Mapping protein–protein interactions by double-REDOR-filtered magic angle spinning NMR spectroscopy

REDOR-based experiments with simultaneous ¹H–¹³C and ¹H?¹⁵N dipolar dephasing are explored for investigating intermolecular protein–protein interfaces in complexes formed by a U–¹³C,¹⁵N-labeled protein and its natural abundance binding partner. The application of a double-REDOR filter (dREDOR) results in a complete dephasing of proton magnetization in the U–¹³C,¹⁵N-enriched molecule while the proton magnetization of the unlabeled binding partner is not dephased. This retained proton magnetization is then transferred across the intermolecular interface by ¹H–¹³C or ¹H–¹⁵N cross polarization, permitting to establish the residues of the U–¹³C,¹⁵N-labeled protein, which constitute the binding interface. To assign the interface residues, this dREDOR-CPMAS element is incorporated as a building block into ¹³C–¹³C correlation experiments. We established the validity of this approach on U–¹³C,¹⁵N-histidine and on a structurally characterized complex of dynactin’s U–¹³C,¹⁵N-CAP-Gly domain with end-binding protein 1 (EB1). The approach introduced here is broadly applicable to the analysis of intermolecular interfaces when one of the binding partners in a complex cannot be isotopically labeled.     

Leucoagglutinin induces differential proliferation of lymphocyte subsets

In this study we have established culture conditions that allow the preferential and rapid expansion of either T cell receptor (TCR)⁺/CD3⁺16^? T lymphocytes or TCR^?/CD3^?16^? natural killer (NK) cells, or the non-selective outgrowth of both subsets. Optimal proliferation of lymphocytes was obtained using a combination of irradiated allogeneic peripheral blood lymphocytes (PBL) and irradiated Epstein Barr virus (EBV) transformed lymphoblastoid B cell lines (B-LCL). Addition of 1μg/ml leucoagglutinin to the culture medium induced a preferential outgrowth of TCR^?/CD3^?16^? T lymphocytes. The proportion of TCR^?/CD3^?16^? NK cells was decreased to 5% or less, although still a 2000-fold multiplication of TCR^?/CD3^?16^? NK cells was obtained at day 13. Without leucoagglutinin a 1000-fold increase of about 70% pure TCR^?/CD3^?16^? NK cells was obtained at day 13. Intermediate concentrations of leucoagglutinin (0.1–0.3μg/ml) resulted in a non-selective expansion of both NK cells and T cells. Irrespective whether leucoagglutinin was added or not, the number of TCR⁺/CD3⁺8⁺ lymphocytes increased more rapidly relative to the TCR⁺/CD3⁺4⁺ lymphocytes resulting in an increased TCR⁺/CD3⁺8⁺ population size. Also under limiting dilution conditions leucoagglutinin increased the frequency of proliferating cells. In contrast to the preferential outgrowth of TCR⁺/CD3⁺8⁺ lymphocytes in bulk cultures, approximately 80% of the clones generated was TCR⁺/CD3⁺4⁺, demonstrating a growth promoting effect of TCR⁺/CD3⁺4⁺ lymphocytes on TCR⁺/CD3⁺8⁺ lymphocytes in PBL bulk cultures.     

Simultaneous Influx and Efflux of Nitrate during Uptake by Perennial Ryegrass

Experiments with intact plants of Lolium perenne previously grown with ¹⁴NO₃⁻ revealed significant efflux of this isotopic species when the plants were transferred to solutions of highly enriched ¹⁵NO₃⁻. The exuded ¹⁴NO₃⁻ was subsequently reabsorbed when the ambient solutions were not replaced. When they were frequently replaced, continual efflux of the ¹⁴NO₃⁻ was observed. Influx of ¹⁵NO₃⁻ was significantly greater than influx of ¹⁴NO₃⁻ from solutions of identical NO₃⁻ concentration. Transferring plants to ¹⁴NO₃⁻ solutions after a six-hour period in ¹⁵NO₃⁻ resulted in efflux of the latter. Presence of Mg²⁺, rather than Ca²⁺, in the ambient ¹⁵NO₃⁻ solution resulted in a decidedly increased rate of ¹⁴NO₃⁻ efflux and a slight but significant increase in ¹⁵NO₃⁻ influx. Accordingly, net NO₃⁻ influx was slightly depressed. A model in accordance with these observations is presented; its essential features include a passive bidirectional pathway, an active uptake mechanism, and a pathway for recycling of endogenous NO₃⁻ within unstirred layers from the passive pathway to the active uptake site.     

