Biochemical Characterization of an Acetylcholine-hydrolyzing Enzyme from Bean Seedlings

Abstract

An acetylcholine hydrolyzing enzyme was prepared and purified (40 times) from dwarf bean hypocotyl hooks. The purity of the enzyme was proved by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined to be 65,000 daltons. Enzyme activity was the highest at pH 8.0 and between 30 and 36 C. The enzyme had an apparent affinity constant (Kin) for acetylcholine of 460/micromolar. The affinity for substrate analogs increased from butyrylthiocholine to propionylthiocholine to acetylthiocholine. The enzyme activity was inhibited by choline, neostigmine, physostigmine, manganese, and calcium. Magnesium had no influence on the enzyme activity. We conclude that the enzyme from dwarf beans is an acetylcholinesterase (EC 3.1.1.7).Acetylcholine is one of the several naturally occurring esters of choline in animals and plants. Although its only well understood function is that of a neurotransmitter (25) it is found in nonnervous tissues of multicellular animals, protozoa, bacteria, and all plants so far investigated (1 1, 17). The physiological function of acetylcholine in plants and other nonnervous organisms remains unclear and controversial. Besides the transmitter function of acetylcholine in the nervous systems of animals there are many other reactions of acetylcholine in nervous or nonnervous tissues of many organisms which are not understood. To overcome this ignorance it is necessary to investigate the pathways of synthesis and degradation, especially in plant tissues. Riov and Jaffe (27) isolated the enzyme cholinesterase (EC 3.1.1.8) from mung bean roots (Phaseolus aureus, Roxb.), Kasturi and Vasantharajan (21) identified it in peas (Pisum sativum L.), and Wettlauffer (29) isolated it from etiolated hypocotyl hooks of dwarf beans (Phaseolus vulgaris L.).We report data which characterize an acetylcholine-hydrolyzing enzyme from green hypocotyl hooks of dwarf beans. MATERIALS AND METHODSDwarf beans (P. vulgaris, var. St. Andreas, erste Ernte, grunhulsig) were grown in a constant temperature room at 20 C ± I C, in plastic tanks covered with a glass plate. The substrate (moist Vermiculite) was sterilized and the light conditions were 16 h red light (Philips fluorescent tubes TL 40 W/ 15 with Plexiglas filter, Rohm No. 501 red, 4.8 w m 2) and 8 h dark. The dwarf bean seedlings were harvested after 7 days and the hook part excised above the cotyledons.The hooks were homogenized in 10 mm K-phosphate (buffer A, pH 7.0) using 4 ml/g fresh weight, with an Ultra-Turrax 'This work was supported by a grant from the Deutsche Forschungsgemeinschaft.homogenizer. After stirring for 30 min the homogenate was filtered through two layers of a 55-[Lm mesh nylon net. The residue was resuspended in 2 volumes (w/v) of 10 mm K-phosphate and 4% ammonium sulfate (buffer B, pH 7.0) and stirred for I h. It was then filtered again through a 55-,um mesh nylon net. The filtrate, which contained the solubilized enzyme, was centrifuged for 30 min at 40,000g. Ammonium sulfate was added to the supernatant fluid to...

Cholinergic constituents in plants: Characterization and distribution of acetylcholine and choline

“…Although cholinergic systems have not been identified in higher plants, two of these four components have been established. AChE has been found to be ubiquitous in plants (Riov and Jaffe 1973, Fluck and Jaffe 1974b, Kasturi and Vasantharajan 1976, Mansfield et al 1978, Ernst and Hartmann 1980, Gupta and Maheshwari 1980, Miura et al 1982. Choline acetyltransferase has been detected in a number of plants (Barlow and Dixon 1973, Hadacova et al 1981, Smallinan and Maneckjee 1981.…”

Acetylcholinesterase from oat Seedlings. I. Preliminary biochemical characterization of the enzyme

“…Like in many other cases (Riov and Jaffe 1973, Kasturi and Vasantharajan 1976, Ernst and Hartmann 1980, Gupta and Maheshwari 1980, this enzyme was inhibited much more effectively by neostigmine than by eserine. Its activity was also inhibited by Ca 2 § (Ernst and Hartmann 1980). Similarly to other plant ChE the affinity of oat enzyme for substrate analogs increased from BTCh to PTCh to ATCh (Riov and Jaffe 1973, Ernst and Hartmann 1980, Gupta and Maheshwari 1980, Roshchina 1988) and its highest activity was between 30 and 36~ (Kasturi andVasantharajan 1976, Ernst andHartmann 1980).…”

“…Oat cholinesterase, however, in some biochemical properties, is similar to other plant ChE. Like in many other cases (Riov and Jaffe 1973, Kasturi and Vasantharajan 1976, Ernst and Hartmann 1980, Gupta and Maheshwari 1980, this enzyme was inhibited much more effectively by neostigmine than by eserine. Its activity was also inhibited by Ca 2 § (Ernst and Hartmann 1980).…”

“…5). Also choline which stimulates (Riov andJaffe 1973, Vackov~ et al 1984) or inhibits (Ernst and Hartmann 1980) ChE from different plant species was without influence on oat enzyme. Furthermore, the oat enzyme differs from all the other ones (Riov and Jaffe 1973a,b, Kasturi and Vasantharajan 1976, Ernst and Hartmann 1980, Vackovfi et al 1984 in having a comparatively low optimum pH, 7.2.…”

“…Also choline which stimulates (Riov andJaffe 1973, Vackov~ et al 1984) or inhibits (Ernst and Hartmann 1980) ChE from different plant species was without influence on oat enzyme. Furthermore, the oat enzyme differs from all the other ones (Riov and Jaffe 1973a,b, Kasturi and Vasantharajan 1976, Ernst and Hartmann 1980, Vackovfi et al 1984 in having a comparatively low optimum pH, 7.2. This is important in so far as the optimum pH obtained in other experiments lies within the pH range in which both ACh and ATCh are unstable (Tretyn and Kendrick 1991) and become rapidly nonenzymatically hydrolyzed.…”