Comparison of salt- and heat-induced alterations of protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803

Protein synthesis of the cyanobacterium Synechocystis spec. PCC 6803 decreases after a 684 mM NaCl salt shock. Qualitative changes were observed during the shock and the subsequent adaptation process using one-dimensional polyacrylamide electrophoresis. Proteins of apparent molecular masses of 13.0, 14.2, 16.6, 20.0, 21.0, 23.0, 33.0, 47.0, 52.0, 65.0 and 72.0 kDa are synthesized at enhanced rates after salt stress. The proteins of 14.2, 21.1 and 52.0 kDa are transiently induced during the first hours of the adaptation phase, while the other proteins are also synthesized at enhanced rates in salt-adapted cells. The proteins of 14.2, 23.0, 33.0 and 65.0 kDa are also induced by heat shock (43°C). Heat shock proteins of about 88.0, 75.0, 58.0, 17.5 and 13.8 kDa, in contrast, are induced by heat shock but not by salt. Two-dimensional polyacrylamide electrophoresis showed that the induced salt and heat shock proteins in some cases consisted of isoforms of different isoelectric points.Abbreviations IP isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride     

A facile method to study DNA synthesis in the cyanobacteriumSynechocystis PCC 6803

For the efficient study of replication of DNA, the cyanobacteriumSynechocystis PCC 6803 was first permeabilized by eitherl--lysophosphatidylcholine (LPC) or lysozyme-EDTA treatment. Permeability of the treated cells was evidenced by the incorporation of exogenously added³²P-TTP into DNA. In cells permeabilized by treatment with either method, the³²P-TTP incorporation at 30°C was appreciably higher than that in untreated control cells and increased with time for about 4 h. In addition, treated cells became permeable to proteins such as DNase I and micrococcal nuclease, which entered cells and degraded the newly synthesized DNA. Lysozyme/EDTA-treated cells not only incorporated³²P-TTP more efficiently than did LPC-permeabilized cells, but were capable of uptake and synthesis of exogenously supplied cyanobacterial plasmids isolated fromSynechocystis 6803. This capacity of lysozyme/EDTA-treatedSynechocystis to catalyze replication of exogenous DNA will allow the facile identification of DNA replication origins and their related regulatory sequences.     

Response ofWestiellopsis prolifica andAnabaena sp. to salt stress

Summary The response of a salt-tolerantWestiellopsis prolifica ARM 366 and a sensitiveAnabaena C-10 to NaCl was studied. While the former could tolerate up to 400mm NaCl, the latter was highly sensitive to concentrations above 50mm NaCl. Under salt stress, the tolerantW. prolifica showed an increased nitrogen demand as exemplified by high nitrogenase activity, and growth inhibition at higher concentrations of NaCl did not appear to be a direct consequence of inhibition of nitrogen fixation. One of the striking responses to Na⁺ challenge by the tolerantW. prolifica was the excess production of extracellular polysaccharides which adsorbed the bulk of the Na⁺. The influx of Na⁺ into the cell was comparatively small.

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Resumen Se estudió la respuesta al NaCl deWestiellopsis prolifica ARM 366, organismo tolerante a la sal, y deAnabaena C-10 que es sensible. El primero pudo tolerar concentraciones de hasta 400mm NaCl, sin embargo Anabaena C-10 se mostró extremadament sensible a concentraciones superiores a 50mm de NaCl. Bajo condiciones de estrés salinoW. prolifica mostró un incremento en la demanda de N₂, como se deduce de la elevada actividad nitrogenásica; la inhibición del crecimiento a concentraciones superiores de NaCl no se pudo relacionar directamente con la inhibición de la fijación de nitrógeno. Una de las respuestas más sorprendentes deW. prolifica frente al Na⁺ fue la producción de un exceso de polisaccaridos extracelulares que absorbieron gran parte del Na⁺. El flujo de Na⁺ hacia la célula fue, pues, comparativemente pequeño.

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Résumé On a étudié la réponse au NaCl d'une souche halo-tolérante deWestiellopsis prolifica (ARM 366) et, d'une souche halo-sensible d'Anabaena (C-10). Alors que la première tolère jusqu'a 400mm NaCl, la seconde est hautement sensible aux concentrations supérieures à 50mm. Soumise à un choc de salinité, la souche halo-tolérante deW. prolifica présente une demande d'azote accrue, comme le montre une activité nitrogénase élevée, et l'inhibition de la croissance par une forte concentration en NaCl ne parait pas être la conséquence directe d'une inhibition de la fixation d'azote. Une réponse remarquable deW. prolifica à l'agression par Na⁺ est la production en excès de polysaccharides exo-cellulaires, qui absorbent la plus grande partie du Na⁺. La pénétration de Na⁺ dans la cellule est relativement faible.

