The absolute activity of choline-esterase

Abstract

The present investigation has been rendered possible by the results obtained in two separate, and at first apparently disconnected, fields of enquiry. One of us (Stedman, 1926), in a study of the relationship between chemical constitution and physiological action, had ascertained the molecular arrangement which was responsible for the characteristic pharmacological activity of physostigmine and, partly in conjunction with collaborators (1929-1931), had used this discovery to synthesize and investigate a number of compounds possessing physiological activities similar to that of physostigmine. During the same period, Loewi and Navratil (1926), in pursuing the well-known work of the former author on the humoral transmission of nerve action, discovered that small but pharmacologically active amounts of acetyl choline, as well as of the so- called vagus substance, underwent enzymic decomposition when treated with extracts of certain tissue, and that this decomposition was inhibited by the addition to such extracts of physostigmine, which discoveries were subsequently confirmed and extended by Engelhart and Loewi (1930), Matthes (1930), and other investigators. It thus became evident that the synthetic drugs should share with physostigmine the property of preventing the destruction of acetyl choline by blood and tissue extracts, provided this property was responsible, wholly or in part, for the physio­ logical activity of the alkaloid. Another problem was, however, involved. No evidence was available as to the identity of the enzyme concerned in the process. It appeared probable that the destruction of acetyl choline was brought about by a simple esterase such as is known to exist in the liver, in blood serum, and in other tissues; nevertheless, the possibility remained that the effect was due to a specific enzyme. Stedman and Stedman (1931 b , 1932) attempted to solve this problem indirectly by examining the influence of their synthetic drugs upon various ester­hydrolysing enzymes, and were able to demonstrate that, while these substances exerted a marked inhibitory action on liver and serum esterases, they were without such influence on other ester-hydrolysing enzymes such as pancreatic lipase and kidney phosphatase. The drugs appeared to exert a specific inhibitory effect on simple esterases, and to this extent the experiments supported the view that these simple esterases were respon­sible for the destruction of acetyl choline originally observed by Loewi and Navratil. When, however, Stedman, Stedman, and Easson (1932) developed a technique for following the hydrolysis of acetyl choline by chemical methods, it was found that liver esterase was entirely without action on this ester, although the presence in horse serum of an enzyme which brought about its hydrolysis could be readily demonstrated. These and other results, in particular the purification of the enzyme, clearly proved that the hydrolysis of acetyl choline was brought about by a specific agent. To this new enzyme, which also catalyzes the hydrolysis of butyryl choline, the above-mentioned authors gave the name of choline-esterase. From the foregoing account of the relationship between Loewi’s work and that of the present authors, it will be clear that physostigmine and the analogous synthetic drugs will not only inhibit liver esterase but will also exert a similar action on choline-esterase. Experimental verification of this was, in fact, made in this laboratory shortly after the discovery of the specific nature of choline-esterase, but publication was postponed until the qualitative results could be supplemented by quantitative ones. When such a detailed examination of the drugs was commenced, their inhibitory activity towards choline-esterase was found to be much greater than towards liver esterase. The amount of inhibitor required to produce a large inhibition of the enzyme proved, in fact, to be so minute that there appeared to be a possibility of utilizing this property of the drugs for determining the absolute rate of hydrolysis of a choline ester by choline-esterase, a possbility which has been developed in the present investi­gation.