Presence Of Manganous Ions Research Articles

Nanomedicine-Leveraged Intratumoral Coordination and Redox Reactions of Dopamine for Tumor-Specific Chemotherapy

Great efforts have been made in investigating the neurotoxicity of dopamine (DA) in the presence of manganous ions. In contrast, here, we probe the possibility of DA-based cancer chemotherapy by le...

A rotating disk study of gold dissolution by bromine

Gold dissolution with bromine was studied using the rotating disk technique with Geobrom™ 3400 as a source of bromine. The parameters studied were speed of rotation, lixiviant concentration, pH, temperature, sulfuric acid and hydrochloric acid concentrations, and the concentrations of various cations (i.e., copper, iron, zinc, aluminum, manganese, potassium, and sodium) and anions (i.e., chloride, bromide, sulfate, nitrate, and iodide). According to the Lavich plot and activation energy, gold dissolution is controlled by a chemical reaction rate. Copper, iron, and manganese in their highest oxidation states, as well as aluminum, zinc, sodium, and potassium, have no effect on the rate of gold dissolution. The presence of manganous ion substantially decreases the gold dissolution rate. The kinetic performance of bromine was found to be dramatically better than the performance of cyanide and thiourea.

Potentiation of contraction in bullfrog ventricle strips by manganese and nickel.

Sodium-lack contractures by strips of bullfrog ventricle were found to be increased in the presence of manganous ion (1-10 mM). In addition, peak force development was usually attained earlier in the presence of manganous ion and the rate of relaxation was decreased by nickel (0.7-2.0 mM), although the latter cation did not potentiate contractural force. Both manganese and nickel had only depressant effects on potassium-excess contractures, as well as on electrically stimulated twitches. Depressant effects of manganese and nickel on sodium-lack contractures were also found. These were smaller, the lower the extracellular sodium concentration and the higher the extracellular calcium concentration. When these well-known negative inotropic effects of the divalent cations were suppressed in sodium-free, calcium-rich media, their potentiating effects were unmasked, resulting in marked augmentation by these cations of potassium-excess contractures and of twitches, along with slowing of relaxation. Experimental maneuvers that have been reported to bring about entry of manganese into these cardiac cells did not increase the observed positive inotropic effect. It thus seems probable that these ions act on the membrane of the ventricle fiber. Also, in view of earlier evidence that they neither affect myofibrillar function nor induce calcium release from mitochondria, it is provisionally concluded that the mechanism of their potentiating effect on mechanical activation is due to their inhibition of calcium extrusion from the fibers, described in the accompanying paper.

The role of the nuclear gene "mitochondrial mutability control" (MMC1) in the process of mutability of the mitochondrial genome by different mutagens in Saccharomyces cerevisiae.

The accumulation of respiratory deficient (RD) mutants in Saccharomyces cerevisiae depended upon the mutagens used and upon the presence of the nuclear gene previously identified as MMC1 (one) which we showed to control the spontaneous and the erythromycin-induced RD mutability. In this paper data are reported about the accumulation of RD mutants in the presence of manganous ions (Mn++) and UV which was higher in the mmc1 (one) than in MMC1 strains. We found that the characters 'low spontaneous' and 'low induced' RD mutability by erythromycin, manganous ions and UV, were controlled by the same genetic determinant. In the presence of manganous ions, also the frequency of antibiotic resistant mutants capR and eryR was higher in the mmc1 strains. Moreover, the accumulation of RD mutants in the presence of berenil, 5-fluorouracil and basic fuchsin was higher in the mmc1 than in MMC1 strains. In contrast, RD mutants accumulation by acriflavine and ethidium bromide treatments did not appear affected by the MMC1 genetic constitution.

The reactivation of nitrate reductase from spinach (Spinacea oleracea L.) inactivated by NADH and cyanide: effects of peroxidase and associated systems

