ATPase activity and conformational changes in the regulation of actin.

The eukaryotic microfilament system is regulated in part through the nucleotide- and cation-dependent conformation of the actin molecule. In this review, recent literature on the crystal and solution structures of actin and other actin-superfamily proteins is summarized. Furthermore, the structure of the nucleotide binding cleft is discussed in terms of the mechanism of ATP hydrolysis and P(i) release. Two distinct domain movements are suggested to participate in the regulation of actin. (1) High-affinity binding of Mg(2+) to actin induces a rearrangement of side chains in the nucleotide binding site leading to an increased ATPase activity and polymerizability, as well as a rotation of subdomain 2 which is mediated by the hydroxyl of serine-14. (2) Hydrolysis of ATP and subsequent release of inorganic phosphate lead to a butterfly-like opening of the actin molecule brought about by a shearing in the interdomain helix 135-150. These domain rearrangements modulate the interaction of actin with a variety of different proteins, and conversely, protein binding to actin can restrict these conformational changes, with ultimate effects on the assembly state of the microfilament system.     

Study of pyoverdine type and production by Pseudomonas aeruginosa isolated from cystic fibrosis patients: prevalence of type II pyoverdine isolates and accumulation of pyoverdine-negative mutations

The lungs of cystic fibrosis patients are frequently colonized by Pseudomonas aeruginosa, which produces high-affinity fluorescent peptidic siderophores, pyoverdines. Three pyoverdines which differ in their peptide chain and are easily differentiated by isoelectric focusing exist, only one being produced by a given strain. P. aeruginosa isolates from cystic fibrosis patients of a German hospital were analyzed by sequential, pulse-field gel electrophoresis (PFGE) and for pyoverdine production and type. Only producers of type I and type II pyoverdine were found. There was a perfect correlation between the type of pyoverdine produced and the clonality determined by PFGE. PFGE clone C, the most prevalent among cystic fibrosis patients, and found in an aquatic environment, produced type II pyoverdine. Pyoverdine-negative mutants seemed to increase as a function of the lung colonization time, but retained the capacity to take up pyoverdines. Most isolates that took up type II pyoverdine were also able to utilize type I pyoverdine as judged by growth stimulation experiments. No correlation was observed between the loss of pyoverdine production and mucoidy.     

Sugar utilization in the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324: starch degradation to acetate and CO2 via a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming)

The hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324, rather than the type strain VC16, was found to grow on starch and sulfate as energy and carbon source. Fermentation products and enzyme activities were determined in starch-grown cells and compared to those of cells grown on lactate and sulfate. During exponential growth on starch, 1 mol of glucose-equivalent was incompletely oxidized with sulfate to approximately 2 mol acetate, 2 mol CO2 and 1 mol H2S. Starch-grown cells did not contain measurable amounts of the deazaflavin factor F420 (<0.03 nmol/mg protein) and thus did not show the F420-specific green-blue fluorescence. In contrast, lactate (1 mol) was completely oxidized with sulfate to 3 mol CO2 by strain 7324, and lactate-grown cells contained high amounts of F420 (0.6 nmol/mg protein). In extracts of starch-grown cells, the following enzymes of a modified Embden-Meyerhof pathway were detected: ADP-dependent hexokinase (ADP-HK), phosphoglucose isomerase, ADP-dependent 6-phosphofructokinase (ADP-PFK), fructose-1,6-phosphate aldolase, glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAP:FdOR), phosphoglycerate mutase, enolase, and pyruvate kinase (PK). Specific activities of ADP-HK, ADP-PFK, GAP:FdOR, and PK were significantly higher in starch-grown cells than in lactate-grown cells, indicating induction of these enzymes during starch catabolism. Pyruvate conversion to acetate involved pyruvate:ferredoxin oxidoreductase and ADP-forming acetyl-CoA synthetase. The findings indicate that the archaeal sulfate reducer A. fulgidus strain 7324 converts starch to acetate via a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming). This is the first report of growth of a sulfate reducer on starch, i.e. on a polymeric sugar.     

Movement patterns of the bush cricket Platycleis albopunctata in different types of habitat: matrix is not always matrix

Abstract.  1. Inter-patch movement is usually assumed to be homogeneous across a landscape. As the intervening area between suitable patches is usually richly textured, it cannot be assumed to be uniform in real landscapes.
2. In an experimental mark-and-resight study, the movement behaviour of the bush cricket Platycleis albopunctata in four habitat types as well as at the border between two of these habitat types was observed.
3. Analysis of recapture data indicated differences in mortality risk (or emigration rates) between habitat types.
4. When released at the border between suitable habitat and a crop field, P. albopunctata did not show a consistent preference for the suitable habitat. This suggests that the crop field is at least temporarily attractive for P. albopunctata .
5. Movement in suitable habitat was not always different from movement in the matrix, and movement between different types of matrix also differed.
6. The results indicate that the movement behaviour of P. albopunctata is influenced not only by suitability for breeding but also by structural resistance and other factors (e.g. food availability or habitat-specific mortality risk).     

