Michael P. Ready

Dodecandrin, a new ribosome-inhibiting protein from Phytolacca dodecandra

Dodecandrin, a newly discovered ribosome-inhibiting protein, has been isolated and purified from the leaves of the African endod plant, Phytolacca dodecandra. Dodecandrin has a molecular weight of approx. 29 000. It cross-reacts with antiserum prepared against pokeweed antiviral protein from Phytolacca americana and exhibits similar requirements for antiribosomal activity. It is more basic than pokeweed antiviral protein, and comparison of the first 30 amino-terminal residues of the two proteins reveals 83% homology. This level of homology is greater than that between pokeweed antiviral protein and pokeweed antiviral protein S, another antiviral protein found in P. americana. Such conservatism in sequence, coupled with the high efficiency of the proteins in deactivating ribosomes and with their abundance in plant tissue, suggests that they serve an important function in the life of the plant, probably as a defense against infection.

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K+ and 3 mM Mg2+ retards the reaction, while 20 mM K+ and 2 mM Mg2+ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300-400 mol/mol per min. in each case. The Km for ribosomes is 1 microM in the presence of low salt and 0.2 microM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pKa value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.

Crystallization of an endochitinase from Hordeum vulgare L. seeds

Higher plants contain several constitutively expressed proteins for protection against infections by viruses, bacteria and fungi. Here we report the crystallization of a polypeptide with antifungal activity, a 26,000 dalton endochitinase from barley (Hordeum vulgare L.) seeds, in a form suitable for high-resolution X-ray analysis. Crystals were grown by vapor diffusion under several different conditions. The best crystals, obtained with ammonium sulfate as the precipitant, belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2), with cell dimensions a = b = 62.9 A and c = 96.0 A. The cell dimensions are consistent with one endochitinase molecule per asymmetric unit, and the crystals diffract to at least 2.0 A resolution.