Computer Simulation of Assembly and Co-operativity of Hexameric AAA ATPases

AAA ATPases form a functionally diverse superfamily of proteins. Most members form homo-hexameric ring complexes, are catalytically active only in the fully assembled state, and show co-operativity among the six subunits. The mutual dependence among the subunits is clearly evidenced by the fact that incorporation of mutated, inactive subunits can decrease the activity of the remaining wild type subunits. For the first time, we develop here models to describe this form of allostery, evaluate them in a simulation study, and test them on experimental data. We show that it is important to consider the assembly reactions in the kinetic model, and to define a formal inhibition scheme. We simulate three inhibition scenarios explicitly, and demonstrate that they result in differing outcomes. Finally, we deduce fitting formulas, and test them on real and simulated data. A non-competitive inhibition formula fitted experimental and simulated data best. To our knowledge, our study is the first one that derives and tests formal allosteric schemes to explain the inhibitory effects of mutant subunits on oligomeric enzymes.     

Electrochemical identification of microbially mediated hydrogen sulfide oxidation

The process of H₂S oxidation by the phototrophic bacteriaThiocapsa roseopersicina andChlorobium phaeobacteroides, respectively, was monitored using a Pt-glass-Ag⁰, Ag₂S electrode combination without liquid junction. Due to the resulting pe(pH) and pH₂S plottings three steps can be distinguished: oxidation of H₂S to an S(0) state, oxidation of S (0) to SO₄ ^2–, and oxidation of the remaining H₂S directly to SO₄ ^2–. Differences between the investigated bacteria exist with respect to their individual oxidation strategies.Thiocapsa apparently stops oxidizing H₂S at pH₂S 7.5 (e.g. 10^–7.5M H₂S) and shifts to the utilization of the intracellularly stored S (0). In contrastChlorobium utilizes its extracellularly stored sulfur parallel to the extracellular H₂S fraction. The corresponding Pt-sensor responses (pe₇ values) were found to be similar to the corresponding partial redox equilibria (p₇ values) of H₂S oxidation stoichiometries as proposed by Van Niel (1931) and Trüper (1964). It is concluded that the recording of pe enables investigators to understand (and control) in situ redox processes, independent of their thermodynamic equilibration, only bound to changes of electroactivity vs. sensor.     

Continuous acetone-butanol production with direct product removal

Summary To eliminate the product inhibition and increase the productivity of butanol formation, a continuously operated membrane bioreactor was connected to a four-stage mixer-settler cascade. Clostridium acetobutylicum was cultivated in this reactor. Butanol was selectively extracted with butyric acid saturated n-decanol from the cell-free cultivation medium, and the butanol-free medium was refed into the reactor. Due to the high boiling point of decanol, the recovery of butanol from the decanol solution is easy. The partition coefficient and selectivity of butanol in the cultivation medium-decanol-system is sufficiently high for removing it from the medium. Direct contact of the cells with the decanol phase causes cell damage. However, decanol is practically insoluble in the fermentation medium, thus the contact of the cell-free medium with the solvent phase does not influence of cell growth and product formation. At a dilution rate of D _z=0.1 h^-1, the butanol productivity was increased by removing butanol from the medium by a factor of four. A further increase was prevented by a contaminant of the technical decanol, which was identified by GC-MS-analysis as 1-,3-hexandiol.Symbols D dilution rate, h^-1 - D _eff effective dilution rate (Eq. 3), h^-1 - D _Ex extraction dilution rate (Eq. 3), h^-1 - D _g dilution rate of cell suspension in reactor-filter-system, h^-1 - E degree of extraction (Eq. 3), l - P product concentration in medium after extraction, g l^-1 - P _O product concentration in reactor, g l^-1 - R _P productivity and product formation rate, g l^-1 h^-1 - q _p S specific product formation coefficient with regard to the cell growth rate, l - V _F volume of cell suspension in filter module, l - V _g volume of the cell suspension in reactor and in filter module V _g =V _R +V _F , l - V _R volume of cell suspension in ractor, l - v _O cell free feed rate, l h^-1 - v ₁ flow rate of cell suspension leaves the reactor, l h^-1 - v _E flow rate of decanol through the extractor, l h^-1 - v _w flow rate of the cell free medium through the filter modul, l h^-1 - X cell mass concentration, g l^-1 - specific growth rate of the cells, h^-1 Dedicated to Professor Dr. H. J. Rehm on the occasion of his 60th birthday     

