Membrane biochemistry (Membr Biochem )


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    Membrane biochemistry
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    ABSTRACT: Sodium regulation of ligand binding to the dopamine transporter of rat and/or bovine striata was investigated using a filtration binding assay. In low Na+ phosphate or bicarbonate-buffered sucrose (300 mOsm), the tissue exhibited high affinity for [3H]cocaine which was reduced by the addition of Na+ in a dose-dependent manner. However, [3H]GBR 12935 binding was insensitive to Na+ in these physiological buffers. Although binding of [3H]GBR 12935 was displaced by cocaine in a manner consistent with competitive displacement, a non-linear affinity shift of the displacement of [3H]GBR 12935 by cocaine suggests that the two ligands bind to distinct sites. Binding of both radioligands was suppressed when measured in sodium-free 50 nM Tris-sucrose and increased with the addition of Na+. Scatchard analysis indicated that Bmax for [3H]cocaine binding in Tris plus 120 mM NaCl reached the same level as in the physiological buffers. In Krebs-Ringer buffer with phosphate, bicarbonate or Tris, which contained 120 nM NaCl, both [3H]cocaine and [3H]WIN 35428 binding exhibited lower affinities than in Na(+)-deficient phosphate buffer. It is suggested that the cation form of Tris binds to the dopamine transporter and that the Tris-receptor complex does not bind [3H]cocaine or [3H]GBR 12935. Na+ displaces Tris, forming a Na(+)-receptor complex which binds these ligands. Thus, it is suggested that the Na(+)-dependent binding of cocaine to the dopamine transporter is observed only in Tris.
    Membrane biochemistry 07/2009; 10(3):129-44.
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    ABSTRACT: We investigated the potential ability of p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD) to discriminate between M1 and M3 muscarinic receptor subtypes using Chinese hamster ovary cells stably transfected with the genes encoding the two receptors. Both radioligand binding and functional assays were utilized for this purpose. In contrast to initial reports of a 14-fold selectivity of this antagonist for M3 versus M1 receptors, we have detected a qualitatively similar selectivity that was markedly smaller in magnitude.
    Membrane biochemistry 07/2009; 9(4):293-300.
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    ABSTRACT: Diabetic patients present alterations in the activity of a number of enzymes of the plasma membrane. The aim of this study was to verify if the modifications of the enzymatic activities in diabetes mellitus are associated with structural alterations of the cellular membrane. By means of the freeze-fracturing technique, we studied the structure of erythrocyte membranes from 15 insulin-dependent diabetic patients (24-43 years) and 15 age-matched healthy subjects (26-47 years). The kinetic properties of the Na+/K(+)-ATPase of the same membranes were also investigated. The Na+/K(+)-ATPase of the erythrocyte plasma membrane shows an uncompetitive inhibition in the diabetic subjects. As for the freeze-fracturing results, the intramembrane particles of the erythrocyte membranes from diabetic patients appear more clustered with respect to those obtained from controls. The uncompetitive inhibition of the enzyme suggests the presence of conformational modifications of the protein. This hypothesis is supported by the freeze-fracture results which indicate that the integral protein constituents of the membrane in diabetes tend to aggregate. Modifications of the interactions between the enzymatic subunits and the membrane lipid environment might be at the basis of the Na+/K(+)-ATPase alteration in diabetes.
    Membrane biochemistry 07/2009; 10(2):71-9.
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    ABSTRACT: The uncoupling of Ca2+ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu3+ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Ca2+ and at 25 degrees C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca2+, 3 mM ATP, and at 25 degrees C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu3+. Digest past TD2 in the presence of 5 mM Ca2+ and 3 mM AMP-PNP (a non-hydrolyzable ATP analog) at 25 degrees C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca2+ transport from ATPase activity is possible by tryptic digestion of the (Ca2+ + Mg2+)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.
    Membrane biochemistry 07/2009; 10(4):191-201.
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    ABSTRACT: Human placenta was used to investigate the effects of chronic methadone use during pregnancy on villus tissue opioid receptors. Patients included in this investigation received 35-60 mg methadone per day. Methadone-exposed placenta villus tissue had no detectable opioid receptor binding sites measured by tritiated opioid agonists. In vitro release of acetylcholine and hCG from trophoblast tissue of methadone-exposed placentas was not modulated by opioids. Absence of opioid receptor binding sites and their two mediated responses in trophoblast tissue of placentas obtained from patients with documented chronic methadone use during pregnancy indicate that the receptors were down regulated or desensitized.
    Membrane biochemistry 07/2009; 10(2):91-8.
