Choline acetyltransferase from a temperature-sensitive mutant of caenorhabditis elegans.
A temperature dependent paralytic mutant of C. elegans was isolated and mapped to be an allele of the cha-1 gene that has been shown to be the structural gene for acetyl-CoA: choline-O-acetyltransferase (EC 2.3.1.6; ChAT) (Hosono et al., J. Exp. Zool.235, 409-421, 1985; Rand and Russell, Genetics106, 227-248, 1984). In crude extracts from the mutant, ChAT activity was present when assayed at a permissive temperature but not detectable at a temperature that provoked abnormal phenotypes. The mutant ChAT was purified to a specific activity of 2.9 nmol of product min (-1) per mg of protein at 10 degrees C and its enzymatic properties were studied by comparison with the wild-type enzyme. The temperaturesensitivity of the mutant ChAT was so remarkable that no activity was detected over 20 degrees C. This inactivation at higher temperature appeared to be partly reversible. The Km values of the mutant enzyme for choline and acetyl-CoA were about twice of those in the wild-type enzyme, but increased 10- to 20-fold in the presence of high salt concentrations. The mutant enzyme was also more sensitive to sulfhydryl reagents. These findings indicate that depending upon changes in the physical environment, the mutant ChAT may lose the normal-conformation leading to inactivation.
Sassa
T
T
Department of Biochemistry, School of Medicine, Kanazawa University, Kanazawa, Ishikawa 920, Japan.
Hosono
R
R
Kuno
S
S