Rat hypothalamic extract inhibits vasopressin-stimulated Na+-K+-ATPase activity in the rat renal medulla

Arginine vasopressin stimulates Na⁺-K⁺-ATPase activity located in the rat thick ascending limb of s'Henle loop. Mammalian hypothalamus appears to produce a factor capable of inhibiting Na⁺-K⁺-ATPase activity in a variety of tissues. The effect of a purified rat hypothalamic extract with and without AVP on rat renal Na⁺-K⁺-ATPase activity was evaluated by a cytochemical technique. The hypothalamic extract alone failed to affect basal Na⁺-K⁺-ATPase activity throughout renal segments after 10 min exposure. Na⁺-K⁺-ATPase activity stimulated by AVP (1–10 fmol l^?1) for 10 min was inhibited by rat hypothalamic extract over the concentration range 10^?7–10^?3 U ml^?1 in a dose-dependent manner. Complete inhibition of AVP-stimulated Na⁺-K⁺-ATPase activity occurred at a hypothalamic extract concentration of 10^?3 U ml^?1. Only Na⁺-K⁺-ATPase activity located in the renal medullary thick ascending limb was influenced by the rat hypothalamic extract.     

Effect of Hypoxia on Na+-K+-Cl− Cotransport in Cultured Brain Capillary Endothelial Cells of the Rat

Abstract: The effect of hypoxia on Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring ⁸⁶Rb⁺ uptake as a tracer for K⁺. RBECs expressed both Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na⁺,K⁺-ATPase activity by 38.9%, whereas it significantly increased Na⁺-K⁺-Cl^? cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin-induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time-dependent decrease of cellular ATP content (by ～65%) along with a complete inhibition of Na⁺,K⁺-ATPase and a coordinated increase of Na⁺-K⁺-Cl^? cotransport activity (up to 100% above control values). Oligomycin augmentation of Na⁺-K⁺-Cl^? cotransport activity was not observed in the presence of 2-deoxy-d -glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na⁺,K⁺-ATPase activity is reduced, RBECs have the ability to increase K⁺ uptake through Na⁺-K⁺-Cl^? cotransport.     

The effects of FMRFamide, serotonin, and acetylcholine on the isolated crop-gizzard of the earthworm, Lumbricus terrestris

We examined the effects of FMRFamide, serotonin, and acetylcholine on the isolated crop-gizzard of the earthworm, Lumbricus terrestris. FMRFamide caused a decrease in contraction amplitude with a threshold between 10⁻⁹ and 10⁻⁸ M and a biphasic change in contraction rate. The contraction rate increased with a threshold between 10⁻⁸ and 10⁻⁷ M and decreased with a threshold between 10⁻⁶ and 10⁻⁵ M. Serotonin decreased the contraction rate with a threshold between 10⁻⁸ and 10⁻⁷ M and the amplitude with a threshold between 10⁻⁸ and 10⁻⁷ M. Acetylcholine increased the contraction rate with a threshold between 10⁻⁸ and 10⁻⁷ M and caused a biphasic change in contraction amplitude. Amplitude rose with a threshold between 10⁻⁹ and 10⁻⁸ M and decreased with a threshold between 10⁻⁶ and 10⁻⁵ M. These results suggest that all three neurotransmitters may play a role in controlling the movement of the digestive tract in L. terrestris.     