     

Monoclonal antibody GOM-2 binds to blood group B-Ley active glycolipid antigens on human gastric cancer cells,KATO-III

The antigen structure of a mouse monoclonal antibody, GOM-2, established by immunization with KATO-III human gastric cancer cells, was examined. GOM-2 reactive glycolipids were prepared from KATO-III cells and treated with endoglycoceramidase. Structural studies of ten GOM-2 reactive oligosaccharides by a combination of glycosidase digestions, methylation, and affinity chromatography on anUlex europeus agglutinin I (UEA-I) column revealed that nine of them had a Y-related B-active difucosylated determinant (B-Le^y) and one had a B-active determinant. Affinity chromatography of the purified and modified oligosaccharides on an immobilized GOM-2 column demonstrated that GOM-2 has a novel binding specificity; it binds tightly to the biantennary structure carrying the B-Le^y determinant at the termini or the branched structure carrying the B-Le^y structure at two nonreducing termini.Abbreviations UEA-I Ulex europeus agglutinin I - PNA Arachis hypogaea agglutinin - Fuc l-fucose - Gal d-galactose - Glcol glucitol - GlcNAc N-acetyl-d-glucosamine - TBS 10mm Tris-HCl, pH 7.8, containing 150mm NaCl - PBS 10mm sodium phosphate buffer, pH 7.5, containing 150mm NaCl - HPLC high performance liquid chromatography.     

sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

l-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of d-glucose 6-phosphate to l-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989–998, 2004). One of the forms with a ~50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as a ~65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified ~1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive ~65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.Anirban Chatterjee and Krishnarup Ghosh Dastidar contributed equally.     

Cloning and characterization of the chlorophyll biosynthesis gene chlM from Synechocystis PCC 6803 by complementation of a bacteriochlorophyll biosynthesis mutant of Rhodobacter capsulatus

A bacteriochlorophyll a biosynthesis mutant of the purple photosynthetic bacterium Rhodobacter capsulatus was functionally complemented with a cosmid genomic library from Synechocystis sp. PCC 6803. The complemented R. capsulatus strain contains a defined mutation in the bchM gene that codes for Mg-protoporphyrin IX methyltransferase, the enzyme which converts Mg-protoporphyrin IX to Mg-protoporphyrin IX methylester using S-adenosyl-l-methionine as a cofactor. Since chlorophyll biosynthesis also requires the same methylation reaction, the Synechocystis genome should similarly code for a Mg-protoporphyrin IX methyltransferase. Sequence analysis of the complementing Synechocystis cosmid indicates that it contains an open reading frame exhibiting 29% sequence identity to BchM. In addition, expression of the Synechocystis gene in the R. capsulatus bchM mutant via the strong R. capsulatus puc promoter was shown to support nearly wild-type levels of bacteriochlorophyll a synthesis. To our knowledge, the Synechocystis sequence thus represents the first chlorophyll biosynthesis gene homolog of bchM. The complementing Synechocystis cosmid was also shown to code for a gene product that is a member of a highly conserved family of RNA binding proteins, the function of which in cyanobacteria remains undetermined.     

Na,K-ATPase and the development of Na+ transport in rat distal colon

Summary Na, K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na⁺ transport during early postnatal development. The maximum Na⁺-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (I _sc ^N ) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats.I _sc ^N was entirely inhibited by ouabain; the inhibitory constant was 0.1mm in 10-day-old (young) and 0.4mm in 90-day-old (adult) rats. The affinity of the Na, K pump for Na⁺ was higher in young (11mm) than in adult animals (19mm). The Na, K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (K _I ) was not changed during development (0.1–0.3mm). Specific [³H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (K _D ) was 8 and 21 m in young and adult rats, respectively. The Na⁺ turnover rate per single Na, K pump, which was calculated fromI _sc ^N and estimated density of binding sites per cm² of tissue was 500 in adult and 6400 Na⁺/min·site in young rats. These data indicate that the very high Na⁺ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na⁺ of a single isoform of the Na, K pump. The development of Na⁺ extrusion across the basolateral membrane is not directly regulated by corticosteroids.     

