Nickel in plants: I. Uptake kinetics using intact soybean seedlings

The absorption of Ni²⁺ by 21-day-old soybean plants (Glycine max cv. Williams) was investigated with respect to its concentration dependence, transport kinetics, and interactions with various nutrient cations. Nickel absorption, measured as a function of concentration (0.02 to 100 μm), demonstrated the presence of multiple absorption isotherms. Each of the three isotherms conforms to Michaelis-Menten kinetics; kinetic constants are reported for uptake by the intact plant and for transfer from root to shoot tissues. The absorption of Ni²⁺ by the intact plant and its transfer from root to shoot were inhibited by the presence of Cu²⁺, Zn²⁺, Fe²⁺, and Co²⁺. Competition kinetic studies showed Cu²⁺ and Zn²⁺ to inhibit Ni²⁺ absorption competitively, suggesting that Ni²⁺, Cu²⁺, and Zn²⁺ are absorbed using the same carrier site. Calculated K_m and K_i constants for Ni²⁺ in the presence and absence of Cu²⁺ were 6.1 and 9.2 μm, respectively, whereas K_m and K_i constants were calculated to be 6.7 and 24.4 μm, respectively, for Ni²⁺ in the presence and absence of Zn²⁺. The mechanism of inhibition of Ni²⁺ in the presence of Fe²⁺ and Co²⁺ was not resolved by classical kinetic relationships.     

Cadmium uptake kinetics in intact soybean plants

The absorption characteristics of Cd²⁺ by 10- to 12-day-old soybean plants (Glycine max cv Williams) were investigated with respect to influence of Cd concentration on adsorption to root surfaces, root absorption, transport kinetics and interaction with the nutrient cations Cu²⁺, Fe²⁺, Mn²⁺, and Zn²⁺. The fraction of nonexchangeable Cd bound to roots remained relatively constant at 20 to 25% of the absorbed fraction at solution concentration of 0.0025 to 0.5 micromolar, and increased to 45% at solution concentration in excess of 0.5 micromolar. The exchangeable fraction represented 1.4 to 32% of the absorbed fraction, and was concentration dependent. Using dinitrophenol as a metabolic inhibitor, the `metabolically absorbed' fraction was shown to represent 75 to 80% of the absorbed fraction at concentration less than 0.5 micromolar, and decreased to 55% at 5 micromolar. At comparatively low Cd concentrations, 0.0025 to micromolar 0.3, root absorption exhibited two isotherms with K₂ values of 0.08 and 1.2 micromolar. Root absorption and transfer from root to shoot of Cd²⁺ was inhibited by Cu²⁺, Fe²⁺, Mn²⁺, and Zn²⁺. Analyses of kinetic interaction of these nutrient cations with Cd²⁺ indicated that Cu²⁺, Fe²⁺, Zn²⁺, and possibly Mn²⁺ inhibited Cd absorption competitively suggesting an involvement of a common transport site or process.     

Glutamine synthetase of pea leaves: divalent cation effects, substrate specificity, and other properties

Purified glutamine synthetase from pea seedlings was most active with Mg²⁺ as the metal activator, but Mn²⁺ and Co²⁺ were 45 to 60% and 30 to 45% as effective, respectively, when assayed at the optimal pH for each cation. The Mg²⁺ saturation curve was quite sigmoid, and evidence indicates that MgATP is the active ATP substance. Co²⁺ also gave a sigmoidal saturation curve, but when Mn²⁺ was varied only slightly sigmoidal kinetics were seen. Addition of Mn²⁺, Ca²⁺, or Zn²⁺ at low concentrations sharply inhibited the Mg²⁺ -dependent activity, partially by shifting the pH optimum. Addition of Co²⁺ did not inhibit Mg²⁺-dependent activity. The nucleotide triphosphate specificity changed markedly when Co²⁺ or Mn²⁺ replaced Mg²⁺. Using the Mg²⁺-dependent assay, the Michaelis constant (Km) for NH₄⁺ was about 1.9 × 10⁻³ M. The Km for l-glutamate was directly proportional to ATP concentration and ranged from 3.5 to 12.4 mm with the ATP levels tested. The Km for MgATP also varied with the l-glutamate concentration, ranging from 0.14 mm to 0.65 mm. Ethylenediaminetetracetic acid activated the enzyme by up to 54%, while sulfhydryl reagents gave slight activation, occasionally up to 34%.     

