Nitrate and nitrite reductases in the crude extract of aerobically grown Rhizobiwn meliloti were determined with methylviologen as electron donor at pH 7. Nitrate reductase was detected in the cells grown in the medium that did not contain nitrate, and in the presence of nitrate the specific activity increased about 2-fold.
Nitrite reductase was induced by nitrate and produced ammonia from nitrite.
In nitrate reducing cells, two kinds of 0, labile nitrate reductase were found. One enzyme had optimal pH at 7 and was stabilized to 0, by treating with DEAE-Toyopearl650~. The other had optimal pH at 9 and was stabilized by the addition of dithiothreitol and EDTA.
Nitrate reductase stabilized by DEAE-Toyopearl650~ treatment was purified 3,360-fold from crude extract. The purified enzyme showed a single protein band in polyacrylamide gel electrophoresis, and there was no absorption peak in the visible region. It had a molecular weight of 64,OOO in SDS PAGE and 58,000 on Sephadex G-100 gel filtration. K, for nitrate was 0.9 mM. It was inhibited by pchloromercuribenzoate, cyanide, and a,a'dipyridyl.
Nitrate usually inhibits nodulation and nitrogen-fixing activity of legume-Rhizobia symbiosis. Correlation between nitrate-reducing ability of Rhizobia and the efficiency of symbiotic nitrogen fmation has been discussed, and many studies of nitrate reductase (NaR) of Rhizobia were done with aerobically grown, anaerobically grown and symbiotic state bacteria. However, most studies of NaR of Rhizobia were done with intact cells or crude enzyme extract because NaR of Rhizobia was very unstable.
Extraction and partial purification of NaR from aerobically grown Rhizobia was initially reported by NICHOLAS et al. (1962) with Bradyrhizobium japonicum. NaR from aerobically grown Rhizobia was purified only 11-fold so far by LOWE and EVANS (1963) with B. japonicum. However, with the same strain, 3. japonicum CC705, the molecular weight of NaR was reported to be 70,000 and 170,000 by, KENNEDY et al. (1975) and DANIEL andGRAY (1976), respectively.
Assimilatory NaR of plant or fungi was studied precisely and we can get much information about the characteristic of the enzyme. However, as STEPHENS and NEYRA (1983) described, assimilatory NaR of aerobically grown bacteria is generally unstable, and we have little knowledge about that. With crude enzyme extract of Azotobacter chroococum GUERRERO et al. (1973) reported that the molecular weight of NaR was 100,000 and TORTOLERO et al. (1 975) showed that reduced ferredoxin could mediate the reduction of nitrate by NaR. NaR from Rhodopseudomonas capsulata, ALEF and KLEMME (1979), is the only enzyme that was purified to a homogeneous state from aerobically grown bacterium but its specific activity was very low.
As for NaR from anaerobically grown or symbiotic state Rhizobia, DANIEL and GRAY (1976) purified the enzyme 9-fold from the extract of anaerobically grown B. japonicum and showed its molecular weight to be 69,000 and KENNEDY et al. (1975) purified NaR 13fold from the bacteroids of B. japonicum. Instability of NaR of Rhizobia seems to make its purification and characterization difficult.
In addition to the characteristics of NaR, its induction has been studied also. DANIEL and GRAY (1976) showed with B. japonicum that the specific activity of NaR was constant