Properties of PBCV WAL (wall associated) lysin

Properties of PBCV WAL (wall associated) lysin

Hypothesis was lysozyme-like enzyme which bound to cell walls

Original purification protocol (dissociate from "wall fraction" (cell residue) with high salt)
(compare isolation procedure to that of ribosomal initiation factors)
WAL lysin acts on cells of all endosymbiotic Chlorella (and a few others), but not on all Chlorella

This contrasts with intact virions which are host specific

pH optimum around neutral

Can be purified and stored @ pH 4.5

Requires moderate salt concentration

Stable in the fridge (months), loses activity above 50C

Stimulated by divalent cations, calcium and strontium work well

Resistant to PMSF, DTT, IAm, hydrogen peroxide, borate, bisulfite

Inhibited by polycations, sarkosyl, EGTA, diethyl pyrocarbonate, proteases, antibody to PBCV

Fails to hydrolyze a collection of umbelliferyl sugars

Alginate lyase is the only commercial enzyme with (weak) lysin activity

Lysin doesn't hydrolyze alginate (Chlorella walls are the only lysin substrate)

Salt activates lysin in PBCV virions - viscosity indicates some DNA release

Conditions (high salt) which activate lysin in virions resemble those which extract lysin from "wall".

Protein composition of lysin preps ("WAL lysin" enriched in virions, not in cell residue)

Native gels of lysin preps

Gel diffusion assay of lysin - Overlaying native gels (above) on Chlorella agarose shows no digestion

Protamine sulfate effectively removes DNA from lysin preps

Gel filtation of lysin prep (comes off in void volume of G200)

Gel filtration of most recent prep

Lysin components do not bind detectably to cell walls