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    ABSTRACT: Impairment of transport and metabolism of retinal pigment epithelium (RPE) has been recognized to play a role in the development of diabetic macular edema. To understand the mechanism(s) of action of high glucose levels in alteration of RPE metabolism, primary cultures of RPE cells were used as an in vitro model of diabetic retinopathy/maculopathy. RPE cells were grown with 5 mM (control) or 40 mM glucose (a monosaccharide that enters the cells), or 40 mM sucrose (a disaccharide that does not enter the cells), and the extent of Na(+)-dependent active transport of an osmolyte ([3H]-myo-inositol, MI, 10 microM) into cells was determined. While 40 mM glucose down-regulated 3H-MI transport, 40 mM sucrose stimulated it, compared to 5 mM glucose feeding. Addition of 1 mM amiloride, an inhibitor of Na+/H+ exchanger, in the incubation media, significantly inhibited MI transport. Cells treated with high sucrose or high glucose were more sensitive toward amiloride inhibition, compared to controls. Inhibition of either pump or leak pathway alone was not sufficient to completely inhibit MI transport, but simultaneous inhibition of both pathways, by amiloride and ouabain (1 mM each), strongly inhibited osmolyte accumulation. The strongest inhibition of uptake occurred when 150 mM NaCl in the incubation media was replaced by 150 mM choline-Cl, and the percent inhibition of uptake, with choline-Cl, was highest with sucrose-fed cells, compared to normal or high glucose-fed cells. Imposition of a pH gradient [pHi (6.1) less than pH0 (8.0)] across the cell membrane, a condition that stimulates Na+/H+ exchange activity, also reduced MI accumulation. Cellular water content, measured by the extent of [3H]-3-O-methyl glucose uptake, in the presence of balanced salt solution (BSS), BSS containing half the ionic strength (hypotonic solution), or BSS containing 20 mM K+, for induction of cell swelling, varied when cells were fed with various sugars. Cells fed with high glucose were less sensitive toward media tonicity compared to normal. These results suggested that in cultured RPE cells, changes in Na+/H+ exchanger activity (intracellularly or extracellularly), through its inhibition by amiloride, its activation via intracellular acidification, or perhaps by chronic feeding with high sucrose or high glucose, affected the Na(+)-dependent active accumulation of MI. A metabolic factor involved in the development of diabetic macular edema is perhaps associated with glucose-induced alterations in Na+ fluxes (e.g., changes in Na+/H+ exchanger activity), which can secondarily influence osmolyte accumulation, impairment of pump-leak balance, and/or intracellular pH.(ABSTRACT TRUNCATED AT 400 WORDS)
    Membrane biochemistry 07/2009; 9(4):279-92.
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    ABSTRACT: N-(4'-pyridoxyl)sphingosine was synthesized and characterized as a stable compound for specialized delivery of a bioactive lipid. It was found to be facilely taken up by hepatocytes although by a mechanism more typical for lipids than the one used by natural vitamin B6. Some of the N-(4'-pyridoxyl)sphingosine was metabolically acted upon inside the cell to release pyridoxal 5'-phosphate and sphingosine, but formation of pyridoxal 5'-phosphate from the synthetic compound was poor compared with natural vitamin forms of B6, which may partly be due to entrapment within cell membranes and to constraints at the level of cytosolic pyridoxal kinase which is responsible for phosphorylation of the vitamin. Unlike the parent long-chain base, the B6 conjugate was not particularly cytotoxic. Furthermore, the compound was neither an activator nor inhibitor of the respiratory burst of human neutrophils. These findings identify N-(4'-pyridoxyl)sphingosine as an interesting tool for studies of the cellular transport, metabolism, and functions of both vitamin B6 and sphingosine.
    Membrane biochemistry 07/2009; 10(1):53-9.
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    ABSTRACT: Cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) are the enzymes catalyzing the last step of the de novo pathway for phosphatidylcholine and phosphatidylethanolamine synthesis, respectively. A major limitation for the complete characterization of the reactions catalyzed by the two enzymes derives from their poor stability in detergent-containing buffers. CPT is heavily inactivated, when native membranes are solubilized using a series of detergents, whereas EPT activity is better preserved during solubilization. An investigation of the factors which could play a role in preserving both enzymes from inactivation was carried out. The dramatic loss of enzymatic activities occurring upon dilution of solubilized membranes with detergent-containing buffers can be reduced by supplementing the dilution medium with phospholipids. The addition of Mn2+ ions to the dispersion buffer increases the stability of both enzymes. The procedure previously described for solubilizing EPT from rat brain microsomes has been modified on the basis of this evidence. Microsomes were solubilized in buffered detergent solutions containing Mn2+ ions and both CPT and EPT were partially purified in their active form by anion-exchange chromatography.
