The in vivo equivalence of a cell-free system for RNA processing and transport

The equivalence of messenger RNA released (transported) from isolated rat liver nuclei to three selected media, with messenger RNA normally released to liver cytoplasm $\text{in vivo}$, has been evaluated by competitive DNA: RNA hybridization. Near normal nuclear restriction was exhibited by nuclei in media fortified with ATP, salts, spermidine and dialyzed cytosol. The RNA transport in the latter system was markedly inhibited by colchicine as was also the transport of RNA $\text{in vivo}$. Both nuclear restriction and sensitivity of the RNA transport to colchicine in media lacking spermidine and cytosol deviated significantly from the $\text{in vivo}$ norm. The results emphasize the importance of establishing the $\text{in vivo}$ equivalence in cell-free systems designed to study RNA synthesis, processing and transport.     

The occurrence of phosphatidyl choline exchange protein in leaves

The transfer of phosphatidyl choline between liposomes was stimulated by the protein fractions from spinach leaves, etiolated and greening leaves of $\text{Avena}$ seedlings. This is confirmed by the transfer of [¹⁴C]phosphatidyl choline or spin-labeled phosphatidyl choline between donor and acceptor liposomes. ESR spectrum changes also indicated that no spin-labeled phosphatidyl choline was released from donor liposomes by spinach leaf protein unless acceptor liposomes were present. [¹⁴C]phospholipids were transferred from liposomes to both spinach chloroplasts and $\text{Avena}$ etiochloroplasts by phosphatidyl choline exchange protein from germinated castor bean endosperms and further from liposomes to spinach chloroplasts by spinach leaf protein. These results support the view that phosphatidyl choline in the plastid is supplied from the synthesis site, the endoplasmic reticulum, by phospholipid exchange protein.     

The inactivation of acetylcholinesterase by trimethyloxonium ion, an active-site-directed methylating agent

Trimethyloxonium ion inactivates acetylcholinesterase from the electric eel and acetylcholinesterase on the surface of human red blood cells. Tetramethylammonium ion, which is a competitive inhibitor of acetylcholinesterase, protects against this inactivation. Trimethyloxonium ion does $\text{not}$ inactivate the system that transports choline into the red blood cell. We conclude that trimethyloxonium ion is an affinity-labeling reagent for acetylcholinesterase and that red blood cell acetylcholinesterase is probably not a component of the choline transport system.     

Methionine transport in S37 cells. Substrate-dependent function of amino acid transport system A in exchange processes

Methionine had been observed to interact with two principal transport systems for amino acids in mammalian cells, the A and L systems. The present study of methionine transport and of exchange processes through system A arose in the course of a study to define the specificity of a transinhibition effect caused by cysteine.Methionine uptake through two transport systems in the S37 cell was confirmed by the occurrence of a biphasic double-reciprocal plot for labeled methionine uptake. Preloading cells with methionine stimulated labeled histidine uptake through both systems A and L. Efflux of labeled methionine from cells was stimulated by histidine in a biphasic manner, so that both systems A and L can be used for exchange when methionine is the intracellular amino acid. Aminocycloheptanecarboxylic acid elicited exchange efflux of labeled methionine only through system L. α-Aminoisobutyric acid and $\text{N-}\text{methyl}\text{-α-}\text{aminoisobutyric acid}$ both stimulated efflux of labeled $\text{N-}\text{methyl}\text{-α-}\text{aminoisobutyric acid}$ from S37 cells. These findings are interpreted a showing that transport system A is capable of functioning as an exchange system depending upon the identity of intracellular and extracellular substrates available.     

Respiratory control and mitochondrial monovalent cation permeability of isolated liver cells

Uncoupling agent releases the respiratory control of rat hepatocytes to approximately the same degree as in isolated mitochondria indicating that mitochondria $\text{in situ}$ possess a low H⁺ conductance as $\text{in vitro}$. Mitochondria also have no detectable natural K⁺ conductance since the ionophore, valinomycin, is required for K⁺ ions to uncouple. Na⁺ but not K⁺ or choline inhibits the uncoupled respiration of liver cells. This is consistent with operation of neutral mitochondrial Na⁺ for H⁺ exchange $\text{in vivo}$. These results indicate a considerable similarity between certain functional and permeability properties of mitochondria $\text{in vitro}$ and $\text{in situ}$. These similarities form the basis for discussion of the role of mitochondrial ion transport in metabolic regulation.     

