Trends in the fertilization ofStreptomyces coelicolor A3(2)

SCP1 and SCP2 (in the SCP2^* state) fertility plasmids ofStreptomyces coelicolor A3(2) elicit recombination in SCP1⁺×SCP1^- or SCP2^*×SCP2^- crosses. The rate is essentially constant (c. 10^-4) if referred to the plasmid-less parent, irrespective of extreme variations in the parent balance. In interrupted matings the alleles of the plasmid-less parent gradually increase in frequency in successive samples. The mobilization of the chromosome of the plasmid-less strain appears to be the primary event in merozygote formation.     

A study on Na+-coupled oxidative phosphorylation: ATP formation supported by artificially imposed ΔpNa and ΔpK inVibrio alginolyticus cells

Addition of Na⁺ to the K⁺-loadedVibrio alginolyticus cells, creating a 250-fold Na⁺ gradient, is shown to induce a transient increase in the intracellular ATP concentration, which is abolished by the Na⁺/H⁺ antiporter, monensin. The pNa-supported ATP synthesis requires an additional driving force supplied by endogenous respiration or, alternatively, by a K⁺ gradient (high [K⁺] inside). In the former case, ATP formation is resistant to the protonophorous uncoupler. Dicyclohexylcarbodiimide and diethylstilbestrol, but not vanadate, completely inhibit Na⁺ pulse-induced ATP formation. The data agree with the assumption that Na⁺-ATP-synthase is involved in oxidative phosphorylation inV. alginolyticus. Interrelation of H⁺ and Na⁺ cycles in bacteria is discussed.Abbreviations and electrochemical gradients of H⁺ and Na⁺, respectively - transmembrane electric potential difference - pH, pNa, and pK concentration gradients of H⁺, Na⁺, and K⁺, respectively - CCCP carbonyl cyanidem-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diesthylstilbestrol - HQNO 2-heptyl-4-hydroxyquinolineN-oxide - Tricine N[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Expression of an Escherichia coli flagellin gene, hag48, in the presence of a Salmonella H1-repressor

Summary An Escherichia coli K12 flagellin gene, hag48, was found to be expressed in the presence of the Salmonella rh1 gene product. The strains which had hag48 on a chromosome or on an F factor were constructed from strains H2-e,n,x ^on-off rh1 ⁺ and fixed H2-e,n,x ^on rh1 ⁺ in which rh1 ⁺ is contranscribed with H2 in its on state. Motility of these strains in semisolid medium was inhibited by anti-H48 serum and motile clones (swarms) that escaped from it were hag mutants in case of the hag48 e,n,x ^on-off strain tested. H48 flagellin was detected by electrophoresis, though its amount was less than e,n,x flagellin, from all the strains that were nonmotile in the presence of anti-H48 serum.     

Polymorphism and mapping of the class II genes in the rat: RT1.B,RT1.D,and RT1.H,a new DP-like region

The major histocompatibility complex of the rat (RTI) encodes the class II molecules involved with antigen presentation and cell to cell communication. The organization of these class II genes has been studied by Southern blot hybridization using genomic DNA from inbred and recombinant rat strains digested with various restriction endonuclease and hybridized under stringent conditions with probes for mouse class II and human class II genes. Analysis of the restriction fragment length polymorphisms has mapped the class II genes relative to each other. We have confirmed the order of the - and -chain genes in the RT1.B region, mapped the RT1.D region relative to RT1.B and showed that it has - and -chain loci, and identified a new HLA-DP-like locus, RT1.H, to the RT1.A side of RT1. B. The RT1. H and RT1.H genes map to the region around the recombination point in R22, and there appears to be a hot spot of recombination in RT1.H. The H and D genes have high levels of polymorphism; B , B ,and H have intermediate levels of polymorphism, and D has a low level of polymorphism.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

Involvement of cyclic AMP and its receptor protein in the sensitivity of Escherichia coli K 12 toward serine: excretion of 2-ketobutyrate, a precursor of isoleucine.

