Cholinesterases from Plant Tissue

Fluck, Richard A.; Jaffe, M. J.

doi:10.1104/pp.54.5.797

Cited by 8 publications

(1 citation statement)

References 13 publications

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“…The flow rate was 18 ml/h and fractions of 3 ml were collected. To avoid overlapping with the other protein bands, we did not use all of the fractions which showed acetylcholine hydrolyzing activity (only 10 fractions, no 32-42, were used). Ammonium sulfate was added to 80% saturation to the collected fractions and they were stored overnight at 4 C. The precipitated proteins were then centrifuged for 30 min at 40,000g, the pellet redissolved in 3-5 ml buffer (depending on the protein concentration) and dialyzed for 24 h against buffer A.…”

Section: Methodsmentioning

confidence: 99%

Biochemical Characterization of an Acetylcholine-hydrolyzing Enzyme from Bean Seedlings

Ernst¹,

Hartmann²

1980

Plant Physiol.

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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...

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Section: Methodsmentioning

confidence: 99%