Effect of different phospholipids on the reconstitution of two functions of the lactose carrier ofEscherichia coli

Summary The lactose carrier was extracted from membranes ofEscherichia coli and transport activity reconstituted in proteoliposomes containing different phospholipids. Two different assays f for carrier activity were utilized: counterflow and membrane potential-driven uptake. Proteoliposomes composed ofE. coli lipid or of 50% phosphatidylethanolamine–50% phosphatidylcholine showed very high transport activity with both assays. On the other hand, proteoliposomes containing asolectin, phosphatilcholine or 25% cholesterol/75% phosphatidylcholine showed good counterflow activity but poor membrane potentialdriven uptake. The discrepancy between the two types of transport activity in the latter group of three lipids is not due to leakiness to protons, size of proteoliposomes, or carrier protein content per proteoliposome. Apparently one function of the carrier molecule shows a broad tolerance for various phospholipids, while a second facet of the membrane protein activity requires very restricted lipid enviroment.     

Reaction of the glucose carrier of erythrocytes with sodium tetrathionate: Evidence for inward-facing and outward-facing carrier conformations

Summary Sodium tetrathionate reacts with the glucose carrier of human erythrocytes at a rate which is greatly altered in the presence of competitive inhibitors of glucose transport. Inhibitors bound to the carrier on the outer surface of the membrane, either at the substrate site (maltose) or at the external inhibition site (phloretin and phlorizin), more than double the reaction rate. Inhibitors bound at the internal inhibition site (cytochalasin B and androstenedione), protect the system against tetrathionate. After treatment with tetrathionate, the maximum transport rate falls to less than one-third, and the properties of the binding sites are modified in unexpected ways. The affinity of externally bound inhibitors rises: phloretin is bound up to seven times more strongly and phlorizin and maltose twice as strongly. The affinity of cytochalasin B, bound at the internal inhibition site, falls to half while that of androstenedione is little changed. The affinity of external glucose falls slightly. Androstenedione prevents both the fall in transport activity and the increase in phloretin affinity produced by tetrathionate. An inhibitor of anion transport has no effect on the reaction. The observations support the following conclusions: (1) Tetrathionate produces its effects on the glucose transport system by reacting with the carrier on the outer surface of the membrane. (2) The carrier assumes distinct inward-facing and outward-facing conformations, and tetrathionate reacts with only the outward-facing form. (3) The thiol group with which tetrathionate is presumed to react is not present in either the substrate site or the internal or external inhibitor site. (4) In binding asymmetrically to the carrier, a reversible inhibitor shifts the carrier partition between inner and outer forms and thereby raises or lowers the rate of tetrathionate reaction with the system. (5) Reaction with tetrathionate converts the carrier to an altered state in which the conformation at all three binding sites is changed and the rate of carrier reorientation is reduced.     

Isolation and cryopreservation of O-methylthreonine-resistant Rosa cell lines altered in the feedback sensitivity of l-threonine deaminase

O-Methylthreonine (OMT) inhibits the growth of plated Rosa cells (ID₅₀6·10^-6M). Isoleucine is able to reverse efficiently and specifically this OMT toxicity. From OMT-resistant colonies occurring at a frequency of 1.58·10^-7 variants per cell plated at 10^-4M OMT, the variant strains OMT^R-1 and OMT^R-2 were isolated, cloned via protoplasts and characterized. Both variants were ten times more resistant to OMT than the wildtype and were cross-resistant to another isoleucine analog, dl-4-thiaisoleucine. The resistant variants retained their resistance after storage for three years in liquid nitrogen. Both resistant strains were stable for several months when subcultured in the absence of OMT although it was shown in a reconstitution experiment that wildtype cells overgrow OMT^R-2 variant cells if co-cultivated for many passages in drug-free medium. One case of instability was observed upon long-term subculturing in drug-free medium: the strain OMT^R-1D^* partially lost phenotypic properties. Resistance to OMT was followed qualitatively by a new method based on inhibition-zone formation in cell suspensions plated in agar medium. The OMT-resistant variants showed a reduction in sensitivity of the enzyme l-threonine deaminase to feedback inhibition by isoleucine, a decreased stability of l-threonine deaminase when stored at-18°C or incubated at +55°C and a two- to threefold increase of the free isoleucine pool within the cells. The genetical events and the biochemical mechanisms which might lead to the observed stable and biochemically defined character are discussed with particular reference to the high ploidy level of the Rosa cell line.Abbreviations OMT l-O-methylthreonine - TD l-threonine deaminase     

Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ-Aminobutyric Acid in Vivo

A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.     

The amount and nature of glutathione transferases in rat liver microsomes determined by immunochemical methods

The amount and nature of glutathione transferases in rat liver microsomes were determined using immunological techniques. It was shown that cytosolic glutathione transferase subunits A plus C, and B plus L were present at levels of 2.4 ± 0.6 and 1.5 ± 0.1 μg/mg microsomal protein, respectively. These levels are 10-times higher than those for non-specific binding of cytosolic components judging from the distribution of lactate dehydrogenase, a cytosolic marker. The possibility that a portion of these glutathione transferases is functionally localized on the endoplasmic reticulum is discussed. A previously described microsomal glutathione transferase which is distinct from the cytosolic enzymes is present in an amount of 31 ± 6 μg/mg microsomal protein.     

