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A Hausinger

Publications and source records attributed to A Hausinger.

6 recordsLinked to original sources
PubMed1996

Two synpatic vesicle proteins of 25 kDa: a comparison of the molecular properties and tissue distribution of svp25 and o-rab3.

Two synaptic vesicle proteins of the electric ray Torpedo--svp25 and o-rab3--are compared with respect to their biochemical properties and tissue distribution. On SDS-PAGE both proteins migrate to the same position of about 25 kDa. As revealed by application of monospecific antibodies and subcellular fractionation both proteins comigrate and cofractionate with the synaptic vesicle compartment. o-Rab3 and svp25 can be separated by lectin chromatography; svp25 is highly glycosylated and binds to concanavalin A sepharose. Upon deglycosylation using glycopeptidase F and O-glycosidase its apparent molecular mass is reduced to about 14 kDa. Partial amino acid sequences obtained by direct microsequencing of purified and deglycosylated svp25 revealed that svp25 is a novel protein that has not yet been characterized in molecular terms. Whereas svp25 was detected in all brain areas investigated, the expression of o-rab3 was found to be restricted to specific regions. An immunoblot analysis demonstrates an exclusive association of both proteins with neural tissues. Our results suggest that cholinergic synaptic vesicles from electric ray electric organ contain at least two membrane-associated proteins of an apparent molecular mass of 25 kDa, the membrane associated o-rab3 and the membrane integral protein svp25. The two proteins can be separated by lectin chromatography for assessment of their biochemical properties.

Animals↗
PubMed1995

Calcium-dependent endogenous proteolysis of the vesicle proteins synaptobrevin and synaptotagmin.

The synaptic vesicle integral protein synaptobrevin/VAMP is a target of the clostridial metalloproteases tetanus toxin and botulinum toxins. We provide evidence that synaptobrevin can also be cleaved by an endogenous protease. As revealed by Western blotting proteolysis is calcium-dependent, results in the formation of an 8 kD peptide that becomes apparent within 10 min. Proteolysis can be inhibited by the chelating agents EGTA and EDTA, whereas other protease inhibitors failed to prevent degradation. In addition, a proteolytic degradation of the synaptic vesicle specific protein synaptotagmin could be observed. Other proteins including the synaptic vesicle proteins synapsin I and synaptophysin remained unaltered. Partial calcium-dependent degradation of select synaptic vesicle proteins may play a role in the life cycle of the organelle.

Animals↗
PubMed1995

Inhibition by clostridial neurotoxins of calcium-independent [3H]noradrenaline outflow from freeze-thawed synaptosomes: comparison with synaptobrevin hydrolysis.

Clostridial neurotoxins are known to inhibit regulated, i.e. calcium-dependent exocytosis. In the present study we have investigated their potential role in also inhibiting calcium-independent exocytosis. Synaptosomes from rat forebrain were preloaded with [3H]noradrenaline and permeabilized reversibly by freezing in Ca(2+)-free potassium glutamate containing dimethyl sulfoxide and the toxins to be assayed. Subsequently, outflow of radioactivity was measured in isotonic calcium-free potassium glutamate. The synaptic vesicle protein synaptobrevin-2/VAMP-2 and its toxin-dependent degradation were analysed by Western blotting. The light chain of tetanus toxin reduced the synaptosomal outflow of radioactivity, whereas the activity of the heavy chain was at the detection limit. The respective activities of the dichain toxins from Clostridium tetani and C. botulinum A, B and E were enhanced by pretreatment with dithiothreitol. Reduced single-chain tetanus toxin was less potent than reduced dichain tetanus toxin. Pretreatment with ethylene diamine tetraacetic acid as an inhibitor of Zn(2+)-proteases abolished the actions of the tetanus toxin light chain and of the reduced dichain toxins. Hydrolysis of synaptobrevin-2/VAMP-2 was obtained with tetanus toxin light chain, reduced dichain tetanus toxin and C. botulinum B toxin. Its hydrolysis by single-chain tetanus toxin was less pronounced, and it was absent with botulinum toxins A and E. It is concluded that clostridial neurotoxins can not only inhibit calcium-dependent release but also affect calcium-independent outflow from synaptosomes. Since this is accompanied by selective intrasynaptosomal proteolysis of synaptobrevin, calcium-independent outflow may at least in part involve the vesicular release apparatus.

Animals↗
PubMed1993

Synaptic vesicle life cycle and synaptic turnover.

Cholinergic synaptic vesicles contain a mixture of soluble low molecular mass constituents. Besides acetylcholine these include Ca2+, ATP, GTP, small amounts of ADP and AMP, and also the diadenosine polyphosphates Ap4A and Ap5A. In synaptic vesicles isolated from the electric ray these diadenosine polyphosphates occur in mmol concentrations and might represent a novel cotransmitter. The membrane proteins of cholinergic synaptic vesicles presumably are identical to those in other types of electron-lucent synaptic vesicles. A presumptive exception are the transmitter-specific carriers. The life cycle of the synaptic vesicle in intact neurons and in situ was investigated by analysis of all cytoplasmic membrane compartments that share membrane integral proteins with synaptic vesicles. The results suggest that the synaptic vesicle membrane compartment might originate from the trans-Golgi network and, after cycles of exo- and endocytosis in the nerve terminal, might fuse into an endosomal membrane compartment early on retrograde transport. Tracer experiments using membrane proteins and soluble contents suggest that the synaptic vesicle membrane compartment does not intermix with the presynaptic plasma membrane on repeated cycles of exo- and endocytosis if low frequency stimulation is applied. A cDNA has been isolated from the electric ray electric lobe that codes for o-rab3, a small GTP-binding protein highly homologous to mammalian rab3. While abundant in the nerve terminals of the electric organ and at the neuromuscular junction this protein occurs only in limited subpopulations of nerve terminals in electric ray brain. Immunocytochemical analysis using the colloidal gold technique and a monospecific antibody against o-rab3 suggests that the GTP-binding protein remains attached to recycling synaptic vesicles. No evidence was found for a major contribution of an intraterminal endosomal sorting compartment involved in synaptic vesicle recycling.

Acetylcholine↗
PubMed1993

The synaptic vesicle-associated G protein o-rab3 is expressed in subpopulations of neurons.

The distribution of o-rab3--a synaptic vesicle-associated low-molecular-weight GTP-binding protein--was studied in various neural tissues of the electric ray Torpedo marmorata. o-rab3 was shown to be associated selectively with isolated cholinergic synaptic vesicles derived from the electric organ. Gel filtration of cholinergic synaptic vesicles using Sephacryl S-1000 column chromatography demonstrated a copurification of o-rab3 with the synaptic vesicle content marker ATP and with SV2--a synaptic vesicle transmembrane glycoprotein. Indirect immunofluorescence using antibodies against o-rab3 and SV2 and a double labeling protocol revealed an identical distribution of both antigens in the cholinergic nerve terminals within the electric organ and at neuromuscular junctions. An immunoelectron microscopic analysis demonstrated the presence of o-rab3 at the surface of the synaptic vesicle membrane. In the CNS immunofluorescence of o-rab3 and SV2 overlap only in small and distinct areas. Whereas SV2 has an overall only in small and distinct areas. Whereas SV2 has an overall distribution in nerve terminals of the entire CNS, o-rab3 is restricted to a subpopulation of nerve terminals in the dorsolateral neuropile of the rhombencephalon and in the dorsal horn of the spinal cord. Our results demonstrate that the synaptic vesicle-associated G protein o-rab3 is specifically expressed only in subpopulations of neurons in the Torpedo CNS.

Animals↗