Hydrolysis of the 5'-p-nitrophenyl ester of TMP by oligoribonucleases (ORN) from Escherichia coli, Mycobacterium smegmatis, and human.

Escherichia coli oligoribonuclease (EcoORN), encoded by the orn gene, is a 3'-5' exonuclease that degrades short single-stranded oligoribonucleotides to rNMPs in the final step of RNA degradation. The orn gene is essential in E. coli, but not in higher organisms, and close homologues are present in other genomes from the beta and gamma subdivisions of the Protobacteriaceae, including many pathogenic species. We report here the expression in E. coli of orn and homologues from Mycobacterium smegmatis and human, and large-scale purification of the three enzymes. All three were found to promote the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of Michaelis-Menten parameters (k(cat)=100-650 min(-1), K(M)=0.4-2.0 mM, at pH 8.00 and 25 degrees C, with 1 mM Mn(2+)). Hydrolysis by pNP-TMP by all three enzymes depended on a divalent metal ion, with Mn(2+) being preferred over Mg(2+) as cofactor, and was inhibited by Ni(2+). The concentration dependency of Mn(2+) was examined, giving K(Mn) values of 0.2-0.6 mM. The availability of large amounts of the purified enzymes and a simple spectrophotometric assay for ORN activity should facilitate large-scale screening for new inhibitors of bacterial oligoribonucleases.

Preliminary characterisation and extraction of anterior adhesive secretion in monogenean (platyhelminth) parasites.

Secreted anterior adhesives, used for temporary attachment to epithelial surfaces of fishes (skin and gills) by some monogenean (platyhelminth) parasites have been partially characterised. Adhesive is composed of protein. Amino acid composition has been determined for seven monopisthocotylean monogeneans. Six of these belong to the Monocotylidae and one species, Entobdella soleae (van Beneden et Hesse, 1864) Johnston, 1929, is a member of the Capsalidae. Histochemistry shows that the adhesive does not contain polysaccharides, including acid mucins, or lipids. The adhesive before secretion and in its secreted form contains no dihydroxyphenylalanine (dopa). Secreted adhesive is highly insoluble, but has a soft consistency and is mechanically removable from glass surfaces. Generally there are high levels of glycine and alanine, low levels of tyrosine and methionine, and histidine is often absent. However, amino acid content varies between species, the biggest differences evident when the monocotylid monogeneans were compared with E. soleae. Monogenean adhesive shows similarity in amino acid profile with adhesives from starfish, limpets and barnacles. However, there are some differences in individual amino acids in the temporary adhesive secretions of, on the one hand, the monogeneans and, on the other hand, the starfish and limpets. These differences may reflect the fact that monogeneans, unlike starfish and barnacles, attach to living tissue (tissue adhesion). A method of extracting unsecreted adhesive was investigated for use in further characterisation studies on monogenean glues.