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R B Kelly

Publications and source records attributed to R B Kelly.

At least 145 records · Page 8Linked to original sources
PubMed1981

Identification of minor components of coated vesicles by use of permeation chromatography.

Coated vesicles are thought to be vehicles for the intracellular transport of membranes. Clathrin is the major protein component of coated vesicles. Minor components of these organelles can be identified in highly purified preparations if they can be shown to copurify with clathrin. To show copurification we have made use of the relatively uniform diameter of coated vesicles (50-150 nm) to fractionate conventionally purified coated vesicles according to size in glass bead columns of 200-nm pore size. We have found that bovine brain coated vesicles prepared by the standard procedure of Pearse can be contaminated with large membrane fragments that are removed by permeation chromatography on such glass bead columns. Gel electrophoretic analysis of column fractions shows that only three major polypeptide chains, and a family of polypeptides with molecular weights close to 100,000 are always in constant ratio to clathrin, and are unique to fractions containing coated vesicles. Two other major polypeptides that appear to be components of coated vesicles are also present in other membrane fractions. We have also used permeation chromatography to monitor artifactual membrane trapping during vesicle isolation. Pure radiolabeled synaptic vesicle membranes were added to bovine brain tissue before homogenization. Considerable amounts of the added radioactivity could be recovered in the fractions conventionally pooled in the preparation of coated vesicles. After permeation chromatography, the radioactivity in the coated vesicle peak was reduced essentially to background.

Animals↗
PubMed1980

Antibodies to synaptic vesicles purified from Narcine electric organ bind a subclass of mammalian nerve terminals.

Antibodies were raised in rabbits to synaptic vesicles purified to homogeneity from the electric organ of Narcine brasiliensis, a marine electric ray. These antibodies were shown by indirect immunofluorescence techniques to bind a wide variety of nerve terminals in the mammalian nervous system, both peripheral and central. The shared antigenic determinants are found in cholinergic terminals, including the neuromuscular junction, sympathetic ganglionic and parasympathetic postganglionic terminals, and in those synaptic areas of the hippocampus and cerebellum that stain with acetylcholinesterase. They are also found in some noncholinergic regions, including adrenergic sympathetic postganglionic terminals, the peptidergic terminals in the posterior pituitary, and adrenal chromaffin cells. They are, however, not found in many noncholinergic synapse-rich regions. Such regions include the molecular layer of the cerebellum and those laminae of the dentate gyrus that receive hippocampal associational and commissural input. We conclude that one or more of the relatively small number of antigenic determinants in pure electric fish synaptic vesicles have been conserved during evolution, and are found in some but not all nerve terminals of the mammalian nervous system. The pattern of antibody binding in the central nervous system suggests unexpected biochemical similarities between nerve terminals heretofore regarded as unrelated.

Adrenal Medulla↗
PubMed1980

An antiserum specific for cholinergic synaptic vesicles from electric organ.

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle-specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.

Acetylcholine↗
PubMed1979

Nerve terminal proteins of the rabbit visual relay nuclei identified by axonal transport and two-dimensional gel electrophoresis.

The proteins in nerve terminals can be uniquely identified by two-dimensional gel electrophoresis of proteins labeled during synthesis in the cell body and then transported intra-axonally to the terminals. We have explored the potential of the identification procedure by comparing the proteins which are transported from the retina to the lateral geniculate nucleus (LGN) and the superior colliculus (SC) of the rabbit. We have been able to identify between 150 and 200 proteins which ate common to both LGN and SC nerve terminals, very few of which are present at significantly different concentrations in one nucleus relative to the other. The similarity between proteins sent from the retina along two neural pathways subserving different functions illustrates the subtlety of biochemical changes that must underlie physiological differences. Only a small fraction of the labeled proteins are major proteins of the relay nuclei as judged by Coomassie-staining, and some of these arise from in situ nonspecific labeling with blood-borne radioactivity, rather than by transport to the terminals. We have shown that about 5 times more proteins are transported at fast than at intermediate transport rates. More than 50% of the fast proteins turn over rapidly and are gone in 24 h. Few intermediate proteins turn over rapidly. Since only 6% of the proteins in the relay nuclei (at 36 h) could not be detected in the optic tract at that time, transsynaptic labeling by breakdown and resynthesis must be small, if it occurs at all.