Utilization of sodium-dependent high affinity choline uptake in vitro as a measure of the activity of cholinergic neurons in vivo.

Previous reports indicate that alterations of activity of cholinergic neurons $\text{in vivo}$ are followed by parallel changes in sodium-dependent high affinity choline uptake $\text{in vitro}$. These results are consistent with the proposal that this portion of choline uptake is regulatory in the synthesis of ACh. These results also suggest the possibility of utilizing sodium-dependent high affinity choline uptake as a measure of the relative state of cholinergic activity $\text{in vivo}$. In this study, we administer a number of drugs reported to alter turnover and release of ACh (both are measures of cholinergic activity $\text{in vivo}$, and subsequently examine sodium-dependent high affinity choline uptake $\text{in vitro}$. Administration of pentobarbital, chloral hydrate, morphine, physostigmine, Δ⁹ THC, hemicholinium-3 and oxotremorine, drugs which decrease ACh turnover and release, caused a reduction in choline uptake. Conversely, administration of pentylenetetrazol, atropine, scopolamine, and haloperidol, drugs which increase ACh turnover and release, caused an increase in choline uptake $\text{in vitro}$. These findings support the proposal that sodium-dependent high affinity choline uptake can be used as a relative measure of the activity of cholinergic neurons $\text{in vivo}$.     

Glutamine-requiring mutants of Bacillus subtilis.

Two glutamine-requiring (Gln^?) mutants of $\text{Bacillus subtilis}$ SMY were deficient in glutamine synthetase activity $\text{in vitro}$. The Gln^? mutants sporulated poorly unless glutamine was provided at high concentrations. The differential rate of histidase synthesis following induction was 4- to 6-fold higher in the Gln^? mutants than in wild-type cells. In addition, glucose repression of utilization of alternative carbohydrates appeared to be partially relieved in the Gln^? mutants.     

The effects of removal of sodium ions from the mucosal solution on sugar absorption by rabbit ileum

Net absorption and accumulation of d-galactose, β-methyl d-glucose and low concentrations of $\text{3-O-}\text{methyl}\text{-}\text{d}\text{-}\text{glucose}$ by sheets of rabbit ileum are observed even when Na⁺ in the mucosal solution is replaced by choline. This indicates that active sugar transport can occur in the direction opposite to the brush-border Na⁺ gradient.     

Analysis of microquantities of choline and its esters utilizing gas chromatography-chemical ionization mass spectrometry.

Gas chromatography-chemical ionization mass spectrometry has been applied successfully in the analysis of choline and its esters. This approach serves to extend further the potential of existing gas chromatographic procedures which are capable of the microestimation of choline esters following their N-demethylation by either chemical or physical means. Typical fragmentation patterns with ions at $\text{m}\text{e}\text{= 72}$ and $\text{m}\text{e}\text{= (M + 1)}$ were obtained for each choline ester derivative. When methane was used as the reactant gas, the above fragments were approximately of equal abundance for each ester. Use of isobutane as reactant gas yielded almost 80% of the (M + 1) fragment, and only approximately 5% of the fragment ion at $\text{m}\text{e}\text{= 72}$. Recovery of all fragments was linear for nondeuterated as well as deuterated analogs of choline ester derivatives. Recovery, as evident from the analysis of records of relative ratios of injected isotopic variants of these esters, indicated that this analysis of choline esters using chemical ionization mass spectrometry coupled with gas chromatography is quantitative and highly reproducible.     