    Membrane biochemistry 07/2009; 10(1):43-52.
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    ABSTRACT: This investigation shows the effect of a Ca2+ addition on the structural and physicochemical properties of microvillus plasma membranes obtained from human placenta. Ca2+ addition induces an increase in microviscosity, as shown by the increase of order parameter and rotational correlation time of 5-and 16-doxylsterate derivatives and by the increase of fluorescence polarization of diphenylhexatriene. All the effects were obtained in a wide temperature range. The morphometric analysis of the ultrastructural images shows that the vesicle profiles of syncytiotrophoblast membranes decrease both area and form factor (FF) in the presence of Ca2+ with respect to the controls. The freeze-fracture results also show that Ca2+ induces an enhanced tendency to IMP clusterization. The Ca2+-induced changes were observed in both E and P faces. Our results underline the important role of Ca2+ in the cell membrane structure per se and in modulating interactions between cytoplasmic and extracellular microenvironments. The results of morphometric analysis of the ultrastructural images agree with biochemical data showing an increased stability induced by calcium on plasma membranes.
    Membrane biochemistry 07/2009; 7(4):193-206.
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    ABSTRACT: We have used 6-dodecanoil-2-dimethylaminonaphtalene (Laurdan) to study the membrane fluidity of Vesicular Stomatitis Virus (VSV) during virus activation at acidic pH 5.8). The fluorescence properties of Laurdan provide a unique possibility to study lipid organization because of the different excitation and emission spectra of this probe in the gel and liquid crystalline phase. Acidification to pH 5.8 (the pH which triggers VSV fusion with target membranes) generates a decrease in VSV membrane fluidity that could be reversed perfectly after neutralization. We conclude that lipid reorganization of the VSV membrane in the endocytic vesicles is needed for virus activation.
    Membrane biochemistry 01/1993; 10(4):203-12.
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    ABSTRACT: In the present study lactose permease mutants were isolated which recognize the monosaccharide, L-arabinose. Although the wild-type permease exhibits a poor recognition for L-arabinose, seven independent mutants were identified by their ability to grow on L-arabinose minimal plates. When subjected to DNA sequencing, it was found that all seven of these mutants were single-site mutations in which alanine 177 was changed to valine. The wild type and valine 177 mutant were then analyzed with regard to their abilities to recognize and transport monosaccharides and disaccharides. Free L-arabinose was shown to competitively inhibit [14C]-lactose transport yielding a Ki value of 121 mM for the Val177 mutant and a much higher value of 320 mM for the wild-type. Among several monosaccharides, D-glucose as well as L-arabinose inhibited lactose transport in the Val177 mutant to a significantly greater extent, while D-arabinose and D-xylose only caused a slight inhibition. On the other hand, kinetic studies with sugars which are normally recognized by the wild-type permease such as [14C]-galactose and [14C]-lactose revealed that the Val177 mutant and wild-type strains had similar transport characteristics for these two sugars. Overall, these results are consistent with the notion that the Val177 substitution causes an enhanced recognition for particular sugars (i.e. L-arabinose) but does not universally affect the recognition and unidirectional transport for all sugars. This idea is further supported by the observation that site-directed mutants containing isoleucine, leucine, phenylalanine, or proline at position 177 also were found to possess an enhanced recognition for L-arabinose.
    Membrane biochemistry 01/1993; 10(1):61-70.
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    ABSTRACT: Synthesis of gap junction proteins (GJPs) and of collagenases in the rat uterus has been studied under two physiological conditions: various stages of the estrus cycle, and the early pregnancy period. The synthesis has been studied by incubating uterine horns in a short-term tissue culture medium containing radioactively-labeled amino acids, followed by a double antibody immunoprecipitation of the labeled proteins. After exposure of the media to either anti-collagenase IgG(s) or anti-GJPs IgG(s), the final immunoprecipitation was achieved with the use of goat anti-rabbit IgG. Collagenase(s) synthesis was found to reach the peak, during the estrus cycle, at the proestrus stage, while GJP synthesis reached the maximum during the estrus stage. In the preimplantation, pregnant, rat uterus the syntheses of both the proteins reached the respective peak activities on day 4 of pregnancy, about 24 h before the expected time of ovum implantation. A study of the literature reveals that this time coincides with a spurt in exposure of the progesterone dominated uterus to estradiol.
    Membrane biochemistry 01/1993; 10(3):163-9.
