No abstract

Onconase 0 (ONC) is a homolog of ribonuclease A (RNase A) that has unusually high conformational stability and is toxic to human cancer cells in vitro and in vivo. ONC and its amphibian homologs have a C-terminal disulfide bond, which is absent in RNase A. Replacing this cystine with a pair of alanine residues greatly decreases the conformational stability of ONC. In addition, the C87A/C104A variant is 10-fold less toxic to human leukemia cells. These data indicate that the synapomorphic disulfide bond of ONC is an important determinant of its cytotoxicity. ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Angiogenin (ANG), a homologue of bovine pancreatic ribonuclease A (RNase A), promotes the growth of new blood vessels. The biological activity of ANG is dependent on its ribonucleolytic activity, which is far lower than that of RNase A. Here, the efficient heterologous production of human ANG in Escherichia coli was achieved by replacing two sequences of rare codons with codons favored by E. coli. Hypersensitive fluorogenic substrates were used to determine steady-state kinetic parameters for catalysis by ANG in continuous assays. The ANG pH-rate profile is a classic bell-shaped curve, with pK(1) = 5.0 and pK(2) = 7.0. The ribonucleolytic activity of ANG is highly sensitive to Na(+) concentration. A decrease in Na(+) concentration from 0.25 to 0.025 M causes a 170-fold increase in the value of k(cat)/K(M). Likewise, the binding of ANG to a tetranucleotide substrate analogue is dependent on [Na(+)]. ANG cleaves a dinucleotide version of the fluorogenic substrates with a k(cat)/K(M) value of 61 M(-1) s(-1). When the substrate is extended from two nucleotides to four or six nucleotides, values of k(cat)/K(M) increase by 5- and 12-fold, respectively. Together, these data provide a thorough picture of substrate binding and turnover by ANG.