Purification and Characterization of Thiol Proteinase as a Nitrate Reductase-Inactivating Factor from Leaves ofHordeum distichumL.

摘要

A thiol proteinase was purified 6,400-fold from leaves ofHordeum distichumL. by a sequence of ammonium sulfate fractionation, gel filtration, ion exchange chromatography, hydrophobic chromatography and chromatofocusing. This enzyme also had nitrate reductase (NR)-inactivating activity, which was associated with proteolytic activity in almost constant proportions during the purification procedures. Its molecular weight was estimated as 74,000 by gel filtration, and it had an isoelectric point of 4.05 and an apparent Kmof 0.83 mg ml−1for azocasein. The respective optimum pH for proteolytic and NR-inactivating activities were 6.0 and 7.0. On electrophoresis, the proteinase gave a major band that coincided with both activities; it also produced a faint band associated with no activity.Our purified thiol proteinase inactivated FMNH2-NR and MVH-NR as well as NADH-NR, but it had only a slight effect on NADH cytochromecreductase activity. This enzyme also inactivated glutamine synthetas