Identification of a third isoform of Na+, K+-ATPase activity in rat brain synaptosomes

³H-ouabain binding and ouabain-inhibitable ⁸⁶Rb⁺ (K⁺) uptake were investigated as a means to identify a third isoform of Na⁺, K⁺-ATPase in crude synaptosome preparations. The specific binding of low concentrations (10 nM and 1 uM) of ³H-ouabain, in crude synaptosome preparations, was markedly inhibited by K⁺ (0.5–5 mM). Accordingly, ⁸⁶Rb⁺ (K⁺) uptake, in the presence of 5 mM K⁺ was not sensitive to inhibition by low concentrations (10⁻¹¹–10⁻⁷ M) of ouabain. Higher concentrations (10⁻⁶–10^−2.6 M) of ouabain resulted in a biphasic inhibition of K⁺ uptake, which distinguished the activities of the presumed alpha 2 and alpha 1 isozymes of Na⁺, K⁺-ATPase. Reduction of K⁺ (1.25 mM and 0.5 mM) in the incubation, resulted in the observation of a third component of ouabain- sensitive K⁺ uptake. This Na⁺, K⁺-ATPase activity, which was defined, pharmacologically, as very sensitive (VS) to ouabain, exhibited IC₅₀s of 3.6 nM and 92 nM at 1.25 mM K⁺ and 0.5 mM K⁺, respectively. Inhibition of ouabain binding and VS-dependent K⁺ uptake, at a high, physiological cocentration (5 mM) of K⁺, suggests that VS may be an inactive isoform of brain Na⁺, K⁺-ATPase under resting conditions.     

A novel strategy for the assignment of side-chain resonances in completely deuterated large proteins using 13C spectroscopy

The assignment of the aliphatic ¹³C resonances of trimeric Bacillus Subtilis chorismate mutase, a protein with a molecular mass of 44 kDa, consisting of three 127-residue monomers is presented by use of two-dimensional (2D) ¹³C-start and ¹³C-observe NMR experiments. These experiments start with ¹³C excitation and end with ¹³C observation while relying on the long transverse relaxation times of ¹³C spins in uniformly deuterated and ¹³C,¹⁵N-labeled large proteins. Gains in sensitivity are achieved by the use of a paramagnetic relaxation enhancement agent to reduce ¹³C T ₁ relaxation times with little effect on ¹³C T ₂ relaxation times. Such 2D ¹³C-only NMR experiments circumvent problems associated with the application of conventional experiments for side-chain assignment to proteins of larger sizes, for instance, the absence or low concentration of the side-chain ¹H spins, the transfer of the side-chain spin polarization to the ¹H^N spins for signal acquisition, or the necessity of a quantitative reprotonation of the methyl moieties in the otherwise fully deuterated side-chains. We demonstrate that having obtained a nearly complete assignment of the side-chain aliphatic ¹³C resonances, the side-chain ¹H chemical shifts can be assigned in a semiautomatic fashion using 3D ¹⁵N-resolved and ¹³C-resolved NOESY experiments measured with a randomly partially protonated protein sample. We also discuss perspectives for structure determination of larger proteins by using novel strategies which are based on the ¹H,¹H NOEs in combination with multiple residual dipolar couplings between adjacent ¹³C spins determined with 2D ¹³C-only experiments.     

