Effects of metal ions on activity of plasmin

The effects of some metal ions on amidolytic and fibrinogenolytic activities of highly purified human plasmin were investigated in vitro. In the presence of Zn²⁺, Cu²⁺, Cd²⁺, and Au⁺ in the incubation mixture at the concentrations of 1×10⁻⁵−1×10⁻³ M, the anidolytic plasmin activity was strongly inhibited, whereas Ca²⁺ and Mg²⁺ at the same concentrations were not effective. The analysis of the kinetic study has shown that Zn²⁺ or Cu²⁺ acts as mixed-type inhibitors of plasmin activity. The inhibition of amidolytic plasmin activity by Zn²⁺ and Cu²⁺ was reduced in the presence of EDTA, histidine, or albumin. Incubation of plasmin with Zn²⁺ or Cu²⁺ (at the concentration of 5×10⁻⁴ M) resulted in complete loss of its proteolytic action on fibrinogen, whereas Cd²⁺ and Au⁺ under the same conditions only partially inhibited this process.     

The role of N-acyl groups in the inhibitory activity of LH-RH analogues

Inhibitory analogues of luteinizing hormone-releasing hormone (LH-RH) were prepared with formyl-D-Trp¹, acetyl-D-Trp¹, valeryl-D-Trp¹, tartaryl-D-Trp¹, diacetyl-tartaryl-D-Trp¹, acetyl-Gly¹, and acetyl-Sar¹ successively replacing the position one in the analogue [D-Trp¹, D-p-Cl-Phe², D-Trp³, D-Phe⁶, D-Ala¹⁰]-LH-RH. The formyl-D-Trp¹ and acetyl-D-Trp¹ analogues yielded 100% blockade of ovulation at the 10 μg dose; the others were less potent and inhibited ovulation at the 50 μg dose. The inhibitory potency seems to correlate with the polarity of the acyl group.     

Molecular analysis of the mechanism of potassium uptake through the TRK1 transporter of Saccharomyces cerevisiae

The TRK-HKT family of K⁺ transporters mediates K⁺ and Na⁺ uptake in fungi and plants. In this study, we have investigated the molecular mechanism involved in the movement of alkali cations through the TRK1 transporter of Saccharomyces cerevisiae. The model that best explains the activity of ScTRK1 is a cotransport of two K⁺ or Rb⁺, both of which bind the two binding sites of ScTRK1 with very high affinities in K⁺-starved cells. Na⁺ can be transported in the same way but it exhibits a much lower affinity for the second binding site. Therefore, only at critical concentration ratios between K⁺ and Na⁺, or Rb⁺ and Na⁺, the transporter takes up Na⁺ together with K⁺ or Rb⁺. Mutation analyses suggest that the two binding sites are located in the P fragment of the first MPM motif of the transporter, and that Gln⁹⁰ is involved in these binding sites. ScTRK1 can be in two states, medium or high affinity, and we have found that Leu⁹⁴⁹ is involved in the oscillation of the transporter between these two states. ScTRK1 mediates active K⁺ uptake. This is not Na⁺-coupled and direct coupling of ScTRK1 to a source of chemical energy seems more probable than K⁺-H⁺ cotransport.     

A partial sequence of ionic changes associated with the acrosome reaction of Strongylocentrotus purpuratus

