Detection of low molecular weight copper(II) and zinc(II) binding ligands in ultrafiltered milks—the citrate connection

Low molecular weight zinc(II) and copper(II) binding ligands were detected in ultrafiltered human, bovine, and goat milk by the application of the method of modified gel chromatography. Human milk contains at least three detectable low molecular weight copper binders, whereas bovine and goat milk contain at least two. All three milks show two copper binding peaks with the same elution volumes. Zinc chromatograms were less specific than copper. Zinc showed only a single detectable low molecular weight binding ligand common to all three milks. Elution volumes for both zinc(II) and copper(II) citrate and picolinate systems were measured. Elution volumes of both copper(II) and zinc(II) citrate complexes are identical to elution volumes of an intense peak observed with all three milks; it is reasonable to assume that at least part of this peak corresponds to citrate. Human milk alone has a relatively intense binding peak for copper(II) at the same elution volume as the glutamate complex. Human and goat milk have another low intensity copper(II) binding ligand peak at the same elution volume; a number of amino acid complexes have binding peaks at this position. No peak characteristic of the zinc(II) or copper(II) picolinate systems could be found with any of the milks.     

Effects of zomepirac and indomethacin on renal blood flow and electrolyte excretion in anesthetized monkeys

Zomepirac sodium is a new inhibitor of prostaglandin cyclooxygenase with an $\text{in vitro}$ potency equivalent to indomethacin. Since inhibitors of prostaglandin synthesis have marked effects on renal hemodynamics, zomepirac may be expected to reduce renal blood flow (RBF) in a manner similar to indomethacin. This study compares the effects of zomepirac and indomethacin on RBF and electrolyte excretion in anesthetized Rhesus monkeys. Each experiment consisted of a control period followed by 3 or 4 drug treatment periods in which increasing doses of zomepirac (0.5 to 20 mg/kg) or indomethacin (0.5 to 10 mg/kg were given. Indomethacin (5 mg/kg) reduced RBF by 22% and the higher dose (10 mg/kg) reduced RBF by an additional 13%. Zomepirac had little effect on RBF in doses as high as 20 mg/kg. At any given dose the mean plasma concentration of zomepirac was equal to or greater than indomethacin. Peak indomethacin concentration was 48 μg/ml after the 10 mg/kg dose while the peak zomepirac, after 20 mg/kg, was 158 μg/ml. Neither drug had a significant effect on either glomerular filtration rate or excretion rate of sodium or potassium. Thus, zomepirac had only minimal effects on RBF while indomethacin decreased RBF of anesthetized monkeys in a manner qualitatively similar to its effect in other species. The minimal renal effects caused by zomepirac relative to indomethacin in this primate may indicate a therapeutic advantage for zomepirac in man.     

Hydroxyl free radical production in iron-cysteine solutions and protection by zinc

Hydroxyl radicals (OH^?) can be formed on incubation of an oxygenated solution of ferrous sulphate and cysteine. This has been demonstrated by esr using the spin trap DMPO (5,5-dimethyl-1-pyrroline-1-oxide), catalase, and the radical scavengers ethanol and propan-2-ol. Hydroxyl radicals are not formed when excess zinc sulphate is present. These results provide support for the pro-oxidant action of iron and cysteine and a possible protective role for zinc.     

Spectroscopic studies on the binding of iron, terbium, and zinc by apoferritin

Ultraviolet difference spectroscopy has been used to study Fe (III)-apoferritin complexes formed after addition of Fe (II) to apoferritin in air. At constant iron, the recorded spectra varied with time after Fe (II) addition and with the number of iron atoms/molecule (protein concentration). The results indicate that after production of an initial complex, rearrangement or migration of Fe (III) atoms occurs, with polynuclear species forming as end-product, probably by hydrolytic polymerization. The presence of Tb3+ or Zn2+ ions affected the Fe (III) spectra and their development in different ways. The combined data suggest that more than one site, or processes, are involved in ferritin iron-core formation and that some of the metal sites are clustered.     

