Purification and Properties of the Inducible Cholinesterase of Pseudomonas Fluorescens (Goldstein)

Abstract

The inducible cholinesterase produced by the Goldstein strain of 3Pseudonzonas $uorescelzs was purified t o a state of electrophoretic homogeneity. The enzyme, which resembles a n acetylcholinesterase in its s~abstrate specificity, has a high affinity for acetylcholiale a i d propionylcholine. The estinaated values of K, a t pH 7.4 and 37 "C are 1.4 X 10-6 11.1 for acetylchoIiile and 2.0 X llf for propicanylchdine. The bacterial cholinesterase reacts very slowly with tetraethyl pyrophosghate (TEPP) and diisopropyl phosphorofluoridate (DFP) but coinparatively rapidly with ethyl N,N-dimethylphosphoran1idocyanidate (Tabual). Birnolecnlar rate constants range from 7.7 mol-I min-l for TEFP to 7.4 X lo4 mol-1 min-1 for Tabun. The reactions of the chdinesterase depend upon the ionic state of groups ill the enzyme whose pK, values are in the same range as those reported for other esterases. The results suggest that the enzyme inay be similar in structure t o other cholinesterases, and that both histidine and serine may be involved in its activities.

Acetylcholinesterase from rat red cells and cholinesterase ofPseudomonas aeruginosa: Different types of inhibition by atropine

“…The cholinesterase of Pseudomonas aeruginosa showed a substrate saturation curve similar to other cholinesterases, either animal (6) or bacterial (3,5). Inhibition was observed when the substrate was in excess.…”

“…However, little attention was focused upon bacterial cholinesterase, which was originally described by Goldstein (1) in Pseudomonasfluorescens. Cholinesterase activity was also demonstrated in Pseudomonas aeruginosa (2,3)which, apparently has different properties from those described for the enzyme isolated frOm Pseudomonasfluorescens (3)(4)(5). Accord.ing to Fitch (4) the cholinesterase activity of Pseudomonasfluorescens was not inhibited by atropine,~ which was shown as an inhibitor of animal cholinesterases (6,7).…”

Localization of Cholinesterase in Pseudomonas aeruginosa Strain K

“…The ChE of P. aeruginosu strain K resembles that of P.Jluorescens (Laing et al, 1967) and P. aeruginosa strain A-16 (Tani et al, 1975a, 6 ) in its activity on several choline esters (except for a somewhat higher activity on propionylcholine) and in its inhibition by an excess of acetylcholine. The latter property and the ability of the bacterial ChEs to decompose acetyl-P-methylcholine indicate their similarity to brain (Myers, 1953) and erythrocyte (Myers, 1952) acetylcholinesterases as opposed to human blood plasma pseudo-cholinesterase (Myers, 1952(Myers, , 1953.…”

“…The bacteria were disintegrated by passage through a French pressure cell at 136 MPa and, after centrifugation at 16300g for 30 min, the supernatant was used as the crude enzyme preparation. The total activity of ChE in this preparation was 7.4 units with a specific activity of 0-009 units (mg protein)-l. Partial purification of the ChE was obtained by the following procedure (Laing et al, 1967;Gilboa-Garber et al, 1973): removal of nucleic acids with 0.7% (w/v) streptomycin sulphate at 0 "C, precipitation of foreign proteins by 40% saturation with (NH4),S04 and by acidification to pH 5 with 2 Macetic acid and concentration of the ChE by 60% saturation with (NH,),SO, at pH 7-5 following overnight dialysis against 0.1 M-Tris/HCl buffer pH 7.5 at 4 "C. The specific activity of the partially purified enzyme (670/, yield) was 0.084 ChE units (mg protein)-' (9.33-fold enrichment).…”