Characterization of the Effects of Divalent Cations on the Coupled Activities of the H-ATPase in Tonoplast Vesicles

The substrate requirement of the H⁺-ATPase in purified corn root tonoplast vesicles was investigated. The coupled activities, ATP hydrolysis and proton pumping, were simultaneously supported only by Mg²⁺ or Mn²⁺. The presence of Ca²⁺ or Ba²⁺ did not significantly affect the coupled activities. The addition of Cd²⁺, Co²⁺, Cu²⁺, and Zn²⁺ inhibited both the hydrolysis of Mg-ATP and the proton transport. However, the inhibition of proton pumping was more pronounced. Based on equilibrium analysis, both ATP-complexed and free forms of these cations were inhibitory. Inhibition of the hydrolysis of Mg-ATP could be correlated to the concentrations of the ATP-complex of Zn. On the other hand, the free Cu²⁺ and Co²⁺ were effective in inhibiting hydrolysis. For proton pumping, the ATP complexes of Co²⁺, Cu²⁺, and Zn²⁺ were effective inhibitors. However, this inhibition could be further modulated by free Co²⁺, Cu²⁺, and Zn²⁺. While the equilibrium concentrations of Cd-ATP and free Cd²⁺ were not estimated, the total concentration of this cation needed to inhibit the coupled activities of the H⁺-ATPase was found to be in the range of 10 to 100 micromolars. The presence of free divalent cations also affected the structure of the lipid phase in tonoplast membrane as demonstrated by the changes of emission intensity and polarization of incorporated 1,6-diphenyl-1,3,5-hexatriene. The differential inhibition caused by these cations could be interpreted by interactions with the protogenic domain of the membrane as previously proposed in “indirect-link” mechanism.     

Levels and sub-cellular distribution of physiologically important metal ions in neuronal cells cultured from chick embryo cerebral cortex

Mg²⁺, Ca²⁺, Mn²⁺, Zn²⁺, and Cu content of neurons from chick embryo cortex cultivated in chemically defined serum free growth medium was determined by energy dispersive X-ray fluorescence and atomic absorption spectroscopy. The intracellular volume of cultured neurons was determined to be 2.73 l/mg. Intracellular Mn²⁺, Fe²⁺, Zn²⁺, and Cu²⁺ in the cultivated neurons were 100–200 times the concentrations in the growth medium: Mg²⁺ and Ca²⁺ were 0.9 and 1.7 mM respectively, around 20 fold higher than in growth medium. Mg²⁺, Fe²⁺, Cu²⁺ and Zn²⁺ concentrations in neurons were in the range of ca. 300–600 M, approximately 2–3 times the values previously reported in glial cells; Ca²⁺ and Mn²⁺ content of the neurons were higher by 5 and 10 fold respectively compared to glial cells. In neurons, the subcellular distribution of Fe²⁺, Cu²⁺, and Mn²⁺ follows the rank order: cytosol>microsomes>mitochondria; for Zn²⁺ the distribution differs as following: cytosol >mitochondria>microsomes. Determination of the superoxide dismutase activities in the cultivated neurons indicated that the Mn²⁺ linked activity predominates whereas, the Cu-Zn dependent enzyme is dominant in glial cells. Enrichment of the culture medium with Mn²⁺ to 2.5 M enhanced the Mn-SOD by approximately 33% but Cu²⁺–Zn²⁺ enzyme activity was not modified. The high Mn²⁺ content, the capacity to accumulate Mn²⁺, and the predominancy of the Mn–SOD form observed in neurons is in accord with a fundamental functional role for this metal ion in this type of brain cells.     