Nitrate reductase of spinach (Spinacea oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH2-dependent activity. Ferricyanide, a purely chemical oxidant, produced rapid maximal reactivation (100%) which was 90% complete in less than 3 min. Reactivation occurred slowly and less completely (30-75% in 30 or 60 min) when the enzyme was incubated with pure horseradish peroxidase alone, depending on using one or 20 units and time. Addition of glucose and glucose oxidase to generate hydrogen peroxide increased reactivation slightly (10-15%) with 20 units of peroxidase but more (30-50%) with one unit and to 75-90% of ferricyanide values. Adding catalase decreased reactivation by more than half either with or without glucose oxidase. Glucose and glucose oxidase alone did not cause reactivation. Addition of superoxide dismutase increased reactivation from 50-75% of ferricyanide values with one unit of peroxidase alone but had no effect on greater reactivation obtained in the presence of glucose oxidase. The addition of p-cresol and manganese together increased reactivation with one unit of peroxidase and in the presence of glucose oxidase by about double, but omission of manganese had no effect. However, as shown previously, although trivalent manganese was formed, the residual presence of manganous ions inhibited reactivation. Nevertheless, peroxidase systems either alone or with additionally generated hydrogen peroxide can induce substantial reactivation of nitrate reductase in physiologically relevant conditions.

EFFECT OF MILK CONSTITUENTS ON THE HYDROLYSIS OF LACTOSE BY LACTASE FROM Kluyveromyces fragilis

ABSTRACTLactase activity in cold milk and whey is controlled by the ionic environment; maximum activity occurred in the presence of manganous ions. Of the major cations present in milk, sodium and calcium were inhibitory while potassium and magnesium were activating. Milk proteins strongly activated lactase in potassium phosphate buffer and the effect was enhanced in the presence of manganous ions; however the activation was masked by the milk salts in dairy products. Lactase activity in whey heated to 85°C was about 50% higher than in unheated whey, but no changes were observed in heated milk. There was no evidence for a thermolabile lactase inhibitor among the whey proteins.

Electron spin resonance studies on phaeomelanins

ESR spectra of synthetic melanins formed from dopa and from 5-S-cysteinyldopa and of melanins isolated from human melanomas were studied. Alkali-soluble melanin synthesized from cysteinyldopa showed a signal similar to that of insoluble melanin synthesized from dopa, but also a simple fine structure. Trichochromes C and F synthesized from 5-S-cysteinyldopa had a broader signal than dopamelanin, and in addition a hyperfine structure that could be explained by the presence of manganous ions. An insoluble eumelanin from a human melanoma with a sulphur content of 5% showed an ESR spectrum consisting of one line like dopamelanin. An alkali-soluble phaeomelanin from a human melanoma with a sulphur content of 10% showed a very broad signal with a hyperfine structure that could be explained by the presence of manganous ions.

Evidence for a critical glutamyl and an aspartyl residue in the function of pig heart diphosphopyridine nucleotide dependent isocitrate dehydrogenase.

The pH dependence of the maximum velocity of the reaction catalyzed by diphosphopyridine nucleotide (DPN) dependent isocitrate dehydrogenase indicates the requirement for the basic form of an ionizable group in the enzyme-substrate complex with a pK of 6.6. This pK is unaltered from 10 to 33 degrees C, suggesting the ionization of a carboxyl rather than an imidazolium ion. The enzyme is inactivated upon incubation with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide in the presence of glycinamide or glycine ethyl ester. This inactivation is dependent on pH and the rate constant (k) increases as the pH is decreased in the range 7.3 to 6.25. A plot of 1/(H+) vs. 1/k suggests that the enzyme is inactivated as a result of the modification of a single ionizable group in this pH range. The coenzyme DPN and substrate alpha-ketoglutarate do not affect the rate of inactivation. In contrast, manganous ion (2 mM) and isocitrate (60 mM) produce a sevenfold decrease in the rate constant. The allosteric activator ADP (1 mM) does not itself influence the rate of inactivation; however, it reduces the concentration of Mn2+ (1 mM) and isocitrate (20 mM) required to produce the same decrease in the inactivation constant. These observations imply that the modification occurs at the substrate-binding site. Experiments employing [1-14C]glycine ethyl ester show a net incorporation of 2 mol of glycine ethyl ester per subunit (40 000), concomitant with the complete inactivation of the enzyme. The radioactive modified enzyme, after removal of excess reagent by dialysis, was exhaustively digested with proteolytic enzymes. High voltage electrophoretic analyses of the hydrolysate at pH 6.4 and 3.5 yield two major radioactive spots with approximately equal intensity, which correspond to gamma-glutamylglycine and beta-aspartylglycine, the ultimate products of reaction with glutamic and aspartic acids, respectively. Modification in the presence of manganous ion and isocitrate results in significant reduction in the incorporation of radioactivity into the two dipeptides. These results suggest that carbodiimide attacks one glutamyl and one aspartyl residue per subunit of the enzyme and that the integrity of these residues is crucial for the enzymatic activity.