Adenosine deaminase and immunodeficiency: An in vitro model

We have examined the effect of adenosine and EHNA, a competitive inhibitor of adenosine deaminase (ADA), upon the ability of human peripheral blood lymphocytes to respond to mitogen. Addition of adenosine at concentrations greater than 10 μm (10^?5m) resulted in inhibition of lymphocyte proliferation at 48 hr of culture, provided that the culture medium was relatively free of ADA activity. The actual concentrations of adenosine remaining in inhibited cultures at the time of harvest were considerably lower than those added initially. EHNA alone also inhibited PHA response (and to a lesser extent PWM and Con A responses), but only at high concentrations. Noninhibitory concentrations of EHNA and adenosine together acted synergistically to produce profound inhibition of lymphocyte proliferation. This may provide an in vitro model to explore further the mechanism of the immunodeficiency associated with deficiency of ADA. Adenosine deaminase activity in stimulated cultures did not differ significantly from that found in unstimulated cultures, and the activity per protein or per DNA actually decreased in stimulated versus unstimulated cultures.     

The acute and subacute effects of cadmium on calcium homeostasis and bone trace metals in the rat

The effect of acute and subacute administration of cadmium chloride on calcium homeostasis and the trace metal content of the bone was investigated in the male rat. A single subcutaneous injection of cadmium chloride (1.5 mg Cd⁺⁺/kg) produced a decreased plasma concentration of calcium and a decrease in the femur concentration of both calcium and zinc. Repeated administration of cadmium chloride (1.5 mg Cd⁺⁺/kg daily, for 28 days) caused a marked hypocalciuria that persisted throughout the period of cadmium treatment. There was an accompanying increased excretion of alkaline phosphatase into the urine, and plasma inorganic phosphate was also elevated in these animals. Both of these effects are considered to be evidence of kidney damage.A possible mechanism for this cadmium-induced effect may involve a disturbance of the renal biotransformation of vitamin D, and decreased bioavailability of the essential trace metals due to metallothionein synthesis and excessive loss into the urine.     

A spectroscopic and electrochemical study of the interaction between copper (II) glycylglycyl-L-histidine-N-methylamide and iron (III) tetra-p-sulfophenylporphine

A binuclear complex has been produced by the reaction of an iron porphyrin (sodium tetra-p-sulfophenylporphine iron (III)-FeTPPS) with a copper metallo-tripeptide (copper (II) glycylglycyl-L-histidine-N-methylamide-CuGGH) in aqueous solution. The system has been characterized by electron spin resonance (ESR) spectroscopy, optical absorption spectroscopy, and electrochemical methods. Room-temperature ESR spectra of the copper complex and low-temperature ESR spectra of the iron porphine provide evidence for the formation of a binuclear complex. These findings are supported by absorption spectroscopy and electrochemical studies, and lead to a value of ca. 2 X 10(-3) M-1 (at room temperature) for the equilibrium constant for complex formation. The relevance of this system to the enzymic active site of mammalian cytochrome c oxidase is discussed.     

Frequency and intensity of chick distress calls: Effects on maternal foodcalling in chickens

The food call of broody domestic hens was used to measure maternal response to four frequency components found in chick distress calls (2,3,4 and 5 kHz) and to variations in distress call intensity (0—86 dB). Foodcalling increased significantly with frequency of the pure-tone test pulse; response to a taped distress call occurred between 40 dB and 86 dB intensity with a maximum at 60–65 dB. The results suggest that the mother uses the higher frequency components in recognizing the distress call, but responds maximally within a specific intensity range. The selective advantage of such behaviour is discussed.     

Isolation of a low molecular weight zinc binding ligand from human milk

A low molecular weight zinc binding compound from human milk has been purified by ultrafiltration, gel filtration, and ion-exchange chromatography. Evidence is provided that this compound is citrate. A higher amount of citrate-bound zinc was found in human milk than in cow's milk. It is suggested that the therapeutic value of human milk for patients with the genetic disorder of zinc metabolism acrodermatitis enteropathica (AE) derives from a greater content of bioavailable zinc citrate in human than in cow's milk.     

Circular dichroism (CD) studies on yeast enolase: activation by divalent cations

The effect of divalent cations on the near ultraviolet circular dichroism (CD) spectrum of yeast enolase showed that calcium, magnesium, and nickel ions produced identical changes. This was interpreted as indicating that the cations bound to the same sites on the enzyme and produced identical changes in tertiary structure. There was no effect of magnesium ion on the far ultraviolet spectrum. Evidently magnesium ion has no effect on the secondary structure. Substrate bound to the enzyme when the above cations were present although calcium permits no enzymatic activity. The CD spectral difference produced by the substrate was nearly the reverse of that produced by the metal ions. Glycolic acid phosphate, a competitive inhibitor lacking carbon-3, produced no effect, indicating carbon-3 was necessary for the CD spectral changes. The CD and visible absorption spectra of nickel and cobalt bound to various sites on the enzyme showed that the binding sites were octahedral or distorted octahedral in coordination and that the ligands appeared to be oxyligands: water molecules, hydroxyl or carboxyl groups. Examination of the effects of substrate and two compounds thought to be "transition state analogues" showed that these perturbed the "conformational" sites of the enzyme. The "catalytic" and "inhibitory" sites did not appear to be very CD active.     