Relationships among several of the ion movements associated with the acrosome reaction of S. purpuratus were investigated. Egg jelly initiates ⁴⁵Ca²⁺ and ²²Na⁺ uptake, and K⁺ and H⁺ efflux. H⁺ efflux and ²²Na⁺ uptake occur with approximately equivalent stoichiometries as rapidly as the appearance of acrosomal rods, perhaps reflecting a linked process. Most K⁺ loss, as measured either by ⁴²K⁺ efflux or K⁺-ion-selective electrodes, occurs after the acrosome reaction is complete. Since an elevation of seawater K⁺ (from 10 to 15 mM) or the addition of 0.5 mM tetraethylammonium (TEA), an inhibitor of K⁺ channels, inhibits the acrosome reaction half-maximally, K⁺ movements or alterations of K⁺-dependent membrane potentials may regulate the triggering by jelly. Most, but not all, of the ⁴⁵Ca²⁺ influx is inhibited with a mixture of 10 μM FCCP, 1 mM CN^?, and 2 μg/ml oligomycin, suggesting that the mitochondria store most of the Ca²⁺. The extracellular Na⁺ concentration affects Ca²⁺ fluxes: sperm placed into 5 mM Na⁺ seawater have enhanced ⁴⁵Ca²⁺ uptake, but do not undergo the acrosome reaction, unless 30 mM Na⁺ is also added. Low Na⁺ concentrations lead to spontaneous triggering, by allowing for both Ca²⁺ influx and Na⁺-dependent H⁺ efflux. At least one early Ca²⁺ requirement precedes the Na⁺ and H⁺ movements, as inferred from attempts at reversing the inhibitors of jelly induction of the acrosome reaction. When sperm are incubated with jelly in the absence of Ca²⁺, then washed and incubated with jelly in the presence of Ca²⁺, the acrosome reaction is triggered only upon the second incubation. However, when sperm are mixed with jelly in the presence of the other inhibitors (verapamil, TEA, 5 mM Na⁺ seawater, low pH, or elevated K⁺), they are altered so that even upon subsequent washing, jelly-mediated triggering is no longer possible. This suggests the existence of an intermediate state in the reaction pathway, that follows an event for which Ca²⁺ is required, but that precedes the Na⁺ and H⁺ movements, which are inhibited by all inhibitors of the acrosome reaction. These data are used to develop a partial sequence of ionic changes associated with the triggering mechanism.     

pH titration studies of 13C reductively methylated N-terminal serine of glycophorin AM

The ¹³C resonances of N^α,N-[¹³C]dimethylserine of partially ¹³C reductively methylated glycophorin A^M were monitored as a function of pH at 45°C. For comparison, limited data are also presented for the pH dependence of the ¹³C resonances of N^α,N- [¹³C]dimethylserine of fully ¹³C reductively methylated deglycosylated glycophorin A^M. The ‘major’ component of N^α,N- [¹³C]dimethylserine of glycophorin A^M did not titrate, whereas the ‘minor’ component titrated with a pK_a of 7.80 (Hill coefficient of 0.95). Similar results are also indicated for the N^α,N- [¹³C]dimethylserine resonances of ¹³C reductively methylated deglycosylated glycophorin A^M.     

Distribution and characterization of the opioid octapeptide met5-enkephalin-arg6-gly7-leu8 in the gastrointestinal tract of the rat

Summary The distribution and characterization of the opioid octapeptide met⁵-enkephalin-arg⁶-gly⁷-leu⁸ (met⁵-enk-arg⁶-gly⁷-leu⁸) within the gastrointestinal tract of the rat has been determined by immunohistochemistry and radioimmunoassay by use of a newly developed antibody to met⁵-enk-arg⁶-gly⁷-leu⁸. With both techniques, met⁵-enk-arg⁶-gly⁷-leu⁸-immunoreactivity (met⁵-enk-arg⁶-gly⁷-leu⁸IR) was detected in all regions of the gastrointestinal (GI) tract except the esophagus. The highest concentration of immunoreactive met⁵-enk-arg⁶-gly⁷-leu⁸ was observed in the colon, while intermediate concentrations were found in the stomach, duodenum, jejunum, and ileum. Immunostained somata were observed chiefly in the myenteric plexus; immunostained processes were present primarily in the myenteric plexus and the circular muscle layer. This distribution pattern is similar to that previously observed with antiserum to met⁵-enkephalin-arg⁶-phe⁷ (met⁵-enk-arg⁶phe⁷). Chromatographic analysis of met⁵-enk-arg⁶-gly⁷leu⁸-immunoreactive peptides extracted from the GI tract revealed the presence of an immunoreactive peptide of high molecular weight which accounted for approximately three-quarters of met⁵-enk-arg⁶-gly⁷-leu⁸-IR in both stomach and colon. These findings suggest a role for peptides related to the octapeptide met⁵-enk-arg⁶-gly⁷-leu⁸ in the regulation of GI function.     