The kinetics of ionophore X-537A-mediated transport of manganese through dipalmitoylphosphatidylcholine vesicles A 1H-NMR study

We have studied the kinetics of ionophore X-537A-mediated transport of manganese ions into small unilamellar vesicles formed from dipalmitoylphosphatidylcholine. To follow the transport we used the paramagnetic effect of manganese on the ¹H-NMR signal from choline trimethylammonium groups on the inner phospholipid monolayer. The transport of only one manganese ion produces an intravesicular concentration which is high enough (approx. 1 mM) to substantially broaden this signal. The observed signal thus arises predominantly from those vesicles which contain no manganese. Therefore, as manganese is transported into the vesicles the observed signal decreases in intensity, but does not broaden. The initial time-dependence of the intensity of the signal, $\text{S(t)}$, can be approximated by the simple first-order rate law: $\text{S(t) = S}\text{(O)}\text{exp}\text{(?K′t)}$, where $\text{K′}$ is the probability per unit time for the transport of a manganese ion from the external medium to the intravesicular space. From the dependence of $\text{K′}$ on the ionophore X-537A concentration we conclude that manganese is transported into the vesicles via both 1 : 1 and 2 : 1 complexes with ionophore X-537A. At low ratios of ionophore X-537A to vesicles transport via the 1 : 1 complex predominates; at high ratios transport via the 2 : 1 complex predominates. From the dependence of $\text{K′}$ on manganese concentration we determined that under our conditions the equilibration of ionophore X-537A between vesicles is much faster than the transport of manganese through the vesicles. Lastly, from the dependence of $\text{K′}$ on temperature, we conclude that the ionophore X-537A-mediated transport of manganese into the dipalmitoylphosphatidylcholine vesicles is very sensitive to the gel-liquid crystalline phase transition.     

Effect of ammonia and glutamine on macromolecule synthesis and breakdown during sporulation of Saccharomyces cerevisiae.

The effect of two known inhibitors of sporulation in yeast, ammonia and glutamine, on certain biochemical events during sporogenesis have been studied using sporulating $\text{a}\text{α}$ and non sporulating $\text{α}\text{α}$ cells. Both strains gave similar results on the increase in dry cell weight, protein and RNA breakdown and the suppression of the intensive RNA and protein syntheses occurring after 4 hours. The inhibitory effect of ammonia and glutamine on RNA and protein syntheses is reversible under the same conditions which do so for sporulation.     

Origin of the labile sulfide in the iron-sulfur proteins of Escherichiacoli

A method has been devised for measuring the abundance of sulfur-34 in the hydrogen sulfide released upon the acidification of $\text{Escherichia}\text{coli}$ cells. Evidence is presented, based on the rate at which the hydrogen sulfide is released from the cells as well as the total amount released, that this hydrogen sulfide originates from the iron-sulfur proteins present in the cells. The sulfur-34 abundance in this hydrogen sulfide which was isolated from cells grown with [sulfane-³⁴S]thiocystine, a compound which can differentially label $\text{in}\text{vivo}$ the sulfur-34 abundance of cysteine and hydrogen sulfide, shows cysteine sulfur and not hydrogen sulfide to be the origin of the sulfide sulfur of iron-sulfur proteins in aerobically grown $\text{E.}\text{coli}$     

Ammonium (methylammonium) transport by Klebsiella pneumoniae

Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 100 μ}\text{M}$, $\text{V = 40 μ}\text{mol/min}$ per g dry weight at 15°C) is competitively inhibited by ammonium (apparent $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7 μ}\text{M}$). The low $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase.     

Transport of α-aminoisobutyrate by cells and membrane vesicles of Pseudomonas fluorescens

The transport of α-aminoisobutyrate into Pseudomonas fluorescens NCIB 8865 and membrane vesicles prepared from this organism has been studied. Uptake by cells was mediated by two active transport systems with different apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values, while transport into membrane vesicles was mediated by a single component. The effect of inhibitors on the energy-coupling mechanism for α-aminoisobutyrate transport in these systems suggests that a membrane potential may play a significant role in supporting α-aminoisobutyrate transport. The magnitude of the membrane potential in the vesicle system, and the sensitivity of its generation to inhibitors, has been measured using ¹³⁷Cs in the presence of valinomycin. Direct attempts to demonstrate a proton-symport mechanism for α-aminoisobutyrate transport were negative.     

