Alkaline treatment of muscle microsomes releases amphiphilic and hydrophilic forms of acetylcholinesterase.
To obtain information about the mode of attachment of amphiphilic monomers of acetylcholinesterase (AChE) in sarcoplasmic reticulum (SR) of skeletal muscle, attempts were made to release the enzyme by alkaline hydroxylamine. About half of the activity measured in microsomes preincubated with 0.5% (w/v) Triton X-100 is detached by incubation of SR with bicarbonate buffer (pH 10.5). Addition of 1 M hydroxylamine to the alkaline buffer did not improve enzyme solubilization. Molecular forms of 16S (A12), 10.5S (G4) and 4.0S (G1) are separated by sedimentation analyses of Triton X-100 or bicarbonate-solubilized AChE. Monomeric AChE, released under alkaline conditions (G1A), displays amphiphilic properties. G1A, but not G4 and A12, forms are retained in a phenyl-Sepharose column and this allows its separation from hydrophilic forms. Isolated monomers extracted with Triton X-100 (G1D) or alkaline buffer showed identical kinetic behaviour. The two forms reacted with lectins in a similar manner. However, thermal inactivation experiments revealed that about 90 and 40% of the activity in the G1D and G1A forms were lost by heating at 50 degrees C, following the same rate constant (k = 0.130 min-1). Addition of Triton X-100 to the G1A form leads to an increase of its thermal sensitivity, the enzyme being fully inactivated very rapidly (k = 0.230 min-1). The results suggest that the hydrophobic moiety of the enzyme might be exposed or hidden depending on the environmental hydrophobicity. Changes in the composition of the solvent will determine the final conformational state of the protein.
Moya-Quiles
M R
MR
Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Murcia, Spain.
Villalba-Sánchez
J
J
Muñoz-Delgado
E
E
Vidal
C J
CJ
D000110:Acetylcholinesterase / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013816:Thermodynamics
D002800:Cholinesterase Inhibitors; D006898:Hydroxylamines; D007527:Isoenzymes; D019811:Hydroxylamine; D000110:Acetylcholinesterase