Summary A relationship between serine-induced growth sensitivity and the cAMP-CAP complex is established. Mutants of Escherichia coli K 12 deficient either in the cya or crp gene function exhibit a resistant phenotype on serine media although they harbor a relA allele normally leading to sensitivity toward serine. The presence of a crp ^* allele in a cya rela background restores the sensitivity phenotype, while the analysis of serine resistant mutants selected from a crp ^* cya relA strains shows that the mutation leading to resistance is located at, or very near, the crp gene, giving a more or less Crp^- phenotype. In addition crp ^* cya relA strains excrete large quantities of 2-ketobutyrate when grown on glucose M63 medium. This excretion is unambiguously linked to the presence of the crp ^* allele and is correlated with an enhanced threonine deaminase activity. Besides, the complex regulation exerted on the acetolactate synthase activities is discussed.     

Increased yield of a lysozyme after self-cloning of the gene in Streptomyces coelicolor “Müller”

Summary Streptomyces coelicolor Müller DSM3030 excretes a lysozyme comprising both -1,4-N-acetyl-and -1,4-N,6-O-diacetyl muramidase activities. The lysozyme is named Cellosyl. Gene libraries have been established using genomic DNA from the wild-type strain, S. coelicolor DSM3030, and from an overproducing mutant, S. coelicolor HP1, which exhibits about a twofold increase in lysozome production. The lysozyme-encoding genes (cel) from both strains were detected by oligodeoxynucleotide hybridization. The nucleotide sequence of the cel genes isolated from both strains was shown to be identical. The different levels of lysozyme production could not be correlated with any mutations at the cel gene locus. The cel gene isolated from the wild-type strain could not be expressed in some other species of Streptomyces. However, self-cloning of the cel gene into S. coelicolor DSM3030 and HP1 resulted in a 2.5-fold increase in lysozyme production.     

Structural characterization of a novel mono-sulfated gangliotriaosylceramide containing a 3-O-sulfatedN-acetylgalactosamine from rat kidney

A novel mono-sulfated glycosphingolipid based on the gangliotriaose core structure was isolated from rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, mild alkaline methanolysis, column chromatographies with anion exchangers and silica beads. The structure was characterized by compositional analysis, FTIR spectroscopy, methylation analysis,¹H-NMR spectroscopy and negative-ion liquid secondary ion mass spectrometry (LSIMS) using the intact glycolipid and its desulfation product. The two dimensional chemical shift correlated spectroscopy provided information on the sugar sequence as well as anomeric configurations, and indicated the presence of a 3-O-sulfatedN-acetylgalactosamine within the molecule. Negative-ion LSIMS with high- and low-energy collision-induced dissociation defined the sugar sequence and ceramide composition, confirming the presence of a sulfatedN-acetylgalactosamine at the non-reducing terminus. From these results, the complete structure was proposed to be HSO₃-3GalNAc1-4Gal1-4Glc1-1Cer (Gg₃Cer III³-sulfate, SM2b). Abbreviations: Abbreviations for sulfated glycolipids [17] follow the modifications of the nomenclature system of Svennerholm for gangliosides [37], and the designation of the other glycosphingolipids follows the IUPAC-IUB recommendations [38]. Cer, ceramide; LacCer, lactosylceramide, Gal1-4Glc1-1Cer; Gg₃Cer, gangliotriaosylceramide, GalNAc1-4Gal1-4Glc1-1Cer; Gg₄Cer, gangliotetraosylceramide, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; iGb₄Cer, isoglobotetraosylceramide, GalNAc1-3Gal1-3Gal1-4Glc1-1Cer; Gb₄Cer, globotetraosylceramide, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; SM4s, galactosylceramide sulfate, GalCer I³-sulfate; SM3, lactosylceramide sulfate, LacCer II³-sulfate; SM2a, Gg₃Cer II³-sulfate; SM2b, Gg₃Cer III³-sulfate; SB2, Gg₃Cer II³,III³-bis-sulfate; SM1a, Gg₄Cer II³-sulfate; SM1b, Gg₄Cer IV³-sulfate; SB1a, Gg₄Cer II³,IV³-bissulfate; GLC, gas-liquid chromatography; GC-MS, gas chromatography-mass spectrometry; DQF, double quantum filtered; COSY, chemical-shift-correlated spectroscopy; LSIMS, liquid secondary ion mass spectrometry; CID, collision-induced dissociation; MS/MS, tandem mass spectrometry.     

