The choline-containing lipoteichoic acid (LTA, Forssman Antigen) of Streptococcus pneumoniae suppresses the activity of the pneumococcal autolysin, an N-acetyl-muramoy-L-alanine-amidase (amidase) in aqueous solution [Höltje and Tomasz (1975) Proc. Natl Acad. Sci. USA 72, 1690-1694]. The interaction between LTA and enzyme was used to establish a purification by affinity chromatography on LTA-Sepharose. The amidase could be eluted from the column with choline only.This implies that binding of the enzyme to LTA is mediated via the choline residues of the LTA. Upon binding to the LTA-Sepharose, the amidase converted from the applied E-form (an inactive from of the amidase) to the active C-form, a process which up to now was known to be mediated only by the pneumococcal choline-containing wall teichoic acid.
Similar interactions between LTA and amidase seemed to occur in membrane fractions derived from cholinegrown cells: the membrane-associated enzyme was present in the C-form and could be detached completely with choline, suggesting that the amidase is bound to the membrane attached LTA rather than being a membrane protein itself. This was supported by the absence of amidase of amidase activity in membrane fractions derived from ethanolamine-grown pneumococci, in which choline containing LTA is absent.
The LTA-Sepharose-associated amidase was not inhibited, but retained its activity. The enzyme was also not inhibited by lipase-digested LTA. Both are conditions where the LTA is not present in micelles, unlike in aqueous solution. Therefore, mere binding to the LTA is probably not responsible for the inhibitory effect, but inhibition is a manifestation of an inaccessibility of the substrate for the amidase when bound to micellar LTA.
When the interactions between choline and amidase were investigated, it was found that high choline concentrations (2%) inhibited the enzyme completely. Even in vivo, 2% choline in the culture medium led to phenotypically amidase-deficient pneumococci. Furthermore, in vitro, low choline concentrations (0.1%) suppressed the wallmediated conversion. On the other hand, with high choline concentrations (2%) conversion took place in the absence of cell walls. Depending on how the amidase has been converted, the apparent Mr of the resulting C-amidase was different: the cell-wall-converted enzyme was of high Mr, Whereas the choline-converted and the LTA-Sepharose-eluted enzyme showed an apparent low molecular mass known for the E-form, when analyzed on sucrose gradiens. Prolonged dialysis of the low molecular mass C-amidase resulted in ‘re-conversion’ to the inactive E-form, suggesting that the activity of the C-form is dependent on enzyme-attached choline molecules.
The interactions between choline and amidase led to an improved affinity chromatography system for the enzyme on choline-Sepharose.