A comparison of regulatory effects of chloride on nitrate uptake, and of nitrate on chloride uptake into Pisum sativum seedlings

Net nitrate uptake, ³⁶ClO^?₃/NO^?₃ influx and ³⁶Cl^? influx into Pisum sativum L. cv. Feltham First seedlings have been examined following growth in culture medium containing different combinations of chloride and nitrate. When young (6 days old) seedlings, that had been grown in the absence of N were used, nitrate accumulation stimulated net nitrate uptake and ³⁶ClO^?₃/NO^?₃ influx (r²= 0.99) while chloride accumulation inhibited nitrate uptake and ³⁶ClO^?₃/NO^?₃ influx (r²= 0.65). When nitrate was provided during growth there was no effect of chloride pretreatment on net nitrate uptake and there was little effect of total [NO^?₃+ Cl^?]_i on ³⁶ClO^?₃/NO^?₃ influx (r²= 0.26). A direct effect of Cl^? on ³⁶ClO^?₃/NO^?₃ influx was only found when seedlings had been starved of N for more prolonged periods (14 days). When moderate chloride was supplied during growth, ³⁶Cl^? influx was insensitive to nitrate or chloride accumulated, but significantly correlated with log_e [NO^?₃+ Cl^?]_i (r²= 0.75). When trace amounts of Cl^? were supplied during growth ³⁶Cl^? influx was inhibited by (a) NO^?₃ in the external medium and (b) Cl^? pretreatment, but was insensitive to NO^?₃ pretreatment. The sensitivity of ³⁶Cl^? influx to external nitrate was not found following Cl^? pretreatment in the absence of nitrate. The possibility that there are two populations of chloride carriers which differ in their sensitivity to external nitrate is discussed. Tentative schematic models to account for the regulation of nitrate and chloride uptake are proposed in the context of current hypotheses for regulation of ion transport and control systems theory.     

Effect of the chelation of metal cation on the antioxidant activity of chondroitin sulfates

The antioxidant potencies of chondroitin sulfates (CSs) from shark cartilage, salmon cartilage, bovine trachea, and porcine intestinal mucosa were compared by three representative methods for the measurement of the antioxidant activity; DPPH radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging activity. CSs from salmon cartilage and bovine trachea showed higher potency in comparison with CSs from shark cartilage and porcine intestinal mucosa. Next, CS from salmon cartilage chelating with Ca²⁺, Mg²⁺, Mn²⁺, or Zn²⁺ were prepared, and their antioxidant potencies were compared. CS chelating with Ca²⁺ or Mg²⁺ ions showed rather decreased DPPH radical scavenging activity in comparison with CS of H⁺ form. In contrast, CS chelating with Ca²⁺ or Mg²⁺ ion showed remarkably enhanced superoxide radical scavenging activity than CS of H⁺ or Na⁺ form. Moreover, CS chelating with divalent metal ions, Ca²⁺, Mg²⁺, Mn²⁺, or Zn²⁺, showed noticeably higher hydroxyl radical scavenging activity than CS of H⁺ or Na⁺ form. The present results revealed that the scavenging activities of, at least, superoxide radical and hydroxyl radical were enhanced by the chelation with divalent metal ions.     

Characterization of surface alloantigens of murine neutrophils

Serological analysis of highly purified (>97%) mouse peritoneal exudate neutrophils using a protein-A rosetting technique, showed that these cells possessed the surface phenotype: Ig^–, Thy-1^–, Ly-1^–, Ly-2^–, Ly-3^–, Ly-4⁺, Ly-5⁺, Ly-6⁺, Ly-7^–, Ia^–, FcR⁺ and C3R⁺.     

Effect of Liposomes Containing Various Divalent Cations on the Release of Acetylcholine from Synaptosomes

Abstract: The effect of increasing the cytoplasmic levels of various divalent cations on the release of [³H]acetylcholine ([³H]ACh) from synaptosomes was investigated. Synaptosomes prepared from rat brain and prelabeled with [³H]choline were incubated with liposomes containing Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Sr²⁺, or Ba²⁺. This treatment allows the transfer of the aqueous contents of the liposomes to the cytoplasm of the synaptosomes. The efflux of radioactivity subsequent to this treatment was measured, and the relative proportions of [³H]ACh and [³H]choline were determined. The release of radioactivity from synaptosomes incubated with liposomes containing Mg²⁺, Mn²⁺, or Co²⁺ was not altered when compared with synaptosomes incubated either without liposomes or with liposomes containing isotonic K⁺/Na⁺. Synaptosomes incubated with liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺, however, released significantly more radioactivity than did controls. Moreover, the released radioactivity consisted almost entirely of [³H]ACh. Liposomes containing either Ca²⁺ or Sr²⁺ were equally effective in promoting the release of [³H]ACh from synaptosomes, whereas liposomes containing Ba²⁺ were 2.5 times more effective in promoting the release of [³H]ACh than were liposomes containing either Ca²⁺ or Sr²⁺. Since liposomes introduce their aqueous contents into cytoplasm via a mechanism not involving plasma membrane channels, the increased release of [³H]ACh caused by liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺ is attributable to an increase in the intrasynaptosomal concentration of these ions, and not to their passage through calcium channels.     

