Abstract
Purification of the pneumococcal N-acetylmuramyl-L-alanine amidase to biochemical homogeneity.
Highlights
MATERIALS AND METHODSDiplococcus pneumoniae R 36 A was grown at 37” in a synthetic medium (Cd,.) containing either choline or ethanolamine as described previously [11]
Participation of the bacterial peptidoglycan hydrolases in several important physiological phenomena has been suggested by several authors (l-4)
In the case of the pneumococcal autolysin, an N-acetylmuramyl+alanine amidase, experimental evidence has been obtained for the key role this enzyme plays in the penicillin-induced lysis and death of pneumococci
Summary
Diplococcus pneumoniae R 36 A was grown at 37” in a synthetic medium (Cd,.) containing either choline or ethanolamine as described previously [11]. One unit of amidase activity (U) was defined as the amount of enzyme that catalyzes the hydrolysis (solubilization) of 1 pg of cell wall material in 10 min. Under standard assay conditions enzyme activity was determined by measurement of the amount of radioactivity that was solubilized from [aH]choline cell walls after preincubation at 0’ for 5 min [12] and incubation at 37” for 10 min. The 220 gl reaction mixture contained: 10 ~1 of [8H]choline cell walls (0.15 mg dry weight/ml; lOa cpm/ml) and 10 pl of enzyme (about 0.5 unit) in 200 pl of 0.05 M Tris-maleate buffer, pH 6.9. 10 ml of toluene-based scintillation fluid was added and the samples were counted as described above. Procedures for the preparation of cell walls and lipoteichoic acid have been described previously [12, 15]
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