Molecular characterization of HLA-A28*, a novel HLA product,and its relationship to HLA-A28 and HLA-A2

The HLA-A28^* molecule expressed by the B-cell line IDF is serologically distinct and intermediate between HLA-A28 and HLA-A2. Comparative tryptic peptide mapping of biosynthetically labeled HLA-A28^*, A28, and A2 molecules showed that HLA-A28^* is also chemically distinct. Reverse-phase high pressure liquid chromatographic analysis of tryptic peptides labeled with ³H-arginine and ³H-lysine revealed that A28^*. A28, and A2 share 65% of their tryptic peptides. Multiple differences were observed between A28^* and both A28 and A2. No peptides unique to A28^* were detected and 25 peptides were shared with both A28 and A2. These results show that A28^* is a novel HLA product that is closely related to A28 and A2. Tryptic peptide map comparisons of these molecules labeled separately with 11 amino acids confirm these results. The data suggest that HLA-A28 ^* may have arisen from a genetic exchange event involving HLA-A28 and -A2. These data are consistent with the hypothesis that A28^* is identical with A28 in the first extracellular domain ( ₁) and identical with A2 in the second domain ( ₂).Abbreviations used in this paper EDTA ethylenediaminetetraacetic acid - HPLC high-pressure liquid chromatography - MHC major histocompatibility complex - NP40 Nonidet P40 - PMSF phenylmethylsulphonylfluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TPCK L(tosylamido-2-phenyl) ethyl chloromethyl ketone - Tris tris (hydroxymethyl)-aminomethane - A alanine - C cysteine - D aspartic acid - E glutamic acid - G glycine - H histidine - K lysine - L leucine - M methionine - N asparagine - Q glutamine - R arginine - S serine - T threonine - V valine - W tryptophan - Y tyrosine     

Ultraviolet reactivation of the single stranded DNA phage phiX 174.

Summary When UV-irradiated X174 was grown in pre-irradiated host cells of various strains, ultraviolet reactivation (UVR) was observed only in recombination proficient strains such as E. coli C (uvrA ⁺ recA ⁺) and HF4704 (uvrA ^- recA ⁺), but not in the recombination deficient strain HF4712 (uvrA ⁺ recA ^-). By increasing the multiplicity of infection, no rise in the amount of such reactivation was observed. From the study of the neutral and alkaline sucrose gradient sedimentation patterns of DNA samples extracted from unirradiated cells infected with unirradiated phage, it appears that after the conversion of the viral single stranded (SS) DNA to the double stranded form (DS), nicks or scissions were produced on it within all three strains, which were ultimately sealed up in the recA ⁺ but persisted within the recA ^- host cells. When UV-irradiated phage infected unirradiated host cells, such nicking of the DS DNA appeared to be much more extensive in uvrA ⁺ recA ⁺, but slightly reduced in uvrA ⁺ recA ^- and severely suppressed in uvrA ^- recA ⁺ strains. When the host cells were also UV-irradiated, the conversion of the infecting viral SS DNA to DS DNA as well as its subsequent nicking were reduced in all the three strains to a much greater extent. Although nicking of the DS DNA molecule is an essential step even in the normal intracellular replication of X DNA, the production and the sealing up of such nicks appear not to have any positive correlation with UVR of these phages. A drastic reduction in nicking due te pre-irradiation of the host cells might, however, mean slowing down of the replication of the damaged parental RF molecules which would facilitate their repair perhaps through recombination with the homologous parts of the host genome.     