An investigation of heavy meromyosin-ADP binding equilibria by proton release measurements.

The interaction of magnesium-ADP with skeletal muscle heavy meromyosin has been studied by measuring the accompanying release of protons. Total pH changes of the order of 0.03 were involved, and measurements were performed with a discrimination of some ten-thousandths of a pH unit. At pH 8.0 and 25 degrees C about 0.5 mol of protons per mol of heavy meromyosin is released at saturation. A stoichiometry of binding close to 2 mol of ADP per mol of protein was found, with a binding constant, obtained from the proton release titration curve (pH 8.0, 25 degrees C), of 2 X 10(5) M-1. At 5 degrees C the release of protons per mole is slightly greater, and the binding constant is somewhat increased, reflecting a negative enthalpy of binding. Similar proton release behavior is observed in the presence of manganous ions in place of magnesium. The liberation of protons is thus unrelated to the temperature-dependent isomerization of myosin in the presence of substrate. Alkylation of a reactive thiol group (SH1) does not change the proton liberation at pH 8.0. From the pH dependence of proton release, the association constant of heavy meromyosin with magnesium-ADP at other pH values can be inferred and shows an appreciable rise as the pH increases. The pH-proton release profile also allows the pK of the ionizing groups perturbed by the ligand to be deduced. At least two groups ionizing above pH 7 and one below are involved. Their pK's in the unperturbed state are assigned as 8.5, 9.3, and about 6.6, respectively; they are displaced in the complex to about 8.0, 9.1, and 6.3. A relation to the pH-activity profile of myosin ATPase is indicated. The pH-proton release profile is somewhat changed when the SH1 group is alkylated. Measurements with potassium-ADP, in the absence of magnesium, show that at pH 8.0 there is no proton release but rather a sizeable proton absorption (about 0.5 mol of protons per mol of heavy meromyosin). The association constant derived from the titration curves (pH 8.0, 25 degrees C) is 3 X 10(4) M-1.

Inhibition of Muscle Pyruvate Kinase by Creatine Phosphate

Abstract At neutral pH, concentrations of creatine phosphate comparable to those found in skeletal muscle and heart are highly inhibitory toward rabbit skeletal muscle pyruvate kinase. Creatine phosphate appears to be competitive with phosphoenolpyruvate with a Ki of 2.0 mm. That it is not a simple competition, however, is indicated by the following evidence. The inhibition is increased in the presence of high concentrations of ADP. ATP, a known inhibitor of pyruvate kinase, acts synergistically with creatine phosphate; that is, in the presence of both ATP and creatine phosphate the inhibition is much greater than the sum of the actions of the inhibitors separately. No synergism is seen between ATP and the inhibitory substrate analogue, 2-phosphoglyceric acid. The characteristics of the creatine phosphate effect are quite distinct from the inhibition noted previously with phenylalanine. Phenylalanine inhibition is maximal above pH 8.0 whereas creatine phosphate is more potent at neutral pH. Alanine, which reverses phenylalanine inhibition, has no effect on creatine phosphate inhibition. Phenylalanine does not inhibit the Mn2+-activated enzyme whereas creatine phosphate inhibition is more potent in the presence of manganous ion. Rabbit erythrocyte pyruvate kinase is only slightly inhibited by creatine phosphate. In the presence of the activator, fructose 1,6-diphosphate, the inhibition is much more pronounced. The results are discussed in relation to the regulation of muscle glycolysis and it is suggested that falling creatine phosphate levels may be one of the primary factors contributing to the increase in glycolytic flux that accompanies muscle contraction.

Spin-labelled phosphofructokinase and its interactions with ATP and metal-ATP complexes as studied by magnetic-resonance methods.

