Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-1 encoding the 67-kDa subunit reveals homology to other ATPases

The vacuolar membrane of Neurospora crassa contains a H+-translocating ATPase composed of at least three subunits with approximate molecular weights of 70,000, 60,000, and 15,000. Both genomic and cDNA clones encoding the largest subunit, which appears to contain the active site of the enzyme, have been isolated and sequenced. The gene for this subunit, designated vma-1, contains six small introns (60-131 base pairs) and encodes a hydrophilic protein of 607 amino acids, Mr 67,121. Within the sequence is a putative nucleotide-binding region, consistent with the proposal that this subunit contains the site of ATP hydrolysis. This 67-kDa polypeptide shows high homology (62% identical residues overall and 84% in the middle of the protein) to the analogous polypeptide of a higher plant vacuolar ATPase. The hypothesis that the vacuolar ATPase is related to F0F1 ATPases is strongly supported by the finding of considerable homology between the 67-kDa subunit of the Neurospora vacuolar ATPase and both the alpha and beta subunits of F0F1 ATPases.     

Short-term leaf elongation kinetics of maize in response to salinity are independent of the root

The essentiality of roots to the short-term responses of leaf elongation to salinity was tested by removing the roots of maize (Zea mays L.) from the shoots and comparing the initial short-term response of leaf elongation to that with intact plants. Eightday-old seedlings growing in solution culture were treated with 80 millimolar NaCl and their leaf elongation rate (LER) was monitored with a linear variable differential transformer connected to a computerized data aquisition system. Initially, LER of intact plants was sharply reduced by salinity, then rose rapidly to reach a new steady-state rate about 1.5 hours after salinization. The new steady-state rate of salinized intact plants was about 80% of the control rate. When the roots of nonsalinized plants were excised under the surface of the nutrient solution, excision did not disturb the steady-state LER. When these shoots were salinized, they responded in a manner nearly identical to that of intact plants, indicating that roots are not essential for the modulation of short-term LER of salt-stressed plants.     

Antibacterial activity of some α- substituted 2-Methyl-5-nitrofurans

The minimum inhibitory concentration values against Gram-negative and Gram-positive bacteria were determined and compared for a selected group of synthesized alpha-substituted 2-methyl-5-nitrofuran derivatives. In vitro oxidation of thiols to disulfides by 2-(iodomethyl)-5-nitrofuran indicated that oxidation of enzyme-thiol groups to disulfide bonds was a possible mode of action; but was discounted by noninhibition of thiol enzymes by these compounds. Electron-microscopic studies of the morphology of bacteria after treatment with these derivatives showed the formation of unusual elongation, branching and atypical rod shapes in E. coli, while S. aureus manifested multibud formation with some cytoplasmic protrusions. The possible mode of action of these compounds is discussed.     

Elucidation of the 2-aminoethylphosphonate biosynthetic pathway in Tetrahymena pyriformis

The biosynthetic reaction pathway leading to the natural product, 2-aminoethylphosphonate in Tetrahymena pyriformis has been elucidated. Incubation of [32P]PEP and [14C]PEP with T.pyriformis cellular homogenate fortified with Mg2+ and alanine/pyridoxal phosphate, yielded 2-aminoethylphosphonate as the minor reaction product (2-5% yield) and phosphoglycerate and pyruvate plus orthophosphate as the major products. Inclusion of thiamine pyrophosphate in the reaction mixture increased the yield of 2-aminoethylphosphonate by a factor of 10. Incubation of phosphonoacetaldehyde or phosphonopyruvate in the cellular homogenate also provided 2-aminoethylphosphonate. The cellular homogenate catalyzed the transformation of phosphonoacetaldehyde to 2-aminoethylphosphonate in an ca. 80% yield. However, the maximum yield of 2-aminoethylphosphonic acid obtained by use of phosphonopyruvate was only 15%. The major reaction pathways induced by treatment of phosphonopyruvate with the cellular extract involved its competitive conversion to PEP and pyruvate plus orthophosphate.     

Thermodynamic bookkeeping when nucleotides bind. Applications of the theory of linked functions

The thermodynamic theory of linked functions was used to determine the numbers of modifier ions involved when nucleotides dissociate. Nucleotide dissociation constants, obtained spectrophotometrically using Dowex-1 resin as a model system, were plotted on log/log paper with respect to the modifier concentrations. The slopes of the lines represent the net number of modifier molecules/ions involved in the dissociation. Varying numbers of nucleotides are bound to the resin because the resin capacity is determined by the total number of charges bound. The nucleotides bind to the resin at comparable diffusion-limited rates, irrespective of how tightly they bind. When ATP binds at pH 6.8, 4 chlorides, 4 formates, 2 succinates or 1.4 citrates are displaced, indicating that the fully charged (ATP4-) nucleotide binds. By comparing ATP, ADP and AMP it was possible to evaluate the contributions of the adenosine moiety and each phosphate to the binding. Between pH 2 and 3, where ATP has two negative charges, ATP binds largely as the trianion, displacing 2.7 chlorides and 0.7 protons. In the presence of 4 mM magnesium, 0.58 magnesiums facilitate the dissociation by chelating 58% of the liberated ATP. Calcium behaved similarly to magnesium but aluminum, at pH 6.8, promoted the binding of ATP as an (A1.ATP)3- complex with the concomitant liberation of three chloride ions. These experimental thermodynamic stoichiometries were found to be independent of the concentrations of the other modifiers present. Thermodynamic linkage stoichiometries can be evaluated from log K vs. log (modifier) plots when a direct determination of modifier binding is impossible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-2 encoding the 57-kDa polypeptide and comparison to vma-1