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    ABSTRACT: Lectins from Lens culinaris and Arachis hypogaea immobilized on polyacrylamide beads were used for selective isolation of glycosylated surface membrane domains of adult Schistosoma mansoni worms, and the method was compared with the membrane isolation procedure developed with polycationic (Affi-Gel) beads. The lentil lectin proved to be suitable for interaction with surface membrane components: an increment in the specific activities of tegumental phosphohydrolases was observed in the bound fraction with respect to that observed in a total worm homogenate. A characteristic polypeptide pattern on gel electrophoresis was also seen, more restricted than that obtained with the bound Affi-Gel fraction. Immobilized peanut lectin was not successful as a method for isolating membrane material from the tegument of adult worms. Solubilization and dissociation of the lentil lectin-bound enzyme markers was achieved after addition of detergent and competing sugars. Glycosylation of the solubilized enzymes was further confirmed by affinity chromatography with fresh lentil lectin-coated beads. These results, together with histochemical evidences, suggest that the active sites of some of these enzymes are located within or close to the cytoplasmic leaflet of the surface tegumental membranes, and allow us to propose a model for the double surface membrane complex where some proteins may be crossing the two bilayers.
    Membrane biochemistry 01/1993; 10(3):155-61.
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    ABSTRACT: We have shown that in bovine iris sphincter membranes G proteins are involved in coupling muscarinic-, PGF2 alpha-, endothelin- and platelet-activating factor receptors to the activation of phospholipase A2 and the release of arachidonic acid. GTP gamma S and GTP gamma S plus carbachol stimulated arachidonic acid release in the membranes in a dose- and time-dependent manner. Nucleotide stimulation was specific to GTP gamma S, since GDP, GDP beta S and ATP had no effect. The stimulatory effect of GTP gamma S plus carbachol was blocked by atropine and it required the presence of physiological concentrations of Ca2-. AIF4-, which bypasses the receptor and directly activates the G protein, induced arachidonic acid liberation in the intact iris sphincter, but was ineffective in the membranes. Addition of GTP gamma S plus carbachol to sphincter muscle membranes prelabeled with [3H]inositol or 3H-arachidonic acid resulted in the formation of lysophosphatidylinositol and the liberation of arachidonic acid, thus suggesting the involvement of phospholipase A2. In vitro treatment of the iris membranes with pertussis toxic inhibited arachidonic acid release by the agonists. This is in contrast to the pertussis toxin-insensitive G protein that activates phospholipase C in this tissue (22). These data demonstrate that in the iris sphincter a G protein is involved in the step between receptor activation and the activation of phospholipase A2, and that arachidonic acid release in this tissue is mediated by a pertussis-toxin-sensitive G protein-coupled phospholipase A2. Thus, GTP can regulate arachidonic acid release and its subsequent conversion into eicosanoids by stimulating its formation.
    Membrane biochemistry 01/1993; 10(1):29-42.
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    ABSTRACT: The present study examines the hormonal regulation of the syntheses of gap junction proteins and estrogen receptor activation factors in the rat uterus. Ovariectomy and the depletion of estradiol from the system exerted negative influence on the synthesis of both the proteins. At the same time exposure of the ovariectomized rats to exogenous estradiol resulted in the restoration of protein synthesis back to the control level. A transient peak in the synthesis of the two proteins was observed on day 2 following ovariectomy. This increased activity was not observed in rats subjected to adrenalectomy along with ovariectomy. Furthermore, exposure of the ovariectomized plus adrenalectomized rats to progesterone clearly emphasized the point that the increase in the protein synthesis observed on day 2 post-ovariectomy was due to progesterone released from the adrenals. The results are indicative of a bi-hormonal involvement in the control of the syntheses of the two proteins, estrogen receptor activation factors and gap junction proteins in the rat uterus.
    Membrane biochemistry 01/1993; 10(2):119-27.
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    ABSTRACT: The addition of fMet-Leu-Phe or phorbol 12-myristate 13-acetate to human neutrophils stimulates phospholipase D activity as evidenced by the release of phosphatidic acid and the generation of diacylglycerol, and in the presence of ethanol the formation of phosphatidyl ethanol. The activation of phospholipase D by either the chemotactic factor or active phorbol ester is inhibited by the tyrosine kinase inhibitor erbstatin. The fMet-Leu-Phe-induced stimulation of this enzyme is greatly potentiated in cells which have been preincubated with low concentrations of lipopolysaccharide and serum. The presence of serum is essential for the potentiation by low concentrations of lipopolysaccharide. Moreover, the monoclonal antibody MY4(IgG2b) against CD14 inhibits the potentiation by the low concentration of lipopolysaccharide. These data suggest three important points. First, a tyrosine kinase step is necessary for the activation of phospholipase D. This suggests that the phospholipase D enzyme needs to be phosphorylated on tyrosine residues to be activated. Second, low concentrations of lipopolysaccharide, in the presence of serum, can potentiate the stimulated activity of this enzyme. Third, the priming action of the lipopolysaccharide-serum complex is mediated by CD14.