Increased Activity of Chromatin-bound Ribonucleic Acid Polymerase from Soybean Hypocotyl with Spermidine and High Ionic Strength

Optimal activity of chromatin-bound RNA polymerase from soybeans is obtained with 1 mm Mn²⁻, but only when high ionic strength or polyamines are included in the medium. Such inclusion does not increase the Mg²⁺ activation of the polymerase, but it does lower the concentration needed for optimum activity from 10 mm to 1 mm. Mg²⁻ activation is inhibited by added Mn²⁺, and the inhibition is relieved by high ionic strength or spermidine. The RNA polymerase with either cation is almost entirely polymerase I at low and high ionic strength as evidenced by insensitivity to α-amanitin. Treatment of soybean seedlings with 2,4-dichlorophenoxyacetic acid does not change these characteristics; although the activity rises 3- to 4-fold.     

Metal Ions and Electrolytes Regulate the Dissociation of Heme from Human Hemopexin at Physiological pH

The stability of the hemopexin-heme (Hx-heme) complex to dissociation of the heme prosthetic group has been examined in bicarbonate buffers in the presence and absence of various divalent metal ions. In NH₄HCO₃ buffer (pH 7.4, 20 mm, 25 °C) containing Zn²⁺ (100 μm), 14% of the heme dissociates from this complex (4.5 μm) within 10 min, and 50% dissociates within 2 h. In the absence of metal ions, the rate of dissociation of this complex is far lower, is decreased further in KHCO₃ solution, and is minimal in NaHCO₃. In NH₄HCO₃ buffer, dissociation of the Hx-heme complex is accelerated by addition of divalent metals with decreasing efficiency in the order Zn²⁺ > Cu²⁺ ≫ Ni²⁺ > Co²⁺≫Mn²⁺. Addition of Ca²⁺ prior to addition of Zn²⁺ stabilizes the Hx-heme complex to dissociation of the heme group, and addition of Ca²⁺ after Zn²⁺-induced dissociation of the Hx-heme complex results in re-formation of the Hx-heme complex. These effects are greatly accelerated at 37 °C and diminished in other buffers. Overall, the solution conditions that promote formation of the Hx-heme complex are similar to those found in blood plasma, and conditions that promote release of heme are similar to those that the Hx-heme complex should encounter in endosomes following endocytosis of the complex formed with its hepatic receptor.     

Physiology of genotypic differences in zinc and copper uptake in rice and tomato

Summary Excised roots of rice (Oryzae sativa L.) cv IR26 absorbed both Zn²⁺ and Cu²⁺ from 0.01 mM to 0.50 mM external solutions at rates twice those of cv M101 over a 30-min period. However, the latter have a two-fold greater affinity (1/Km) for Zn²⁺ and Cu²⁺ than do those of the former. Zinc²⁺ and Cu²⁺ mutually and competitively inhibited uptake of each other, indicating that both micronutrient cations are absorbed through the same uptake mechanism or carrier sites. Further, these differences in uptake rates are restricted to roots but they cannot be explained by variations in root surface areas. Excised roots of tomato (Lycopersicon esculentum L.) cv Kewalo absorbed Zn²⁺ and Cu²⁺ much more rapidly than did cv Sel 7625-2. Uptake of each cation was competitively and reciprocally inhibited by the other, so Zn²⁺ and Cu²⁺ are seemingly accumulated through the same uptake system in tomato also. Tomato cultivars Kewalo and Sel 7625-2 did not differ with regard to affinities of the root apices for Zn²⁺ and Cu²⁺; however. Vmax values for Zn²⁺ and Cu²⁺ uptake by roots of cv Kewalo were three-fold greater than those for cv Sel 7625-2. Journal Series 2991 of the Hawaii Institute of Tropical Agriculture and Human Resources. Supported by USDA/CSRS Grants Program in Tropical and Subtropical Agriculture (83-CSRS-2-2245).     