Identification of the ubiquitin interfacial residues in a ubiquitin-E2 covalent complex

One of the key intermediates formed during the protein ubiquitination cycle is a covalent complex between ubiquitin (Ub) and the conjugation enzyme, UBC1. In order to probe the interface between these two proteins we have formed the covalent complex in situ (in the NMR tube) using Ub, the catalytic domain of UBC1, UBC1450, an activation enzyme, E1, and Mg²⁺-ATP. The size of the Ub-UBC1450 complex (25 kDa) and its relatively short lifetime ( 4 h) makes assignment of the backbone resonances in the covalent species difficult. In order to monitor the formation and identify the interface in the complex we have used fast ¹H-¹⁵N HSQC spectra to monitor the decay of ¹H-¹⁵N correlations as a function of time until the complex formed reached about 90%. The residual peak intensities were used to probe the surface of interaction between Ub and UBC1450 and provided a clear surface of interaction on Ub.     

The R gene product of bacteriophage lambda is the murein transglycosylase

Summary The radioactively labeled proteins synthesised in Escherichia coli minicells infected by bacteriophage R and R ⁺ were compared by polyacrylamide gel electrophoresis. R mutants, which have lost the ability to lyse host cells, lack a polypeptide of molecular weight 17.5 kD corresponding to the molecular weight of murein transglycosylase — a bacteriolytic enzyme from lysates which we have described previously. It has been shown by direct comparison using radio-labeled enzyme that transglycosylase comigrates with the R gene product. The enzyme was endetectable in induced cultures of E. coli W3350 su ^o (cI857 Ram5) and C600 (cI857 acR301), while it was present in a R _– ⁺ mutant lysate. We conclude that the transglycosylase is the R gene product.Abbreviations Muropeptide CA GlcNac-1-4-1,6-anhydro-MurNac-L-Ala-D-Glu-msA₂pm-D-Ala - muropeptide CB GlcNac-MurNac-GlcNac-1,6-anhydro-MurNac in which the carboxyl groups of MurNac and 1,6-anhydro-MurNac are substituted by the tetrapeptide L-Ala-D-Glu-msA₂pm-D-Ala - muropeptide C3 dimer of the two units GlcNac-MurNac-L-Ala-D-Glu-msA₂pm-D-Ala which are connected by D-D peptide bond between D-Ala and msA₂pm - GlcNac N-acetyl-D-glucosamine - MurNac N-acetylmuramic acid - msA₂pm meso-diaminopimelic acid - rivanol 6,9-diamino-2-ethoxyacridine lactate - SDS sodium dodecyl sulfate     

Chloramphenicol resistance in Streptomyces coelicolor A3(2): Possible involvement of a transposable element

Summary The transfer of a Chl element, causing resistance to chloramphenicol in Streptomyces coelicolor A3(2), was studied in NF x SCP1^– superfertile crosses. When the Chl element is on the donor side (NF) its transfer to the recombinant cells was virtually total as if the element acted as a second concomitant transfer origin. When the Chl element was on the recipient side (SCP1^–) it was never displaced by the immigrant chromosome even when the region facing chl ⁺ was selected for. A fraction of the original Chl^– mutants presented a requirement for arginine (ArgB^–). A Chl^– mutant gave rise spontaneously to ArgB^– derivatives at high frequency. The same ArgB^– requirement come out at high frequency among Chl^– derivatives from a cross NFChl^– x SCP1^–Chl⁺ in which neither parent required arginine or produced spontaneously arginineless derivatives. It is suggested that the Chl element is a transposable element (Tn) presumably associated with insertion sequences (IS). The insertional inactivation of the Chl element may be accompanied or followed by a deletion in the adjacent ArgB gene.     

