Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

The septins are a conserved family of GTP-binding proteins that, in the baker''s yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified.Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.     

Interaction of extracellular Pseudomonas lipase with alginate and its potential use in biotechnology

Summary Extracellular Pseudomonas lipase is able to interact directly or indirectly with alginate as deduced from the following results: (i) During adsorption chromatography of exolipase the enzyme adsorbed quantitatively to glass beads in the absence of alginate, but not after its preincubation in the presence of the polysaccharide; pretreatment of glass beads with alginate did not prevent enzyme adsorption. (ii) In the presence of alginate exolipase was much more resistant to heat inactivation than in its absence. (iii) In the presence of alginate the increase in exolipase activity caused by the non-ionic detergent Triton X-100 was drastically reduced. (iv) Exolipase could be rapidly and almost completely harvested from cell-free culture fluid of P. aeruginosa 5940 by ethanolic coprecipitation with alginate. After dissolving the coprecipitate in detergent-containing buffer exolipase and polysaccharide could be easily separated by ion-exchange chromatography on DEAE-Sephadex A-25. The coprecipitation method was also successfully applied to exolipases produced by Pseudomonas sp., Chromobacierium viscosum and Rhizopus delamar, thus suggesting potential use of this method in biotechnology.     

Isolation of aClostridium thermocellum gene encoding a thermostable β-1, 3-glucanase (laminarinase)

Summary AClostridium thermocellum gene directing the synthesis of a thermostable -glucanase was localized on a 1.9-kb DNA fragment by subcloning intoEscherichia coli plasmid vectors. The enzyme was highly efficient in degrading glucans with alternating -1, 3- and -1,4-linkages such as lichenan and barley glucan. It was also active towards the -1, 3-glucan laminarin, but lacked activity on cellulosic substrates and -glucans. The enzyme was therefore classified as -1, 3-glucanase (laminarinase) and the corresponding gene was designatedlicA. With barley -glucan as substrate the enzyme had a pH optimum around pH 6.5 and a temperature optimum at 65°C. It was stable for several hours at 60°C in the absence of substrate.     

Protein phosphorylation in intact coronary endothelial cells by bradykinin

In cultured coronary endothelial cells obtained from guinea pig hearts, bradykinin (10(-6) M) stimulated the 32Pi-incorporation into 5 substrate proteins with molecular weights corresponding to 27, 32, 60, 86 and 100 kDa. The time course of phosphorylation of the 60, 86 and 100 kDa proteins was rapid (within 30 s), but transient (max. within 1-2 min.), while the 32Pi incorporation into the 27 and 32 kDa protein was delayed but increased within 10 minutes. Ca+(+)-ionophore A 23187 (10(-5) M) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-5) M) both mimicked the effects of the bradykinin induced phosphorylation pattern. While A 23187 enhanced the phosphorylation of the 27, 60 and 100 kDa substrates, TPA increased the 32Pi-incorporation into the 32 and 86 kDa proteins. Furthermore the time course of protein phosphorylation elicited by A 23187 and TPA showed marked similarities to those obtained with bradykinin. Our findings are consistent with the view, that stimulation of coronary endothelial bradykinin-receptors activates both Ca+(+)-dependent protein kinases and protein kinase C.     

Elastic Torques about Membrane Edges: A Study of Pierced Egg Lecithin Vesicles

The shape of mechanically pierced giant vesicles is studied to obtain the elastic modulus of Gaussian curvature of egg lecithin bilayers. It is argued that such experiments are governed by an apparent modulus, ¯κ_app, not the true modulus of Gaussian curvature, ¯κ. A theory of ¯κ_app is proposed, regarding the pierced bilayer vesicle as a closed monolayer vesicle. The quantity measured, i.e. ¯κ_app/κ, where κ is the rigidity, agrees satisfactorily with the theory. We find ¯κ_app = -(1.9 ± 0.3) · 10^-12 erg (on the basis of κ = (2.3 ± 0.3) · 10^-12 erg). The result may have implications for bilayer fusion.