Toxoplasma gondii: decreased resistance to intracellular bacteria in mice

The effect of sublethal inocula of Toxoplasma gondii on the course of listeriosis and salmonellosis in mice was investigated. Intravenous injection of T. gondii 24 hr after inoculation of Listeria monocytogenes increased mortality from 16% (L. monocytogenes alone) to 68% (L. monocytogenes + T. gondii) (P less than 0.001). Multiplication of L. monocytogenes in spleens also was increased significantly in mice given T. gondii. By 3 days after infection, mice that had received T. gondii and L. monocytogenes had approximately 10 times the number of L. monocytogenes per spleen compared to mice receiving L. monocytogenes alone. Similarly, mortality and the number of bacteria in spleens were increased in mice injected with Salmonella typhimurium and then inoculated with T. gondii. An in vitro assay of macrophage listeriacidal activity was used to investigate the mechanism of this decreased resistance. Peritoneal macrophages from mice injected with T. gondii were less bactericidal than macrophages from uninfected mice. Delayed hypersensitivity responses to L. monocytogenes antigen were markedly suppressed in mice injected with T. gondii. T. gondii infection appears to suppress both macrophage and T-lymphocyte function and may result in decreased resistance to infections caused by intracellular bacteria.     

Voltage dependence of theChara proton pump revealed by current-voltage measurement during rapid metabolic blockade with cyanide

Summary It is generally agreed that solute transport across theChara plasma membrane is energized by a proton electrochemical gradient maintained by an H⁺-extruding ATPase. Nonetheless, as deduced from steady-state current-voltage (I-V) measurements, the kinetic and thermodynamic constraints on H⁺-ATPase function remain in dispute. Uncertainties necessarily surround long-term effects of the relatively nonspecific antagonists used in the past; but a second, and potentially more serious problem has sprung from the custom of subtracting, across the voltage spectrum, currents recorded following pump inhibition from currents measured in the control. This practice must fail to yield the trueI-V profile for the pump when treatments alter the thermodynamic pressure on transport.We have reviewed these issues, using rapid metabolic blockade with cyanide and fitting the resultant whole-cellI-V and difference-current-voltage (dI-V) relations to a reaction kinetic model for the pump and parallel, ensemble leak. Measurements were carried out after blocking excitation with LaCl₃, so that steady-state currents could be recorded under voltage clamp between –400 and +100 mV. Exposures to 1mm NaCN (CN) and 0.4mm salicylhydroxamic acid (SHAM) depolarized (positive-going)Chara membrane potentials by 44–112 mV with a mean half time of 5.4±0.8 sec (n=13). ATP contents, which were followed in parallel experiments, decayed coincidently with a mean half time of 5.3±0.9 sec ([ATP] _t=0, 0.74±0.3mm; [ATP] _t=x , 0.23±0.02mm). Current-voltage response to metabolic blockade was described quantitatively in context of these changes in ATP content and the consequent reduction in pump turnover rate accompanied by variable declines in ensemble leak conductance. Analyses ofdI-V curves (±CN+SHAM) as well as of families ofI-V curves taken at times during CN+SHAM exposures indicated a stoichiometry for the pump of one charge (H⁺) transported per ATP hydrolyzed and an equilibrium potential near –420 mV at neutral external pH; under these conditions, the pump accounted for approximately 60–75% of the total membrane conductance nearV _m. Complementary results were obtained also in fitting previously publishedI-V data gathered over the external pH range 4.5–7.5 Kinetic features deduced for the pump were dominated by a slow step preceding H⁺ unloading outside, and by recycling and loading steps on the inside which were in rapid equilibrium. These characteristics predict, in marked contrast to the situation forNeurospora, that cytoplasmic acid loads inChara should shift the pumpI-V curve negative-going along the voltage axis with little change in maximum current output at positive voltages.     

Inhibition of hexose transport in the human erythrocyte by 5, 5′-dithiobis(2-nitrobenzoic acid): Role of an exofacial carrier sulfhydryl group

Summary The sulfhydryl reagent 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1mm DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10mm cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120mm accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 m cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfydryl, but reaction of this group may not always lock the carrier in an outward-facing conformation.     

Glial uptake system of GABA distinct from that of taurine in the bullfrog sympathetic ganglia

The kinetics and specificity of GABA and taurine uptake were studied in the bullfrog sympathetic ganglia. GABA uptake system consisted of simple saturable component and taurine uptake system consisted of two saturable components exclusive of non-saturable influx. Taurine unaffected GABA uptake while GABA inhibited taurine uptake competitively with theK _i/K_m ratio of 38. GABA (5.14 M) uptake was inhibited by -aminovaleric acid and slightly by 2,4-diaminobutyric acid (5 mM, each) among ten structural analogs. Taurine uptake under high-affinity conditions was most strongly suppressed by hypotaurine and -alanine competitively with theK _i/K_m ratio of 1.0 and 1.9, respectively. Autoradiography showed that glial cells were heavily labeled by both [³H]GABA and [³H]taurine. These results suggest that GABA is transported by a highly specific carrier system distinct from the taurine carrier and that taurine, hypotaurine, and -alanine may share the same high-affinity carrier system in the glial cells of the bullfrog sympathetic ganglia.