Inactivation of the rabbit parotid Na/K/Cl cotransporter by N-ethylmaleimide

Summary The inactivation of the rabbit parotid Na/K/Cl cotransporter by the irreversible sulfhydryl reagent N-ethylmaleimide (NEM) is studied by monitoring its effect on high affinity bumetanide binding to the carrier. NEM reduces the number of bumetanide binding sites with no significant change in the affinity of those remaining. NEM also reduces KCl-dependent²²Na flux via the cotransporter by the same factor as the reduction in bumetanide binding sites. Both bumetanide and its analogue furosemide can protect against the effect of NEM. The concentration range over which this protection occurs is in good agreement with affinities of these two compounds for the high affinity bumetanide binding site (2.6 and 85 m, respectively), indicating an association of this site with the site of action of NEM. Also consistent with this hypothesis are the observations that (i) sodium and potassium, both of which are required for high affinity bumetanide binding, increase the rate of inactivation of binding by NEM and (ii) chloride, at concentrations previously shown to competitively inhibit bumetanide binding, protects the cotransporter against NEM. The effects of NEM on bumetanide binding are mimicked by another highly specific sulfhydryl reagent, methyl methanethiolsulfonate. The apparent rate constant for inactivation of high affinity bumetanide binding by NEM is a hyperbolic function of NEM concentration consistent with a model in which the inactivation reaction is first order in [NEM] and proceeds through an intermediate adsorptive complex. The data indicate that the presence of a reduced sulfhydryl group at or closely related to the bumetanide binding site is essential for the operation of the parotid Na/K/Cl cotransporter.     

Ionic dependence of bumetanide binding to the rabbit parotid Na/K/Cl cotransporter

Summary The Na/K/Cl-dependent component of the binding of the loop diuretic bumetanide to basolateral membrane vesicles from the rabbit parotid is studied. A Scatchard analysis indicates that this binding is due to a single high-affinity site withK _D =3.2±0.3 m (n=9) at 100mm sodium, 100mm potassium and 5mm chloride. When KCl-dependent²²Na transport and tracer [³H]-bumetanide binding are monitored simultaneously as a function of (unlabeled) bumetanide concentration it is found that theK _0.5 for bumetanide inhibition of both processes are identical indicating that the high-affinity bumetanide binding site studied here is identical with a bumetanide-inhibitory site on the Na/K/Cl cotransport system previously identified in this preparation (R.J. Turner, J.N. George and B.J. Baum,J. Membrane Biol. 94:143–152, 1986). High-affinity bumetanide binding exhibits a hyperbolic dependence on both [Na] and [K] consistent with Na/bumetanide and K/bumetanide binding stoichiometries of 11 andK _0.5 values of approximately 33mm for sodium and 23mm for potassium. In contrast, the dependence on [Cl] is biphasic, with bumetanide binding increasing from 0 to 5mm chloride and decreasing toward baseline levels thereafter. Scatchard analysis of this latter inhibitory effect of chloride indicates a competitive interaction with bumetanide in agreement with earlier indications that bumetanide inhibits Na/K/Cl cotransport at a chloride site. However, studies of the effects of various anions on bumetanide binding and²²Na transport show a poor correlation between the specificities of these two processes, suggesting that the inhibitory chloride site is not a chloride transport site.     

Role of phospholipids in the binding of bumetanide to the rabbit parotid Na/K/Cl cotransporter

