A role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals.

Amphiphysin, a major autoantigen in paraneoplastic Stiff-Man syndrome, is an SH3 domain-containing neuronal protein, concentrated in nerve terminals. Here, we demonstrate a specific, SH3 domain-mediated, interaction between amphiphysin and dynamin by gel overlay and affinity chromatography. In addition, we show that the two proteins are colocalized in nerve terminals and are coprecipitated from brain extracts consistent with their interactions in situ.

The function of dynamin in endocytosis.

Temperature-sensitive shibire mutants of Drosophila melanogaster become rapidly paralyzed upon a shift to the restrictive temperature, which is due to a block in synaptic vesicle endocytosis. The shibire gene encodes the GTPase dynamin. Recent studies have shown that dynamin forms rings at the neck of invaginated clathrin-coated pits, and have suggested that a conformational change in the ring, which correlates with GTP hydrolysis, plays an essential role in vesicle fission.

Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells.

There is strong evidence to indicate that proteins of the synaptobrevin family play a key role in exocytosis. Synaptobrevin 1 and 2 are expressed at high concentration in brain where they are localized on synaptic vesicles. Cellubrevin, a very similar protein, has a widespread tissue distribution and in fibroblasts is localized on endosome-derived, transferin receptor-positive vesicles.

rbSec1A and B colocalize with syntaxin 1 and SNAP-25 throughout the axon, but are not in a stable complex with syntaxin.

rbSec1 is a mammalian neuronal protein homologous to the yeast SEC1 gene product which is required for exocytosis. Mutations in Sec1 homologues in the nervous systems of C. elegans and D.

Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals.

The mechanisms through which synaptic vesicle membranes are reinternalized after exocytosis remain a matter of debate. Because several vesicular transport steps require GTP hydrolysis, GTP-gamma S may help identify intermediates in synaptic vesicle recycling. In GTP-gamma S-treated nerve terminals, we observed tubular invaginations of the plasmalemma that were often, but not always, capped by a clathrin-coated bud.

p145, a major Grb2-binding protein in brain, is co-localized with dynamin in nerve terminals where it undergoes activity-dependent dephosphorylation.

Three major rat brain proteins were recently found to bind the SH3 domains of Grb2: synapsin I, dynamin, and a novel 145-kDa protein (p145) (McPherson, P. S., Czernik, A.

Solvent/detergent-treated plasma suppresses shear-induced platelet aggregation and prevents episodes of thrombotic thrombocytopenic purpura.

Two children with congenital chronic relapsing thrombotic thrombocytopenic purpura (TTP) have episodes every 3 weeks. These relapses can be prevented by the infusion of normal fresh-frozen plasma (FFP) without concurrent plasmapheresis. We conducted a study to determine whether the exposure of normal plasma to agents that inactivate human immunodeficiency virus and other viruses destroys the component necessary for the effective treatment of this type of TTP that requires only plasma infusion to prevent or reverse relapses.

Interaction of Grb2 via its Src homology 3 domains with synaptic proteins including synapsin I.

Grb2 is a 25-kDa adaptor protein composed of a Src homology 2 (SH2) domain and two flanking Src homology 3 (SH3) domains. One function of Grb2 is to couple tyrosine-phosphorylated proteins (through its SH2 domain) to downstream effectors (through its SH3 domains). Using an overlay assay, we have identified four major Grb2-binding proteins in synaptic fractions.

Photoaffinity labeling of the ryanodine receptor/Ca2+ release channel with an azido derivative of ryanodine.

Ryanodine receptors/Ca2+ release channels play an important role in regulating the intracellular free calcium concentrations in both muscle and nonmuscle cells. Ryanodine, a neutral plant alkaloid, specifically binds to and modulates these Ca2+ release channels. In the work described here, we characterize the interaction of a tritium-labeled, photoactivable derivative of ryanodine (3H-labeled 10-O-[3-(4-azidobenzamido)propionyl]ryanodine ([3H]ABRy)) with the ryanodine receptor of skeletal, cardiac, and brain membranes.

Radioimmunoassay for the calcium release channel agonist ryanodine.

A novel photo-activatable derivative of ryanodine, 9-hydroxy-21-(4-azidobenzoyloxy)-9-epiryanodine, has been synthesized and conjugated to keyhole limpet hemocyanin for the production of antibodies with high affinity and specificity to ryanodine. The anti-ryanodine antibodies reacted specifically on immunoblots with the azido-ryanodine compound covalently conjugated to bovine serum albumin. A radioimmunoassay specific for ryanodine was developed using the anti-ryanodine antibodies, and a dissociation constant for ryanodine of 1 nM was determined.

Protonation and free energy changes associated with formation of QBH2 in native and Glu-L212-->Gln mutant reaction centers from Rhodobacter sphaeroides.

Formation of the quinol QBH2 in Glu-L212-->Gln mutant [EQ(L212)] reaction centers (RCs) from Rhodobacter sphaeroides was investigated by measuring the proton uptake (using dyes), UV absorption changes, and free energy changes associated with the two-electron reduction of QB. The advantage of using the EQ(L212) RCs for these studies is that the individual protonation steps can be kinetically resolved and analyzed; conclusions reached regarding the mechanism of formation of QBH2 are expected to apply also to native RCs. The proton uptake by EQ(L212) RCs was strongly biphasic: the fast phase was essentially concomitant with the second electron transfer to QB- (approximately 1 ms at pH 7.

Pathway of proton transfer in bacterial reaction centers: role of aspartate-L213 in proton transfers associated with reduction of quinoneto dihydroquinone.