Sodium transport in Na+-rich Chlorella cells

Summary The rate of Na⁺/Na⁺ exchange as measured with ²⁴Na⁺ in Na⁺-rich cells of Chlorella pyrenoidosa is governed by a single rate constant and saturates with increasing external Na⁺ concentration. The K _mvalue for this process is 0.8 mM Na⁺ and the maximum rate of exchange in illuminated cells is about 5 pmoles cm^-2 sec^-1. These values contrast with a K _mof 0.18 mM K⁺ and maximum rate of about 17 pmoles K⁺·cm^-2·sec^-1 for net K⁺ influx. Although the Na⁺/Na⁺ exchange was only slightly sensitive to light it was inhibited by the uncouplers CCCP and DNP and by the energy transfer inhibitor DCCD. This inhibition of the rate of Na⁺/Na⁺ exchange was not accompanied by a loss of internal Na⁺. Both the effect of external K⁺ on ²⁴Na⁺ influx into Na⁺-rich cells and the inhibition of net K⁺ uptake by the presence of external Na⁺ indicates that Na⁺/Na⁺ and K⁺/Na⁺ exchanges share the same carrier and that the external site of this carrier has a three to four times higher affinity for K⁺ over Na⁺.     

Cationic interactions with Na+-H+ exchange and passive Na+ flux in cardiac sarcolemmal vesicles

Na⁺-H⁺ exchange and passive Na⁺ flux were investigated in cardiac sarcolemmal vesicles as a function of changing the ionic composition of the reaction media. The inclusion of EGTA in the reaction medium resulted in a potent stumulation of Na⁺ uptake by Na⁺-H⁺ exchange. It was found that millimolar concentrations of Mg²⁺ and Li⁺ were capable of inhibiting Na⁺-H⁺ exchange by 80%. One mechanism by which these ions may inhibit intravesicular Na⁺ accumulation by Na⁺-H⁺ exchange is via an increase in Na⁺ efflux. An examination of Na⁺ efflux kinetics from vesicles pre-loaded with Na⁺ revealed that Na⁺, Ca²⁺, Mg²⁺ and Li⁺ could stimulate Na⁺ efflux. Na⁺-H⁺ exchange was potently inhibited by an organic divalent cation, dimenthonium, which screens membrane surface charge. This would suggest that Na⁺-H⁺ exchange occurs in the diffuse double layer region of cardiac sarcolemma and this phenomenon is distinctly different from other Na⁺ transport processes. The results in this study indicate that in addition to a stimulation of Na⁺ efflux, the inhibitory effects of Mg²⁺, Ca²⁺ and Li⁺ on Na⁺-H⁺ exchange may also involve a charge dependent screening of Na⁺ interactions with the membrane.     

Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina

The uptake of K⁺ and Ca²⁺ in Dunaliella salina is mediated by two distinct carriers: a K⁺ carrier with a high selectivity against Na⁺, Li⁺, and choline⁺ but not towards Rb⁺, K⁺, Cs⁺, or NH₄⁺, and a Ca²⁺ carrier with a high selectivity against Mg²⁺. The latter is specifically blocked by La³⁺ and by Cd²⁺. Apparent K_m values for K⁺ and Ca²⁺ uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K⁺ and Ca²⁺ uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H⁺-ATPases and protonionophores inhibit K⁺ and Ca²⁺ uptake and accelerate K⁺ efflux. The results suggest that an H⁺-ATPase in the cell membrane provides the driving force for K⁺ and Ca²⁺ uptake. Efflux measurements from ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ loaded cells suggest that part of the intracellular K⁺ and most of the intracellular Ca²⁺ is nonexchangeable with the extracellular pool. Correlations between phosphate and K⁺ contents and the effect of phosphate on K⁺ efflux suggest intracellular associations between K⁺ and polyphosphates. On the basis of these results, it is suggested that: (a) K⁺ and Ca²⁺ uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H⁺-ATPase of the plasma membrane. (b) Part of the intracellular K⁺ is associated with polyphosphate bodies, while most of the intracellular Ca²⁺ is accumulated in intracellular organelles in the algal cells.     