Animals↗
PubMed1979

Topological organization of proteins in an intracellular secretory organelle: the synaptic vesicle.

Intact synaptic vesicles prepared from the electric organ of the marine elasmobranch Narcine brasiliensis have eight major polypeptides demonstrable on sodium dodecyl sulfate gels. Six of these copurify with the synaptic vesicles during isolation of vesicles by chromatography on CPG-3000 and, by this criterion, are specific to vesicles. The other two are either shared by many membrane or are contaminants. One of these proteins comigrates with actin. Three different approaches were used to determine which proteins were exposed on the external, cytoplasmic surface of the vesicle and which were internal. The first was susceptibility to the proteases trypsin, Streptomyces griseus protease, and Pronase; the second was labeling by the membrane-impermeable reagent diazotized [125I]iodosulfanilic acid; and the third was iodination catalyzed by lactoperoxidase. In general, the three approaches give the same result: six of the eight proteins are on the external, cytoplasmic surface and two are accessible only after the vesicles are lysed by freezing and thawing or by detergents. Five of the vesicle-specific proteins are external and one is internal. The actin-like protein is internal. Proteins involved in the interaction of vesicles with the presynaptic membrane during exocytosis might be expected to be vesicle specific and external.

Acetylcholine↗
PubMed1978

The effects of vinblastine on acinar cells of the exorbital lacrimal gland of the rat.

The effects of vinblastine treatment on acinar cells of the rat exorbital lacrimal gland were studied by electron microscopy. Experimental animals of both sexes were given single intraperitoneal injections of (1) vinblastine (4 mg/kg body weight) at 1 to 24 h before sacrifice; (2) pilocarpine (20 mg/kg b.w.) for 1 h; or (3) vinblastine for 1 h followed by pilocarpine for 1 h. Vinblastine treatment caused a number of changes including autophagocytosis, formation of intracisternal granules, and alteration of secretory granules. These changes varied in extent and onset between male and female rats. In addition, the Golgi apparatus was reduced in size and dispersed throughout the cytoplasm. Mitotic figures were commonly observed. Moreover, vinblastine inhibited the pilocarpine-stimulated degranulation of the acinar cells. In view of the known anti-microtubular action of vinblastine, these results suggest that microtubules are involved in various aspects of the transport, packaging, and secretion of exportable proteins in the lacrimal gland. Additionally, autophagocytosis and alteration of secretory granules may partially result from the interaction of vinblastine with membranes.

Animals↗
PubMed1978

Purification of synaptic vesicles from elasmobranch electric organ and the use of biophysical criteria to demonstrate purity.

We have purified cholinergic synaptic vesicles from the electric organs of two related marine elasmobranchs, Torpedo californica and Narcine brasiliensis, to a specific activity higher than had previously been obtained. We have demonstrated the homogeneity of the vesicles by biophysical criteria. The purification scheme consisted of differential centrifugation, flotation equilibrium in sucrose density gradients, and permeation chromatography on glass bead columns of average pore size 3000 A. Our criteria for purity were that bound acetylcholine, bound nucleotide triphosphate, protein, and lipid--phosphorus behave identically when vesicles were analyzed by procedures which depend on vesicle size, density, and charge. Contaminants were not detected when vesicles were fractionated by preparative and analytical sedimentation, by preparative equilibrium sedimentation using glycerol density gradients, or by electrophoresis in Ficoll density gradients. Pure synaptic vesicles, which have been purified 290-fold from the initial homogenate, contain per mg of protein: 8 mumol of acetylcholine, 3 mumol of ATP, and 7 mumol of lipid phosphorus. These procedures may be of general value in the purification of membrane vesicles.

