Purification and properties of the assimilatory nitrite reductase from barley Hordeum vulgare leaves

Abstract

The assimilatory nitrite reductase (ferredoxin: nitrite oxidoreductase, EC 1.7.7.1) from barley (Hordeum vulgare L.) leaves has been purified over 1500-fold with a recovery of 30% and a specific activity of 84 mumol of nitrite reduced/min per mg of protein. The purification procedure includes (NH4)2SO4 fractionation, ion-exchange and molecular-sieve chromatographies and, finally, ferredoxin-Sepharose-4B affinity chromatography. The enzyme appears homogeneous by polyacrylamide gel electrophoresis and consists of a single polypeptide chain with an Mr of 61 000. The absorption spectrum of the pure enzyme was typical of a haem-containing protein. The enzyme showed low thermostability and was specific for ferredoxin (Km 0.4 microM), although reduced Methyl Viologen (Km 120 microM) was also effective. The same Km value for nitrite (250 microM) was obtained with both electron carriers. Cyanide acted as a powerful pure competitive inhibitor of enzyme with respect to nitrite (Ki 40 microM). Thiol-blocking agents also caused considerable inhibition, but only the ferredoxin-driven activity was significantly inhibited by sulphite and hydroxylamine.