The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain IV. Rate constant determination

The expressions for the kinetic constants corresponding to the steady state model for hydrolysis of ATP catalyzed by (Na⁺ + K⁺)-ATPase proposed recently are analyzed with the object of determining the rate constants. The theoretical background for the necessary procedures is described. The results of this analysis are: (1) A small class (four) of rate constants are determined directly by the previously published values of the kinetic constants. (2) For a somewhat larger class of rate constants upper and lower bounds may be established. For several rate constants the upper and lower bounds differ by less than a factor 1.6 (for the ‘(Na⁺ + K⁺)-enzyme’, i.e. the enzyme activity with K⁺ and millimolar substrate concentration) and 1.2 (for the ‘Na⁺-enzyme’, i.e. the activity at micromolar substrate concentrations). (3) Experiments on inhibition by K⁺ of the Na⁺-enzyme at various Mg²⁺ concentrations are reported and analyzed. With the additional assumption that the rate constants governing the addition to ATP of Mg²⁺ is independent of whether or not ATP is bound to an enzyme molecule, a set of consistent values for all the 23 rate constants in the mechanism may be obtained. (4) The values of some rate constants lend further support to the contention discussed in a previous paper that the enzyme hydrolyzes ATP along two kinetically distinct pathways, depending on the presence of K⁺ and on the concentration of substrate, without the necessity of having more than one active substrate site per enzyme unit at any time. (5) The results show that while the two enzyme forms, the ‘Na⁺-enzyme’ E₁ and the “K⁺-enzyme” E₂K, add substrate with (second order) rate constants of the same order of magnitude (differing only by a factor of four in favor of the former), the rate constants for the reverse processes differ by a factor of 100, being largest for the K⁺-enzyme. This is the main reason for the large difference in the Michaelis constants for the two forms reported previously. (6) Compatibility of the model with the well-known rapid dephosphorylation of the phosphorylated enzyme in the presence of K⁺ requires the presence, at non-zero steady state concentration, of an enzyme-potassium-phosphate intermediate, which is acid labile and is therefore not detected as a phosphorylated enzyme using conventional methods.     

Chromosomal locations of two DNA segments that flank ribosomal insertion-like sequences in Drosophila: Flanking sequences are mobile elements

We report the chromosomal locations of two repetitive DNA sequences that flank ribosomal insertion-like sequences in Drosophila melanogaster. The chromocentric region of D. melanogaster contains many copies of sequences that are homologous to type 1 ribosomal insertions. These insertion-like elements are interspersed with other DNA segments that we call flanking sequences. Two distinct flanking sequences derived from the same cloned DNA molecule pDmI 101, the HindIII fragments 101E and 101F, were studied. Whole genome Southern blots with DNA from the D. melanogaster stocks Oregon R (P2), gt-1, and gt-X11 showed complex restriction patterns that differed substantially between the three stocks. This and other data show that flanking sequences are members of diverged repetitive sequence families. In situ hybridization to salivary gland chromosomes of gt-1 and gt-X11 showed that both sequences are homologous to the chromocenter and to about 5 to 8 (101E) or 25 to 30 (101F) euchromatic sites in each stock. Most, if not all, of these sites differed in gt-1 and gt-X11. Both 101E and 101F are homologous to the chromocenter and very few euchromatic bands in D. simulans, but 101F is homologous to numerous bands in D. mauritiana. We conclude that the flanking sequences represented by 101E and 101F are mobile elements within the genome of Drosophila. These two sequences differ in several structural features from mobile DNA elements previously described in this organism.We dedicate this paper to Professor W. Beermann at the occasion of his 60th birthday     

Operation of the glucose-fatty acid cycle after glycogenolysis induced by treatment with nicotinic acid.

