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     

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 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.     

Hen egg white lysozyme fibrillation: a deep‐UV resonance Raman spectroscopic study

Amyloid fibrils are associated with numerous degenerative diseases. The molecular mechanism of the structural transformation of native protein to the highly ordered cross‐β structure, the key feature of amyloid fibrils, is under active investigation. Conventional biophysical methods have limited application in addressing the problem because of the heterogeneous nature of the system. In this study, we demonstrated that deep‐UV resonance Raman (DUVRR) spectroscopy in combination with circular dichroism (CD) and intrinsic tryptophan fluorescence allowed for quantitative characterization of protein structural evolution at all stages of hen egg white lysozyme fibrillation in vitro. DUVRR spectroscopy was found to be complimentary to the far‐UV CD because it is (i) more sensitive to β ‐sheet than to α ‐helix, and (ii) capable of characterizing quantitatively inhomogeneous and highly light‐scattering samples. In addition, phenylalanine, a natural DUVRR spectroscopic biomarker of protein structural rearrangements, exhibited substantial changes in the Raman cross section of the 1000‐cm^–1 band at various stages of fibrillation. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)