Multiplicity and control of alcohol dehydrogenase isozymes in various strains of wine yeasts

An investigation has been made of selffertile (homothallic) progeny which are frequently encountered in matings of heterothallic Phytophthora species. The pattern of segregation of self-sterility from these homothallic isolates during vegetative growth, asexual and sexual reproduction has been studied in some detail.Heterokaryosis was shown not be the cause of this secondary homothallism in a number of cases investigated since homothallism could be transmitted by single uninucleate zoospores.The homothallic cultures (A¹ A²) showed different degrees of stability. One culture, obtained as a very fertile sector from an original A¹ A² gave only A¹ A² phenotypes on repeated single zoospore analysis, whilst two other sectors from the same A¹ A² culture gave in addition to A¹ A², some A² and a few A¹ phenotypes. The A² types derived from this homothallic were also unstable, giving some A¹ A² and occasional A¹ types as well as A² types in single zoospore cultures. This contrasts with the stability of the parental A² culture.Self-fertility was transmitted through the oospore to the sexual progeny. Most progeny were A¹ A² but heterothallic A¹ and A² types segregated.It is suggested that the self-fertile condition was due to the presence of an extra chromosome containing the mating type locus, the A¹ A² type being Aaa. A cytological investigation to test this trisomic hypothesis is being reported separately.     

Influence of lanthanum on the accumulation of trace elements in chloroplasts of cucumber seedling leaves

Influence of La³⁺ on the accumulation of trace elements (⁷⁵Se, ⁵⁶Co, ⁸³Rb, ⁴⁸V, ^95mTc, and ⁶⁷Ga) in chloroplasts of cucumber seedling leaves was studied by a radioactive multitracer technique. At the same time, chloroplast contents of different concentrations of La³⁺ treatment were calculated. It was observed that chloroplast contents peaked at 0.02 mM La³⁺ treatment and that the uptake and distribution of these trace elements in chloroplasts of cucumber seedling leaves are different under different La³⁺ treatments. With the increase of lanthanum concentrations from 0.002 to 2 mM, the uptake percentages of ⁷⁵Se, ⁵⁶Co, and ⁸³Rb presented an obvious increase and then sharply decreased in contrast to the nonlanthanum treatment, whereas there appeared a sharp decrease and then restored control level in the uptake of ⁴⁸V. The other two trace elements, namely ^95mTc and ⁶⁷Ga, were accumulated only in the presence of 0.02 mM La³⁺. The results indicate that lanthanum treatments to growing the cucumber lead to the change of trace element uptake in the chloroplasts of leaves, which suggest that lanthanum might influence the accumulation of trace elements in chloroplasts of cucumber seedling leaves by regulation of various ion transport mechanisms, thus affecting the photosystem of leaves.     

Conformational analyses of cyclic hexapeptide analogs of somatostatin containing arylalkyl peptoid and naphthylalanine residues

We report the conformational analysis by ¹H‐NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of peptoid analogs of the cyclic hexapeptide c‐[Phe¹¹‐Pro⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] L‐363,301 (the numbering refers to the positions in native somatostatin). The compounds c‐[Phe¹¹‐Nphe⁶‐Nal⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nphe ⁶ ‐ Nal ⁷ analog 1 ), c‐[Nal¹¹‐Nphe⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nal ¹¹ ‐ Nphe ⁶ analog 2 ) and c‐[Phe¹¹‐Nnal⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nnal ⁶ analog 3 ), where Nphe denotes N‐benzylglycine and Nnal denotes N‐(1‐naphthylmethyl)glycine, are subjected to SAR studies in order to investigate the influence of the bulky naphthyl aromatic ring on the conformation. The Nal ¹¹ ‐ Nphe ⁶ and Nphe ⁶ ‐Nal ⁷ analogs exhibit potent binding to the hsst2, hsst3 and hsst5 receptors, whereas the Nnal ⁶ analog has decreased binding affinity to all receptors but is more selective towards the hsst2 than the other two analogs and L‐363,301. The conformational search employing distance geometry, energy minimization and molecular dynamic simulations gives insight into the conformational flexibility of these analogs. The molecules adopt both cis and trans orientations of the peptide bond between residues 11 and 6. The cis isomers of these analogs adopt type II′ β‐turns with d ‐Trp in the i+1 position and type VIa β‐turns with the cis peptide bond between residues 6 and 11. The results of free and distance restrained molecular dynamics simulations at 300 K indicate that the Nphe ⁶ ‐Nal ⁷ and Nal ¹¹ ‐Nphe ⁶ compounds adopt a preferred backbone conformation which can be described as ‘folded’ about residues 7 and 10. The Nnal ⁶ analog, which binds less effectively to the hsst receptors, has a more flexible backbone structure than the Nal ¹¹ ‐Nphe ⁶ and Nphe ⁶ ‐Nal ⁷ analogs and prefers a ‘flat’ structure with regard to the orientations about Phe⁷ and Thr¹⁰ during molecular dynamics simulations. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