Crystal structures of the active and alloxanthine-inhibited forms of xanthine dehydrogenase from Rhodobacter capsulatus.

Xanthine dehydrogenase (XDH), a complex molybdo/iron-sulfur/flavoprotein, catalyzes the oxidation of hypoxanthine to xanthine followed by oxidation of xanthine to uric acid with concomitant reduction of NAD+. The 2.7 A resolution structure of Rhodobacter capsulatus XDH reveals that the bacterial and bovine XDH have highly similar folds despite differences in subunit composition. The NAD+ binding pocket of the bacterial XDH resembles that of the dehydrogenase form of the bovine enzyme rather than that of the oxidase form, which reduces O(2) instead of NAD+. The drug allopurinol is used to treat XDH-catalyzed uric acid build-up occurring in gout or during cancer chemotherapy. As a hypoxanthine analog, it is oxidized to alloxanthine, which cannot be further oxidized but acts as a tight binding inhibitor of XDH. The 3.0 A resolution structure of the XDH-alloxanthine complex shows direct coordination of alloxanthine to the molybdenum via a nitrogen atom. These results provide a starting point for the rational design of new XDH inhibitors.     

Cell Adhesion Molecule L1 in Folded (Horseshoe) and Extended Conformations

We have investigated the structure of the cell adhesion molecule L1 by electron microscopy. We were particularly interested in the conformation of the four N-terminal immunoglobulin domains, because x-ray diffraction showed that these domains are bent into a horseshoe shape in the related molecules hemolin and axonin-1. Surprisingly, rotary-shadowed specimens showed the molecules to be elongated, with no indication of the horseshoe shape. However, sedimentation data suggested that these domains of L1 were folded into a compact shape in solution; therefore, this prompted us to look at the molecules by an alternative technique, negative stain. The negative stain images showed a compact shape consistent with the expected horseshoe conformation. We speculate that in rotary shadowing the contact with the mica caused a distortion of the protein, weakening the bonds forming the horseshoe and permitting the molecule to extend. We have thus confirmed that the L1 molecule is primarily in the horseshoe conformation in solution, and we have visualized for the first time its opening into an extended conformation. Our study resolves conflicting interpretations from previous electron microscopy studies of L1.     

Stridulatory pattern generation in acridid grasshoppers: metathoracic interneurons in Stenobothrus rubicundus (Germar 1817)

Stridulation was elicited in tethered gomphocerine grasshoppers of the species Stenobothrus rubicundus in order to identify interneurons of the stridulation pattern generator, and describe their morphological and physiological properties. Nine types of such neurons could be found and characterized; eight of those could additionally be compared to corresponding neuron types previously known from other species. As shown in detail for one selected type, the neurons of the stridulation pattern generator are very similar in their anatomical appearance, and possess similar physiological qualities at least in two species with similar stridulation patterns. Stridulation interneurons of species with largely different stridulatory motor patterns have a similar morphology, but show a different activation timing throughout the stridulation. Nevertheless, special properties such as resetting or initiation capability of certain stridulation interneurons seem to be conserved throughout the species. The results suggest that the stridulation pattern generator of different species consists of a uniform set of interneurons that change their activity pattern to produce species-specific song movements.     

Serratia marcescens forms a new type of cytolysin

Most Serratia marcescens strains produce a new type of cytolysin (hemolysin) which is also found in other Serratia species. The hemolytic polypeptide ShlA (M(r) 162 101) is secreted across the outer membrane through the help of the ShlB protein which also involves conversion of an inactive precursor in an hemolytically active form. Both proteins are synthesized with signal sequences which are released during export across the cytoplasmic membrane. Mutants expressing inactive ShlB derivatives are impaired in activation and secretion suggesting a tight coupling between both processes. The region of ShlA for activation and secretion is confined to the N-terminal 16% of the polypeptide which contains the sequence NPNG which is also found in the Proteus hemolysin, the Bordetella pertussis filamentous hemagglutinin and two highly expressed outer membrane proteins of Haemophilus influenzae. Substitution of the first asparagine (N) residue by isoleucine converts the Serratia hemolysin into an inactive secretion incompetent form. It is concluded that this region is recognized by ShlB for activation and secretion of ShlA. The Serratia hemolysin forms defined pores in erythrocyte membranes.     