Summary It was recently reported (Turner, R.J., George, J.N., 1990,J. Membrane Biol. 113:203–210) that the high affinity bumetanide binding site of the rabbit parotid Na/K/Cl cotransporter could be extracted from a basolateral membrane preparation from this gland using relatively low concentrations of the non-ionic detergent Triton X-100. At the detergent: protein ratios required for complete membrane solubilization bumetanide binding activity in this extract was lost but could be recovered by the addition of crude soybean lipids. In the present paper the ability of various purified lipids to restore high affinity bumetanide binding activity in detergent solubilized rabbit parotid basolateral membranes is studied. We show that the effect of exogenous lipid on the detergent-inactivated bumetanide binding site is to increase the affinity of binding without affecting the number of binding sites. Of the 11 lipid species tested, several relatively minor, negatively charged membrane phospholipids are the most effective in restoring binding activity (phosphatidylserine phosphatidylglycerol > phosphatidylinositol > cardiolipin). while the major mammalian plasma membrane lipid components phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol are without effect. In addition, we show that in the presence of these minor lipids the affinity of bumetanide binding is considerably increased over that observed in the native membrane (e.g.,K _d 0.06 m in membranes extracted with 0.3% Triton and treated with 0.15% wt/vol phosphatidylserine,vs. K _d 3 m in native basolateral membranes). This dramatic dependence of bumetanide binding affinity on the presence of certain lipid species suggests that the properties of the bumetanide binding proteinin situ may be quite dependent on the minor lipid content of the plasma membrane. This effect may account for the relatively large variations in bumetanide binding affinity observed from tissue to tissue.     

The Occurrence of Biogenic Calcian Struvite, (Mg, Ca)NH4PO4.6H2O, as Intracellular Crystals in Paramecium

ABSTRACT. Intracellular crystals are conspicuous refractile "inclusion bodies" commonly found in many protozoans, but very few have been identified mineralogically. We have isolated crystals from axenically grown mass cultures of Paramecium tetraurelia , and purified them using differential centrifugation. The crystals' structure and chemistry were analyzed using x-ray powder diffraction and energy-dispersive electron microprobe techniques. The morphology was studied by means of scanning electron microscopy. The crystals were identified as the orthorhombic mineral, calcian struvite, (Mg, Ca)NH₄PO₄.6H₂O. Struvite from P. tetraurelia exhibited a variety of crystal habits, including hemimorphic forms, epitactic overgrowths and several types of twins. A linear correlation between computed hydration number and Mg content suggests that the crystal composition may reflect the range of conditions under which struvite nucleation and growth occur. The mineral struvite also occurs in association with guano and other rich organic products, and can be biologically induced to precipitate extracellularly. Extracellular struvite has been well characterized in pathogenic calculi (kidney stones) of humans and cats, where precipitation is enhanced by bacterial urease activity that produces ammonia in the urinary tract. This is the first study demonstrating that struvite is also biologically controlled to form as an intracellular mineral. These crystals may have formed within lipid-rich, membrane-bound vesicles in Paramecium .     

Preparation and characterization of the layered borophosphates M(H2O)2[B2P2O8(OH)2]·H2O (M = Fe, Co, Ni)

The isotypic layered transition metal borophosphates M^II(H₂O)₂[B₂P₂O₈(OH)₂]·H₂O (M^II = Fe, Co, Ni) were prepared under hydrothermal conditions. Their crystal structures were determined by single-crystal X-ray diffraction data and revealed an isotypic relationship to Mg(H₂O)₂[B₂P₂O₈(OH)₂]·H₂O, a structure containing wavy 6³ nets formed by tetrahedral phosphate and hydrogenborate groups interconnected in an alternating fashion by sharing common apices. The crystalline compounds were also characterized by chemical analyses, scanning electron microscopy, energy dispersive X-ray analyses, thermal analyses, IR-spectroscopy and magnetic susceptibility measurements.     

Microwave synthesis, spectroscopy, thermal analysis and crystal structure of an one-dimensional polymeric {[Cu(4,4′-bipy)(H2O)3(SO4)] · 2H2O}n complex

A metal organic-inorganic coordination framework formulated as {[Cu(4,4′-bipy)(H₂O)₃(SO₄)] · 2H₂O}_n (1) (where 4,4′-bipy = 4,4′-bipyridine) has been successfully prepared by microwave synthesis. The title complex has been characterized by single crystal X-ray crystallography, FT-IR spectroscopy and thermal analysis. Complex (1) is an one-dimensional (1D) polymer in which 4,4′-bipy acts as a bridging ligand supporting the formation of infinite [Cu(4,4′-bipy)(H₂O)₃(SO₄)] chains. The packing diagram shows that a 3D network is formed via hydrogen bonds. The infrared spectra and thermographic data are consistent with the chemical formula.     