An OFF–ON–OFF type fluorescent probe based on a naphthalene derivative for Al3+ and F− ions and its biological application

A novel fluorescent probe‐based naphthalene Schiff, 1‐(C2‐glucosyl‐ylimino‐methyl)‐naphthalene‐2‐ol (L) was synthesized by coupling d ‐glucosamine hydrochloride with 2‐hydroxy‐1‐naphthaldehyde. It exhibited excellent selectivity and highly sensitivity for Al³⁺ in ethanol with a strong fluorescence response, while other common metal ions such as Pb²⁺, Mg²⁺, Cu²⁺, Co²⁺, Ni²⁺, Cd²⁺, Fe²⁺, Mn²⁺, Hg²⁺, Li⁺, Na⁺, K⁺, Fe³⁺, Cr³⁺, Zn²⁺, Ag⁺, Ba²⁺ and Ca²⁺ did not cause the same fluorescence response. The probe selectively bound Al³⁺ with a binding constant (K_a) of 5.748 × 10³ M^?1 and a lowest detection limit (LOD) of 4.08 nM. Moreover, the study found that the fluorescence of the L ? Al³⁺ complex could be quenched after addition of F^? in the same medium, while other anions, including Cl^?, Br^?, I^?, NO₂^?, NO₃^?, ClO₄^?, CO₃^2?, HCO₃^?, SO₄^2?, HSO₄^?, CH₃COO^?, PO₄^3?, HPO₄^2?, S^2? and S₂O₃^2? had nearly no influence on probe behaviour. Binding of the [L ? Al³⁺] complex to a F^? anion was established by different fluorescence titration studies, with a detection limit of 3.2 nM in ethanol. The fluorescent probe was also successfully applied in the imaging detection of Al³⁺ and F^? in living cells.     

Super-high-affinity binding site for [3H]diazepam in the presence of Co2+, Ni2+, Cu2+, OR Zn2+

Chloride salts of Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Cr³⁺, Mn²⁺, Fe²⁺, and Fe³⁺ had no effect on [³H]diazepam binding. Chloride salts of Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ increased [³H]diazepam binding by 34 to 68% in a concentration-dependent fashion. Since these divalent cations potentiated the GABA-enhanced [³H]diazepam binding and the effect of each divalent cation was nearly additive with GABA, these cations probably act at a site different from the GABA recognition site in the benzodiazepine-receptor complex. Scatchard plots of [³H]diazepam binding without an effective divalent cation showed a single class of binding, with a Kd value of 5.3 mM. In the presence of 1 mM Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺, two distinct binding sites were evident with apparent Kd values of 1.0 nM and 5.7 nM. The higher-affinity binding was not detected in the absence of an effective divalent cation and is probably a novel, super-high-affinity binding site.     

Ion pathways in renal brush border membranes

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na⁺/H⁺ or Li⁺/H⁺ exchange was demonstrated by diluting Na₂SO₄ or Li₂SO₄ loaded vesicles into Na⁺- or Li⁺-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K⁺ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na⁺ conductance smaller than electroneutral Na⁺/H⁺ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na⁺ conductance greater than the H⁺ conductance was suggested. K⁺ gradients also induced changes of pH, at about 10% of the Na⁺ or Li⁺ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K⁺ was a low affinity substrate for the Na⁺/H⁺ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K⁺ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K⁺ conductance. Inward Cl^? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl^? conductance greater than the H⁺ conductance and a Cl^?/OH^? exchange were present. The rate of Na⁺/H⁺ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent K_m for Na⁺ or Li⁺ of about 10 mM and an E_A of 14.3 kcal per mol. A 61-fold Na₂SO₄ gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H⁺ and Cl^? pathways could alter the effects of the Na⁺/H⁺ exchange.     