Rabbit skeletal muscle phosphofructokinase has been specifically modified at its most reactive thiol group with the nitroxide spin label, 4‐(2‐iodoacetamido)‐2,2,6,6–tetramethyl piperidinooxyl. The specific activity and allosteric kinetics are only slightly altered in the modified enzyme. The electron spin resonance spectrum of the labelled enzyme at pH 7.5, in the absence of stabilising ligands, indicates that the label has moderate mobility, with a rotational correlation time, τR, of 3.3 ns, which is greater than the mobility of the smallest fully active oligomer of 340000 molecular weight (τR= 90 ns). The spectrum is sensitive to pH and to the binding of ATP and its magnesium and manganous complexes. The binding of the metal complexes produces an immobilisation of the label (τR= 4.7 ns). In addition to this, MnATP causes a 74% quenching of the signal height as a result of dipolar spin‐spin interaction. At pH 7.5, MnATP and ATP bind competitively to the labelled enzyme with dissociation constants of 540 μM and 1.3 mM respectively. The binding of MgATP is sigmoidal with [S]0.5= 400 μM and a Hill interaction coefficient of 2.0. A two‐state model is proposed to account for the electron spin resonance spectral changes, in which the cooperative interactions are due to metal‐ATP binding at catalytic sites, and the dipolar interactions are due to metal‐ATP binding at a separate regulatory site closer to the spin label. The longitudinal relaxation rate of water protons at 35 MHz is not detectably altered by the presence of the spin label on the enzyme. An analysis of enhancement data obtained in the presence of manganous ions and ATP indicates that the enhancement of the ternary complex, ɛt, is unchanged compared with the native enzyme, but that the dissociation constant for Mn ATP increases from 260 μM in the native enzyme to 480 μM in the labelled enzyme. The quenching of the electron spin resonance spectrum and the enhancement in the ternary complex of enzyme with MnATP have together been used to estimate the manganese‐nitroxide distance. The longitudinal and transverse relaxation rates of the H‐2, H‐8, and H‐1′ protons in MgATP bound to the spin‐labelled enzyme are increased in comparison with the native enzyme due to the dipolar spin‐spin interactions. This allows the proton‐nitroxide distances to be estimated. The distances calculated from the dipolar interactions have been used to obtain a self‐consistent three‐dimensional model of the regulatory site in relation to the spin label.

Bacteriology of manganese nodules. IV. Induction of an MnO2-reductase system in a marine bacillus.

Bacillus 29, isolated from a ferromanganese nodule from the Atlantic Ocean, was shown to possess an MnO(2)-reductase system which is induced in the presence of manganous ion. Maximal activity of the enzyme system was induced in about 5 hr in the presence of 4.35 mm MnSO(4) and was minimally dependent on the presence of either glucose or peptone and oxygen. Induction of optimal activity required the simultaneous presence of glucose and peptone. At least 30% of maximal activity was induced in 5 hr in the presence of 0.4 mum MnSO(4). Actinomycin D (5 mug/ml) or chloramphenicol (35 mug/ml), when added to the induction medium, inhibited approximately 90% of MnO(2)-reductase synthesis and incorporation of uracil-2-(14)C or leucine-1-(14)C. Cell-free extracts having MnO(2)-reductase activity were prepared by sonic disruption of cell suspensions of induced Bacillus 29. Such extracts used glucose metabolism as a source of electrons. They had an average specific activity of 1.15 nmoles of Mn(II) produced per mg of protein per hr at 25 C. They had a temperature optimum of 18 C for reductase activity and retained 50% of their activity at 4 C, the approximate temperature of the natural habitat of the organism. Extracts were stable for several days at 4 C but rapidly lost over 50% of their activity on freezing and thawing. Over 90% of the activity of the extract could be destroyed by heating in a boiling-water bath for 5 min. At a concentration of 1 mm, HgCl(2) and atebrine dihydrochloride inhibited MnO(2)-reductase activity by at least 50%, but sodium azide was ineffective. The MnO(2)-reductase activity of induced cells and extracts from them was no greater in the absence of oxygen than in its presence, confirming an earlier observation that MnO(2) and O(2) do not compete as terminal electron acceptors in the respiratory activity of this organism.