Genetically polymorphic α-l-fucosidase (FUCA1) isozymes detected in blood plasma

The main isozyme patterns of desialylated blood plasma or serum -l-fucosidase (FUCA) were found to be almost identical to those of semen, urine, placental extracts, and leukocyte lysates, when detected by polyacrylamide gel isoelectric focusing, and activity staining using the fluorogenic substrate 4-methylumbelliferyl--l-fucopyranoside. Three phenotypes (1, 2-1, and 2) determined from plasma samples were identical to the phenotypes from urine and leukocyte lysates from the same individuals. A population study of plasma samples collected from 485 Japanese individuals indicated that the frequencies of the FUCA11 ^* and FUCA12 ^* alleles were 0.7505 and 0.2495, respectively. The mean plasma enzyme activities (+SD) of the three phenotypes were 318.8 ± 116.7 nmol/ml per h for type 1, 268.0 ± 108.3 nmol/ml per h for type 2-1, and 233.2 ± 84.4 nmol/ml per h for type 2. The mean activities of types 1 and 2 suggest that, on average, the FUCA11 ^* gene product in plasma has about 1.4 times the activity of FUCA12 ^*.     

Revertants from RNase III negative strains of Escherichia coli

Summary E. coli strains carrying the rnc-105 allele do not show any level of RNase III in extracts, grow slower than rnc ⁺ strains at temperatures up to 45°C and fail to grow at 45°C. Revertants which can grow at 45°C were isolated. The vast majority of them still do not grow as fast as rnc ⁺ strains and did not regain RNase III activity. The mutation(s) which caused them are suppressor mutations (physiological suppressors) which do not map in the immediate vicinity of the rnc gene. A few of the revertants regain normal growth, and contain normal levels of RNase III. They do not harbor the rnc-105 allele and therefore are considered to be true revertants. By using purines other than adenine it was possible to isolate rnc ⁺ pur ^- revertants from an rnc ^- pur ^- strain with relative ease. They behaved exactly like the true rnc ⁺ revertants isolated from rnc ^- strains at 45°C.A merodiploid strain which contains the rnc ⁺ gene on an episome behaves exactly like an rnc ⁺ strain with respect to growth and RNA metabolism, eventhough its specific RNase III activity is about 60% of that of an rnc ⁺ strain; thus the level of RNase III is not limiting in the cell.The rnc ^- strains show a characteristic pattern of transitory molecules, related to rRNA, 30S, 25S, p23 and 18S, which are not observed in rnc ⁺ strains. This pattern is unchanged in rnc ^- strains and in the revertants which are still lacking RNase III, regardless of the temperature in which RNA synthesis was examined (30° to 45°C). On the other hand, in the rnc ⁺ strains as well as in the true revertants and the rnc ⁺/rnc ^- merodiploid, the normal pattern of p16 and p23 is observed at all temperatures. These findings suggest that all the effects observed in RNase III^- strains are due to pleiotropic effects of the rnc-105 allele, and that the enzyme RNase III is not essential for the viability of the E. coli cell.     

Molecular cloning of the phosphate (inorganic) transport (pit) gene of Escherichia coli K12. Identification of the pit+ gene product and physical mapping of the pit-gor region of the chromosome

Summary The pit ⁺ gene, encoding the phosphate (inorganic) transport system of Escherichia coli, was isolated from a library of E. coli genes inserted in the cosmid vector pHC79. A 25.5-kb chromosomal DNA fragment was shown also to carry the gor locus encoding glutathione oxidoreductase. Physical mapping placed the two genes about 10 kb apart, confirming bacteriophage P1 mapping of the 77-min region. Subcloning and deletion analysis indicated that the entire pit ⁺ gene was located within a 2.2-kb Sal1-Ava1 fragment. The pit ⁺ gene product was identified by SDS-polyacrylamide gel electrophoresis as a 39-kdal inner membrane protein by two methods: (i) ³⁵S-methionine-labelling of minicells carrying pit ⁺ plasmids or plasmids from which all or part of the pit ⁺ gene was deleted. (ii) Overproduction of the Pit protein using a thermoinducible runaway replication plasmid.Complementation of the pit-1 mutant allele using a unit-copy-number pit ⁺ plasmid indicated that the pit-1 mutation is recessive.Strains carrying a multicopy pit ⁺ plasmid show a 10-fold increase in the initial rate of phosphate uptake; however there is no change in the steady-state level of ³²Pi accumulation.Abbreviations kb kilobase-pairs - kdal kilodalton - Pi inorganic phosphate - G3P sn-glycerol-3-phosphate - LB Luria broth - Tc tetracycline - Cm chloramphenicol - Ap ampicillin - UV ultraviolet light - TE 10 mM Tris.HCl, pH 8.0, 1 mM EDTA - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Ionic effects on bumetanide binding to the activated Na/K/2Cl cotransporter: Selectivity and kinetic properties of ion binding sites