Effects of Metal Ions on the Conformation and Activity of Acutolysin D from Agkistrodon Acutus Venom

Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A ^1% ₂₈₀) of acutolyisn D have been determined to be 47,850 ± 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca²⁺-binding sites and two Zn²⁺-binding sites determined by atomic absorption spectrophotometer. Zn²⁺ is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn²⁺ significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)₂ precipitate formation. Ca²⁺ is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca²⁺ markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu²⁺, Co²⁺, Mn²⁺ or Tb³⁺ and inhibited completely by Cd²⁺, is enhanced by Mg²⁺. The fluorescence intensity of the protein decreases in the presence of Cu²⁺, Co²⁺, Cd²⁺ or Mn²⁺, but neither for Ca²⁺, Mg²⁺ nor for Tb³⁺. Zn²⁺, Ca²⁺, Mg²⁺, Cu²⁺, Mn²⁺, Co²⁺ and Tb³⁺ have slight effects on its secondary structure contents. In addition, Cd²⁺ causes a marked increase of antiparallel β-sheet content from 45.5% to 60.2%.     

Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) : Orientation of Active Site and Activation by Ca and Mg

Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/v) sodium deoxycholate, whereas in fractions enriched in inside-out (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP₂ phospholipase C was dependent on Ca²⁺ with maximum activity at 10 to 100 μm free Ca²⁺ and half-maximal activation at 0.1 to 1 μm free Ca²⁺. In the presence of 10 μm Ca²⁺, 1 to 2 mm MgCl₂ or MgSO₄ further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) inhibited the enzyme activity. The stimulatory effect by Mg²⁺ was observed also when 35 mm NaCl was included. Thus, the PIP and PIP₂ phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP₂ as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca²⁺, with maximum activity at 1 mm CaCl₂, and could not be further stimulated by Mg²⁺.     

The Activity of Ribulose Diphosphate Carboxylase in Extracts of Gonyaulax polyedra in the Day and the Night Phases of the Circadian Rhythm of Photosynthesis

The ribulose 1,5-diphosphate carboxylase from Gonyaulax polyedra Stein. has a half-life of about four hours in buffer, but can be stabilized by the addition of 50% glycerol. The optimum pH is 7.8 to 8.0 and the optimum Mg²⁺ concentration is 3 mm. Heavy metal ions (Cu²⁺, Hg²⁺, Ni²⁺, Zn²⁺), EDTA, pyrophosphate, and adenosine triphosphate were strongly inhibitory. Ribulose 1,5-diphosphate carboxylase from Gonyaulax was not cold-sensitive or activated by light activation factor from tomato or Gonyaulax. No difference in the activity of this enzyme was detected when extracts prepared at the maximum and the minimum of the circadian rhythm of photosynthesis were compared. The Km of HCO₃⁻ was also the same (16 to 19 mm).     

Partial Purification and Properties of d-Glucosamine 6-Phosphate N-Acetyltransferase from Phaseolus aureus

d-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for d-glucosamine-6-P. Neither d-glucosamine nor d-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 × 10⁻²m EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg²⁺ was required for maximum activity of the enzyme. Mn²⁺ and Zn²⁺ were inhibitory, while Co²⁺ had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg²⁺ on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 × 10⁻⁴m and 7.1 × 10⁻⁴m were obtained for d-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 × 10⁻⁴mp-hydroxymercuribenzoate, and this inhibition was partially reversed by l-cysteine; indicating the presence of sulfhydryl groups at or near the active site of the enzyme.     

Separation and partial characterization of multiple ribonucleic Acid polymerases from soybean hypocotyl

Two major peaks of RNA polymerase activity have been routinely separated by diethylaminoethyl cellulose chromatography following solubilization from soybean (Glycine max L. var. Wayne) chromatin. The relative amounts of these two peaks depend upon the manner in which the chromatin is purified. Pelleting the chromatin through dense sucrose solutions results in not only a loss of total solubilized RNA polymerase activity but also a selective loss of the α-amanitin-sensitive form of the enzyme. Peak I elutes from a diethylaminoethyl cellulose column at a KCl concentration of approximately 0.27 m, is insensitive to α-amanitin and rifamycin, and has Mg²⁺ + Mn²⁺ optima of 5 mm and 1.25 mm, respectively. The enzyme is inhibited by KCl concentrations of about 0.03 m or greater. Peak II elutes from the column at a KCl concentration of approximately 0.35 m, is sensitive to α-amanitin, insensitive to rifamycin, and has Mg²⁺ + Mn²⁺ optima of 2 mm and 1.0 mm, respectively. Activity is inhibited by KCl concentrations of about 0.06 m or greater. Both enzymes prefer denatured calf thymus DNA, but peak II exhibits a stronger preference.     

