Control of the production and partial characterization of repressible extracellular 5′-nucleotidase and alkaline phosphatase in Neurospora crassa

Abstract

A new species of orthophosphate repressible extracellular 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) was found to be released into mycelial culture media when a wild type strain of Neurospora crassa was grown on limiting amounts of phosphate. The production of 5′-nucleotidase and extracellular acid and alkaline phosphatase was inhibited by the addition of rifampicin when it was added at the later stage of mycelial growth, but not when it was added at a very early stage. The 5′-nucleotidase and extraceUular alkaline phosphatase were partially purified and characterized, pH optimum of the former was 6.8 and that of the latter was higher than 10.0. The 5′-nucleotidase activity was inhibited by ethylenediaminetetraacetate (EDTA) and ZnCl 2 at pH 6.8 and stimulated by MnC1 2 and CoCl 2 at pH 4.0. Alkaline phosphatase activity was stimulated by EDTA, MgCl 2, CoCl 2 and MnCl 2. 5′-nucleotidase hydrolyzed various 5′-nucletides but not 3′-nucleotides or other various phosphomono- and diester compounds. Alkaline phosphatase hydrolyzed all the phosphomonoester compounds tested. Mutants, nuc-1 and nuc-2, which were originally isolated by the inability to utilize RNA or DNA as a sole source of phosphate, were unable to produce 5′-nucleotidase or six other repressible enzymes reported previously. These mutants showed no or significantly reduced growth on orthophosphate-free nucleotide media depending on the number of conidia inoculated, mainly because of loss of ability to produce these repressible extracellular phosphatases.