The Journal of Medical Microbiology, Vol 30, Issue 2 119-127, Copyright © 1989 by Society for General Microbiology
Separation and properties of the haemolysins and extracellular enzymes of Listeria monocytogenes and L. ivanovii
R. Barclay, D. R. Threlfall and I. Leighton
Department of Plant Biology, University of Hull.
Desalted ammonium-sulphate (0-65%) precipitates from the cell-free
supernates of 16-24-h cultures of Listeria monocytogenes Boldy and L.
ivanovii (previously L. monocytogenes) Type 5 were eluted through Sephadex
G-200. The enzyme activities gave rise to two main peaks. The first peak
(approximate mol. wt of protein 150,000) contained only phosphatase
activity (assayed by hydrolysis of 4-nitrophenylphosphate at pH 5.0 and
7.0). The second peak (approximate mol. wts of proteins 40,000-60,000)
contained the haemolysin activity and the following hydrolytic activities
(assay substrates are given in parentheses): phospholipase C (phosphatidyl
choline and 4-nitrophenyl-phosphoryl-choline); phosphodiesterase
(bis-4-nitrophenyl-phosphate); acid phosphatase (4-nitrophenylphosphatase);
and esterases and lipases (4-nitrophenyl acetate, naphthyl-acetate and
-oleate, triacetin and triolein). DEAE-Sephadex chromatography of
appropriate fractions from the Sephadex G-200 purification step separated
the first peak into two phosphatases and resolved the second peak into its
constituent activities. Polyacrylamide gel electrophoresis showed that the
individual fractions from the DEAE-Sephadex step consisted of mixtures of
protein. The effects of pH and potential activators and inhibitors on the
active proteins purified by DEAE-Sephadex chromatography were examined.