Bacteriology of Manganese Nodules

Bacillus 29, isolated from a ferromanganese nodule from the Atlantic Ocean, was shown to possess an MnO2-reductase system which is induced in the presence of manganous ion. Maximal activity of the enzyme system was induced in about 5 hr in the presence of 4.35 mm MnSO4 and was minimally dependent on the presence of either glucose or peptone and oxygen. Induction of optimal activity required the simultaneous presence of glucose and peptone. At least 30% of maximal activity was induced in 5 hr in the presence of 0.4 μm MnSO4. Actinomycin D (5 μg/ml) or chloramphenicol (35 μg/ml), when added to the induction medium, inhibited approximately 90% of MnO2-reductase synthesis and incorporation of uracil-2-14C or leucine-1-14C. Cell-free extracts having MnO2-reductase activity were prepared by sonic disruption of cell suspensions of induced Bacillus 29. Such extracts used glucose metabolism as a source of electrons. They had an average specific activity of 1.15 nmoles of MnII produced per mg of protein per hr at 25 C. They had a temperature optimum of 18 C for reductase activity and retained 50% of their activity at 4 C, the approximate temperature of the natural habitat of the organism. Extracts were stable for several days at 4 C but rapidly lost over 50% of their activity on freezing and thawing. Over 90% of the activity of the extract could be destroyed by heating in a boiling-water bath for 5 min. At a concentration of 1 mm, HgCl2 and atebrine dihydrochloride inhibited MnO2-reductase activity by at least 50%, but sodium azide was ineffective. The MnO2-reductase activity of induced cells and extracts from them was no greater in the absence of oxygen than in its presence, confirming an earlier observation that MnO2 and O2 do not compete as terminal electron acceptors in the respiratory activity of this organism.

Chromic acid oxidation of aromatic alcohols

The mechanism of the chromic acid oxidation of di- and tri-aryl carbinols has been studied in 80 wt% acetic acid containing sulfuric acid. The reactions are cleanly second-order and give benzophenones in the case of the secondary alcohols and benzophenones plus phenols in the case of the tertiary alcohols. Electron-donating substituents in the tertiary alcohol appear predominantly in the phenol component and the rate-controlling step is believed to be a 1,2-aryl shift. The reaction constant for the migration is ρ+ = −1.44 and that for the overall reaction is ρ+ = −0.879. The presence of manganous ions does not alter these values although it lowers the overall rate of reaction. Although an analogous 1,2-hydride shift mechanism can be written for the oxidation of the secondary alcohols, there are enough points of difference between the oxidations of the secondary and tertiary systems to make this appear unlikely.

PHOTOCHEMISTRY AND METAL CATALYSIS: STUDIES ON A FLAVIN SENSITIZED: OXIDATION OF ASCORBATE*

Summary This paper constitutes a study of the interaction between ascorbate, diketogulonate, manganous ion, oxygen and illuminated flavins (or other sensitizers). When ascorbate is chosen as the substrate, the resulting photoxidation assumes a peculiar two‐step course. This happens because traces of ascorbate inhibit the photoxidation of diketogulonate and oxygen retards the photoxidation of small amounts of ascorbate. Sensitized photo‐reactions which are inhibited by high concentrations of oxygen are affected by the addition of various organic compounds. Of these, the herbicides 3‐(p‐clilorophenyl)‐1,1‐dimethylurea (CMU) and 3‐(3, 4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU) interfere nearly exclusively with flavin sensitized photoreactions, while other inhibiting compounds tested, such as tryptophane and tyrosine, do not display a similar selectivity for only one dye. A second even more rigid specificity concerns the role of manganous ions. Diketogulonate is oxidized through the action of light excited flavin or of traces of alkali in the dark only when Mn++ is available. No other metal tested had this ability.The response of flavin sensitized photoreactions to the addition of CMU or DCMU varies with the substrate being oxidized. With ascorbate and 2, 3‐diketogulonate as substrates in the presence of manganous ions, CMU reverses the inhibition by oxygen, but with ethylenediamine tetraacetic acid (EDTA) or triethylene tetramine (TETA) as substrates CMU acts as an inhibitor itself. With one exception all effects of CMU on flavin sensitized reactions, be they anaerobic bleaching or aerobic oxidations, were inhibitory. The one exception, the apparent promotion of manganese catalyzed photoxidations, may come about through the inhibition of competing reactions such as the quenching of light‐excited dyes by oxygen. At the same time it IS noteworthy that the manganese dependent photoxidation is refractory against CMU as inhibitor.The conclusion is inescapable that herbicides like CMU form stable charge transfer complexes with excited flavin molecules. But simple complex formation is not sufficient to explain all of our findings on the basis of the two most frequently proposed reaction mechanisms for sensitized light reactions. We have had to modify them to make them fit the experiments here summarized.

