The Ribonucleolytic Activity of Angiogenin

Abstract

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.

Intraspecies Regulation of Ribonucleolytic Activity

“…RI was then added at equimolar concentrations (100 nM) and incubated with a labeled ribonuclease at 25 °C for 5 min. A 50-fold molar excess of human angiogenin (5 μM) (purified as described previously (39)) was added to scavenge dissociated RI, and the change in fluorescence was measured at various time points. To ensure that the stability of the proteins was maintained over the extended duration of the experiment, additional data points were monitored under the same conditions, only without the addition of angiogenin.…”

Motoneurons Secrete Angiogenin to Induce RNA Cleavage in Astroglia

“…Angiogenin is a secreted RNase of the pancreatic RNase A superfamily, showing rRNA and tRNA substrate cleavage, and defined by its potency to induce neovascularization (Fett et al, 1985;Shapiro et al, 1986;St Clair et al, 1987). Despite retaining only 10 Ϫ5 -to 10 Ϫ6 -fold the activity of RNase A, the weak RNase activity of angiogenin is critical to its angiogenic activity (Shapiro and Vallee, 1989;Leland et al, 2002). The majority of mutations that segregate with ALS do not significantly alter the secondary structure or stability of the protein, but rather disrupt its RNase function or subcellular distribution (Moroianu and Riordan, 1994;Leland et al, 2002;Greenway et al, 2006;Crabtree et al, 2007).…”

A new RNase sheds light on the RNase/angiogenin subfamily from zebrafish

“…In ZF-RNase-5 this helix, which encompasses residues [24][25][26][27][28][29][30][31][32][33][34][35], has a first turn in a 3 10 conformation, and terminates with a turn in a π helix conformation with main chain hydrogen bonds Met31-Ile36 and Ser32-Lys35 and Lys35 in the Lα conformation. In the corresponding region (residues 23-36) of ZF-RNase-1 [4] the network of hydrogen bonds (Ile30-Ile37 and Gly31-Lys36) is spatially conserved.…”

“…Thus the topology of this fragment is substantially unchanged with respect to ZF-RNase-5, although the insertion of the two residues formally breaks the helix at the level of Ile30 (Figure 2 and supplemental Figure S3). In the case of ZF-RNase-3 (residues [23][24][25][26][27][28][29][30][31][32][33][34][35] [4] the first turn of the helix is highly distorted.…”

“…This is not surprising, given its ability to act as an angiogenin (see below), and the typical low RNase activity of angiogenins. For measuring the activity, a continuous, very sensitive assay was chosen [24], previously proposed [25] as particularly suitable for measuring the low activity values of angiogenins and for a comparison of the activities of several RNases/angiogenins.…”