Summary The loop diuretic bumetanide binds specifically to the Na/K/2Cl cotransporter of many cell types including duck erythrocytes. Membranes isolated from these erythrocytes retain the ability to bind bumetanide when cells are exposed to cotransport activity stimuli prior to membrane isolation. An extensive study of the effects of ions on specific [³H]bumetanide binding to such membranes is presented here and compared to the activity of these ions in supporting transport function in intact cells. Both Na⁺ and K⁺ enhanced bumetanide binding in a saturable manner consistent with a single-site interaction. The K _m for each ion was dependent on the concentration of the other cation suggesting heterotropic cooperative interactions between the Na⁺ and K⁺ binding sites. Na⁺ and K⁺ were partially replaceable, with the selectivity of the Na⁺ site being Na⁺ > Li⁺ > NH ₄ ⁺ ; N-methyl-d-glucamine⁺, choline⁺ and tetramethylammonium⁺ also supported a small amount of specific binding when substituted for Na⁺. The selectivity of the K⁺ site was K⁺ Rb⁺ > NH ₄ ⁺ > Cs⁺; N-methyl-d-glucamine⁺, choline⁺ and tetramethylammonium⁺ were inactive at this site. The results of transport experiments revealed a slightly different pattern. Li⁺ could partially substitute for Na⁺ in supporting coteansport, but other monovalent cations were completely inactive. The order of potency at the K⁺ site was NH ₄ ⁺ > K⁺ Rb⁺ > Cs⁺ other monovalent cations. The effect of Cl^- on bumetanide binding was biphasic, being stimulatory at low [Cl^-] but inhibitory at high [Cl^-]. As this implies the existence of two Cl^- binding sites (termed Cl _H and Cl _L for the high- and low- affinity sites, respectively) each phase was examined individually. Cl^- binding to Cl _H could be described by a rectangular hyperbola with a K _m of 2.5 mm, while kinetic analysis of the inhibition of bumetanide binding at high [Cl^-] revealed that it was of a noncompetitive type (K _i = 112.9 mm). The selectivity of anion binding to the two sites was distinct. Cl _H was highly selective with Cl^- > SCN^- > Br^-; F^-, NO ₃ ^- , ClO ₄ ^- , MeSO ₄ ^- , gluconate^- and SO ₄ ^2- were inactive. The efficacy of anion inhibition of binding to Cl _L was ClO ₄ ^- > I^- > SCN^- > NO₃ > Cl^-; F^-, MeSO ₄ ^- , gluconate^-, and SO ₄ ^2- were inactive. Thus, Cl _H is much more selective than Cl _L and largely accounts for the specificity of the system with respect to anion transport. SO ₄ ^- , NO ₃ ^- , I^-, SCN^- and ClO ₄ ^- did not support cotransport when bound to Cl _L and the latter three anions were inhibitory. Mg²⁺ was found to stimulate binding at a narrowly defined peak around 1.5 mm, but was inhibitory at higher concentrations. Other divalent cations caused a similar inhibition of bumetanide binding but did not exert a stimulatory effect at 1.5 mm. Divalent cations have little effect on cotransport in intact cells at concentrations up to 20 mm, suggesting that their effects on diuretic binding reflect interactions at internally disposed sites. Bumetanide binding was optimal at a pH of 7.8–8.1 and declined sharply as the pH was lowered towards 6. The titration curve correlated well with the effect of pH on cotransport in intact cells; the inhibitory effect of low pH suggests that protonation of the cotransporter may inhibit its function.We thank Drs. Brad Pewitt, John Westley and Mrinalini Rao for discussion, Sara Leung and Artelia Watson for their excellent technical assistance, and Dr. R.J. Turner for his gift of [³H] bumetanide. This work was supported in part by Cystic Fibrosis Center grant #CF RO11 7-04.     

