PubMed Health⌕ Search

Biomedical subjects

B I Kanner

Publications and source records attributed to B I Kanner.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and expression of a rat brain L-glutamate transporter.

Synaptic transmission of most vertebrate synapses is thought to be terminated by rapid transport of the neurotransmitter into presynaptic nerve terminals or neuroglia. L-Glutamate is the major excitatory transmitter in brain and its transport represents the mechanism by which it is removed from the synaptic cleft and kept below toxic levels. Here we use an antibody against a glial L-glutamate transporter from rat brain to isolate a complementary DNA clone encoding this transporter. Expression of this cDNA in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and transport shows the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8-9 putative transmembrane alpha-helices. Database searches indicate that this protein is not homologous to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters. This protein therefore seems to be a member of a new family of transport molecules.

Amino Acid Sequence↗
PubMed1992

A monoclonal antibody against a Na(+)-L-glutamate cotransporter from rat brain.

A monoclonal mouse IgM antibody (Z8E9) was raised against the Na(+)-L-glutamate cotransporter from rat brain. In a preparation of brain plasma membrane vesicles, Z8E9 binds specifically to a polypeptide with an apparent molecular weight of 70,000 and inhibits Na+ gradient-dependent L-glutamate cotransport (up to 50%) in brain membrane vesicles. In the membrane vesicles, the antibody does not alter the membrane permeability for Na+ and K+ nor the Na+ gradient-dependent uptake of gamma-aminobutyric acid. Kinetic experiments showed that Z8E9 does not alter the K0.5 values for L-glutamate and Na+ activation of L-glutamate transport. However, an apparent cooperativity observed for L-glutamate activation was increased, and the Vmax of L-glutamate transport was decreased. Immunostaining of rat cerebellum identified antigenic sites of Z8E9 in Golgi epithelial cells and astrocytes (by light and electron microscopy), whereas no labeling at nerve terminals was detected. The data suggest that a component of a Na(+)-L-glutamate cotransporter subtype has been identified that is specific for glia cells in brain.

Amino Acid Transport System X-AG↗
PubMed1992

Comparative analysis of sodium-dependent L-glutamate transport of synaptosomal and astroglial membrane vesicles from mouse cortex.

Uptake of [3H]L-glutamate into membrane vesicles prepared from either mouse cortical astrocyte cultures or synaptosomes was found to be an electrogenic sodium- and potassium-dependent transport process with saturable uptake kinetics. Pharmacological differences were revealed by using a variety of substrate analogues. L-trans-PDC inhibited the synaptosomal glutamate transport 2-4-fold stronger than the astroglial uptake. The substrate analogues DL-threo-beta-hydroxy-aspartate, DL-aspartate-beta-hydroxamate, L-aspartate and D-aspartate inhibited glutamate transport of astroglial and neuronal membrane vesicles in a distinctive manner, whereas D-glutamate, quisqualate and dihydrokainate had no effect in either case. Immunoblotting and immunocytochemical labeling with antibodies against the rat brain glutamate transporter revealed the selective reaction of a band at about 75 kDa mol. wt. and a specific pattern of astrocyte immunostaining.

Amino Acid Transport System X-AG↗
PubMed1992

An [Na+ + K+]coupled L-glutamate transporter purified from rat brain is located in glial cell processes.

