Purification and Characterization of Thiol Proteinase as a Nitrate Reductase-Inactivating Factor from Leaves of Hordeum distichum L.

A thiol proteinase was purified 6,400-fold from leaves of Hordeumdistichum L. by a sequence of ammonium sulfate fractionation,gel filtration, ion exchange chromatography, hydrophobic chromatographyand chromatofocusing. This enzyme also had nitrate reductase(NR)-inactivating activity, which was associated with proteolyticactivity in almost constant proportions during the purificationprocedures. Its molecular weight was estimated as 74,000 bygel filtration, and it had an isoelectric point of 4.05 andan apparent K_m of 0.83 mg ml^–1 for azocasein. The respectiveoptimum pH for proteolytic and NR-inactivating activities were6.0 and 7.0. On electrophoresis, the proteinase gave a majorband that coincided with both activities; it also produced afaint band associated with no activity. Our purified thiol proteinase inactivated FMNH₂-NR and MVH-NRas well as NADH-NR, but it had only a slight effect on NADHcytochrome c reductase activity. This enzyme also inactivatedglutamine synthetase. (Received September 16, 1983; Accepted January 26, 1984)