Relations of enzymes inAspergillus repens grown under sodium chloride stress

Aspergillus repens, a salt-pan isolate, was halotolerant. When grown for 72 h (log phase) and 144 h (beginning of stationary phase) in a medium containing 2m sodium chloride, the activities of invertase, malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH), and glutamate dehydrogenase (GDH) were found to have increased. Control cultures grown in a medium devoid of 2m NaCl failed to show such changes. The activities of MDH, G6PDH, and GDH increased with rising concentrations of Na⁺ (as NaCl) when added up to 100mm in vitro. At higher concentrations they decreased. Changes in kinetic constants, Km and Vmax of these enzymes, as well as their de novo synthesis, were found to be some of the responses to NaCl stress-mediated changes.     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.     

Isolation of an abnormally phosphorylated erythrocyte membrane band 3 glycoprotein from patients with myotonic muscular dystrophy

Summary A fraction of erythrocyte Band 3 (M _r , 93,000) glycoprotein that demonstrates decreased autophosphorylation in membranes from myotonic muscular dystrophy patients is demonstrated. Sequential affinity chromatography of Triton X-100 solubilized erythrocyte membrane proteins separated three specifically retained glycoprotein fractions on a Ricin Communis I-Sepharose 4B column. One fraction contains a portion of the major sialoglycoprotein (apparentM _r , 78,000) and is specifically eluted from the column by 10mm NaCl and 100mm d-galactose (10/100). The two other glycoprotein fractions are eluted by 100mm NaCl, 10mm d-galactose (100/10) and 100mm NaCl, 100mm d-galactose (100/100). The composition of both fractions contains greater than 95% Band 3 (apparentM _r , 93,000) glycoprotein.The quantities of glycoprotein in each fraction obtained from erythrocytes of myotonic dystrophy patients did not differ from the quantities obtained from control erythrocytes. Following endogenous protein kinase incubations of ghosts with [-³²P]ATP, the specific [³²P] phosphorylation of the 10/100 and 100/10 fractions are identical. The 100/100 fraction, which makes up approximately 3% of the total erythrocyte membrane protein, demonstrates a different pattern for myotonic dystrophy patients; specific phosphorylation was reduced by 50% relative to activity in control experiments. These findings are consistent with previous experiments that demonstrated decreased autophosphorylation of the glycoprotein portion of Band 3 (Roses & Appel, 1975,J. Membrane Biol. 20: 51) and are consistent with the autosomal dominant mode of inheritance in this disease.     

Na+-stimulated nitrate uptake with increased activity under osmotic upshift in Synechocystis sp. strain PCC 6803

In the non-diazotrophic cyanobacterium Synechocystis sp. strain PCC 6803, an osmolality of 30 and 40 mosmol/kg sorbitol and NaCl resulted in 3.5- and 4.5-fold increase of nitrate uptake, respectively. The NaCl-stimulated uptake was abolished by treatment with chloramphenicol. At 25 mosmol/kg or higher, NaCl induced higher nitrate uptake than sorbitol suggesting an ionic effect of Na⁺. The nitrate uptake in Synechocystis showed K _s and V _max values of 46 μM and 1.37 μmol/min/mg Chl, respectively. Mutants disrupted in nitrate and nitrite reductase exhibited a decreased nitrate uptake. Ammonium, chlorate, and dl-glyceraldehyde caused a reduction of nitrate uptake. Dark treatment caused a drastic reduction of uptake by 70% suggesting an energy-dependent system. Nitrate transport was sensitive to various metabolic inhibitors including those dissipating proton gradients and membrane potential. The results suggest that nitrate uptake in Synechocystis is stimulated by Na⁺ ions and requires energy provided by the functioning electron transport chain.     

A continuous culture study of the regulation of extracellular protease production inVibrio SA1