Polyclonal antibodies were generated against the major polypeptide (73,000 mol. wt) present in a highly purified preparation of the [Na+ + K+]coupled L-glutamate transporter from rat brain. These antibodies were able to selectively immunoprecipitate the 73,000 mol. wt polypeptide as well as most of the L-glutamate transport activity--as assayed upon reconstitution--from crude detergent extracts of rat brain membranes. The immunoreactivity in the various fractions obtained during the purification procedure [Danbolt et al. (1990) Biochemistry 29, 6734-6740] closely correlated with the L-glutamate transport activity. Immunoblotting of a crude sodium dodecyl sulphate brain extract, separated by two-dimensional isoelectric focusing-sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the antibodies recognized one 73,000 mol. wt protein species only. Deglycosylation of the protein gave a 10,000 reduction in molecular mass, but no reduction in immunoreactivity. These findings establish that the 73,000 mol. wt polypeptide represents the L-glutamate transporter or a subunit thereof. The antibodies also recognize a 73,000 mol. wt polypeptide and immunoprecipitate L-glutamate transport activity in extracts of brain plasma membranes from rabbit, pig, cow, cat and man. Using the antibodies, the immunocytochemical localization of the transporter was studied at the light and electron microscopic levels in rat central nervous system. In all regions examined (including cerebral cortex, caudatoputamen, corpus callosum, hippocampus, cerebellum, spinal cord) it was found to be located in glial cells rather than in neurons. In particular, fine astrocytic processes were strongly stained. Putative glutamatergic axon terminals appeared non-immunoreactive. The uptake of glutamate by such terminals (for which there is strong previous evidence) therefore may be due to a subtype of glutamate transporter different from the glial transporter demonstrated by us.

Amino Acid Transport System X-AG↗
PubMed1979

Solubilization and reconstitution of the catecholamine transporter from bovine chromaffin granules.

The catecholamine transporter from bovine chromaffin granules has been solubilized by using low concentrations of sodium cholate in the presence of phospholipids. The functional solubilized protein has been incorporated into liposomes after removal of the detergent either by gel filtration or by dialysis. Reserpine-sensitive accumulation against a concentration gradient is achieved by artifically imposing a pH gradient across the membrane. In the reconstituted system adenosine 5'-triphosphate (ATP) serves as an energy source only at higher detergent concentrations. The proton-translocating adenosine triphosphatase (ATPase) is solubilized in parallel with the increasing efficiency of ATP as an energy source. Several criteria are proposed to distinguish between carrier-mediated (reserpine sensitive) and unmediated transport in the reconstituted system. The reserpine-sensitive process shows affinity and ss presented in this communication provide further support for the contention that concentrative uptake in biogenic amine storage vesicles is driven by a transmembrane pH gradient, which, in the native system, is generated by a proton-translocating ATPase. Moreover, the assays described provide a tool for the isolation and purification of the transport protein.

Adenosine Triphosphatases↗
PubMed1978

Active transport of L-glutamate by membrane vesicles isolated from rat brain.

Membrane vesicles, isolated after osmotic shock of synaptosomal rat brain fractions, actively accumulate L-glutamate. This process requires the presence of external sodium ions and internal potassium ions and is driven by artifically imposed ion gradients as the sole energy source. Either an Na+ gradient (out is greater than in) or a K+ gradient (in is greater than out) or both can be utilized to concentrate L-glutamate inside the vesicles. Transport is enhanced by valinomycin or by external thiocyanate ions and is about 50% inhibited by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone. This transport thus appears to be stimulated by a membrane potential (interior negative). The glutamate transporter, the Km of which has been determined to be 3 micrometer, is specific for L-glutamate. The transport process is unaffected by ouabain but is strongly inhibited by p-hydroxymercuribenzoate as well as by nigericin, which collapses the energizing ion gradients across this membrane. Unlike the sodium dependent, but potassium independent active accumulation of gamma-aminobutyric acid in these vesicles (Kanner, B.I. (1978) Biochemistry 17, 1207) active L-glutamate uptake is not dependent on the presence of small monovalent anions in the external medium. The results provide direct evidence for Na+-coupled electrogenic active L-glutamate transport by rat brain membrane vesicles. The dependence on internal potassium ions is discussed.

Animals↗
PubMed1978

Role of a transmembrane pH gradient in epinephrine transport by chromaffin granule membrane vesicles.