The intramembranous segment of glycophorin A has been localized to a 35-amino acid peptide. This has been isolated by a new procedure in which acid-insoluble peptides of a tryptic digest of detergent-purified glycophorin A are fractionated by countercurrent distribution. Amino acid sequence analyses, using both manual and automatic Edman degradation techniques, indicate that this peptide has a unique sequence in contrast to earlier work (J. P. Segrest, I. Kahane, R. L. Jackson, and V. T. Marchesi, 1973, Biochem. Biophys. Res. Commun., 49, 964–969). Ambiguities at three positions have been resolved, and sequencing errors at two additional positions have been corrected. One segment of this peptide has an uninterrupted stretch of 22 uncharged amino acids, and it is likely that this is the part which spans the lipid bilayer of the membrane. The complete 35-residue peptide has an apparent molecular weight in the 6000–8000 range, when analyzed on sodium dodecyl sulfate gels, suggesting that it forms dimers under these conditions. This result is consistent with our earlier proposal that intact glycophorin A molecules exist as dimers in sodium dodecyl sulfate which are stabilized by noncovalent associations between hydrophobic segments of their polypeptide chains.     

Patterns of ribosomal DNA spacer lengths are inherited

The non-transcribed spacer regions in Xenopus laevis ribosomal DNA vary in length, even within a single nucleolar organizer. The pattern of spacer lengths is sufficiently different from one nucleolar organizer to another to allow the pattern to be used as a genetic marker. We have analyzed the spacer patterns of rDNA² from a total of 50 progeny from three separate matings. Spacer patterns were inherited with no detectable change in all but two cases. The reproducibility of the patterns among siblings and their stable inheritance between generations rule out sudden mechanisms for gene evolution, such as the master-slave model, and support more gradual mechanisms.Two animals out of 50 showed marked changes in their rDNA spacer patterns. It is not possible at present to decide which of several possible mechanisms were responsible for the observed changes.     

A quantitative model of Phycomyces phototropism

A quantitative model accounting for phototropism in the wild type and in behavioural mutants of Phycomyces is described.Photomecisms (changes in the sporangiophore's growth velocity in response to changes in light intensity) are produced by a system composed of two sets of linear transducers separated by an adaptation mechanism, the first transducer being the photoreceptor.Phototropism under asymmetrical light distributions is caused by the summation of local photomecisms in the distal half of the sporangiophore, where two bright bands are produced by refraction of the incident light. The photoreceptors turn around the sporangiophore axis; they are approximately adapted to local intensity everywhere except upon entrance to the first bright band. Thus, a continuous photomecism originates at this band while the rest of the sporangiophore remains practically unstimulated.The mutants suffer a reduction in the efficiency of transduction.The behaviour of the wild type and of the mutants has been quantitatively simulated by computer. The predictions from the model fit the experimental results.     

Novel Tools to Analyze the Function of Salmonella Effectors Show That SvpB Ectopic Expression Induces Cell Cycle Arrest in Tumor Cells

In order to further characterize its role in pathogenesis and to establish whether its overproduction can lead to eukaryotic tumor cell death, Salmonella strains able to express its virulence factor SpvB (an ADP-ribosyl transferase enzyme) in a salicylate-inducible way have been constructed and analyzed in different eukaryotic tumor cell lines. To do so, the bacterial strains bearing the expression system have been constructed in a ∆purD background, which allows control of bacterial proliferation inside the eukaryotic cell. In the absence of bacterial proliferation, salicylate-induced SpvB production resulted in activation of caspases 3 and 7 and apoptotic cell death. The results clearly indicated that controlled SpvB production leads to F-actin depolimerization and either G1/S or G2/M phase arrest in all cell lines tested, thus shedding light on the function of SpvB in Salmonella pathogenesis. In the first place, the combined control of protein production by salicylate regulated vectors and bacterial growth by adenine concentration offers the possibility to study the role of Salmonella effectors during eukaryotic cells infection. In the second place, the salicylate-controlled expression of SpvB by the bacterium provides a way to evaluate the potential of other homologous or heterologous proteins as antitumor agents, and, eventually to construct novel potential tools for cancer therapy, given that Salmonella preferentially proliferates in tumors.     

A multi-criteria decision-making model for classifying wood products with respect to their impact on environment

Background, aim and scope  

Although life cycle assessment is frequently used in scientific studies of product comparison, many practitioners are looking for improvements in the normalisation, grouping and weighting of life cycle inventory results. Local conditions, which are well known to local experts, are very important to these steps. The goal of this work was to develop a computer-based decision support system for classifying wood products according to their influence on the environment in their whole life cycle. The model specifically addresses local conditions in the Republic of Slovenia and was developed by Slovenian experts.