Partial Purification And Characterization Of Acetylcholinesterase From Adult Onchocerca Volvulus

ABSTRACT

(' V ) Acetylcholinesterase (AChE) from adult Onchocerca volvulus was extracted in either 5OmM Tris-HCl, containing Triton X-100 (detergent buffer) or plain 50mM Tris-HCl (lowsalt buffer) Gel chromatography on Sephacryl S-300 column, of both the detergent buffer and the low-salt buffer extracts gave two components each./ The two components of the detergent buffer gave purification factors of 12 and 13 folds and yields of 22 and 23% respectively; those of the low-salt buffer had purification factors of 5 and 9 folds and yields of 11 and 19% respectively The two components of the detergent buffer extract had sedimentation coefficient values of 9.5S and 18S while those of the low-salt buffer extract were 11.3S, the major component in terms of activity, and >38S, the minorcomponent . The enzyme showed a marked preference for acetylthiocholine compared to butyry 11hioc-ho 1 ine and it was also inhibited by high concentration of the former substrate. Moreover, the enzyme was inhibited by the general cholinesterases inhibitor, eserine Michae1is-menten constant (Km) of 0 .22mM and 0.28mM and '/max values of 2.7 x 10”3 and 2.5 x 10-3 nmole thiocholine min-1 , were found for the respective components of the detergent buffer extract. The major component of the low-salt buffer- extract gave a Km value of 0 34mM and Vmax value of 1 9 x 10~3 umole thiocholine min " 1 (Vi) The two components of the detergent buffer extract had a molecular weight of 525 KD and 375 KD on Sephacryl S-300; >600 KD and 400 KD were the values obtained for the low-salt buffer extract. In contrast, polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions revealed molecular weight species of 560 KD, 355 K D , 290 KD and 67 KD for the enzymes in the detergent buffer extract; those of the low-salt buffer extract were 500 KD, 280 KD, 220 KD and 58 K D . The crude enzyme was activated by Mg+ + , Mn-+ and Ca++ at concentrations ranging from 10~4M to 10~2M. Mg++ at similar concentrations also activated the purified enzymes. Co++ moderately inhibited the crude enzyme at 10-2M . The enzyme showed maximal activity at pH 8.0 in three buffer systems. Veronal and phosphate buffers were activating. The results indicate that adult O. volvulus AChE is similar to the mammalian AChE and AChE of other nematodes in its catalytic properties.