Partial Purification and Properties of an Alkaline alpha-Galactosidase from Mature Leaves of Cucurbita pepo

A fourth molecular from of α-galactosidase, designated L_IV, an alkaline α-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165-fold. It was active over a narrow pH range with optimal hydrolysis of p-nitrophenyl-α-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. K_m determinations in McIlvaine buffer (200 millimolar Na₂-phosphate, 100 millimolar citric acid, pH 7.5) for p-nitrophenyl-α-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. L_IV was partially inhibited by Ca²⁺, Mg²⁺, and Mn²⁺, more so by Ni²⁺, Zn²⁺, and Co²⁺, and highly so by Cu²⁺, Ag²⁺, Hg²⁺ and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-α-d-galactoside or by myo-inositol, but α-d-galactose was a strong inhibitor. As observed for most other forms of α-galactosidase, L_IV only catalyzed the hydrolysis of glycosides possessing the α-d-galactose configuration at C₁, C₂, and C₄, and did not hydrolyze p-nitrophenyl-α-d-fucoside (α-d-galactose substituted at C₆). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-α-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of L_IV in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of L_IVin vivo. It was possible that the results reflected the ability of these anions, and ethylene-diaminetetraacetate, to restore L_IV activity through coordination with some toxic cation introduced as a buffer contaminant.     

Vanadium Uptake by Plants: Absorption Kinetics and the Effects of pH, Metabolic Inhibitors, and Other Anions and Cations

The kinetics of vanadium absorption by excised barley (Hordeum vulgare L., cv. Eire) roots were investigated with respect to ionic species of V in solution, time and concentration dependence, Ca sensitivity, and interaction with various anions, cations, and pH levels. The role of metabolism in V absorption was also studied using anaerobic treatment (N₂ gas) and chemical inhibitors (NaN₃, KCN, or 2,4-dinitrophenol). Approximately one-third of the labeled V initially taken up by excised roots was desorbed to a constant level after 45 min in unlabeled V solutions. The rate of absorption of labeled V from 5 μm NH₄VO₃ solutions containing 0.5 mm CaSO₄ was constant for at least 3 hours. Omission of Ca resulted in a 72% reduction in V uptake when compared to controls with 0.5 mm CaSO₄. The rate of uptake of V was highest at pH 4 but dropped to a very low level at pH 10. It was relatively constant between the pH levels of 5 and 8 at which the VO₃⁻ ion is the predominant ionic species in solution. The rate of absorption of V was followed as a function of concentrations from 0.5 to 100 μm NH₄VO₃. It was found to be a linear function of concentration and did not follow saturation kinetics. Absorption experiments carried out with labeled V from either N_aVO₃ or NH₄VO₃ sources gave similar results. No anion studied (i.e. HPO₄²⁻, HAsO₄²⁻, MoO₄²⁻, SeO₄²⁻, SeO₃²⁻, CrO₄²⁻, BO₃³⁻, No₃⁻, and Cl⁻) interfered appreciably (i.e. less than 30% inhibition) with the absorption of labeled V. Anaerobic treatment of absorption solution with N₂ gas did not inhibit V absorption by excised roots. The results obtained using chemical inhibitors were not consistent. It was concluded that V is not actively absorbed by excised barley roots.     

Regulation of divalent cations of the membrane-bound pyrophosphatase of Rhodospirillum rubrum,as shown by the hydrolysis of tripositive-pyrophosphate complexes

Tripositive-pyrophosphate [M(III)-PPi] complexes were used to investigate the role of free divalent cations on the membrane-bound pyrophosphatase. Divalent cations remain free and the M(III)-PPi complexes were employed as substrates. Formation of a La-PPi complex was studied by fluorescence, and the fact that Zn²⁺ and Mg²⁺ remain free in the solution was validated. Hydrolysis of La-PPi is stimulated by the presence of fixed concentrations of free Mg²⁺ or Zn²⁺ and this stimulation depends on the concentration of the cations when the La-PPi complex is fixed. The divalent cation stimulation order is Zn²⁺ > Co²⁺ > Mg²⁺ > Mn²⁺ > Ca²⁺ (at 0.5 mm of free cation). With different M(III)-PPi complexes, Zn²⁺ produces the same K _m, for all the complexes and Mg²⁺ stimulates with a different K _m. The results suggest that both Mg²⁺ and Zn²⁺ activate the membrane-bound pyrophosphatase but through different mechanisms.     