Animals↗
PubMed1977

Membranes undergoing phase transitions are preferentially hydrolyzed by beta-bungarotoxin.

beta-Bungarotoxin preferentially hydrolyzes choline phospholipids (dilauroyl, dimyristoyl, dipalmitoyl) at their respective gel to liquid crystalline phase transition temperatures. Cholesterol markedly reduces the rate of phospholipid hydrolysis; at 0.33 mol percent cholesterol:phospholipid, the toxin's phospholipase activity is completely inhibited.

Bungarotoxins↗
PubMed1977

Selective enzymatic hydrolysis of nerve terminal phospholipids by beta-bungarotoxin: biochemical and morphological studies.

beta-Bungarotoxin, a presynaptic protein neurotoxin isolated from the venom of the snake Bungarus multicinctus, modifies release of neurotransmitter at the neuromuscular junction. The toxin has a potent phospholipase activity toward both natural membranes and phospholipid liposomes. Studies of ionic requirements and selective chemical modification demonstrate that beta-bungarotoxin can only modify synaptic transmission under conditions in which the phospholipase is active. Morphological studies on frog cutaneous pectoris neuromuscular junctions incubated with beta-toxin and horseradish peroxidase-conjugated beta-toxin support the hypothesis that beta-toxin specifically interacts with presynaptic plasma membranes and selectively hydrolyzes nerve terminal phospholipids.

Animals↗
PubMed1976

Saturable binding to cell membranes of the presynaptic neurotoxin, beta-bungarotoxin.

Brief exposure to the protein neurotoxin, beta-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using 125I-labelled toxin. At low membrane protein concentration 125I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration 125I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes. In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of 125I-labeled toxin binding sites on brain mitochondrial preparations (3.3 +/- 0.3 pmol/mg) exceeded by a factor of ten the density on liver mitochondrial preparations (0.3 +/- 0.05 pmol/mg). It is also shown that labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude that beta-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.

Animals↗
PubMed1976

The mechanism of beta-bungarotoxin action. I. Modification of transmitter release at the neuromuscular junction.

The protein, beta-bungarotoxin, a presynaptic neurotoxin isolated from the venom of the snake Bungarus multicinctus, is known to inhibit mitochondrial function. Within 30 min after adding the toxin to a rat diaphragmphrenic nerve preparation, the quantal content increased tenfold and the frequency of miniature endplate potentials increased fourfold. No increase in miniature endplate potential frequency was seen in the absence of extracellular calcium. Since mitochondria may be involved in regulating intracellular calcium levels, the rate at which the transmitter release is turned off was studied by measuring delayed release in the presence and absence of toxin. Delayed release is elevated about eightfold by the toxin. If delayed release is due to residual calcium, as has been hypothesized, these data may be explained if the toxin does not alter the amount of calcium which enters the terminal, but rather the rate at which that calcium is removed. Alternatively, a calcium-dependent modification of the release process itself might be produced. The eventual reduction in transmitter output did not appear to result from depletion of the terminal of releaseable packets of transmitter, but does require extracellular calcium.

Animals↗
PubMed1976

beta-Bungarotoxin, a pre-synaptic toxin with enzymatic activity.

beta-Bungarotoxin, a pre-synaptic neurotoxin isolated from the venom of the snake Bungarus multicinctus, has been shown to modify release of neurotransmitter at the neuromuscular junction. In this communication, we demonstrate that beta-bungarotoxin is a potent phospholipase A2 (phosphatide 2-acyl hydrolase, EC 3.1.1.4), comparable in activity with purified phospholipase enzymes from Naja naja and Vipera russellii. The phospholipase activity of beta-bungarotoxin requires calcium and is stimulated by deoxycholate. When strontium replaces calcium, no phospholipase activity is detected. Since neuromuscular transmission is not blocked when calcium is replaced by strontium, it was possible to examine the effects of the toxin on neuromuscular transmission in the presence of strontium. Under these conditions, when the phospholipase activity should be inhibited, the toxin has little or no effect on neuromuscular transmission. If beta-bungarotoxin owes its toxicity in part to its enzymatic activity, then it must be placed in a different class from those toxins which produce their effect by binding passively to an appropriate receptor.

Animals↗