HYDROLYSIS OF CALF THYMUS DEOXYRIBONUCLEATE BY PANCREATIC DEOXYRIBONUCLEASE: II. ISOLATION AND ANALYSIS OF THE MONONUCLEOTIDES AND DINUCLEOTIDES OBTAINED BY ENZYMIC DEGRADATION IN THE PRESENCE OF MANGANOUS IONS

Analysis of the oligonucleotides produced by the exhaustive hydrolysis of calf thymus DNA by pancreatic DNAase in the presence of manganous ions at pH 6.6 shows an increase in the concentration of mononucleotides and dinucleotides which are found to comprise 5% and 49% of the total nucleotide phosphorus. The relative amounts of the isomeric dinucleotides formed during manganous ion activation of the enzyme differ from those obtained for magnesium ion activation. The results are interpreted as indicating a random attack by the enzyme at multiple points in an endonuclease action with certain linkages being more susceptible than others. The evidence suggests that the pPu–pPu linkage is stable and that the increased liberation of purine nucleotides occupying the 5′-terminal phosphate position is due to hydrolysis of pPy–pPu linkages.

HYDROLYSIS OF CALF THYMUS DEOXYRIBONUCLEATE BY PANCREATIC DEOXYRIBONUCLEASE: II. ISOLATION AND ANALYSIS OF THE MONONUCLEOTIDES AND DINUCLEOTIDES OBTAINED BY ENZYMIC DEGRADATION IN THE PRESENCE OF MANGANOUS IONS

Analysis of the oligonucleotides produced by the exhaustive hydrolysis of calf thymus DNA by pancreatic DNAase in the presence of manganous ions at pH 6.6 shows an increase in the concentration of mononucleotides and dinucleotides which are found to comprise 5% and 49% of the total nucleotide phosphorus. The relative amounts of the isomeric dinucleotides formed during manganous ion activation of the enzyme differ from those obtained for magnesium ion activation. The results are interpreted as indicating a random attack by the enzyme at multiple points in an endonuclease action with certain linkages being more susceptible than others. The evidence suggests that the pPu–pPu linkage is stable and that the increased liberation of purine nucleotides occupying the 5′-terminal phosphate position is due to hydrolysis of pPy–pPu linkages.

High oxidation states of manganese in sulphuric acid and caustic alkali media

The mechanism of permanganate reduction in sulphuric acid. and caustic alkali media, on a bubbling smooth platinum electrode was widely investigated. The influence of acidity, temperature change and alkalinity on the behavior of permanganate in the presence of manganous ions was studied. Evidence is given for the presence of two intermediate species in the permanganate reduction process in acid media. Formation of a Mn(III)-sulphuric acid complex was observed, and the conditions under which the redoxy MnO 4 −-Mn(III) system becomes reversible in sulphuric acid media are defined.

THE HETEROGENEITY OF PANCREATIC DEOXYRIBONUCLEASE

The action of crystalline pancreatic deoxyribonuclease on sodium oligonucleotides in the presence of manganous ions has been studied and a pH optimum of 6.6 observed. Inhibition of the enzyme activity by increased ionic strength of the digest occurred. The liberation of products soluble in uranyl acetate – trichloroacetate was found to vary with enzyme concentration and the relative activity of the enzyme on oligonucleotides was best determined by a logarithm-plot method. The activity of the enzyme towards deoxyribonucleic acid or sodium oligonucleotides as substrate was not affected by treatment with acetone. Evidence of heterogeneity in the crystalline enzyme preparation was obtained using paper electrophoresis and chromatography on carboxymethylcellulose. Two fractions were separated that showed different ratios of activity towards the two substrates employed.

THE HETEROGENEITY OF PANCREATIC DEOXYRIBONUCLEASE

The action of crystalline pancreatic deoxyribonuclease on sodium oligonucleotides in the presence of manganous ions has been studied and a pH optimum of 6.6 observed. Inhibition of the enzyme activity by increased ionic strength of the digest occurred. The liberation of products soluble in uranyl acetate – trichloroacetate was found to vary with enzyme concentration and the relative activity of the enzyme on oligonucleotides was best determined by a logarithm-plot method. The activity of the enzyme towards deoxyribonucleic acid or sodium oligonucleotides as substrate was not affected by treatment with acetone. Evidence of heterogeneity in the crystalline enzyme preparation was obtained using paper electrophoresis and chromatography on carboxymethylcellulose. Two fractions were separated that showed different ratios of activity towards the two substrates employed.