Birgit Vennesland

Properties of a nitrate reductase of Chlorella

Abstract 1. An NADH-nitrate oxidoreductase (EC 1.6.6.1) of Chlorella has the unusual property of existing in cell-free extracts mainly in the form of an inactive precursor which can be activated by a variety of procedures. This enzyme is associated with a cytochrome of the b type. 2. The inhibitors, azide, cyanate, thiocyanate and nitrite, react rapidly with the enzyme, with kinetics which show that they are competitive with nitrate. 3. The inhibitors, cyanide and hydroxylamine, react slowly with the reduced form of the enzyme to give an inactive product which can slowly be reactivated in the presence of nitrate. There is at least a superficial similarity between the reactivation of the inhibited enzyme and the activation of the enzyme precursor in fresh extracts. 4. Mammalian cytochrome c , dichlorophenolindophenol and ferricyanide can substitute for nitrate as oxidants for NADH in the presence of the enzyme. This “diaphorase” reaction does not require activation, but is fully active in fresh extracts. It is not inhibited by cyanide, hydroxylamine, azide, cyanate, thiocyanate, or by the substrate, nitrate. Oxidized cytochrome c , on the other hand, inhibits the reduction of nitrate by NADH in the presence of the enzyme. 5. Pyridoxal phosphate inhibits both nitrate reductase and cytochrome c reductase to about the same extent.

Reversible inactivation of nitrate reductase in Chlorella vulgaris in vivo

SummaryThe NADH-nitrate oxidoreductase of Chlorella vulgaris has an inactive form which has previously been shown to be a cyanide complex of the reduced enzyme. This inactive enzyme can be reactivated by treatment with ferricyanide in vitro. In the present study, the activation state of the enzyme was determined after different prior in vivo programs involving environmental variations. Oxygen, nitrate, light and CO2 all affect the in vivo inactivation of the enzyme in an interdependent manner. In general, the inactivation is stimulated by O2 and inhibited by nitrate and CO2. Light may stimulate or inhibit, depending on conditions. Thus, the effects of CO2 and nitrate (inhibition of reversible inactivation) are clearly manifested only in the light. In contrast, light stimulates the inactivation in the presence of oxygen and the absence of CO2 and nitrate. Since the inactivation of the enzyme requires HCN and NADH, and it is improbable that O2 stimulates NADH formation, it is reasonable to conclude that HCN is formed as the result of an oxidation reaction (which is stimulated by light). The formation of HCN is probably stimulated by Mn2+, since the formation of reversibly-inactivated enzyme is impaired in Mn2+-deficient cells. The prevention of enzyme inactivation by nitrate in vivo is in keeping with previous in vitro results showing that nitrate prevents inactivation by maintaining the enzyme in the oxidized form. A stimulation of nitrate uptake by CO2 and light could account for the effect of CO2 (prevention of inactivation) which is seen mainly in the presence of nitrate and light. Ammonia added in the presence of nitrate has the same effect on the enzyme as removing nitrate (promotion of reversible inactivation). Ammonia added in the absence of nitrate has little extra effect. It is therefore likely that ammonia acts by preventing nitrate uptake. The uncoupler, carbonylcyanide-m-chloro-phenylhydrazone, causes enzyme inactivation because it acts as a good HCN precursor, particularly in the light. Nitrite, arsenate and dinitrophenol cause an enzyme inactivation which can not be reversed by ferricyanide in crude extracts. This suggests that there are at least two different ways in which the enzyme can be inactivated rather rapidly in vivo.

Activation of nitrate reductase by oxidation

Abstract The activation of the inactive form of the nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) present in cell-free extracts of Chlorella vulgaris Beijerinck requires an oxidizing agent. Ferricyanide causes conversion of the proenzyme to active enzyme within a few minutes, even at 0°C. Molecular oxygen causes a slow activation which requires many hours at room temperature, and never reaches the high activity level attained with ferricyanide. In unfractionated extracts, CO inhibits the activation by molecular O2. The sensitivity of this activation to CO may account for the in vivo sensitivity of nitrate reduction to CO in these algae.

Reversible inactivation of the nitrate reductase of Chlorella vulgaris Beijerinck.

Abstract The ferricyanide-activated NADH:nitrate oxidoreductase (EC 1.6.6.1) of Chlorella vulgaris Beijerinck, in extracts freed of low molecular weight components, undergoes a reversible inactivation on addition of an ultrafiltrate containing cellular components in the molecular weight range 1000–10 000. This inactivation reaction, which goes virtually to completion, accounts for the fact that the enzyme in the original cell extracts is present almost entirely in the inactive form. NADH or NADPH mimic the effect of the unknown components of the ultrafiltrate. In all cases, the inactivation occurs more readily at pH 7.6 than at pH 6.7, and is prevented by nitrate. The inactivated enzyme can be reactivated with ferricyanide. A partial, reversible inactivation also occurs in the absence of any additions, when extracts freed of low molecular weight components, are brought to a pH of 8.8. The diaphorase component of the nitrate reductase is not inactivated to a substantial degree by any of these procedures. Partially purified, activated nitrate reductase is only slowly and partially inactivated by added NADH or NADPH. The ultrafiltrate alone has no effect on the partially purified, activated enzyme, but enhances the inactivating effect of the reduced pyridine nucleotides. The inactivated enzyme can be reactivated with ferricyanide.