Regulation of glutamine synthetase in fed-batch cultures of Neurospora crassa.

The effect of the nitrogen and carbon sources in the regulation of g$\text{l}$u tamine synthetase has been studied in fed-batch cultures of $\text{Neurospora crassa}$. The limitation of ammonium in an excess of the carbon source, leads to an accumulation of α-ketoglutarate and elevation of glutamine sy$\text{n}$ thetase. The limitation of sucrose in an excess of ammonium results in a decrease in glutamine synthetase activity. These results indicate that the carbon source exerts a positive control in the regulation of glutamine synthetase.     

NADH-dependent glutamate synthase and nitrogen metabolism in Neurospora crassa

The GDH (NADPH) mutant strain $\text{am-1}$ of $\text{N. crassa}$ has sizable pools of glutamine and glutamate under ammonium-limited conditions for which requires an elevated glutamine synthetase activity. Glutamine in the pre$\text{s}$ ence of 2-oxoglutarate, stimulated nicotinamide nucleotide oxidation by crude and purified extracts of the $\text{am-1}$ strain and led to a reductant dependent formation of two molecules of glutamate. Aminooxyacetate did not have any effect on the reaction, whereas azaserine inhibited it completely. It is concluded that in $\text{N. crassa}$ glutamine synthetase and glutamate synthase are responsible for the assimilation of low ammonium concentrations.     

Characterization of glutamine requiring mutants of Bacillus subtilis

Mutants of $\text{B. subtilis}$ 168 which exhibited an absolute requirement for glutamine have been isolated and characterized. Of the two mutants studied in detail, one had normal levels of glutamine synthetase and sporulated normally, the other had reduced glutamine synthetase and was asporogenic. Both mutants were mapped close to the $\text{thy}$ A region of the chromosome by PBS1 transduction.A study of spontaneous revertants selected for glutamine prototrophy (or the sporulation character in the case of the asporogenic mutant) led to the conclusion that there is a relationship between the glutamine requirement and sporulation. However, the influence of glutamine could not be entirely explained by the catalytic properties of glutamine synthetase.     

Properties of carnitine transport in rat kidney cortex slices

The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-¹⁴C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated K_m for L-carnitine was 90 μM and $\text{V = 22}\text{nmol/min}$ per ml intracellular fluid; for D-carnitine, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 166}\text{μM}$ and $\text{V = 15}\text{nmol/min}$ per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$, N₂ atmosphere, KCN, $\text{N-}\text{ethylmaleimide}$, low temperature (4°C) and ouabain. Complete replacement of Na⁺ in the medium by Li⁺ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K⁺ or Ca²⁺ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation.     

Endotoxin-induced alterations in glucose transport in isolated adipocytes

2-Deoxyglucose and $\text{3-O-}\text{methyglucose}$ were used to assess endotoxin-induced changes in glucose transport in rat adipocytes. 6 h after Escherichia coli endotoxin injection insulin-stimulated 2-deoxyglucose uptake was significantly depressed ($\text{V}\text{decreased}\text{, K}{}_{\mathrm{<mtext>m</mtext>}}\text{unaltered}$), phosphorylation of 2-deoxyglucose was seemingly unimpaired; basal 3-methylglucose entry was significantly increased, insulin-stimulated uptake was unaltered. Insulin significantly reduced $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ in control and endotoxin-treated cells. Cytochalasin B-insensitive uptake of both 2-deoxyglucose and 3-methylglucose, a small fraction of total transport, increased significantly in endotoxic cells. Endotoxin reduced spermine- and insulin-stimulated 2-deoxyglucose uptake to a similar extent. Results are consistent with the hypotheses that (1) a site of endotoxin-induced insulin resistance is at the cell membrane level and may reflect a decrease in number or activity of effective carrier units, rather than alterations in affinity, (2) endotoxin does not compromise the hexokinase system, (3) the cell membrane-localized effect of endotoxin on hexose transport is not necessarily mediated by the insulin receptor and (4) the entry of 2-deoxyglucose and 3-methylglucose may involve two separate transport systems.