The role of Asp-L213 in proton transfer to reduced quinone QB in the reaction center (RC) from Rhodobacter sphaeroides was studied by site-directed replacement of Asp with residues having different proton donor properties. Reaction centers (RCs) with Asn, Leu, Thr, and Ser at L213 had greatly reduced (approximately 6000-fold) proton-coupled electron transfer [kAB(2)] and proton uptake rates associated with the second electron reduction of QB (QA- QB- + 2H(+)-->QAQBH2) compared to native RCs. RCs containing Glu at L213 showed faster (approximately 90-fold) electron and proton transfer rates than the other mutant RCs but were still reduced (approximately 70-fold) compared with native RCs.

Light-induced proton uptake by photosynthetic reaction centers from Rhodobacter sphaeroides R-26.1. II. Protonation of the state DQAQB2-.

Proton uptake associated with the two-electron reduction of QB was investigated in reaction centers (RCs) from Rhodobacter sphaeroides R-26.1 using pH-sensitive dyes. An uptake of two protons was observed at pH < or = 7.

Characterization of the major brain form of the ryanodine receptor/Ca2+ release channel.

At least three distinct ryanodine receptor genes appear to be expressed in mammalian brain. We have used biochemical and immunological methods to characterize the major form of ryanodine binding protein purified from brain. [3H]Ryanodine binding to the purified brain receptor is stimulated by Ca2+, ATP, KCl, and phosphorylation and is inhibited by calmodulin, Mg2+, and ruthenium red.

Differential immunohistochemical localization of inositol 1,4,5-trisphosphate- and ryanodine-sensitive Ca2+ release channels in rat brain.

Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive and ryanodine-sensitive intracellular Ca2+ stores is mediated by distinct proteins identified as IP3 receptors (IP3R) and ryanodine receptors (RyR), respectively. We have compared the immunohistochemical localizations of IP3R and RyR in the brain at the light and electron microscopic levels and have also evaluated the distribution of the major brain intracellular Ca(2+)-pumping ATPase. IP3R and RyR occur in overlapping populations of neurons in widespread areas of the brain, but labeling is distinct in a number of areas.

The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle.

The subcellular distribution of the Ca(2+)-release channel/ryanodine receptor in adult rat papillary myofibers has been determined by immunofluorescence and immunoelectron microscopical studies using affinity purified antibodies against the ryanodine receptor. The receptor is confined to the sarcoplasmic reticulum (SR) where it is localized to interior and peripheral junctional SR and the corbular SR, but it is absent from the network SR where the SR-Ca(2+)-ATPase and phospholamban are densely distributed. Immunofluorescence labeling of sheep Purkinje fibers show that the ryanodine receptor is confined to discrete foci while the SR-Ca(2+)-ATPase is distributed in a continuous network-like structure present at the periphery as well as throughout interior regions of these myofibers.

Cortical localization of a calcium release channel in sea urchin eggs.

We have used an antibody against the ryanodine receptor/calcium release channel of skeletal muscle sarcoplasmic reticulum to localize a calcium release channel in sea urchin eggs. The calcium release channel is present in less than 20% of immature oocytes, where it does not demonstrate a specific cytoplasmic localization, while it is confined to the cortex of all mature eggs examined. This is in contrast to the cortical and subcortical localization of calsequestrin in mature and immature eggs.

The brain ryanodine receptor: a caffeine-sensitive calcium release channel.

The release of stored Ca2+ from intracellular pools triggers a variety of important neuronal processes. Physiological and pharmacological evidence has indicated the presence of caffeine-sensitive intracellular pools that are distinct from the well-characterized inositol 1,4,5,-trisphosphate (IP3)-gated pools. Here we report that the brain ryanodine receptor functions as a caffeine- and ryanodine-sensitive Ca2+ release channel that is distinct from the brain IP3 receptor.

Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes.

A high affinity [3H]ryanodine receptor has been solubilized from rabbit brain membranes and biochemically characterized. [3H]Ryanodine binding to rabbit brain membranes is specific and saturable, with a Kd of 1.3 nM.

Pathway of proton transfer in bacterial reaction centers: replacement of serine-L223 by alanine inhibits electron and proton transfers associated with reduction of quinone to dihydroquinone.

The pathway of proton transfer in the reaction center (RC) from Rhodobacter sphaeroides was investigated by site-directed mutagenesis. Ser-L223, a putative proton donor that forms a hydrogen bond with the secondary quinone acceptor QB, was replaced with Ala and Thr. RCs with Ala-L223 displayed reduced electron transfer and proton uptake rates in the reaction QA-QB- + 2H+----QAQBH2.

Abnormalities of von Willebrand factor multimers in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome.

Purpose: To analyze and review von Willebrand factor (vWF) multimeric patterns in patients with single-episode thrombotic thrombocytopenic purpura (TTP), intermittent TTP (episodes at infrequent, irregular intervals), chronic relapsing TTP (episodes at frequent, regular intervals), and the hemolytic-uremic syndrome (HUS).

Patients And Methods: Platelet-poor plasma samples were obtained in EDTA, citrate, or citrate-hirudin-aprotinin-leupeptin from 36 patients with single-episode TTP, eight patients with intermittent TTP, four patients with chronic relapsing TTP, and 26 patients with HUS. The samples were separated by sodium dodecyl sulfate-agarose gel electrophoresis, overlaid with rabbit 125I-anti-human vWF IgG, and analyzed by autoradiography.

Relationship between human development and disappearance of unusually large von Willebrand factor multimers from plasma.

von Willebrand factor (vWF) multimers were examined in fetal, umbilical cord, and neonatal platelet-poor plasma (PPP) specimens. Sixty-five of 65 (100%) fetal PPP samples aged less than 35 weeks and seven of ten (70%) fetal samples aged greater than 35 weeks had unusually large vWF (ULvWF) multimers. Thirty of 46 (65%) cord PPP samples from neonates ranging in gestational age from 34 to 41 weeks had ULvWF.