Der Einfluß der Kulturbedingungen auf den Nicotinamid-Adenin-Dinucleotid(phosphat)-Gehalt in Zellen von Rhodospirillum rubrum

Zusammenfassung In Zellen von R. rubrum war das Verhältnis von oxydiertem zu reduziertem NAD(P) vom Sauerstoffpartialdruck im Medium, der Lichtintensität und der Nährbodenzusammensetzung abhängig. In ruhenden Kulturen unter aeroben Bedingungen im Licht oder im Dunkeln und anaerob bei hoher Lichtintensität, wenn der ATP-Pool in den Zellen groß ist, beobachtete man einen relativ hohen Wert für das Verhältnis von NAD(P)⁺/NAD(P)H. Unter Kulturbedingungen, bei denen der ATP-Gewinn der Zellen gering ist (anaerob Schwachlicht oder anaerob Dunkel), sank das Verhältnis von NAD(P)⁺/NAD(P)H ab. Die niedrigsten Werte für das Verhältnis von NAD(P)⁺/NAD(P)H wurden dementsprechend in anaerober Dunkelkultur, die höchsten in aerober Lichtkultur gefunden.Anaerob im Dunkeln war der NAD(P)H-Spiegel auch vom Substrat abhängig: mit Fructose oder ohne Substrat beobachtete man einen sehr großen NAD(P)H-Pool in den Zellen; nach Zugabe von Acetat, Succinat, Pyruvat oder Malat sank der Spiegel der reduzierten Coenzyme ab.In wachsenden Kulturen (außer anaerob im Dunkeln) nahm die relative Konzentration von NAD⁺ und der NADP⁺-Pool im Vergleich zu ruhenden Zellen stark zu (3-5fach).Änderungen im Verhältnis von NAD⁺/NADH und von NADP⁺/NADPH waren aber nicht unter allen Kulturbedingungen direkt korreliert.Es wird diskutiert, wieweit das Adenylatsystem und das NAD(P)-System einen regulativen Einfluß auf die Bacteriochlorophyll-Synthese und die Morphogenese bei Athiorhodaceae haben.

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The influence of culture conditions on the NAD(P) content of Rhodospirillum rubrum cells

Summary In cells of R. rubrum the ratio of oxidized to reduced NAD(P) depended on the oxygen pressure in the medium, the light intensity, and the composition of the medium. The ratio of NAD(P)⁺/NAD(P)H was high under conditions when the ATP-pool in the cell is large, viz. in resting cultures either kept aerobically in the light or in the dark or kept anaerobically in strong light. The quotient NAD(P)⁺/NAD(P)H decreased under conditions of reduced ATP-synthesis in the cells (anaerobic in dimlight or in the dark). Consequently, the lowest NAD(P)⁺/NAD(P)H value was observed in anaerobic dark cultures, the highest in aerobic light cultures.Under anaerobic conditions in the dark, the NAD(P)H level depended also on the substrate: with fructose or without any substrate, a large NAD(P)H pool was observed; the level of reduced coenzymes decreased upon addition of acetate, succinate, pyruvate, or malate.In growing cultures (except under anaerobic conditions in the dark) the relative concentration of NAD⁺ and the NADP⁺ pool showed a considerable increase (3 to 5 fold), as compared with resting cells. However, the changes in the proportions of NAD⁺/NADH and NADP⁺/NADPH were not directly correlated under all culture conditions.The regulative influence of the adenylate and the NAD(P) systems on the synthesis of bacteriochlorophyll and morphogenesis in Athiorhodaceae is discussed.

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Abkürzungen BChl Bacteriochlorophyll a - NAD(P) NAD-Nucleotide=reduziertes und oxydiertes Nicotinamid-Adenin-Dinucleotid und Nicotinamid-Adenin-Dinucleotidphosphat Herrn Prof. Dr. H. Engel zum 70. Geburtstag gewidmet.     

ADP-ribosylation factor (ARF)-like 4, 6, and 7 represent a subgroup of the ARF family characterization by rapid nucleotide exchange and a nuclear localization signal.

The novel ARF-like GTPase ARL7 is a close relative of ARL4 and ARL6 (71% and 59%) identical amino acids). A striking characteristic of these GTPases is their basic C-terminus which, when fused to the C-terminus of green fluorescent protein (GFP), targets the constructs to the nucleus of transfected COS-7 cells. Full length ARL4 was detected in both nuclear and extranuclear compartments, whereas a construct of ARL4 lacking its C-terminus was excluded from the nucleus. Nucleotide exchange rates of recombinant ARL4, ARL6 and ARL7 were similar and appeared considerably higher than those of other members of the ARF family (ARF1, ARP). It is concluded that ARL4, ARL6 and ARL7 form a subgroup within the ARF family with similar, possibly nuclear, function.