Use of ion chromatography for the determination of selected metals in blood serum of patients with type 2 diabetes

Ion chromatography followed by microwave-induced acid digestion was used to evaluate the serum levels of Fe³⁺, Cu²⁺, Ni²⁺, Zn²⁺, and Mn²⁺ in patients with diagnosed type 2 diabetes and in healthy controls. Recoveries ranged from 98.0% to 102% for Fe³⁺, from 89.9% to 100% for Cu²⁺, from 87.9% to 102% for Zn²⁺, and from 89.6% to 102% for Mn²⁺ were determined by examining samples spiked with various amounts of all the studied ions. The time of mineralization longer than 28 min did not affect the assay values. Precision was assessed at four unique concentrations in replicates of six, on four separate occasions. RSD was determined to be 1.16% for Fe³⁺, 5.20% for Cu²⁺, 2.8% for Zn²⁺, and 3.75% for Mn²⁺. The accuracy results (values of RSD) were as follows: 5.16% for Fe³⁺, 6.35% for Cu²⁺, 4.9% for Zn²⁺, and 7.23% for Mn²⁺.The statistical analysis confirmed that mean concentrations of Fe³⁺ and Zn²⁺ did not differ significantly from analogous values in the control group. Patients who additionally suffered from hypertension had higher copper concentrations compared with diabetic patients. For diabetics the presence of Mn²⁺ was not stated (LOD values amounting to 0.006 μg/mL). Ni²⁺ was not detectable for either the studied group or the control group (LOD=0.006 μg/mL).     

Physical and kinetic properties of sheep liver pyridoxine kinase

Pyridoxine kinase purified from sheep liver was found to consist of a single polypeptide chain with a molecular weight of 60,000 as determined by gel filtration, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric pH of the enzyme was 5.1, and the pH optimum was between 5.5 and 6.0. The enzyme required divalent cations for activity. At cation concentrations of 80 μm, the enzyme activity with each cation was in the order of Zn²⁺ > Mn²⁺ > Mg²⁺. At cation concentrations of 400 μm, the enzyme activity with each cation was in the order of Mn²⁺ > Zn²⁺ > Mg²⁺. Excess free divalent cation inhibited the enzyme. Pyridoxine kinase also required monovalent cations. The enzyme activation was greatest with K⁺, then Rb⁺ and NH₄⁺, whereas the enzyme had very little activity with Na⁺, Li⁺, or Cs⁺. Na⁺ did not interfere with the activation by K⁺. The activation of the kinase by K⁺, NH₄⁺, and Rb⁺ followed Michaelis-Menten kinetics, and the apparent K_m values for the cations were 8.9, 3.7, and 5.3 mm, respectively. Increasing the potassium concentration lowered the apparent K_m value of the enzyme for pyridoxine and had little or no effect on the K_m for ZnATP^2? or the V of the kinase-catalyzed reaction.     

Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a K_m value for dCMP of 0.91mm. dCTP activation required the presence of Mg²⁺ or Mn²⁺ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg²⁺ or Mn²⁺ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).     

Proton-Metal Cation Exchange in the Cell Wall of Nitella flexilis

When protons are exchanging for bivalent cations (Cu²⁺, Zn²⁺, or Ca²⁺) on the carboxylic groups of Nitella flexilis cell wall, the values of the respective global equilibrium constants do not change up to a protonation degree of 80%. These values drastically increase at higher proton concentrations and tend to 3.4, which is the intrinsic pK value of the constitutive α-d-galacturonic acid monomer. These data suggest that the electric field in the matricial polymer and the cation bridges between pairs of negative sites have disappeared.