pH-responsive switching of near-infrared absorption of a diradical complex of Pt and 3,4-diaminobenzoate formed in aqueous solutions

In alkaline aqueous solutions, 3,4-diaminobenzoate (H₂(²L^PDA)⁻) reacts with Pt^II to form a 1:2 (Pt:L) complex that intensely absorbs near-infrared (NIR) light at 713 nm (ε = 8.0 × 10⁴ M⁻¹ cm⁻¹). The absorption disappeared at pH < 3 (in DMSO), showing pH-responsive switching of the NIR absorption. By comparing the NIR-absorbing behavior of this complex to that of a complex, [Pt^II(¹L^ISQ)₂]²⁻, containing the analogous phenylenediamine ligand [(¹L^ISQ)²⁻ = o-diiminobenzosemiquinonate radical], the complex can be formulated as [Pt^II(²L^ISQ)₂]²⁻. The assignment of the entity was consistent with the redox and spectroelectrochemical behaviors and electronic spin resonance (ESR) spectroscopy. First, one-electron oxidation of [Pt^II(²L^ISQ)₂]²⁻ formed an ESR-silent complex assignable to the dimeric complex [{Pt^II(²L^ISQ)(²L^IBQ)}₂]²⁻ [(²L^IBQ) = o-iminobenzoquinone form] in which the two radical centers at were antiferromagnetically coupled. Second, the one-electron reduced complex of [Pt^II(²L^ISQ)₂]²⁻ exhibited an ESR signal attributed to [Pt^II(²L^ISQ)(²L^PDA)]³⁻; 34% of the electronic spin was located at the Pt^II center rather than on the moiety. The pH-responsive switching-off of the NIR absorption was thus rationally explained by oxidation of [Pt^II(²L^ISQ)₂]²⁻ to [{Pt^II(²L^ISQ)(²L^IBQ)}₂]²⁻ by the increase of the rest potential of the solution in the lower pH region.     

Effects of NaCl on ion relations and carbohydrate status of roots and on osmotic regulation of roots and shoots of Atriplex amnicola

Abstract Atriplex amnicola was grown at 25, 200 or 400 mol m³ NaCl. Root tissues at different stages of development were investigated for concentrations of K⁺, Na⁺ and Mg²⁺, and in some cases for Cl^?. Sugar and starch concentrations were measured for plants grown at 25 or 400 mol m³ NaCl. In the ‘slightly vaeuolated’ root tips, Na⁺ was only 40 mol m^?3 at an external concentration of 400 mol m^?3 NaCl. The concentrations of K⁺ were not affected substantially by external NaCl between 25 mol m^?3 and 400 mol m^?3. The ‘highly vacuolated’ root tissues had substantially higher concentrations of K⁺, Na⁺ and Cl^? in plants grown at 200 and 400 mol m ³ NaCl than in plants grown at 25 mol m^?3 NaCl. Concentrations of Cr and of the sum of the cations in recently expanded tissue were similar to those in the bulk of the roots, consisting mainly of old cells. However, the K⁺: Na⁺ decreased with age; at 400 mol m^?3 external NaCl with a K⁺: Na⁺ of 0.012, the K⁺: Na⁺ in recently expanded 12 mm root tips was as high as 1.6, compared with 0.7 for the bulk of the roots. These ion data were used to estimate cytoplasmic and vacuolar concentrations of K⁺ and Na ⁺. Such calculations indicated that between 25 mol m³ and 400 mol m^?3 external NaCl the concentration of the sum of (Na⁺+K⁺) in the cytoplasm was maintained at about 180–200 mol m^?3 (cell water basis). In contrast, the (Na⁺+ K⁺) concentration in the vacuole was 170 mol m^?3 for plants grown at 25 mol m^?3 NaCl and 420 mol 400 mol m^?3 NaCl. The expanding root (issues exhibited greatly decreased soluble sugars and starch between dusk and dawn. Ai both times, sugar and starch concentrations in these tissues were 2.5–4.0 times greater in plants grown at 400 mol m^?3 NaCl compared with plants grown at 25 mol m^?3 NaCl. In contrast, carbohydrate concentrations in expanded root tissues were very similar at 25 and 400 mol m^?3 and showed little diurnal fluctuation. This paper considers the causes for the slower growth of A. amnicola at 400 than at 25 mol m”³ NaCl, using the data for the roots described here, and those for the shoots presented in the preceding paper (Aslam et al., 1986). There is no support for possible adverse effects by high internal ion concentrations. Instead, there may be deficiencies in supply of organic solutes for osmotic regulation; during part of the night a limited supply of such solutes may well restrict the rate of expansion of cells in plants growing at 400 mol m^?3 NaCl. There is insufficient evidence to decide whether this limitation in the expanding tissues is particularly prominent for the roots or for the shoots.     