    Membrane biochemistry 01/1993; 10(2):81-9.
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    ABSTRACT: The solubilization of multilamellar liposomes by metoprolol tartrate (MPL) has been studied as a function of pH, [MPL], [dimyristoylphosphatidylcholine (DMPC)], temperature and lipid composition. The solubilization of liposomes at 37 degrees C by 7.3 nM MPL occurred at different rates at different pH values. MPL completely solubilized by 7.2 mM DMPC liposomes after about 17 h at pH 12, but only a partial solubilization occurred at pH 10 and 11. Between pH 7 and 9 no change in turbidity was observed after 1 week. Addition of cholesterol (CHOL) to DMPC (2:1 mol) had very little effect on solubilization after 24 h, however with DMPC:CHOL (5:1 mol) the decrease in turbidity was observed after 24 h, even though solubilization was much less compared with that of DMPC alone. The rate of solubilization was decreased when dipalmitoylphosphatidylcholine liposomes were employed. Addition of dicetylphosphate (DCP) to DMPC liposomes reduced the rate of solubilization significantly. The solubilization of liposomes by 7.3 mM MPL as a function of [DMPC], indicated that the lower the liposome concentration the greater the effect on solubilization. It is concluded that MPL in the non-ionized form has a solubilizing effect on liposomes, and addition of CHOL or DCP to DMPC has a stabilizing effect against solubilization.
    Membrane biochemistry 01/1993; 10(3):145-54.
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    ABSTRACT: Duramycin increases short-circuit current (Isc) and net Cl- secretion in tracheal epithelium. We measured the intracellular free calcium ([Ca2+]i) response to duramycin using Indo-1 and bovine and canine tracheal cell suspensions, and the effect of an intracellular calcium chelator, BAPTA, and the protein kinase C inhibitor, staurosporine, on the Isc and [Ca2+]i response to duramycin. [Ca2+]i increased in a dose-dependent manner from basal levels of 34 +/- 5 to 949 +/- 136 nM at 5 x 10(-6) M duramycin. Both BAPTA (50 microM) and staurosporine (5-50 nM) pretreatment blunted the increase in Isc and net Cl- secretion produced by duramycin. BAPTA also blunted the rise in [Ca2+]i produced by duramycin (5 x 10(-6) M) in the presence of extracellular calcium (499 +/- 122 nM). In the absence of extracellular calcium, the duramycin-induced (5 x 10(-6) M) rise in [Ca2+]i was blunted from 949 +/- 136 nM (stimulation in the presence of Ca2+) to 621 +/- 122 nM, and was further decreased in the presence of BAPTA to 197 +/- 42 nM. In contrast, staurosporine (50 nM) pretreatment had no effect on the rise in [Ca2+]i produced by duramycin (basal 90 +/- 27 to 861 +/- 110 nM at 5 x 10(-6) M). Duramycin had no effect on [Ca2+]i in human neutrophils. These data demonstrate that duramycin releases calcium from intracellular stores and stimulates the influx of calcium in airway epithelial cells. These data also demonstrate that, in the presence of protein kinase C pathway blockade, an increase in intracellular free calcium is not sufficient for chloride secretion; thus, duramycin-stimulated chloride secretion may depend upon protein kinase C.
    Membrane biochemistry 01/1993; 10(2):107-18.
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    ABSTRACT: In this study the volume-dependent, ouabain-resistant K+ influx and efflux in camel red blood cells were measured with the tracer 86Rb+. The results showed that the camel erythrocytes do not have the Na(+)-K+ cotransport. The cell swelling increases a ouabain-resistant K+ influx and shrinkage decreases it nearly two-fold. The swelling-stimulated K+ influx and efflux were chloride dependent. The anion dependence of K+ influx in swollen cells was as follows: Br- > Cl- > NO3. The pH-dependent curve for swelling-stimulated potassium influx, and the active K+ influx in camel erythrocytes were determined. The findings indicate that camel erythrocytes' potassium transport system has many similarities to other mammalian species.
    Membrane biochemistry 01/1993; 10(2):99-106.