During growth ofVibrio SA1 in a lactate-limited chemostat in the presence of 2mm phenylalanine as an inducer, the rate of production of two proteolytic enzymes, namely an endopeptidase and an aminopeptidase, was dependent upon the dilution rate. An optimum in the rate of synthesis of both proteases was observed at a dilution rate of 0.23 h^-1 and enzyme production only occurred between dilution rates of 0.06 and 0.45 h^-1. Without inducer a low rate of aminopeptidase production was found with an optimum at 0.19 h^-1, but only trace amounts of endopeptidase were detectable in the culture. In the presence of inducer the rate of enzyme production increased with increasing dilution rates over the range 0.06 to 0.23 h^-1 which was explained by an increase in saturation of inducer sites. The progressive decrease in the rate of protease production at higher dilution rates was ascribed to an increasing effect of catabolite repression by the increasing concentration of the growth substrate. It was shown that 5mm cyclic AMP could not relieve catabolite repression caused by glucose or lactate. Repression of protease production also occurred in the presence of higher concentrations (5mm) phenylalanine and other amino acids and by ammonium ions. It is suggested that the energy-status of the cell may play an important role in the regulation of protease synthesis inVibrio SA1.This study was supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Effect of NaCl on kinetics ofd-glucosamine uptake in yeasts differing in halotolerance

The initial rate ofd-glucosamine uptake by the non-halotolerant yeastSaccharomyces cerevisiae was approximately halved as the apparent half saturation constant (K_m) and the apparent maximum velocity (V_max) changed from 6.6mm to 16.4mm and from 22 μmol · g⁻¹ · min⁻¹ to 16 μmol · g⁻¹ · min⁻¹, respectively, when the salinity in the medium was increased from zerom to 0.68m NaCl. Corresponding changes in a high affinity transport system in the halotolerant yeastDebaryomyces hansenii were from 1.1mm to 4.6mm and from 3.1 μmol · g⁻¹ · min⁻¹ to 4.5 μmol · g⁻¹ · min⁻¹, implying a practically unchanged transport capacity. In 2.7m NaCl, K_m and V_max in this system were 24.5mm and 1.1 μmol · g⁻¹ · min⁻¹, respectively, representing a marked decrease in transport capability. Nevertheless, the degree of affinity in this extreme salinity must still be regarded as noteworthy. In addition to the high affinity transport system inD. hansenii, a low affinity system, presumably without relevance ind-glucosamine transport, was observed.     

Further photophysical and photochemical characterization of flavins associated with single-shelled vesicles

Summary In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands ofSqualus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170mm KCl gradient was initially about five-fold higher than in the presence of a 170mm KNO₃ gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4mm bumetanide and 40% by 0.8mm N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170mm NaCl gradient than in the presence of a 170mm NaNO₃ gradient. The effect of chloride was completely abolished by 0.4mm bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound³[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparentK _D of 3.5×10^–6 m and a number of sitesn of 104 pmol/mg protein. At 0.8 m, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600mm or when 1mm bumetanide or furosemide were added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland.     

ATP-sensitive potassium channels in adult mouse skeletal muscle: Characterization of the ATP-binding site

Summary Single K⁺-selective channels were studied in excised inside-out membrane patches from dissociated mouse toe muscle fibers. Channels of 74 pS conductance in symmetrical 160mm KCl solutions were blocked reversibly by 10 m internal ATP and thus identified as ATP-sensitive K⁺ channels. The channels were also blocked reversibly bymm concentrations of internal adenosine, adenine and thymine, but not by cytosine and uracil. The efficacy of the reversible channel blockers was higher when they were present in internal NaCl instead of KCl solutions. An irreversible inhibition of ATP-sensitive K⁺ channels was observed after application of several sulphydryl-modifying substances in the internal solution: 0.5mm chloramine-T, 50mm hydrogen peroxide or 2mm n-ethylmaleimide (NEM). Largeconductance Ca-activated K⁺ channels were not affected by these reagents. The presence of 1mm internal ATP prevents the irreversible inhibition of ATP-sensitive K⁺ channels by NEM. The results suggest that internal Na⁺ ions increase the affinity of the ATP-sensitive K⁺ channel to ATP and to other reversible channel blockers and that a functionally important SH-group is located at or near the ATP-binding site.     

Phenotypic character of the lipid hydroperoxide-resistant mutant: Positive relationship between flocculation and resistance against lipid hydroperoxide inSaccharomyces cerevisiae

A lipid hydroperoxide-resistant mutant was isolated from a strain ofSaccharomyces cerevisiae. The mutant was resistant to 1.5mm tert-butylhydroperoxide and 1.0mm linoleic acid hydroperoxide. It flocculated in a Ca²⁺-dependent manner and the resistance against lipid hydroperoxide was suppressed by mannose, which also inhibited flocculation. A positive relationship between the acquirement of, the flocculent phenotype and resistance against lipid hydroperoxide is suggested. A protein with a molecular weight of 33 kDa was found on the surface of the mutant cell.     

