STUDIES ON THE PYRIDINE NUCLEOTIDE SPECIFICITY OF NITRATE REDUCTASE IN HIGHER PLANTS AND ITS RELATIONSHIP TO SULFHYDRYL LEVEL.

Abstract

1. 1. Nitrate reductase (NAD (P)H: nitrate oxidoreductase, EC 1.6.6.2) present in extracts from 15 of 16 plant species was found to have a specific or preferential requirement for NADH as cofactor. The enzyme present in extracts from soybeans (Glycine max (L) Merr) could utilize either NADH or NADPH. 2. 2. In all extracts, except those from soybeans, optimum nitrate reductase activity was observed at pH of 7.5 when NADH was cofactor. With preparations from soybeans, optimum activity was recorded at pH 6.25 with either NADH or NADPH as cofactor. In other species, the trace of nitrate reductase activity with NADPH as cofactor was maximum at pH 6.25. 3. 3. The relative efficiencies of NADH or NADPH to function as cofactor for the nitrate reductase from soybeans was dependent upon the method of enzyme extraction. NADH was twice as effective as NADPH for the nitrate reductase in extracts of soybeans prepared with a homogenizing medium containing cysteine. In soybean extracts prepared without cysteine, either NADH or NADPH was equally effective as electron donor for nitrate reduction. When corn leaves were the source material, nitrate reductase activity could not be detected when cysteine was omitted from the extracting medium. In extracts from corn leaves prepared with cysteine, the nitrate reductase activity showed a preferential requirement for NADH as cofactor. 4. 4. The increased effectiveness of NADH to serve as cofactor in soybean extracts prepared with cysteine was related to the action of the sulfhydryl group in protecting enzyme binding sites. Soybean tissue extracted with a medium containing cysteine (extracts subsequently subjected to purification procedures, without additional cysteine) gave partially purified preparations with higher sulfhydryl content and activity per unit of protein that comparable preparations extracted in the absence of cysteine. 5. 5. The K m values for substrates NO 3 - or NADH were altered by the inclusion of cysteine during the preparation of the enzyme from soybeans. The K m for NADPH was not affected by the inclusion of cysteine in the extracting medium.