The cytoskeleton of isolated murine primitive erythrocytes

Summary Cytoskeletons of primitive erythrocytes have been isolated from the embryos of day 12 pregnant C57/Bl mice and examined by transmission electron microscopy, immunofluorescence microscopy, and SDS-polyacrylamide gel electrophoresis. Microtubules are the most prominent cytoskeletal component. They are found either singly or organized into loose bundles just under the plasma membrane, but do not form classical marginal bands in most cells. Immunofluorescence with a polyclonal tubulin antiserum confirms this distribution and further reveals numerous mitotic figures among the cells. Rhodamine-conjugated phalloidin and heavy meromyosin labeling reveal that actin is localized in the cortex of the primitive erythrocyte in the form of 6 nm filaments. Antibody directed against avian erythrocyte alpha spectrin demonstrates that spectrin is also found in the cortex. Occasional 10-nm intermediate filaments, observed in the primitve erythrocytes by electron microscopy, are believed to be of the vimentin class based on positive reaction of the cells with vimentin-specific antiserum. In addition, a band in erythrocyte cytoskeletons comigrates in SDS-polyacrylamide gels with vimentin isolated from mouse kidney. Spectrin and actin were also found to be associated with the membrane of primitive erythrocytes when membrane ghost preparations were analyzed by SDS-polyacrylamide gel electrophoresis.     

Evidence of excited state absorption in PS II membrane fragments

Utilizing a two-beam technique in the frequency domain, the pumped absorption of PS II membrane fragments from spinach and of acetonic chlorophyll-a solutions was measured at room temperature. In a very narrow wavelength region (0.2 nm around the pump pulse wavelength) the relative test beam transmission exhibited either a decrease or an increase, respectively, dependent on the intensity of a strong pump beam. In contrast, the transmission changes of chl-a solutions were not affected by the wavelength mistuning between pump and test beam. The data obtained for PS II membrane fragments were interpreted in terms of excited state absorption of pigment-protein clusters within the light-harvesting complex of PS II. The interpretation of the small absorption band as a homogeneously broadened line led to a transversal relaxation time for chlorophyll in vivo of about 1 ps.Abbreviations PS I photosystem I of green plants - PS II photosystem II of green plants - P700 primary donor of PS I - P680 primary donor of PS II     

Analysis of the electron transfer from Pheo to QA in PS II membrane fragments from spinach by time resolved 325 nm absorption changes in the picosecond domain

Absorption changes at 325 nm (delta A325) induced by 15 ps laser flashes (lambda = 650 nm) in PS II membrane fragments were measured with picosecond time-resolution. In samples with the reaction centers (RCs) kept in the open state (P I QA) the signals are characterized by a very fast rise (not resolvable by our equipment) followed by only small changes within our time window of 1.6 ns. In the closed state (PI QA-) of the reaction center the signal decays with an average half-life time of about 250 ps. It is shown that under our excitation conditions (E = 2 x 10(14) photons/cm2 per pulse) subtraction of the absorption changes in closed RCs (delta A closed 325) from those in open RCs (delta A open 325) leads to a difference signal which is dominated by the reduction kinetics of QA. From the rise kinetics of this signal and by comparison with data in the literature it is inferred that QA becomes reduced by direct electron transfer from Pheo- with a time constant of about 350 +/- 100 ps.     

Observations on Echinococcus granulosus of cattle origin in Switzerland

Switzerland is one of the few countries where high fertility rates have been reported in cattle hydatid cysts and where the cattle/dog cycle is the most important for the maintenance of Echinococcus granulosus. The developmental and morphological characteristics of E. granulosus of Swiss cattle origin were studied and compared with that of E. granulosus of domestic animal origin from Great Britain and Australia, countries where bovine hydatid cysts are usually sterile and cattle play little role in the life-cycle of the parasite. Adult E. granulosus of Swiss cattle origin differed markedly in its developmental characteristics compared to other isolates, particularly in its rate of maturation in dogs, producing eggs as early as 35 days post-infection. The morphology of E. granulosus of Swiss cattle origin was characteristic and it could be easily distinguished from other isolates of the parasite. Further, E. granulosus of Swiss cattle origin was found to closely resemble that occurring in cattle in South Africa where high fertility rates have also been reported in bovine hydatid cysts. It is concluded that a strain of E. granulosus exists which is adapted to cattle and that further studies are required to determine whether this strain warrants formal taxonomic status as the species E. ortleppi which was originally described for the parasite of South African cattle origin.     

Calcium domains associated with individual channels can account for anomalous voltage relations of CA-dependent responses.

Computer-assisted modeling of calcium influx through voltage-activated membrane channels predicted that buffer-limited elevation of cytoplasmic free calcium ion concentration occurs within microscopic hemispherical "domains" centered upon the active Ca channels. With increasing depolarization, the number of activated channels, and hence the number of Ca domains, should increase; the single-channel current should, however, decrease, thereby decreasing Ca2+ accumulation in each domain relative to the macroscopic current. Such voltage dependence of the microscopic distribution of Ca2+ may influence relations between total Ca2+ entry and Ca-dependent processes. Ca-mediated inactivation of Ca channels in Aplysia neurons exhibits behavior consistent with the calcium domain hypothesis.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.