Conformational analysis of cyclic angiotensin II analogues

Summary Conformationally restricted cyclic analogues of angiotensin II (ANG II), Asp¹-Arg²-Val³-Tyr⁴-Val⁵-His⁶-Pro⁷-Phe⁸, with a link between positions 3 and 5 have considerable biological activity. It is proposed that the spatial arrangement of the pharmacophore groups of Tyr⁴, His⁶ and Phe⁸ side chains and the C-terminal carboxyl group in ANG II and active analogues is similar. Conformational analysis of ANG II and two cyclic analogues c[Sar¹, Lys³,Glu⁵]ANG II and c[Sar¹,Hcy³,Mpt⁵]ANG II was performed, and a geometrical comparison of the low-energy conformations of these compounds allowed one to propose a model of receptor-bound conformation in terms of the spatial arrangement of the pharmacophore groups. This model is characterised by the close spatial location of the His⁶-Phe⁸ side chains and the Tyr⁴ C-terminal carboxyl group and is stabilised by the electrostatic interaction of Arg² and the C-terminal carboxyl group.Abbreviations ANG II angiotensin II - Hcy homocysteine - Mpt trans-4-mercaptoproline     

Microinjection of arginase into enzyme-deficient cells with the isolated glycoproteins of sendai virus as fusogen

Bumetanide is a potent diuretic drug which has some structural features in common with furosemide. The steady-state exchange of K⁺ and Cl^? was investigated in Ehrlich ascites tumor cells treated with bumetanide. This agent did not alter the cellular content of K⁺ or Cl^? but the self-exchange of both ions was depressed. K⁺ self-exchange was inhibited by 55% at bumetanide concentrations as low as 10^?6 M. Cl^? self-exchange was less sensitive to this drug but at low concentrations (between 10^?6 and 10^?3 M) bumetanide was a more effective inhibitor of Cl^? transfer than furosemide. The steady-state K⁺ flux of cells equilibrated in NO₃^? media was compared with the K⁺ flux in cells treated with 10^?4 or 10^?3 M bumetanide; the Cl^? -sensitive K⁺ exchange was equivalent to the bumetanide-sensitive K⁺ exchange. Since the results suggested that a bumetanide-sensitive (Cl^?, K⁺) cotransport could be operative in steady-state cells, the stoichiometry of the bumetanide-sensitive fluxes was determined by measuring Cl^? and K⁺ fluxes simultaneously in the same cell suspension. At $\text{5 · 10}{}^{\mathrm{?4}}$ and 10^?3 M bumetanide concentrations, the ratio of these fluxes was $\text{0.98 ? 0.07 (}\text{S.E.}\text{)}\text{and}\text{1.04 ? 0.06}$, respectively, consistent with the postulated cotransport mechanism. At 10^?4 and 10^?5 M, however, the ratio of the bumetanide-sensitive Cl^?/K⁺ flux was significantly less than 1.0. Since the magnitude of the bumetanide-sensitive K⁺ flux at 10^?4 M was close to that of the Cl^?-sensitive flux, a ratio of less than 1.0 at this drug level indicates that Cl^? sensitivity and drug sensitivity may not reflect inhibition of the same process under all circumstances.     

