On the deacetylase activity of Vi bacteriophage III particles.

1. Using the complete phage particles as an enzyme, O-acetyl (1 leads to 4)-alpha-D-galacturonan (acetylated pectic acid) as a substrate, and gas-liquid-chromatography for the determination of the acid liberated, the virus-catalysed deacetylation of the polymer was studied. The activity was found to be stable up to about 50 degrees C, and from pH 4.5 to 9, with an optimum at pH 7.8; it was not affected by EDTA, or by 1,10-phenanthroline. The initial reaction velocity (at 37 degrees C) exhibited a simple hyperbolical dependence on the substrate concentration, with Km = 10.5 mM for O-acetyl (independent of virus concentration), and Vmax = 15 nmoles/min and 10(10) plaque forming units. The reaction was, however, rapidly inhibited by a partially deacetylated product (but neither by acetate, nor by pectic acid itself). 2. Using the natural substrate, acetylated (1 leads to 4)-2 amino-2-deoxy-alpha-D-galacturonan (Vi polysaccharide, Vi antigen), and a variety of structural analogues, the following conclusions about the substrate specificity of the Vi phage III deacetylase (acetyl-alpha-1,4-galacturonan acylhydrolase) were reached: (a) acetylated galacturonan is as good a substrate as acetylated aminogalacturonan; (b) of the two substrate diastereomers, acetylated alpha-L-guluronan (also 1 ax leads to 4 ax-linked units, but with axial acetyl residues at C-3), and beta-D-mannuronan (1 eq leads to 4 eq-linkages, and axial acetyl groups at C-2), only the former was acted upon, possibly indicating a specificity for the conformation of the polymer rather than for the configuration of the single residues; (c) all acyl analogues tested, O-monofluoroacetyl, O-propionyl, and O-butyryl galacturonan, were inert, showing a high degree of specificity for O-acetyl; (d) the oligomers, acetylated tri- and digalacturonic acid, as well as methyl-alpha-D-galacturonide, were still deacetylated, although more slowly, demonstrating tolerance of the enzyme of substrate size.

Disruption of Vi bacteriophage III and localization of its deacetylase activity.

It has been shown that particles of Vi bacteriophage III catalyse deacetylation of O-acetyl pectic (polygalacturonic) acid, a structural analogue of Vi polysaccharide (Vi antigen). Using this substrate, and determining the acetic acid liberated by gas-liquid chromatogrphy, a method for the estimation of Vi phage deacetylase activity has been developed. Purified particles of Vi phage III were exposed to a variety of mildly dissociative reagents and conditions, and then tested for plaque-forming and for deacetylase activity. They have also been inspected under the electron microscope. Osmotic shock, and incubation in the presence of ethylenediamine tetraacetic acid (greater than or equil 0-01 M), or of L-arginine (0-25 M), were found to cause disintegration of the virions into empty head capsids, deoxyribonucleic acid, and base plates still carrying the spikes. The mixtures of viral fragments exhibited an increased deacetylase activity. Using zonal sedimentation and ion exchange chromatography, the phage fragments obtained by treatment with ethylenediaminetetraacetic acid have been fractionated and the base plates isolated. Amongst the viral components, these structures showed the highest specific deacetylase activity. They had the shape of six-pointed stars (about 9-5 nm inner, and 14-5 nm outer diam.) with a central hole or plug (approximately 3 nm), carrying six spikes, roughly cylindrical organelles of approx. 11 X 4 nm, one at each of the points. Of the polypeptides of six sizes (P.1, about 153,000 daltons; P.2, 91,000; P.3, 71,000; P.4 56,500; P.6, 22,000), detected in whole Vi phage III virions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, only two, P.2 and P.3 were found in the base plates.