Synthesis, structure and dielectric properties of the 2D K-tetrazole complex [K2(4-TPA)2(H2O)2]n

The 2-D K(I)-tetrazole metal-organic complex, [K₂(4-TPA)₂(H₂O)₂]_n (1), which is constructed by the [K₂O₄N]_n inorganic skeleton chains bridged by the 4-TPA linkers, has been synthesized and characterized by single crystal X-ray crystallography and temperature-dependence dielectric constant(ε) measurement under the alternating electric field, (4-TPA = 2-(4-(1H-tetrazol-5-yl)pyridinium-1-yl) acetate). The ε of temperature dependence remains unchanged almost within the measured temperature range of 90 K to 430 K at 1 M Hz, and the ε of frequency dependence shows a significant decline from 6.7 to 4.6 within the measured frequency range of 200-1 MHz at room temperature. And it is consistent with the low dielectric loss (ε₂/ε₁) behavior, which is attributed to the highly ordered polarization mechanism.     

Dioxouranium complexes as ligand. Synthesis and characterization of sandwich-type polyoxometalates [X2W18(UO2)2{(H2O)3M}2O68] (X = As and P) (M = Co, Cu, Mn, Ni and Zn)

The title complexes are synthesized by the reaction of an unusual ligand of [K₂P₂W₁₈(UO₂)₂O₆₈]¹²⁻ (1) and [KAs₂W₁₈(UO₂)₂O₆₈]¹³⁻ (2) with divalent metal ions of Co^II, Cu^II, Mn^II, Ni^II and Zn^II in 1:2 mole ratio and are characterized by elemental analysis, IR, ³¹P NMR, UV-Vis spectroscopy, TGA, and single crystal structure analysis. Crystals of [P₂W₁₈(UO₂)₂{(H₂O)₃Co}₂O₆₈]¹⁰⁻ (1a) and [As₂W₁₈(UO₂)₂{(H₂O)₃Cu}₂O₆₈]¹⁰⁻ (2b) are orthorhombic space group Cmca. Both 1a and 2b have structures in which two [M(H₂O)₃] (M = Co^II, Cu^II) and two UO₂ groups are sandwiched between two symmetry equivalent (XW₉) (X = P, As) units in a virtual C_i symmetry. In solution, 1a and [P₂W₁₈(UO₂)₂{(H₂O)₃Zn}₂O₆₈]¹⁰⁻ (1d) give two-line P NMR spectra that are consistent with a C_s symmetry structures so, are not consistent with the solid-state structures. The sodium salts of them give one-line P NMR spectra and are consistent with the C_i symmetry of solid-state structures. The uranium atoms have pentagonal-bipyramidal coordination, achieved by three equatorial bonds to the one XW₉ and two bonds to the other. The M atoms have octahedral or square pyramidal coordination, but only one bond to the one XW₉ and one bond to the other.     

Aqueous acid-base chemistry involving dioxovanadium(V) complexes of 2,6-pyridinedimethanol, and the X-ray structures of Na[VO2{2,6-(OCH2)2NC5H3}] · 4H2O and [1-H-2,6-(HOCH2)2NC5H3]Cl

Reaction of NH₄VO₃ with 2,6-pyridinedimethanol in water at 85 °C followed by the room temperature addition of HCl (aq) yields [HVO₂(pydim)]_x (pydim = 2,6-pyridinedimethanolato dianion), as a sparingly soluble off-white solid. This acid may be deprotonated by titration with NaOH (aq), yielding Na[VO₂(pydim)] · 4H₂O, which has been structurally characterized by single-crystal X-ray diffraction. Treating Na[VO₂(pydim)] · 4H₂O with HCl (aq) regenerates [HVO₂(pydim)]_x, but reaction with additional NaOH (aq) displaces the pyridinedimethanolato ligand from the vanadium center. Similarly, treating [HVO₂(pydim)]_x with excess HCl (aq) strips the pyridinedimethanolato ligand from the vanadium center and yields the adduct [H₃(pydim)]⁺Cl⁻ as one component in a mixture of products. This adduct has been structurally characterized by single-crystal X-ray diffraction. The optimum pH range for stable dioxovanadium(V) complexes stabilized by the 2,6-pyridinedimethanolato ligand is at least 1.5-9.4.     