Raman and infrared spectroscopy of the oxo-bridged iron(III) complex, [Cl3Fe-O-FeCl3]-2 as a spectroscopic model for the oxo bridge in hemerythrin and ribonucleotide reductase

Vibrational spectroscopic data were collected on the salt [C5H6N]2[Cl3FeOFeCl3] . C5H5N, which has previously been structurally characterized by X-ray crystallography. The modes associated with the oxo bridge were identified by experiments on the 18O-containing species. Spectra for the mu-16O complex contain Raman bands at 870, 458, and 203 cm-1 that shift to 826, 440, and 198 cm-1 in the mu-18O complex. These are respectively assigned to the asymmetric, symmetric, and angle deformations of the bent Fe-O-Fe moiety. A normal mode vibration analysis based on a simple valence force field for the Fe-O-Fe portion of the molecule provides surprisingly good agreement with these experimental frequencies and their assignments. The vibrational data for this simple inorganic complex confirm the assignment of a resonance Raman band around 500 cm-1 in the oxygen-carrying protein hemerythrin and enzyme ribonucleotide reductase as the symmetric stretch of an oxo bridge between two iron(III) centers.     

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured human fetal aortic smooth muscle cells.

Cultured human fetal aortic smooth muscle cells derived from the abdominal aorta converted benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) $\text{via}$ cytochrome P-450-dependent monooxygenation to metabolites detectable by both a highly sensitive radiometric assay and high pressure liquid chromatography (HPLC). Cells incubated with ³H-BaP transformed this substrate primarily to phenols. ¹⁴C-DMBA was converted to metabolites that cochromatographed with 12-hydroxymethyl-7-methylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz-[a]anthracene, 7,12-dihydroxymethylbenz[a]anthracene, and trans-8,9-dihydrodiol-7,12-DMBA. Exposure of cells in culture to 13 μM 1,2-benz[a]anthracene resulted in increased oxidative metabolism of both BaP and DMBA. In the case of BaP, total phenol formation was increased, while with DMBA all metabilities detected by HPLC were increased. Support for the potential role of metabolism of polycyclic aromatic hydrocarbons by aortic smooth muscle cells in the etiology of atherosclerosis was obtained.     

Application of the Hodgkin-Huxley equations to an electric field model for interaction between excitable cells

We previously described a model for the electrical transfer of excitation from one cell to the next which utilized the electric potential generated in the junctional cleft between the cells. Low-resistance connections between the cells were not used in the model, and it was assumed that the junctional membranes were excitable. This model was analyzed for the static case without capacitances and for the dynamic case in which capacitances were part of the circuit elements. For simplicity, the Na⁺ resistance (R_Na), after a threshold potential was exceeded, was allowed to decrease exponentially (to 1% of its initial value) within 0·25–1·0 ms, and possible changes in the K⁺ resistance were ignored. In this paper, we have incorporated the Hodgkin-Huxley equations into the operation of the lumped membrane units for the electrical equivalent circuit of the cell membrane. The parameters varied are the membrane capacitances, resistances, maximum Na⁺ conductance ($\text{g}{}_{\mathrm{<mtext>Na</mtext>}}$), and the radial cleft resistance (R_jc). We demonstrated that our model worked very well, i.e. the successful transfer of action potentials was achieved, with the membrane units following Hodgkin-Huxley dynamics for changes in g_Na and g_K. The calculations indicate that transmission is facilitated when the junctional units have a higher $\text{g}{}_{\mathrm{<mtext>Na</mtext>}}$ and a lower capacitance and when R_jc is elevated. Lowering the resistance of the junctional membrane units several fold, relative to the surface membrane units, also facilitated transmission; however, the absolute resistance of the junctional membrane was still well above the maximum value that would allow sufficient local-circuit current to flow to effect transmission. Thus, the electric field model provides an alternative means of cell-to-cell propagation between myocardial cells which is electrical in nature but does not require the presence of low-resistance connections between cells.     

