The effect of solubilization on the properties and molecular forms of rat brain acetylcholinesterase.
Attempts were made to solubilize acetylcholinesterase from Wistar rat brains by extraction with dilute buffer, Triton X-100 and proteolytic digestion. About 13% of the total enzyme activity could be solubilized with 30 mM sodium phosphate buffer (pH 7) and the remainder brought into solution with 1% w/v Triton X-100. Storage of the brains in dry toluene for 3-6 months followed by extraction did not improve the yield and resulted in the loss of about half of the enzyme activity. Digestion with trypsin or collagenase was totally ineffective in solubilizing the enzyme from fresh or toluene-stored brains. The enzyme in the buffer and detergent extracts of fresh and toluene-stored brains was very stable when stored at -20 degrees C for several months although some activity was found in a 100,000 g pellet obtained by centrifugation of the thawed extracts. All enzyme preparations showed inhibition by excess substrate and an optimum substrate concentration of 2 mM acetylcholine. The Km of the crude tissue suspension was 80 microM acetylcholine while that of the buffer-soluble enzyme was 91 microM and that of the detergent-solubilized enzyme was 250 microM. Storage of the brains in toluene had little effect on these values. Starch-block electrophoresis and polyacrylamide gel electrophoresis showed up to five bands with different net charge while gradient gel electrophoresis revealed up to eleven forms with molecular weights ranging from 39,000 to 450,000. The electrophoretic pattern obtained depended on the preparation and extraction of the tissue as well as the temperature and the presence of salt, mercaptoethanol and inhibitors. Storage of the tissue in toluene does affect the yield and the properties of acetylcholinesterase obtained from rat brain thus emphasising the need to clearly define the methods and conditions of solubilization when reporting the presence of multiple molecular forms of acetylcholinesterase.
Vidal
C J
CJ
Department of Biochemistry, Chelsea College, Manresa Road, London, SW3 6LX, U.K.
Chai
M S
MS
Plummer
D T
DT