Characteristics of ouabain binding to isolated trout hepatocytes

Summary Na⁺, K⁺ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10^–4 M produced maximal inhibition (95%) of K⁺ uptake and enhanced intracellular Na⁺ accumulation, showing that active fluxes account for a very large proportion of Na⁺ and K⁺ exchanges. Inhibition of the Na–K pump by ouabain was significant at low concentrations (10^–8 M). When external K⁺ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC₅₀) of K⁺ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [³H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K⁺ concentration in the medium, the other linear and independent of external K⁺. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5·10⁵ per cell) and independent of the external K⁺ concentration (7, 0.5 or 0 mM), while the dissociation constant (K_D) decreased from 4.2 M to 7.3 nM when K⁺ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min^–1), as estimated from ouabain-sensitive K⁺ flux, in comparison to those described in other cell types of higher vertebrates. At each external K⁺ concentration studied, the inhibition of K⁺ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside.     

Localization of Sites in the Tail Domain of the Middle Molecular Mass Neurofilament Subunit Phosphorylated by a Neurofilament-Associated Kinase and by Casein Kinase I

Abstract: We have shown previously that a neurofilament (NF)-associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF-M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKI) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKI inhibitor CKI-7, (2) the similarity in substrate specificity of NFAK and authentic CKI, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKI under similar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKI. We have also identified Ser⁵⁰², Ser⁵²⁸, and Ser⁵³⁶ as phosphorylation sites by NFAK/CKI in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser⁵²⁸ and Ser⁵³⁶ and flanking amino acids are highly conserved in higher vertebrate NF-M sequences. Neither Ser⁵⁰² nor Ser⁵³⁶ has been identified previously as NF-M phosphorylation sites.     

The chloroplastchIL gene of the green algaChlorella vulgaris C-27 contains a self-splicing group I intron

ThechiL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome ofChlorella vulgaris strain C-27 contains the first example of a splitchiL gene, which is interrupted by a 951 bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5 and 3 splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522 nt), separating the catalytic core from the 3 splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3 splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.     

Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergenCry j I: relationship between the structures and antigenic epitopes of plantN-linked complex-type glycans

The oligosaccharide structures ofCry j I, a major allergenic glycoprotein ofCryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz¹H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch.Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides asCry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing 1–6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin andClerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides onCry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.Abbreviations Cry j I a major allergenic glycoprotein ofCryptomeria japonica - B-SJA-II Sophora japonica bark lectin II - CTA Clerodendron trichotomum lectin - TFMS trifluoromethanesulfonic acid - HRP horseradish peroxidase     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both ₁-adrenoceptors (₁AR) and ₂-adrenoceptors (₂AR) mediate positive inotropic effects but that only ₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both ₁ AR and ₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of ₁AR and 1/3 of ₂AR, whether or not ₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate ₂AR, we used the ₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the ₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for ₂AR than for - 1 AR; the binding to ₂AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both ₁AR and ₂AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both ₁AR and ₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

Genetic variation in soybean photosynthetic electron transport capacity is related to plastocyanin concentration in the chloroplast

Fifteen ancestral genotypes of United States soybean cultivars were screened for differences in photosynthetic electron transport capacity using isolated thylakoid membranes. Plants were grown in controlled environment chambers under high or low irradiance conditions. Thylakoid membranes were isolated from mature leaves. Photosynthetic electron transport was assayed as uncoupled Hill activity using 2,6-dichlorophenolindophenol (DCIP). Soybean electron transport activity was dependent on genotype and growth irradiance and ranged from 6 to 91 mmol DCIP reduced [mol chlorophyll]^–1 s^–1. Soybean plastocyanin pool size ranged from 0.1 to 1.3 mol plastocyanin [mol Photosystem I]^–1. In contrast, barley and spinach electron transport activities were 140 and 170 mmol DCIP reduced [mol chlorophyll]^–1 s^–1, respectively, with plastocyanin pool sizes of 3 to 4 mol plastocyanin [mol Photosystem I]^–1. No significant differences in the concentrations of Photosystem II, plastoquinone, cytochrome b₆f complexes, or Photosystem I were observed. Thus, genetic differences in electron transport activity were correlated with plastocyanin pool size. The results suggested that plastocyanin pool size can vary significantly and may limit photosynthetic electron transport capacity in certain species such as soybean. Soybean plastocyanin consisted of two isoforms with apparent molecular masses of 14 and 11 kDa, whereas barley and spinach plastocyanins each consisted of single polypeptides of 8 and 12 kDa, respectively.Abbreviations DAP days after planting - DCIP 2,6-dichlorophenolindophenol - LiDS lithium dodecyl sulfate - PPFD photosynthetic photon flux density (mol photons m^–2 s^–1) - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Evolutionary plasticity in prokaryotes: A panglossian view

Enzyme directed genetic mechanisms causing random DNA sequence alterations are ubiquitous in both eukaryotes and prokaryotes. A number of molecular geneticist have invoked adaptation through natural selection to account for this fact, however, alternative explanations have also flourished. The population geneticist G.C. Williams has dismissed the possibility of selection for mutator activity on a priori grounds. In this paper, I attempt a refutation of Williams' argument. In addition, I discuss some conceptual problems related to recent claims made by microbiologists on the adaptiveness of molecular variety generators in the evolution of prokaryotes. A distinction is proposed between selection for mutations caused by a mutator activity and selection for the mutator activity proper. The latter requires a concept of fitness different from the one commonly used in microbiology.