Posttranslational processing of concanavalin A precursors in jackbean cotyledons

Metabolic labeling of immature jackbean cotyledons with 14C-amino acids was used to determine the processing steps involved in the assembly of concanavalin A. Pulse-chase experiments and analyses of immunoprecipitated lectin forms indicated a complex series of events involving seven distinct species. The structural relatedness of all of the intermediate species was confirmed by two-dimensional mapping of 125I-tryptic peptides. An initial glycosylated precursor was deglycosylated and cleaved into smaller polypeptides, which subsequently reannealed over a period of 10-27 h. NH2-terminal sequencing of the abundant precursors confirmed that the intact subunit of concanavalin A was formed by the reannealing of two fragments, since the alignment of residues 1-118 and 119-237 was reversed in the final form of the lectin identified in the chase and the precursor first labeled. When the tissue was pulse-chased in the presence of monensin, processing of the glycosylated precursor was inhibited. The weak bases NH4Cl and chloroquine were without effect. Immunocytochemical studies showed that monensin treatment caused the accumulation of immunoreactive material at the cell surface and indicated that the ionophore had induced the secretion of a component normally destined for deposition within the protein bodies. Consideration of the tertiary structure of the glycosylated precursor and mature lectin showed that the entire series of processing events could occur without significant refolding of the initial translational product. Proteolytic events included removal of a peptide from the surface of the precursor molecule that connected the NH2- and COOH-termini of the mature protein. This processing activated the carbohydrate-binding activity of the lectin. The chase data suggest the occurrence of a simultaneous cleavage and formation of a peptide bond, raising the possibility that annealment of the fragments to give rise to the mature subunit involves a transpeptidation event rather than cleavage and subsequent religation

Inhibition of Vesicular Glutamate Uptake by Rose Bengal-Related Compounds: Structure–Activity Relationship

Synaptic vesicular accumulation of glutamate is a vital initial step in glutamate transmission. We have previously shown that Rose Bengal, a polyhalogenated fluorescein analog, is a potent inhibitor of glutamate uptake into synaptic vesicles. Here, we report the structural features of Rose Bengal required for this inhibition. Various Rose Bengal-related compounds, with systematic structural variations, were tested. Results indicate that the four iodo groups and the phenyl group attached to the xanthene moiety are critical for potent inhibitory activity. Replacement of these groups with two iodo groups and an alkyl group, respectively, results in substantial reduction in potency. Of further interest in creating high potency is the critical nature of the oxygen atom which links the two benzene rings of xanthene. Thus, the phenyl group and multiple iodo groups, as well as the bridging oxygen of xanthene, are crucial elements of Rose Bengal required for its potent inhibitory action.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45424/1/11064_2005_Article_2610.pd

Investigation of G72 (DAOA) expression in the human brain

<p>Abstract</p> <p>Background</p> <p>Polymorphisms at the G72/G30 locus on chromosome 13q have been associated with schizophrenia or bipolar disorder in more than ten independent studies. Even though the genetic findings are very robust, the physiological role of the predicted G72 protein has thus far not been resolved. Initial reports suggested G72 as an activator of D-amino acid oxidase (DAO), supporting the glutamate dysfunction hypothesis of schizophrenia. However, these findings have subsequently not been reproduced and reports of endogenous human G72 mRNA and protein expression are extremely limited. In order to better understand the function of this putative schizophrenia susceptibility gene, we attempted to demonstrate G72 mRNA and protein expression in relevant human brain regions.</p> <p>Methods</p> <p>The expression of G72 mRNA was studied by northern blotting and semi-quantitative SYBR-Green and Taqman RT-PCR. Protein expression in human tissue lysates was investigated by western blotting using two custom-made specific anti-G72 peptide antibodies. An in-depth <it>in silico </it>analysis of the G72/G30 locus was performed in order to try and identify motifs or regulatory elements that provide insight to G72 mRNA expression and transcript stability.</p> <p>Results</p> <p>Despite using highly sensitive techniques, we failed to identify significant levels of G72 mRNA in a variety of human tissues (e.g. adult brain, amygdala, caudate nucleus, fetal brain, spinal cord and testis) human cell lines or schizophrenia/control post mortem BA10 samples. Furthermore, using western blotting in combination with sensitive detection methods, we were also unable to detect G72 protein in a number of human brain regions (including cerebellum and amygdala), spinal cord or testis. A detailed <it>in silico </it>analysis provides several lines of evidence that support the apparent low or absent expression of G72.</p> <p>Conclusion</p> <p>Our results suggest that native G72 protein is not normally present in the tissues that we analysed in this study. We also conclude that the lack of demonstrable G72 expression in relevant brain regions does not support a role for G72 in modulation of DAO activity and the pathology of schizophrenia via a DAO-mediated mechanism. <it>In silico </it>analysis suggests that G72 is not robustly expressed and that the transcript is potentially labile. Further studies are required to understand the significance of the G72/30 locus to schizophrenia.</p

Amyloid Precursor Protein Is Trafficked and Secreted via Synaptic Vesicles

A large body of evidence has implicated amyloid precursor protein (APP) and its proteolytic derivatives as key players in the physiological context of neuronal synaptogenesis and synapse maintenance, as well as in the pathology of Alzheimer's Disease (AD). Although APP processing and release are known to occur in response to neuronal stimulation, the exact mechanism by which APP reaches the neuronal surface is unclear. We now demonstrate that a small but relevant number of synaptic vesicles contain APP, which can be released during neuronal activity, and most likely represent the major exocytic pathway of APP. This novel finding leads us to propose a revised model of presynaptic APP trafficking that reconciles existing knowledge on APP with our present understanding of vesicular release and recycling

Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure.

Uptake of GABA was demonstrated in rat brain synaptic vesicles which were prepared by a new and efficient procedure. The uptake activity co-purified with the synaptic vesicles during the isolation procedure. The purity of the vesicle fraction was rigorously examined by analysis of marker enzymes and marker proteins and also by immunogold electron microscopy using antibodies against p38 (synaptophysin). Contamination by other cellular components was negligible, indicating that GABA uptake by the synaptic vesicle fraction is specific for synaptic vesicles and not due to the presence of other structure possessing GABA uptake or binding activities. GABA uptake was ATP dependent and similar to the uptake of glutamate, which was assayed for a comparison. Both uptake activities were independent of sodium. They were inhibited by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, indicating that the energy for the uptake is provided by an electrochemical proton gradient. This gradient is generated by a proton ATPase of the vacuolar type as suggested by the effects of various ATPase inhibitors on neurotransmitter uptake and proton pumping. Competition experiments revealed that the transporters for GABA and glutamate are selective for the respective neurotransmitters