Characterization of esophageal desalination in the seawater eel,Anguilla japonica

To characterize mechanisms of esophageal desalination, osmotic water permeability and ion fluxes were measured in the isolated esophagus of the seawater eel. The osmotic permeability coefficient in the seawater eel esophagus was 2·10^-4 cm·s^-1. This value was much lower than those in tight epithelial, although the eel esophagus is a leaky epithelium with a tissue resistance of 77 ohm·cm^-2. When the esophagus was bathed in normal Ringer solutions on both sides no net ion and water fluxes were observed. However, when mucosal NaCl concentration was increased by a factor of 3, Na⁺ und Cl^- ions were transferred from mucosa to serosa (desalination). If only Na⁺ or Cl^- concentration in the mucosal fluid was increased by a factor of 3, net Na⁺ and Cl^- fluxes were reduced to 30–40%, indicating that 60–70% of the net Na⁺ and Cl^- fluxes are coupled mutually. The coupled NaCl transport seems to be effective in desalting the luminal high NaCl. The remaining 30–40% of the total Na⁺ and Cl^- fluxes seems to be due to a simple diffusion, because these components are independent of each other and follow their electrochemical gradients, and also because these fluxes remain even after treatment with NaCN or ouabain. A half of the coupled NaCl transport could be explained by a Na⁺/H⁺–Cl^-/HCO ₃ ^- double exchanger on the apical membrane of the esophageal epithelium, because mucosal amiloride and 4.4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited the net Na⁺ and Cl^- fluxes by approximately 30%. The other half of the coupled NaCl transport, which follows their electrochemical gradients, still remains to be explained.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NMDG N-methyl-d-glucosamine - P _Cl Cl^- permeability coefficient - PD transepithelial potential difference - P _Na Na⁺ permeability coefficient - P _osm osinotic permeability coefficient - TALH thick ascending limb of Henle's loop     

The formation,vacuolar localization,and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures

The metabolism of salicylic acid (SA) in tobacco (Nicotiana tabacum L. cv. KY 14) cell suspension cultures was examined by adding [7–¹⁴C]SA to the cell cultures for 24 h and identifying the metabolites through high performance liquid chromatography analysis. The three major metabolites of SA were SA 2-O--D-glucose (SAG), methylsalicylate 2-O--D-glucose (MeSAG) and methylsalicylate. Studies on the intracellular localization of the metabolites revealed that all of the SAG associated with tobacco protoplasts was localized in the vacuole. However, the majority of the MeSAG was located outside the vacuole. The tobacco cells contained an SA inducible SA glucosyltransferase (SAGT) enzyme that formed SAG. The SAGT enzyme was not associated with the vacuole and appeared to be a cytoplasmic enzyme. The vacuolar transport of SAG was characterized by measuring the uptake of [¹⁴C]SAG into tonoplast vesicles isolated from tobacco cell cultures. SAG uptake was stimulated eightfold by the addition of MgATP. The ATP-dependent uptake of SAG was inhibited by bafilomycin A₁ (a specific inhibitor of the vacuolar H⁺-ATPase) and dissipation of the transtonoplast H⁺-electrochemical gradient. Vanadate was not an inhibitor of SAG uptake. Several -glucose conjugates were strong inhibitors of SAG uptake, whereas glutathione and glucuronide conjugates were only marginally inhibitory. The SAG uptake exhibited Michaelis–Menten type saturation kinetics with a K_m and V_max value of 11 M and 205 pmol min^–1 mg^–1, respectively, for SAG. Based on the transport characteristics it appears as if the vacuolar uptake of SAG in tobacco cells occurs through an H⁺-antiport-type mechanism.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation