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B I Kanner

Publications and source records attributed to B I Kanner.

At least 73 records · Page 4Linked to original sources
PubMed1986

Purification and identification of the functional sodium- and chloride-coupled gamma-aminobutyric acid transport glycoprotein from rat brain.

Using the reconstitution conditions developed recently (Radian, R., and Kanner, B. I. (1985) J. Biol. Chem. 260, 11859-11865) we have now purified the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain to apparent homogeneity. A partially purified transporter preparation was passed over wheat germ agglutinin-Sepharose 6MB and non-bound proteins were washed away. The transport activity, as expressed upon reconstitution of the protein into liposomes, was eluted by a solution containing Triton X-100 and N-acetylglucosamine. The specific transport activity was increased almost 400-fold over that of the crude extract. Taking into account an approximately 2.5-fold inactivation during the lectin column chromatography, the actual purification is about 1000-fold. Sodium dodecyl sulfate-polyacrylamide electrophoresis of the active fractions revealed one band of 80 kDa and small amounts of a band which ran at an apparent molecular mass of 160 kDa. The ratio between the two could be experimentally changed such as, for instance, by lyophilization. Polyclonal antibodies were prepared against the 80-kDa band which also cross-reacted with the 160-kDa band, indicating that the latter apparently represents a dimer form of the first. Using Protein A-Sepharose Cl-4B and the antibody against the 80-kDa band, we were able to quantitatively immunoprecipitate the potential gamma-aminobutyric acid transport activity from a crude transporter preparation. The pure transporter preparation exhibited the same features of the transporter in synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogeneity, affinity, and efflux and exchange properties. We conclude that the 80-kDa band represents the gamma-aminobutyric acid transporter.

Animals↗
PubMed1985

Reconstitution and purification of the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain.

The sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain has been highly purified. Synaptic plasma membranes from rat brain were extracted with cholate in the presence of 10% ammonium sulfate. The soluble extract was incorporated into liposomes consisting of asolectin and crude brain lipids. Brain lipids markedly enhanced the transport activity. The resulting proteoliposomes catalyzed sodium- and chloride-coupled gamma-aminobutyric acid transport which, in the presence of internal potassium, was greatly (up to 20-fold) stimulated by valinomycin. Using this transport of the reconstituted system as an assay, the transporter was purified by the following steps. The cholate extract was fractionated by ammonium sulfate. The activity was not precipitated by 50% but could be precipitated by 70% ammonium sulfate. The cholate and ammonium sulfate were removed on a Sephadex G-50 column. Subsequently, the transporter was partially purified on DEAE-cellulose in a mixture of Triton X-100 and octyl glucoside. The active fractions were chromatographed on a hydroxylapatite column in the presence of Triton X-100. Although the increase in specific activity was only up to 100-fold, this was due to partial inactivation. The actual purification was at least 1000-fold. The purified transporter exhibited the same features of the synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogenicity, and a similar affinity. The sodium dodecyl sulfate gel pattern indicated that a major protein ran as a 24-kDa band. This band may represent the gamma-aminobutyric acid transporter.

Animals↗
PubMed1985

Transport of 5-hydroxytryptamine in membrane vesicles from rat basophilic leukemia cells.

Rat basophilic leukemia (RBL) cells were grown as tumors. Membrane vesicles were isolated from them and serotonin transport was measured. Two types of transport were identified. One was sensitive to imipramine and sodium-dependent, while the other was sensitive to reserpine and ATP-dependent. The transport systems exhibit different affinities for serotonin (sodium-dependent, Km 0.22 microM; ATP-dependent, Km 2.6 microM) and are different in their substrate specificity, the former being much more specific. The 5-hydroxytryptamine transport by the reserpine-sensitive system was strongly inhibited by other biogenic amines, like norepinephrine, epinephrine and dopamine, whereas that by the imipramine-sensitive system was not. Upon Ficoll gradient centrifugation the two transport systems were separated. The reserpine-sensitive activity was found much further into the gradient than the imipramine-sensitive one. The latter co-migrated with the receptor of IgE, which is located in the plasma membrane. Characterization of latter showed that in addition to the dependence of 5-hydroxytryptamine influx on external sodium it was also absolutely dependent on external chloride and was strongly stimulated by internal potassium. On the other hand, efflux required external potassium. An alternative potassium independent way of loss of labelled 5-hydroxytryptamine was by exchange. A small but consistent stimulation of influx was observed in the presence of valinomycin, indicating that the process is electrogenic. The reserpine-sensitive system could also be driven in the absence of ATP. This required the imposition of pH gradient (acid inside) and was stimulated by an artificially imposed membrane potential (positive inside).

Adenosine Triphosphate↗
PubMed1985

Thrombin-induced degranulation of cultured bone marrow-derived mast cells: effect on calcium uptake.

The role of calcium in the mechanism of thrombin activation of bone marrow-derived mast cells (BMMC) was explored by measuring the changes in the uptake of 45Ca2+ into quiescent BMMC and into cells stimulated by thrombin or by IgE-antigen. The results indicate that activation of BMMC by either thrombin or IgE-antigen is Ca2+-dependent. One million BMMC, activated by 0.05-5 U thrombin, accumulated 45Ca2+ in a concentration-dependent manner, which levelled off at around 1 U thrombin. Extracellular 45Ca2+ uptake of thrombin-stimulated cells is saturable within 90 seconds and corresponds to the kinetics of histamine release, whereas that of IgE-antigen exposed cells continues unabated for over 5 min. The pattern of 45Ca2+ uptake of IgE-sensitized BMMC exposed to thrombin suggests that the pro-stimulatory locus of thrombin action on the surface membrane is distinct from that of IgE.

Animals↗
PubMed1984

Initial characterization of the calcium channel activated by the cross-linking of the receptors for immunoglobulin E.

It is currently thought that aggregation of the receptor for immunoglobulin E on mast cells, basophils, and a tumor analog, rat basophilic leukemia (RBL) cells, induces an enhanced permeability of the plasma membrane to calcium, thereby initiating degranulation of the cells. Even in the absence of calcium ions, aggregation of the receptor causes depolarization of the plasma membrane (Kanner, B. I., and Metzger, H. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 5744-5748), suggesting that other ions can traverse the putative channel. Direct evidence for this now has been obtained with measurements of increased 22Na+ fluxes in the absence of calcium ions, induced by aggregation of receptors. This reaction was optimally studied in the presence of ouabain. When aggregation of the receptor was induced by reacting the cell-bound IgE with a multivalent antigen, the sodium flux was completely inhibited by univalent hapten. The sodium flux was also completely inhibited by 2 mM calcium. Aggregation-induced 45Ca2+ fluxes were observed in the presence of millimolar concentrations of external Ca2+, but not in its absence. Depolarization of the plasma membrane potential by the addition of potassium to the medium in the presence of calcium did not itself induce degranulation. In fact, aggregation of the receptors for IgE in the presence of high external potassium resulted in a greatly diminished degranulation. These data indicate that the ion channel modulated by aggregation of receptor differs from the voltage-dependent type of calcium channels. We suggest that in the absence of calcium this channel is rather unspecific, but that calcium can modify it to become calcium selective.

Animals↗
PubMed1984

Amino acid sequence of bovine heart coupling factor 6.

The amino acid sequence of bovine heart mitochondrial coupling factor 6 (F6) has been determined by automated Edman degradation of the whole protein and derived peptides. Preparations based on heat precipitation and ethanol extraction showed allotypic variation at three positions while material further purified by HPLC yielded only one sequence that also differed by a Phe-Thr replacement at residue 62. The mature protein contains 76 amino acids with a calculated molecular weight of 9006 and a pI of approximately equal to 5, in good agreement with experimentally measured values. The charged amino acids are mainly clustered at the termini and in one section in the middle; these three polar segments are separated by two segments relatively rich in nonpolar residues. Chou-Fasman analysis suggests three stretches of alpha-helix coinciding (or within) the high-charge-density sequences with a single beta-turn at the first polar-nonpolar junction. Comparison of the F6 sequence with those of other proteins did not reveal any homologous structures.

Adenosine Triphosphatases↗
PubMed1983

Functional asymmetry of the amine transporter from chromaffin granules.

The studies presented in this communication describe the existence of a pH-dependent kinetic barrier in amine translocation in membrane vesicles isolated from chromaffin granules from bovine adrenal medulla. This barrier prevents efflux of amines previously accumulated in the membrane vesicles. In these preparations, once the amine is accumulated, there is no further need for ATP. This would suggest a low permeability of the membrane, both to ions and to the amine. In addition, we show that under conditions which thermodynamically favour efflux this is kinetically blocked. This block is due to a rapid protonation of the transporter in the interior of the vesicle. Net efflux can be induced by agents that bring upon an alkalinization of the internal pH such as ammonium salts or nigericin. On the other hand, the rate of exchange with extravesicular substrate is almost identical at the various pH values tested. The physiological implications of this mechanism are discussed.

Adenosine Triphosphate↗
PubMed1983

Efflux and exchange of gamma-aminobutyric acid and nipecotic acid catalysed by synaptic plasma membrane vesicles isolated from immature rat brain.

The mechanism of gamma-aminobutyric acid translocation in synaptic plasma membrane vesicles from rat brain has been probed by comparing the ion dependency of net efflux with that of exchange. Furthermore the question has been asked if the same mechanism operates for other solutes translocated by this transporter. Dilution-induced efflux of gamma-aminobutyrate from the membrane vesicles is about 3-fold stimulated by externally added gamma-aminobutyrate. Half maximal stimulation is obtained at a gamma-aminobutyrate concentration similar to the Km for gamma-aminobutyrate influx. This stimulation (exchange) is dependent on external sodium but not on external chloride. In contrast to this gamma-aminobutyrate influx is absolutely dependent on the simultaneous presence of sodium and chloride ions (Kanner, B.I. (1978) Biochemistry 17, 1207-1211), while efflux is dependent on the presence of these two ions on the inside (Kanner, B.I. and Kifer, L. (1981) Biochemistry 20, 3354-3358). Nigericin stimulates dilution-induced efflux of gamma-aminobutyrate from potassium loaded vesicles to a larger extent than external gamma-aminobutyrate. gamma-Aminobutyrate further enhances the nigericin-induced stimulation, provided that the vesicles are not preloaded with chloride. Nipecotic acid is transported with the same features as gamma-aminobutyrate and the two solutes behave similar with respect to the ion dependence of net flux and exchange. A model for the translocation cycle is proposed in which at least one of the translocated sodium ions binds to the transporter in its 'outside' conformation after chloride and the solute have bound previously. Conversely, the solute is released from its 'inside' conformation prior to chloride and at least one of the sodium ions.

Animals↗
PubMed1983

Crosslinking of the receptors for immunoglobulin E depolarizes the plasma membrane of rat basophilic leukemia cells.

Aggregation of the receptor for IgE on mast cells, basophils, and a tumor analog, rat basophilic leukemia (RBL) cells, induces a calcium-dependent degranulation of the cells. We have measured the membrane potential (delta psi) of RBL cells during this reaction by using the tetraphenylphosphonium ion (Ph4P+) equilibration technique. We observed a 20-45% reduction in ionophore-sensitive Ph4P+ accumulation. The phenomenon persisted under conditions expected to collapse the mitochondrial membrane potential, consistent with the effect being due to a change in delta psi of the plasma membrane. We estimated that the change reflects a depolarization of 20 mV (from -90 to -70 mV, interior negative). Whereas degranulation fails to occur in the absence of external Ca2+, this was not true of the depolarization, indicating that the latter was not a consequence of secretion. When aggregation of the receptor is induced by reaction of the cell-bound IgE with a multivalent antigen, the secretory reaction can be halted by adding a univalent hapten. In this case, complete repolarization occurs. Equivalent depolarization was observed in the absence of Na+ but was diminished when both Ca2+ and Na+ were absent. Together, the data suggest that aggregation of the receptor opens ion channels and that the latter disappear promptly when the receptors are disaggregated. It is plausible that formation of these channels leads to the entry of Ca2+ and is an early and critical consequence of the aggregation of the receptors, thereby leading to degranulation.

Animals↗
PubMed1982

Binding order of substrates to the sodium and potassium ion coupled L-glutamic acid transporter from rat brain.

Efflux of L-glutamic acid from synaptic plasma membrane vesicles requires external potassium. This requirement is saturated by concentrations of about 15 mequiv/L potassium. In the absence of potassium, L-glutamic acid can be released from the vesicles in the presence of external L-glutamic acid. This stimulation does not require external sodium but is dependent on the external concentration of L-glutamic acid. Half-maximal effects are obtained by concentrations of about 1 microM which are very similar to the apparent Km for L-glutamic acid influx. Efflux of labeled glutamate driven by external sodium plus glutamate requires internal sodium. These findings suggest that the transporter displays an asymmetric behavior toward sodium. This ion dissociates much more slowly than L-glutamic acid on the external surface of the membrane but not on the internal surface. Furthermore, it appears that the transporter translocates potassium in a step distinct from the L-glutamic acid translocation step. The simplest explanation is that upon translocation of sodium and L-glutamic acid and their release to the inside, potassium binds to the transporter, enabling it to return to the outside to allow initiation of a new transport cycle.

Animals↗
PubMed1982

Efflux of L-glutamate by synaptic plasma membrane vesicles isolated from rat brain.

Synaptic plasma membrane vesicles isolated from rat brain were loaded with L-glutamate either passively, by using a freeze-thaw technique, or by active transport. Subsequently the ion dependency of glutamate efflux from these vesicles was studied. With each of the types of loading similar results were obtained. Efflux requires the simultaneous presence of internal sodium ions and external potassium ions. The process is also stimulated by chloride ions, but either internal or external chloride ions cause stimulation. Addition of unlabeled L-glutamate stimulates efflux about 2-fold. It is concluded that efflux of L-glutamate is in many aspects symmetrical with its influx [Kanner, B. I., & Sharon, I. (1978) Biochemistry 17, 3949--3954]. It appears that in order for L-glutamate to interact with the transporter, sodium has to be present on the same side as L-glutamate whereas potassium has to be simultaneously present on the opposite site. The simplest way to account for these and the previous data is to postulate that the L-glutamate transporter catalyzes sodium and L-glutamate cotransport, while it simultaneously catalyzes antiport of potassium.

Animals↗