Enhancement of salt responses in frog gustatory nerve by removal of Ca2+ from the receptor membrane treated with 1-anilinonaphthalene-8-sulfonate

Summary The frog tongue was incubated in 1-anilinonaphthalene-8-sulfonate (ANS) solution and the responses of the glossopharyngeal nerve to various chemical stimuli were measured after the ANS solution was washed out. The responses to galactose, quinine and distilled water were unchanged by the ANS treatment. On the other hand, the responses to the salts, except for CaCl₂, were enhanced in greater or lesser degree after the ANS treatment. The order of relative magnitude of the enhanced response to 100mm salts of monovalent cations was Na⁺>NH ₄ ⁺ >K⁺>Li⁺, while that before the treatment was NH ₄ ⁺ >K⁺>Na⁺>Li⁺. The enhancement of the salt responses was also observed after the tongue was treated with 6-p-toluidinonaphthalene-2-sulfonate or 1,2-cyclohexanediaminetetraacetic acid solution.The enhanced responses to the salts were suppressed to the original level before the ANS treatment by addition of CaCl₂ or SrCl₂. The suppression curve satisfied the Langmuir adsorption isotherm when the suppression was postulated to be responsible for the binding of Ca²⁺ or Sr²⁺ to the receptor membrane treated with ANS. The apparent binding constants for Ca²⁺ and Sr²⁺ in the presence of 100mm NaCl were obtained to be 1.2×10⁴ m ^–1 and 6.7×10³ m ^–1, respectively.The ANS treatment modified the temperature dependence of the salt responses. For example, 100mm KCl solution of low temperature induced a large response after the ANS treatment, while that of 20°C induced only small response.It was concluded that the removal of Ca²⁺ from the gustatory receptor membrane in the frog, which was brought about by the ANS treatment, led to the enhancement of the salt responses. The mechanism on the enhancement of the salt response by the Ca²⁺ removal was discussed.     

H+/ATP stoichiometry for the gastric (K++H+)-ATPase

Summary The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150mm KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639±84 and 619±65 nmol/min×mg protein, respectively, giving an H⁺/ATP ratio of 1.03±0.7. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (<10 nmol/min×mg protein) was exhibited by vesicles in 150mm NaCl at pH 6.1 despite ATP hydrolysis of 187±46 nmol/min×mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H⁺/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K⁺ greater than 10mm. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H⁺/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K⁺+H⁺)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach.     

An endonuclease fromCaenorhabditis elegans: Partial purification and characterization

A deoxyribonuclease was partially purified from the free-living nematodeCaenorhabditis elegans. The DNase functioned as an endonuclease and introduced both single-strand nicks and double-strand breaks into DNA. The enzyme hydrolyzed double-stranded DNA seven times more rapidly than single-stranded DNA. DNase activity was not affected by the addition of divalent cations below 1mm but was inhibited at higher ionic concentrations. In addition, the enzyme was not inhibited in the presence of 10mm EDTA. The enzyme was inhibited by salt concentrations greater than 20mm. Three independent mutations in thenuc-1 gene were shown to reduce nuclease activity to less than 1% of that seen in wild-type organisms. This work was supported by National Institutes of Health Grant AG03161 and a TCU Research Foundation Grant. Some stocks used in these experiments were obtained from theCaenorhabditis Genetics Center, which is supported by Contract NOI-AG-9-2113 between the NIH and the curators of the University of Missouri.     

Isolation of salt-induced periplasmic proteins from Synechocystis sp. strain PCC 6803

Periplasmic proteins were obtained from control cells and salt-adapted cells of the cyanobacterium Synechocystis sp. PCC 6803 using the method of cold osmotic shock. Two of these proteins (PP 1, apparent mol. mass 27.6 kDa, and PP 3, apparent mol. mass 39.9 kDa) were accumulated in high amounts in the periplasm of salt-adapted cells, while the major periplasmic protein (PP 2, apparent mol. mass 36.0 kDa) was accumulated independently from salt. After isolation from gels and partial sequencing, the proteins could be assigned to proteins deduced from the complete genome sequence of Synechocystis. Neither salt-induced periplasmic proteins (PP 1, Slr0924 and PP 3, Slr1485) exhibited sequence similarity to proteins of known function from databases. The major protein (PP 2-Slr0513) showed significant sequence similarities to iron-binding proteins. All proteins included typical leader sequences at their N-terminus. Received: 21 September 1998 / Accepted: 17 December 1998