Purification of secretory granules on a urografin gradient

A procedure is described for the preparation of highly purified secretory granules from rat parotid glands. A 250g supernatant fraction of parotid homogenates is layered over a gradient composed of 20, 30, and 40% solution of Urografin. The secretory granules form a layer at the 30–40% interface. Chemical and enzymatic analysis show the purified granules contain 63 ± 8% of the amylase, 2 ± 0.8% of the succinic dehydrogenase, little or no RNA, and 10% of the protein present in the 250g supernatant fraction.     

Target size analysis of serotonin 5-HT1 and 5-HT2 receptors in bovine brain membranes

Freeze-dried crude synaptic membranes prepared from bovine cerebral cortex and striatum were exposed to high energy gamma ray from the source of 60Co. The size of serotonin 5-HT1 receptors labeled by [3H]serotonin and that of 5-HT2 receptors labeled by [3H]spiperone or [3H]ketanserin was determined by target size analyses. The values were 57,000 daltons, 145,000 daltons and 152,000 daltons for the cerebral cortex and 56,000 daltons, 141,000 daltons and 150,000 daltons for the striatum, respectively. The estimated sizes were deduced by reference to enzyme standards with known molecular masses and which were irradiated in parallel. Our results demonstrate that the molecular entities in situ for 5-HT1 receptors are distinct from those for 5-HT2 receptors, thus supporting data on the existence of two distinct populations of serotonin receptors, hitherto evidenced physiopharmacologically.     

The binding of copper(II) and zinc(II) to oxidized glutathione

1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained.     

Activation of yeast enolase by Cd(II)

Activation of yeast enolase by Cd2+ exhibits properties similar to activation by the physiological cofactor Mg2+. The activity is weakly stimulated, then inhibited by increasing ionic strength. The activity increases, then falls with increasing Cd2+ concentration. The effect of pH on activity produced by Cd2+ is very similar to that produced by Mg2+, except that the Cd2+ profile is shifted one pH unit to more alkaline values, and the maximum activity of the Cd2+-enzyme is about 10% of that of the Mg2+-enzyme. The apparent kinetic parameters of Cd2+ activation show little effect of pH except for inhibition by high concentrations of Cd2+: the apparent Ki increases sharply with pH. This is interpreted as the result of Cd2+ being a less effective "catalytic" metal ion, and Cd2+ being more effective in stabilizing the enzyme at alkaline pH's. The similarity of effects of ionic strength, divalent cation, and pH may be due to interaction with the same six sites per mole of enzyme. We also characterized the dependence of what is believed to be the enzyme-catalyzed enolization of a substrate analog, D-tartronate semialdehyde-2-phosphate (TSP) on similar parameters of pH, ionic strength, etc. The putative enolization is dependent on catalytic metal ion, although the TSP binds to the conformational Cd2+-enzyme complex. The reaction is very slow and very pH dependent, increasing with pH with a midpoint of reaction velocity at pH 8.7. There is a strong qualitative correlation between pH dependencies of reaction velocity of substrate conversion and TSP enolization and absorbance of the enzyme-bound TSP enolate, whether with Mg2+ or Cd2+ as cofactor. The slowness of the Cd2+-TSP reaction is not limited by proton release or any reaction involving covalent bonds to hydrogen. The apparent reaction rate constant increases linearly with Cd2+ concentration. Addition of excess ethylenediaminetetraacetic acid reverses the TSP reaction, but again very slowly. The binding of Cd2+ to the catalytic sites is characterized by low association and dissociation rate constants.     

Cadmium(II)-113 NMR studies of the mechanism of metal ion activation of yeast enolase

Yeast enolase binds one mole of 113Cd2+ per subunit at a site that consists of all oxyligands in a distorted octahedral environment. This "conformational" metal ion's environment undergoes further distortion on addition of substrate/product or analogs. At pH's below the optimum value the shifted resonance tends to break up into several, suggesting the existence of several slowly exchanging intermediate forms. At acid pH's, on addition of one additional mole/subunit of 113Cd2+, which greatly increases catalysis, "conformational" resonance(s) further broadens, suggesting that the second, "catalytic" metal ion increases the rates of interconversion between "conformational" species. At more alkaline pH's, near the optimum pH, the "conformational" peak is sharpened, which suggests that very fast interconversion is occurring. The position of the "catalytic" metal ion resonance also suggests all oxyligands in a distorted octahedral geometry. The "catalytic" resonance is often broadened to the point where it cannot be seen, suggesting rapid changes in its geometry due to interconversion of substrate and product.