Structural studies of a synthetic peptide derived from the carboxyl-terminal domain of RNA polymerase II

The conformation of the repeating heptapeptide unit of the carboxyl-terminal domain of RNA Polymerase II, Y¹S²P³T⁴S⁵P⁶S⁷ has been examined using nuclear magnetic resonance spectroscopy and circular dichroism. Nuclear Overhauser effects and CD spectra for the synthetic 56-residue peptide H₂N-(S²P³T⁴S⁵P⁶S⁷Y^l)₈-COOH in water indicate that the peptide is largely unordered. A small population of folded molecules is observed to contain β-turns located at Ser²-Pro³-Thr⁴-Ser⁵ (SPTS) and Ser⁵-Pro⁶-Ser⁷-Tyr¹ (SPSY). CD and NMR results in 90% TFE also indicate an equilibrium population of structures, but the fraction of turns is higher. Similarities of nuclear Overhauser effects in water and in 90% TFE suggest that the structures in TFE are biologically relevant. Based on these observations, the average structure of a single conformer of the heptapeptide repeat in 90% TFE was obtained by a distance geometry-simulated annealing method, using distance restraints extracted from nuclear Overhauser data. NMR spectra of the 56-mer show signals corresponding to only one repeat indicating that each repeat is in an identical environment. Thus it is possible to obtain an average structure of the heptapeptide repeat from NOE data on the 56-mer. Twenty-seven final structures were calculated and the root mean square deviations between the 27 structure and the mean coordinates was 1.52 Å for the backbone and 2.2 Å for all nonhydrogen atoms. The heptapeptide repeat consists of two overlapping β-turns which are potentially stabilized by hydrogen bonds. The hydroxyl side chains of Ser², Ser⁵, Thr⁴, and Ser⁷ all appear to be equally exposed for potential phosphorylation. The tyrosyl side chain of each repeat is folded inwards to the backbone and can potentially hydrogen bond to the carbonyl oxygen of the tyrosine in the preceding repeat. Iteration of the average structure of the heptapeptide repeat results in a model of the carboxyl-terminal domain with a regular but unusual secondary structure consisting of a series of staggered β-turns. © 1995 Wiley-Liss, Inc.     

Effect of Hypoxia on Na+-K+-Cl− Cotransport in Cultured Brain Capillary Endothelial Cells of the Rat

Abstract: The effect of hypoxia on Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring ⁸⁶Rb⁺ uptake as a tracer for K⁺. RBECs expressed both Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na⁺,K⁺-ATPase activity by 38.9%, whereas it significantly increased Na⁺-K⁺-Cl^? cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin-induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time-dependent decrease of cellular ATP content (by ～65%) along with a complete inhibition of Na⁺,K⁺-ATPase and a coordinated increase of Na⁺-K⁺-Cl^? cotransport activity (up to 100% above control values). Oligomycin augmentation of Na⁺-K⁺-Cl^? cotransport activity was not observed in the presence of 2-deoxy-d -glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na⁺,K⁺-ATPase activity is reduced, RBECs have the ability to increase K⁺ uptake through Na⁺-K⁺-Cl^? cotransport.     

Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu²⁺, Zn²⁺, Fe²⁺ and.Co²⁺) and heavy metals (Hg²⁺and Pb²⁺) ions were studied. All investigated metals produced a larger maximum inhibition of Na⁺/K⁺-ATPase than Mg²⁺-ATPase activity. The free concentrations of the key species (inhibitor, MgATP^{2 ?} , MeATP^{2 ?} ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu²⁺/Fe²⁺ or Hg²⁺/Pb²⁺caused additive inhibition, while Cu²⁺/Zn²⁺ or Fe²⁺/Zn²⁺ inhibited Na⁺/K⁺-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu²⁺/Fe²⁺ or Cu²⁺/Zn²⁺ inhibited Mg²⁺-ATPase activity synergistically, while Hg²⁺/Pb²⁺ or Fe²⁺/Zn²⁺ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na⁺/K⁺-ATPase activity by reducing the maximum velocities (V_max) rather than the apparent affinity (K_m) for substrate MgATP^2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg²⁺-ATPase activity by Zn²⁺, Fe²⁺ and Co²⁺ as well as kinetic analysis indicated two distinct Mg²⁺-ATPase subtypes activated in the presence of low and high MgATP^{2 ?} concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na⁺/K⁺-ATPase with various potencies. Furthermore, these ligands also reversed Na⁺/K⁺-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg²⁺-induced inhibition was not obtained.