Characterization and comparison of a Neurospora crassa RNase purified from cultures undergoing each of three different states of derepression

Abstract

Extracellular RNase N4 from Neurospora crassa is derepressible by limitation of any of the three nutrient elements obtainable from RNA. We have purified and characterized the enzyme from cultures grown under each of the three states of derepression. The purification procedure consisted of an ultrafiltration step, cation-exchange chromatography, and gel filtration. We found only one enzyme (N4) that hydrolyzed RNA at pH 7.5 in the presence of EDTA in culture filtrates from nitrogen-, phosphorus-, or carbon-limited cells. In all three cases, the enzymes were identical by polyacrylamide gel electrophoresis (Mr approximately 9,500) and by gel filtration (Mr approximately 10,000). There were no differences in thermal stability or pH optimum; all three cross-reacted with antibody to the nitrogen-depressed enzyme in interfacial ring and in Ouchterlony tests. Digestion of homopolyribonucleotides indicated that N4 preferentially cleaved phosphodiester bonds adjacent to guanine residues. Results indicate that the enzymes are very similar or identical and are probably products of the same gene. N4 appears to be homologous to guanine-specific RNases from other fungal sources.