Puromycin and cycloheximide as inhibitors of human brain acetylcholinesterase

Brain Research, 86 (1975) 339-342

339

© Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands

Puromycin and cycloheximide as inhibitors of human brain acetylcholinesterase

RONALD ZECH AND GOETZ F. DOMAGK Physiologisch-chemisches Institut der Universitiit, D-34 G6ttingen ( G.F.R.)

(Accepted December 3rd, 1974)

Puromycin and cycloheximide are the 'classical' inhibitors of protein biosynthesis. They have frequently been used to demonstrate protein synthesis being essential for responses of or effects on biological systems. Amongst others Agranoff1, Barondes and Cohen z, and Flexner~ have used metabolic inhibitors such as puromycin or cycloheximide to study their influence on learning and the storage of learned information. It was shown that the inhibition of protein synthesis by either antibiotic did not interfere with learning, but prevented the long-term storage of the acquired information. Therefore, it was concluded that long-term memory was accompanied by protein or peptide formation. An effect of puromycin on acetylcholinesterase activity was observed by Burkhalter3 in chicken muscle organ cultures. He could not find an enzyme inhibition by puromycin, but assumed an inhibition of acetylcholinesterase biosynthesis. Moss et al. s first described a puromycin dependent inhibition of acetylthiocholine hydrolysis by human or bovine erythrocyte membranes (red cell ghosts). Since the kinetics of acetylcholinesterase show marked differences between enzymes from various organs and species, and with the substrate and incubation system used, we were interested in the inhibition of brain acetylcholinesterase by puromycin and cycloheximide. The enzyme was isolated by the method of Morton v from human corpus striatum, sampled 12 h post mortem. Acetylcholinesterase was measured by an automatic titration method and by the thiocholine method of Ellman4. The inhibition constants were calculated according to Krupka 6, Kt being the dissociation constant of the enzyme-inhibitor complex El (reversible, competitive type of inhibition) and K~' being the dissociation constant of the acetylated enzyme-inhibitor complex E-AcI (reversible, non-competitive type of inhibition). The effect of puromycin on acetylcholine hydrolysis by human brain acetylcholinesterase is shown in Fig. 1. A purely non-competitive type of inhibition is seen from the Michaelis plot, the Lineweaver-Burk plot and Dixon plot. The same type of inhibition is found with the Ellman method for acetylthiocholine hydrolysis, even though the kinetic constants are different. The data are listed in Table I. The different

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Fig. 1. Acetylcholine hydrolysis by human brain acetylcholinesterase in the presence o f puromycin (molar concentrations indicated on the curves). A: Michaelis plot. B: Lineweaver-Burk plot. C: D i x o n plot.

kinetic data were shown not to be due to the different substrates (acetylcholine and acetylthiocholine), since both substrates give identical values when tested with the automatic titration method. The difference is due to the redox state of the acetylcholinesterase; the Ellman reagent used in the thiocholine method oxidizes the SHgroups of the acetylcholinesterase, causing an increase in the substrate affinity and a marked decrease of the maximum activity. The degree of inhibitor binding is less affected. Cycloheximide also inhibits human brain acetylcholinesterase in a reversible TABLE I K I N E T I C C O N S T A N T S OF P U R O M Y C I N

AND CYCLOHEXIMIDE INHIBITION OF HUMAN

BRAIN ACETYLCHOLIN m

ESTERASE

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Substrate

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Non-competitive inhibitor constant K (

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Km

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Fig. 2. Acetylcholine hydrolysis by human brain acetylcholinesterase in the presence of cycloheximide. A: Michaelis plot. B: Lineweaver-Burk plot.

purely non-competitive way, as is shown in Fig. 2. The binding of this inhibitor is not as tight as with puromycin; the dissociation constants are listed in Table I. Moss s found an apparent I~0 (concentration which causes 50 ~ inhibition at 10-4 M substrate concentration) for puromycin and red cell acetylcholinesterase of 1.26 x 10-4 M, the type of inhibition being a mixed competitive-non-competitive one. Inhibitor constants were not determined. With purified acetylcholinesterase from erythrocytes we again found a mixed type of puromycin inhibition; the competitive inhibitor constant, K,, was 2.2 x 10-4 and the non-competitive inhibitor constant, K/, was 4.3 x 10-4. Since little is known about the function of red cell acetylcholinesterase, these findings are of minor significance. In contrast, the type of inhibition of the brain acetylcholinesteraseis of great importance. A reversible non-competitive inhibition can only be partly overcome by high substrate concentration, for instance by accumulated acetylcholine at the synaptic junction. The inhibitor concentrations in the brain, which blocked long-term memory, estimated from ref. 2, were about 1 x I0 -a M for puromycin and 1.6 x 10-s M for cycloheximide, but nothing is actually known about the local concentration of these antibiotics at the synapses. An extensive inhibition of brain acetylcholinesterase might well have occurred in addition to the observed block of amino acid incorporation into brain proteins. According to Podleski and Nachmansohn9 the acetylcholine receptor protein in the postsynaptic membrane has some properties very similar to acetylcholinesterase and so puromycin and cycloheximide could possibly have reacted with the acetylcholine receptor in addition to the attack on cholinesterase shown above. This work was supported by SFB 33 (Deutsche Forschungsgemeinschaft) and by Bundesamt ffir Zivilschutz.

1 AGRANOFF,B. W., Memory and protein synthesis, Sci. Amer., 216 (1967) 115-122. 2 BARONDE~,S. H., AND COHEN, H.n., Comparative effects of cycioheximide and puromycin on cerebral protein synthesis and consolidation of memory in mice, Brain Research, 4 (1967) 44-51.

342 3 BURKHALTER,A., Effect of puromycin on cholinesterase activity in embryonic chick intestine in organ culture, Nature (Lond.), 199 (1963) 598-599. 4 ELLMAN, G . L . , COURTNEY, K. D., ANDRES, V., AND FEATHERSTONE,R. M., A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. 5 FLEXNER, L. B., Dissection of memory in mice with antibiotics, Amer. Scientist, 56 (1968) 52-57. 6 KRU~'KA, R . M . , Acetylcholinesterase: trimethylammonium inhibition of deacetylation, Biochemistry, 3 (1964) 1749-1754. 7 MORTON, R. K., Methods of extraction of enzymes from animal tissues. In S. P. COLOWlCK ANt) N. O. KAPLAN(Eds.), Methods in Enzyrnology, Vol. 1, Academic Press, New York, 1955, pp. 25-5 I. 8 Moss, D. R., Moss, D. E., AND FAHRNEV, D., Puromycin as an inhibitor of acetylcholinesterase, Biochim. biophys. Acta (Amst.), 350 (1974) 95-99. 9 POOLESKX,T. R., AND NACHMANSOHN,D., Similarities between active sites of acetylcholine receptor and acetylcholinesterase tested with quinolinium ions, Proc. nat. Acad. Sci. (Wash.), 56 (1966) 1034-1039.