Sodium Transport in Capillaries Isolated from Rat Brain

Abstract: Brain capillary endothelial cells form a bloodbrain barrier (BBB) that appears to play a role in fluid and ion homeostasis in brain. One important transport system that may be involved in this regulatory function is the Na⁺,K⁺-ATPase that was previously demonstrated to be present in isolated brain capillaries. The goal of the present study was to identify additional Na⁺ transport systems in brain capillaries that might contribute to BBB function. Microvessels were isolated from rat brains and ²²Na ⁺ uptake by and efflux from the cells were studied. Total ²²Na ⁺ uptake was increased and the rate of ²²Na ⁺ efflux was decreased by ouabain, confirming the presence of Na⁺,K⁺-ATPase in capillary cells. After inhibition of Na⁺,K⁺-ATPase activity, another saturable Na ⁺ transport mechanism became apparent. Capillary uptake of ²²Na ⁺ was stimulated by an elevated concentration of Na ⁺or H⁺ inside the cells and inhibited by extracellular Na⁺, H⁺, Li⁺, and NH₄⁺. Amiloride inhibited ²²Na ⁺ uptake with a K_i between 10^?5 and 10^?6M but there was no effect of 1 mM furosemide on ²²Na⁺ uptake by the isolated microvessels. These results indicate the presence in brain capillaries of a transport system capable of mediating Na ⁺/ Na ⁺ and Na ⁺/H ⁺ exchange. As a similar transport system does not appear to be present on the luminal membrane of the brain capillary endothelial cell, it is proposed that Na ⁺/H ⁺ exchange occurs primarily across the antiluminal membrane.     

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic ^1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis ⁴. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects ⁵. For transmission between insect hosts, the bacteria colonize the intestine of the nematode''s infective juvenile stage ^6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects ^9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems ⁹.We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila¹⁴. Similar methods have been used to investigate other nematode-bacterium associations ^9,15-18and the approach therefore is generally applicable.The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization ^14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues ^14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication ²²or grinding ²³, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes ^21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization ^17,18, and is less laborious than other methods, including sonication ^22,25-27and individual nematode dissection ^28,29.     

Initial organic products of assimilation of [N]ammonium and [N]nitrate by tobacco cells cultured on different sources of nitrogen

Glutamine is the first major organic product of assimilation of ¹³NH₄⁺ by tobacco (Nicotiana tabacum L. cv. Xanthi) cells cultured on nitrate, urea, or ammonium succinate as the sole source of nitrogen, and of ¹³NO₃⁻ by tobacco cells cultured on nitrate. The percentage of organic ¹³N in glutamate, and subsequently, alanine, increases with increasing periods of assimilation. ¹³NO₃⁻, used for the first time in a study of assimilation of nitrogen, was purified by new preparative techniques. During pulse-chase experiments, there is a decrease in the percentage of ¹³N in glutamine, and a concomitant increase in the percentage of ¹³N in glutamate and alanine. Methionine sulfoximine inhibits the incorporation of ¹³N from ¹³NH₄⁺ into glutamine more extensively than it inhibits the incorporation of ¹³N into glutamate, with cells grown on any of the three sources of nitrogen. Azaserine inhibits glutamate synthesis extensively when ¹³NH₄⁺ is fed to cells cultured on nitrate. These results indicate that the major route for assimilation of ¹³NH₄⁺ is the glutamine synthetase-glutamate synthase pathway, and that glutamate dehydrogenase also plays a role, but a minor one. Methionine sulfoximine inhibits the incorporation of ¹³N from ¹³NO₃⁻ into glutamate more strongly than it inhibits the incorporation of ¹³N into glutamine, suggesting that the assimilation of ¹³NH₄⁺ derived from ¹³NO₃⁻ may be mediated solely by the glutamine synthetase-glutamate synthase pathway.     

Substrate specificity of thermitase,a thermostable serine proteinase fromThermoactinomyces vulgaris