Relations of the Al,B, Ba,Br, Ca,Cl, Cu,Fe, K,Li, Mg,Mn, Na,P, S,Si, Sr,and Zn mass fractions to morphometric parameters in pediatric and nonhyperplastic young adult prostate glands

The variation with age of the 18 trace element mass fractions and some histological characteristics of intact prostate glands of 50 subjects aged 0–30 years was investigated by instrumental neutron activation analysis, inductively coupled plasma atomic emission spectrometry, and a quantitative morphometric analysis. Mean values ± standard error of the mean (M ± SΕΜ) for the mass fractions (in milligrams per kilogram wet tissue) of these trace elements in pre-puberty were: Al 28.5 ± 9.0, B 0.40 ± 0.11, Ba 1.48 ± 0.44, Br 10.5 ± 1.5, Ca 241 ± 30, Cl 3,203 ± 278, Cu 3.51 ± 0.89, Fe 33.7 ± 4.1, K 2,364 ± 145, Li 0.020 ± 0.004, Mg 153 ± 23, Mn 0.46 ± 0.06, Na 2,286 ± 130, P 1,391 ± 100, S 1,698 ± 132, Si 62 ± 11, Sr 0.38 ± 0.08, and Zn 27.6 ± 2.3. During puberty and postpuberty, when there is a significant increase in circulating androgens, the mean values were: Al 7.2 ± 1.4, B 0.21 ± 0.05, Ba 0.25 ± 0.06, Br 5.8 ± 1.0, Ca 433 ± 81, Cl 2,314 ± 201, Cu 1.77 ± 0.13, Fe 20.9 ± 1.6, K 2,585 ± 118, Li 0.0088 ± 0.0014, Mg 232 ± 27, Mn 0.34 ± 0.04, Na 1,875 ± 107, P 1,403 ± 98, S 1,673 ± 73, Si 22.2 ± 3.1, Sr 0.22 ± 0.03, and Zn 93.3 ± 8.9. Mean values (M ± SΕΜ) of percent volumes (%) of the stroma, epithelium and lumen in the prostate before puberty were 73.4 ± 2.6, 20.4 ± 1.7, and 4.45 ± 0.94, respectively, versus 46.5 ± 2.5, 38.5 ± 1.9, and 14.9 ± 1.2 during puberty and postpuberty. This work’s results confirm that the Zn mass fraction in prostate tissue is an androgen-dependent parameter. For the first time it has been demonstrated that the glandular lumen is a main pool of Ca, Mg, and Zn accumulation and that the stroma is a main pool of Al, B, Ba, Br, Cl, Cu, Fe, Mn, Na, and Si accumulation in the normal human prostate, for the age range 0–30 years. It was concluded that the Ca, Mg, and Zn binds tightly within the prostatic fluid, because the volume of glandular lumen reflects the volume of prostatic fluid.     

Syntheses, spectroscopic and structural characterization of some (solvated) binuclear adducts of the form Ag(oxyanion):dpem(:S) (1:1(:x))2 (oxyanion = ClO4, F3CCO2, F3CSO3; dpem = Ph2E(CH2)EPh2 (E = P, As))

Syntheses, spectroscopic (IR, NMR and ESI MS) and single crystal X-ray structural characterizations are reported for a wide variety of adducts of Ag(oxyanion):dpem(:S) (1:1(:n))₂ (oxyanion = ClO₄, F₃CCO₂ (tfa) F₃CSO₃ (tfs); dpem = Ph₂E(CH₂)EPh₂) stoichiometry among which the basic Ag(Ph₂E(CH₂)EPh₂)₂Ag core is diversely perturbed by interactions with anions and solvent molecules. ESI MS and ³¹P NMR spectroscopy indicated that dinuclear species also exist in solution.