Characterization of an ATPase on the inside of rat-liver nuclear envelopes by affinity labeling

Nuclear envelope membranes from rat liver cells contain ATPases, one of which can be inhibited and irreversibly labeled by (S-dinitrophenyl)-6-mercaptopurine riboside triphosphate. Inhibition and covalent substitution of the ATPase are achieved only after disruption of the nuclei, the ATP analogue is inactive on the ATPase activity of whole nuclei or on vesicles of the membrane prepared after a modified heparin method of Bornens and Courvalin. Electron micrographs and scanning micrographs helped to establish the characterization of closed vesicles and intact nuclei. With the aid of (alpha-32P)-labeled, and of the (beta, gamma-32P)-labeled analogue, it was possible to demonstrate the incorporation of the nucleotide into a few protein regions of the nuclear membrane disc electrophoresis pattern.     

Photo-activated inhibition of sulfate equilibrium exchange in human erythrocyte ghosts by a 4-azido-2-nitrobenzoate derivative of phlorizin

Like phlorizin, two glycosidic esters of phlorizin, the 4-azido-2-nitrobenzoate (ANB-phlorizin) and the 2-nitrobenzoate (NB-phlorizin) were found to be effective inhibitors of SO₄^2? equilibrium exchange at the outer but not at the inner membrane surface of the human erythrocyte ghost. After photolysis of ghost suspensions in the presence of extracellular ANB-phlorizin an irreversible inhibition of SO₄^2? exchange was observed, while photolysis of intracellular ANB-phlorizin was without effect. After photolysis in the presence of extracellular or intracellular tritiated ANB-phlorizin gel electrophoresis of the labelled membranes revealed similar locations of binding. These findings suggest that the sidedness of action of ANB-phlorizin could not be related to inaccessibility of the inner membrane surface for the agent but that inhibition occurs via binding to fixed sites at the outer membrane surface that are not associated with a mobile carrier which crosses the membrane.     

Soccer Offside Judgments in Laypersons with Different Types of Static Displays

Four experiments investigated offside decisions in laypersons with different types of static displays. Previous research neglected this group although the majority of assistant referees in soccer games at the amateur level are laypersons. The aims of our research were (a) to investigate the spatial resolution in laypersons’ perception of offside situations, (b) to search for biases in laypersons’ offside judgments, and (c) to develop useful displays for future research. The displays showed the moment when a midfielder passes the ball to a forward moving in the vicinity of a defender. We varied the spatial location of the forward around the defender in eleven steps and participants made their offside decision by pressing a key. Across experiments, displays varied in abstractness (simple shapes, clipart figures, photographs). There were two major findings. Firstly, both accuracy and speed of offside judgments deteriorated when the spatial distance between forward and defender decreased, approaching guessing rate at the smallest distances. Secondly, participants showed a consistent bias in favor of the non-offside response, in contrast to most studies on professional assistant referees. In sum, the results highlight the limited spatial resolution of the visual system and underscore the role of response bias in offside-judgment tasks.     

Evidence that the novobiocin-sensitive ATP-binding site of the heat shock protein 90 (hsp90) is necessary for its autophosphorylation

The 90kDa heat shock protein (Hsp90) is one of the most abundant protein and essential for all eukaryotic cells. Many proteins require the interaction with Hsp90 for proper function. Upon heat stress the expression level of Hsp90 is even enhanced. It is assumed, that under these conditions Hsp90 is required to protect other proteins from aggregation. One property of Hsp90 is its ability to undergo autophosphorylation. The N-terminal domain of Hsp90 has been shown to contain an unusual ATP-binding site. A well-known inhibitor of Hsp90 function is geldanamycin binding to the N-terminal ATP-binding site with high affinity. Recently it was shown that Hsp90 possesses a second ATP-binding site in the C-terminal region, which can be competed with novobiocin. Autophosphorylation of Hsp90 was analysed by incubation with gamma(32)P-ATP. Addition of geldanamycin did not interfere with the capability for autophosphorylation, while novobiocin indeed did. These results suggest that the C-terminal ATP-binding site is required for autophosphorylation of Hsp90.