The specificity of thermitase (EC 3.4.21.14), a microbial thermostable serine proteinase fromThermoactinomyces vulgaris, with several oligo- and polypeptide substrates was investigated. Preferred hydrolysis of peptide bonds with a hydrophobic amino acid at the carboxylic site was observed. The proved carboxypeptidolytic splitting of Leu⁵-enkephalin and bradykinin, as well as the noncleavability of casomorphins by thermitase, can be explained by the position of the glycine and proline residues in these substrates. Major cleavage sites in the oxidized insulin B chain in a 15-min incubation with thermitase at Gln⁴-His⁵, Ser⁹-His¹⁰, Leu¹¹-Val¹², Leu¹⁵-Tyr¹⁶ and in the oxidized insulin A chain at Cys SO₃H¹¹-Ser¹², Leu¹³-Tyr¹⁴, and Leu¹⁶-Glu¹⁷ were observed. Additional cleavages of the bonds His⁵-Leu⁶, Arg²²-Gly²³, Phe²⁴-Phe²⁵, Phe²⁵-Tyr²⁶, and Tyr²⁶-Thr²⁷ in the oxidized B chain and Cys SO₃H⁶-Cys SO₃H⁷ and Tyr¹⁹-Cys SO₃H²⁰ in the oxidized A chain in 2-h incubations with thermitase were also noted. Hydrolysis of salmine A I component in a 10-min incubation was observed mainly at four peptide bonds: Arg⁵-Ser⁶, Ser⁶-Ser⁷, Arg¹⁸-Val¹⁹, and Gly²⁷-Gly²⁸. The cleavage sites of thermitase in both insulin chains were similar to those reported in the studies of subtilisins.     

Rubidium transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

Synechococcus R-2 is a unicellular blue-green alga. The cells will grow on Rb⁺ as a substitute for K⁺ but at a slower rate (t₂～ 15 h versus 12 h). Potassium is not, strictly speaking, an essential element for Synechococcus. Rubidium duxes (using ⁸⁶Rb⁺) are much slower than those of potassium, about 1 nmol m^?2 s^?1 in the light (0.35 mol m^?3 Rb⁺). ⁸⁶Rb⁺ fluxes in the dark are about 0.1 nmol m^?2 s^?1. These fluxes are very slow compared to those of Na⁺ and other ions. Isotopic influx of Rb⁺ can supply sufficient Rb⁺ to keep up with the demands for growth, but the net dux needed to keep up with growth in the light is a large proportion of the total observed dux. Kinetic studies of Rb⁺ uptake versus [Rb⁺] show two uptake phases consistent with a high-affinity and a low-affinity system. Both systems appear to be light-activated. Transport of Rb⁺ appears to be passive at pH_o 10 in the light and dark. There is no case for active transport of Rb⁺ at pH_o 7.5 in the light, but a marginal case for active uptake in the dark (about 3 kJ mol^?1). There is only a small effect of Na⁺ upon Rb⁺ transport. ⁸⁶Rb⁺ should not be used in place of ⁴²K⁺ in K⁺ nutrition studies as the details of Rb⁺ transport are different to those of K⁺ transport.     

Expression of T-cell differentiation antigens on activated Thymocytes

The expression of Thy 1, TL, Lyt1, and Lyt2 antigens on resting and proliferating thymocytes activated by concanavalin A in the presence of interleukin 2 has been studied by conventional complement-dependent cytotoxicity assay. The predominant population of resting thymocytes has a TL⁺Lytl⁺Lyt2⁺ phenotype while the predominant population of proliferating thymocytes has a TL — Lytl⁺Lyt2⁺ phenotype. Using several separation procedures such as agglutination by peanut lectin, BSA density gradient centrifugation, and pretreatment with high dilutions of anti-H-2 serum it was impossible to obtain a 100% pure population of TL⁺Lytl⁺Lyt2⁺ cells, suggesting that the population of resting immature thymocytes contains small subpopulations of phenotypically differentiated cells. The population of proliferating thymocytes is also phenotypically heterogenous and contains cells bearing all phenotypes that were described for different stages of T-cell differentiation, including TL⁺Lytl⁺Lyt2^? and TL⁺Lytl^?Lyt2⁺ with the following approximate frequency: TL⁺Lytl⁺Lyt2⁺—27%, TL⁺Lytl⁺Lyt2^?—8%, TL⁺Lyt1^?Lyt2⁺—4%, TL^?Lytl⁺Lyt2⁺—45%, TL^?Lyt1⁺Lyt2^?—13%, TL^?Lyt1^?Lyt2⁺—3%.