ATP-driven transport and accumulation of epinephrine in chromaffin granule membrane vesicles isolated from bovine adrenal medulla is inhibited by the proton ionophores carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin, but not by valinomycin. Moreover, an artificially imposed pH gradient (interior acid) is able to drive this reserpine-sensitive transport system in the absence of ATP. Dicyclohexylcarbodiimide, an inactivator of the chromaffin granule membrane-bound ATPase, completely inhibits ATP-dependent epinephrine accumulation, but has much less effect when an imposed pH gradient is the driving force for epinephrine transport. The findings provide a strong indication that a pH gradient (interior acid) is the immediate driving force for epinephrine uptake in these storage granules and suggest that ATP-driven epinephrine transport is the result of two processes: (i) generation of a proton electrochemical gradient (interior acid and positive) by the membrane-bound, proton-translocating ATPase; and (ii) pH gradient-driven accumulation of the catecholamine.

Animals↗
PubMed1976

Purification and properties of the proton-translocating adenosine triphosphatase complex of bovine heart mitochondria.

1. The proton-translocating adenosine triphosphatase (ATPase) of bovine heart mitochondria was highly purified by extraction of submitochondrial particles with cholate, fractionation with ammonium sulfate, and sucrose gradient centrifugation in the presence of methanol, deoxycholate, and lysolecithin. 2. The preparation had a very low content of phospholipids, respiratory components, and adenine nucleotide transporter. The ATPase activity (14 o 16 micromoles/min/mg at 30 degrees) was dependent on addition of phospholipids. The purified enzyme was reconstituted with phospholipids, coupling factor 1 (F1), and the oligomycin sensitivity-conferring protein (OSCP) yielding vesicles with highly active 32Pi-ATP exchange (up to 260 nanomoles/min/mg at 30 degrees), and a proton pump driven by ATP. Site III oxidative phosphorylation was reconstituted when purified cytochrome oxidase was included. 3. The 32Pi-ATP exchange of the reconstituted vesicles was sensitive to both rutamycin and dichylohexylcarbodiimide but the ATPase activity was sensitive to rutamycin and not to dicyclohexylcarbodiimide. 4. In sodium dodecyl sulfate-acrylamide gel scans of the complex, the subunits of F1, OSCP, and three other major bands with apparent molecular weights of 32,000, 23,000, and about 11,000 were noted. Three other minor bands with estimated molecular weights of 80,000, 70,000, and 52,000 were also detected. These bands apparently represent residual trace amounts of respiratory components. Quantitative assays of individual respiratory components revealed between 0 and 3% contamination. 5. We conclude that the rutamycin-sensitive ATPase complex functions as a reversible ATP-driven proton pump.

Adenosine Triphosphatases↗
PubMed1975

Differentiation between mutants of Escherichia coli K defective in oxidative phosphorylation.

Hybrid membrane particles from two mutants of Escherichia coli K12, Bv4 and K11, defective in oxidative phosphorylation, have been prepared, in which ATP-driven membrane energization is restored. A soluble factor of mutant K11 was found to have properties similar to parental crude coupling factor, ATPase (EC 3.6.1.3). Membrane particles of this mutant could not be reconstituted by parental coupling factor. Either parental coupling factor, or the soluble factor of mutant K11 could reconstitute both respiration-driven and ATP-driven energization to membrane particles of mutant Bv14 or to parental particles depleted of ATPase. Mutant Bv4 was found to be devoid of coupoing factor activity, while retaining the ability to hydrolyze ATP. Both mutants possess an ATPase with an altered binding to the membrane. Mutant K11 is impaired in respiration-driven amino acid transport, in contrast to mutant Bv4. The three major subunits of parental Escherichia coli ATPase have been isolated and antibodies have been prepared against these subunits. Antibodies against the largest subunit (alpha component) or against the intact catalytic subunits (alpha + beta components) inhibit both ATP-Pi exchange in the parent organism as well as ATP hydrolytic activity in parent and mutants. Antibodies against the two other subunits (beta or gamma components) also inhibit these two reactions, but were found to be less effective. Mutant N144, which lacks ATPase activity, shows no precipitin lines with anti-alpha, anti-beta, anti-gamma, or anti (alpha + beta) preparations. In contrast, mutants Bv4 and K11, exhibit cross-reactivity with all of the antisera.

Acridines↗