Cyanide formation in preparations from Chlorella vulgaris Beijerinck: Effect of sonication and amygdalin addition

SummaryA critical evaluation of a method for recovering HCN from cell extracts is presented. Since crude extracts often bind or metabolize HCN extensively, the HCN recovered by distillation at room temperature represents only the difference between production and consumption. Sonication leads to HCN release from the alga, Chlorella vulgaris Beijerinck. Illumination of extracts at high light intensity in oxygen, with added Mn2+ and peroxidase, also stimulates HCN production. In both processes, the HCN is probably formed by oxidation of nitrogenous precursors. Chlorella extracts cause formation of HCN from added amygdalin. No evidence was found, however, for the presence of cyanogenic glycosides in the algae.

Alcohol dehydrogenase of wheat germ

Abstract Evidence is presented that extracts of wheat germ contain alcohol dehydrogenase(s) catalyzing the reduction of both TPN and DPN by a variety of alcohols. The enzyme reaction is completely inhibited by 10−2 M iodoacetate. Because of the presence of this enzyme activity, the TPN-dependent oxidation of hexose phosphates may be coupled with the reduction of acetaldehyde. A procedure is described for following such coupled reactions by measuring acid formation manometrically in bicarbonate-CO2 mixtures.

Cyanide formation from histidine in Chlorella. A general reaction of aromatic amino acids catalyzed by amino acid oxidase systems.

The formation of HCN from D-histidine in Chlorella vulgaris extracts is shown to be due to the combined action of a soluble protein and a particulate component. Either horse-radish peroxidase (EC 1.11.1.7) or a metal ion with redox properties can be substituted for the particulate component. Ions of manganese and vanadium are especially effective, as are o-phenanthroline complexes of iron. Cobalt ions are less active. The D-amino acid oxidase (EC 1.4.3.3) from kidney and the L-amino acid oxidase (EC 1.4.3.2) from snake venom likewise cause HCN production from histidine when supplemented with the particulate preparation from Chlorella or with peroxidase or with a redox metal ion. The stereospecificity of the amino acid oxidase determines which of the two stereoisomers of histidine is active as an HCN precursor. Though histidine is the best substrate for HCN production, other naturally occurring aromatic amino acids (viz. tyrosine, phenylalanine and tryptophan) can also serve as HCN precursors with these enzyme systems. The relative effectiveness of each substrate varies with the amino acid oxidase enzyme and with the supplement. With respect to this latter property, the particulate preparation from Chlorella behaves more like a metal ion than like peroxidase.

Glyoxylate carboligase of Escherichia coli: some properties of the enzyme.

Abstract Glyoxylate carboligase from Escherichia coli has previously been shown to contain the prosthetic group flavin adenine dinucleotide. The present paper describes some properties of the enzyme, and attempts to shed some light on the function of the flavin. The enzyme is inhibited by heavy metal ions and by p -OH mercuribenzoate and is activated by SH compounds such as cysteine and glutathione. If the assay is carried out at optimal pH with added cysteine, the turnover number of the best preparations at 30 ° is 3440 μmoles CO 2 produced per minute per micromole of flavin. The combination of the flavin-free apoenzyme with flavin adenine dinucleotide follows Michaelis-Menten kinetics, with a K m of about 2 × 10 −7 M . It was not possible to show that the enzyme is bleached by glyoxylate. There was, on the other hand, a rather slow bleaching of the enzyme by added sodium dithionite. The dithionite-treated enzyme is inactive, and its activity can be restored by added O 2 , flavin adenine dinucleotide, or flavin mononucleotide. The product of the carboligase reaction, tartronic semialdehyde, is oxidized when the enzyme reaction is carried out in the presence of oxygen and certain buffers, but no evidence could be obtained that the carboligase participates in this oxidation reaction.

Cyanide formation in preparations from Chlorella and New Zealand spinach leaves: Effect of added amino acids

SummaryAs part of an effort to identify the natural precursor(s) of HCN in the alga Chlorella vulgaris Beijerinck, and in leaves of New Zealand spinach (Tetragonia expansa, Murr.), HCN release was measured after addition of various amino acids to illuminated algal extracts and grana preparations. Histidine is particularly effective as an HCN precursor, both with Chlorella extracts and leaf grana. With the algal extracts, d-histidine is about ten times more effective than l-histidine and histamine, whereas the two isomers (and histamine) are about equally effective with leaf grana. In the presence of leaf grana plus added Mn2+ and peroxidase, l-tyrosine and l-cysteine like-wise cause HCN formation; but these amino acids cause little or no HCN formation in the presence of Chlorella extracts. A stimulation of HCN production by l-histidine was observed with intact Chlorella cells. Because of the limitations of the assay method, the possibility can not be excluded that other substances than histidine may also lead to HCN generation in Chlorella vulgaris, but the results show that histidine has an important role in HCN generation by this species.

The relationship of the Hill reaction to photosynthesis: Studies with fluoride-poisoned blue-green algae

Abstract Fluoride has been shown to inhibit photosynthesis and the quinone Hill reaction when added to cell suspensions of the blue-green alga Plectonema boryanum. The effectiveness of fluoride increases with decreasing pH. The inhibition of the Hill reaction commences at a fluoride concentration which inhibits photosynthesis strongly. At this threshhold concentration of fluoride (about 0.003 m KF at pH 5.2), the Hill reaction becomes dependent on CO2 i.e., the Hill reaction can be almost completely inhibited by removing CO2, and readily reactivated by adding CO2 at low tensions (0.05% CO2 gives 50% reactivation). If the fluoride concentration is increased a little more (to about 0.006 m at pH 5.2) the quinone Hill reaction develops a requirement for chloride as well as for CO2. There is no chloride requirement for the Hill reaction with intact cells tested in the absence of fluoride, but fluoride acts as an apparent antagonist for chloride, so that intact algae supplemented with fluoride show a chloride requirement for the quinone Hill reaction. Nitrate and bromide can substitute for chloride to some extent, just as in the case of the well-known chloride requirement of the Hill reactions obtained with isolated green grana.