Search

NADH nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris has been purified 640-fold with an over-all yield of 26% by a combination of protamine sulfate fractionation, ammonium sulfate fractionation, gel chromatography, density gradient centrifugation, and DEAE-chromatography. The purified enzyme is stable for more than 2 months when stored at minus 20 degrees in phosphate buffer (pH 6.9) containing 40% (v/v) glycerol. After the initial steps of the purification, a constant ratio of NADH:nitrate reductase activity to NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities was observed. One band of protein was detected after polyacrylamide gel electrophoresis of the purified enzyme. This band also gave a positive stain for heme, NADH dehydrogenase, and reduced methyl viologen:nitrate reductase. One band, corresponding to a molecular weight of 100, 000, was detected after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains FAD, heme, and molybdenum in a 1:1:0.8 ratio. One "cyanide binding site" per molybdenum was found. No non-heme-iron or labile sulfide was detected. From a dry weight determination of the purified enzyme, a minimal molecular weight of 152, 000 per molecule of heme or FAD was calculated. An s20, w of 9.7 S for nitrate reductase was found by the use of sucrose density gradient centrifugation and a Stokes radius of 89 A was estimated by gel filtration techniques. From these values, and the assumption that the partial specific volume is 0.725 cc/g, a molecular weight of 356, 000 was estimated for the native enzyme. These data suggest that the native enzyme contains a minimum of 2 molecules each of FAD, heme, and molybdenum and is composed of at least three subunits.

In the presence of NADH and cyanide, NADH-nitrate oxidoreductase (EC 1.6.6.1) from Chlorella vulgaris is converted to an inactive form which is readily reactivated by ferricyanide. Experiments with H14CN indicated that the inactivation process is associated with the firm binding to the protein of 0.066 nmole of cyanide per unit of enzyme inactivated. This cyanide binding was linearly proportional to the amount of inactivation. No firm cyanide binding and no inactivation occurred in the absence of NADH or an equivalent reductant. The bound 14C was released when the enzyme was reactivated by ferricyanide. After the cells had been treated with ammonia for several hours prior to disruption, the crude cell extracts contained the nitrate reductase primarily in the inactive form. Critical examination of the methods of cell disruption suggested that the inactivation of the enzyme had truly occurred in vivo. After 300-fold purification, activation of this in vivo inactivated enzyme resulted in the release of 0.066 nmole of HCN per unit of enzyme activated. We conclude that the inactivation of nitrate reductase in vivo involves the formation of a firmly bound complex of reduced enzyme and cyanide. The reaction between reduced enzyme and HCN may be written: Er + HCN (ka)/⇄/(kd) Er-HCN The measured value for ka was 1.25 x 106 m-1 min-1, for kd, 4.5 x 10-4 min-1, giving a dissociation constant, Kd = 3.6 x 10-10 m.

The oxygen evolved during the reduction of KN 18 O 3 by illuminated Chlorella vulgaris has normal isotope abundance, reflecting the isotope content of the water, and not of the nitrate.

The volume changes undergone by corn (Zea mays L.) mitochondria suspended in solutions of constant or varying osmolarity were studied. Within the range of osmotic pressure from 1.8 to 8.4 atmospheres, corn mitochondria behave as osmometers, if allowance is made for an osmotic "dead space" of about 6.9 mul/mg protein. The final equilibrium volume of mitochondria swollen in solutions containing both ribose and sucrose were shown to depend upon the concentration of impermeable solute (sucrose) present and not upon the concentration of ribose present. Osmotic reversibility was found for mitochondria swollen in isotonic solutions of KCI or ribose. The passive swelling of corn mitochondria may be due to the osmotic flow of water coupled to the diffusion of a permeable solute.

Ribulose diphosphate carboxylase was found to exist in two distinct kinetic forms in spinach leaf extracts. One form displayed an apparent K m for CO 2 in excess of 200 μ m and is likely to be the form purified and studied by many previous workers. However, if leaf extracts were prepared in the presence of Mg 2+ and atmospheric levels of CO 2 , the recently described high-affinity form was obtained. It had a K m for CO 2 of about 20 μ m , was quite stable even at 25 °C, and its properties were consistent with it being the form which operates in photosynthesis in vivo . Mg 2+ was also able to convert the high- K m (CO 2 ) form to the low- K m (CO 2 ) form when it was added to an extract which had been prepared in its absence. Mg 2+ was more effective in causing this conversion if bicarbonate was added as well. This activating effect of bicarbonate is a probable cause of previously reported apparent homotropic effects of bicarbonate on ribulose diphosphate carboxylase activity. It is possible that the apparently high- K m (CO 2 ) form is not intrinsically active and appears to have activity only by virtue of the low- K m (CO 2 ) form produced by contact with Mg 2+ and bicarbonate (or CO 2 ) during the course of the assay. Extracts prepared with ribose 5-phosphate in the absence of Mg 2+ also showed low- K m (CO 2 ) carboxylase activity initially, but the presence of this sugar phosphate was deleterious during storage at 25 °C, where it promoted conversion to the apparently high- K m (CO 2 ) form. Effects on the affinity of ribulose diphosphate carboxylase for CO 2 were paralleled by effects on the activity of the associated ribulose diphosphate oxygenase. Treatments which produced the low- K m (CO 2 ) form of the carboxylase also resulted in high oxygenase activity, and it is possible that the apparently high- K m (CO 2 ) form of the carboxylase has little, if any, oxygenase activity associated with it. The carboxylase and oxygenase activities of the low- K m (CO 2 ) form showed broad and quite similar responses to pH variation, and the oxygenase had a K m for O 2 of 0.22 m m . The stability of the low- K m (CO 2 ) form in the presence of Mg 2+ and bicarbonate was quite sufficient for it to be partially purified by Sepharose chromatography. The significance of the low- K m (CO 2 ) form is discussed with respect to activation of photosynthesis by Mg 2+ .

NITRATE reductase of Chlorella vulgaris is present in crude extracts mainly in an inactive form1–3 which may be converted to an active form by the addition of an artificial oxidant such as ferricyanide4. In crude extracts from which low-molecular weight substances have been removed by gel filtration, added NADH (or NADPH) causes a rapid conversion of the active form of nitrate reductase to the inactive form5. Losada and coworkers have also observed an inactivation of the nitrate reductase from Chlorella fusca6 and Chlamydomonas reinhardi7 by added NADH. A low-molecular weight fraction separated from crude extracts of Chlorella vulgaris also causes an inactivation of nitrate reductase when added back to the extract which had been treated with ferricyanide and passed through a Sephadex column to remove excess reagent5. In all cases the enzyme can be reactivated by the addition of the oxidant, ferricyanide4–8. On purification of the enzyme, Solomonson9 found that two substances must be present simultaneously to cause the reversible inactivation: (1) a reducing substance, preferably NADH, and (2) a substance that behaves like HCN. The molar concentration of HCN needed to effect a rapid and reversible inactivation of the enzyme was of about the same magnitude as the enzyme concentration. Thus, activated, suitably purified nitrate reductase with added excess NADH, provides an exceedingly sensitive, though non-specific test for HCN, as shown in Fig. 1.