectra, but differed in their steady-state fluorescence spectra. Although the wave-lenths of the excitation and emission spectra were similar, the pyrene fluorescence of the non-aged conjugate was ca. 50% quenched relative to the aged conjugate. Nanosecond fluorescence decay studies revealed two principal lifetime components of pyrene fluorescence. Both were longer for the aged (PBP-AChE) than for the non-aged (PBEP-AChE) conjugate and revealed a ca. 50% lower quantum yield for the non-aged as compared to the aged conjugate. A possible interpretation for these results is that in the aged conjugate the organophosphoryl moiety is less acessible to the external medium. Measurement of quenching of pyrene fluorescence in the aged and non-aged conjugates by the peripheral anionic site ligand propidium also indicated marked conformational differences between the two conjugates, and circular polarization of luminescence measurements revealed that propidium itself induced a substantial conformational change in both conjugates. Fluorescence lifetime measurements revealed that whereas propidium had little effect on the decay parameters for the non-aged conjugate it caused a decrease in lifetime and in relative quantum yield for the aged conjugate. PBEPF virtually eliminated cholinesterase activity in dissociated cord and brain cultures. Fluorescence microscopy reveals fine green fluorescent grains distinctly located throughout many neurons and glia. Labelling is much more pronounced in larger and older neurons. No specific fluorescence could be detected in cultures preincubated with nonfluorescent organophosphates.
D000818:Animals; D001665:Binding Sites; D002800:Cholinesterase Inhibitors; D002802:Cholinesterases / Q000032:analysis*; D004557:Electric Organ / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008163:Luminescent Measurements; D009474:Neurons / Q000201:enzymology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D002800:Cholinesterase Inhibitors; D005456:Fluorescent Dyes; D008024:Ligands; D009943:Organophosphorus Compounds; D002802:Cholinesterases