In partially purified preparations of the vacuolar ATPase from Neurospora crassa, the two most prominent components are polypeptides of Mr = 70,000 and 60,000. We previously reported the isolation of the gene vma-1, which encodes the Mr = 70,000 polypeptide, and presented evidence that the polypeptide contains the site of ATP hydrolysis (Bowman, E. J., Tenney, K., and Bowman, B. J. (1988) J. Biol. Chem. 263, 13994-14001). We now report the isolation of a gene (designated vma-2), that encodes the Mr = 60,000 polypeptide. Analysis of the DNA sequence shows that the polypeptide has 513 amino acids and a molecular mass of 56,808 daltons (and will thus be referred to as the 57-kDa polypeptide). It is fairly rich in polar amino acids and has no apparent membrane-spanning domains. The vma-2 gene contains five short introns (55-71 bases), all clustered in the 5' end of the coding region. The gene maps to the right arm of linkage group II, near 5 S RNA gene 3. Thus, it is unlinked to vma-1 and to other known ATPase genes in N. crassa. The 57-kDa polypeptide shows 25% amino acid sequence identity with the vma-1 gene product. It shows essentially the same degree of similarity (25-28%) to both the alpha and beta subunits of F0F1 ATPases. Analysis of specific regions of the 57-kDa polypeptide, however, suggests it may have a function like that of the alpha subunit in F0F1 ATPases. The data indicate that all four types of ATPase polypeptides have evolved from a common ancestor and that the vacuolar-type ATPases have a structure surprisingly similar to that of the F0F1 ATPases.     

Potential antiradiation drugs containing no nitrogen, and related compounds

Capabilities are reported of di- and higher sulfides (RSnR') terminated by sulfinate functions [-S(O)O-] for protecting mice against otherwise lethal effects of ionizing radiation. With the use of congeners, structure-activity correlations are developed for the effects of esterification of the sulfinate function, of changing the length of the chain of sulfur atoms, of reduction to a mercapto sulfinate, and of changing the substituents R and R' to chiral and other types of groups. Neither a trisulfide nor a sulfinate by itself was significantly radioprotective. The key requirement for radio-protection in the series appears to be the presence of a sulfur function (-Sn-) from which a thiol can be engendered by a neighboring-group effect of an electron-donating group; sulfoxide functions may afford alternatives to sulfinate functions as such neighboring groups. The relevance of structure-activity relations to the chemical and biological mechanisms involved in the radioprotective activities is discussed.     

Blue Light-Reducible Cytochromes in Membrane Fractions from Neurospora crassa

We have assayed absorbance changes generated by blue light in plasma membranes, endoplasmic reticulum, and mitochondrial membranes from Neurospora crassa. Light minus dark difference spectra, obtained anaerobically in the presence of ethylenediaminetetraacetate, indicated that b-type cytochromes could be photoreduced in all three membranes. In plasma membranes, a b-type cytochrome with a distinct difference spectrum was photoreducible without addition of exogenous flavin. Addition of riboflavin greatly stimulated the photoreduction of cytochromes in endoplasmic reticulum and mitochondrial membranes. In its spectral characteristics the cytochrome on the endoplasmic reticulum resembled cytochrome b₅ or nitrate reductase, while the cytochrome in mitochondrial membranes had the same spectrum as cytochrome b of the mitochondrial respiratory chain.

Cytochromes in the three membrane fractions reacted differently to blue light in the presence of various inhibitors. Potassium azide inhibited reduction of plasma membrane cytochrome b, with 50% inhibition at 1.0 millimolar. The same concentration of azide stimulated photoreduction of cytochromes in both endoplasmic reticulum and mitochondria. Although photoreduction of cytochromes in all three membranes was inhibited by salicylhydroxamic acid, cytochromes in plasma membranes were more sensitive to this inhibitor than those in endoplasmic reticulum and mitochondria. Cells grown to induce nitrate reductase activity showed an elevated amount of blue light-reducible cytochrome b in the endoplasmic reticulum.

     

Distributions of cell populations within alpha-particle range of plutonium deposits in the rat and beagle testis

Plutonium is not uniformly distributed in testicular tissues; thus some cell populations may receive larger or smaller radiation exposures than would be expected if the nuclide were uniformly distributed. The distributions of cell populations within alpha-particle range of Pu deposits in rat and beagle testes were determined. The data were collected from autoradiographs of testicular tissues containing 241Pu. A cell distribution factor (CDF) was determined for each cell population and is defined as the average number of each cell type within alpha-particle range of each observed Pu deposit relative to the number of each cell type that would be expected within alpha-particle range of each Pu deposit, if the deposits were distributed uniformly. In addition, the percentage of the spermatogonial stem cell population within alpha-particle range of Pu deposits was determined. In rats, the CDF for the spermatogonial stem cells is about 2.2. This value is similar to other enhancement and inhomogeneity factors reported for rodents in the literature. In beagles the CDFs to all cells in the seminiferous epithelium were less than the rats. In addition, the percentage of spermatogonial cells within alpha-particle range of Pu concentrations in the interstitial tissues was a factor of about 3 less in the dog than in the rat. The largest CDFs seen in both species were in the interstitial tissues, particularly for Leydig cells. Because the organization of testicular tissues in the beagle is quite different from rodents but more similar to human, the results from this study suggest that extrapolations from rodents to humans may tend to overestimate the potential for radiation exposure to spermatogonial stem cells as well as the fraction of the spermatogonial stem cell population at risk to exposure from internally deposited 239Pu.