Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli

Osmotic upshock of E. coli cells in NaCl or sucrose medium resulted in a large decrease in the cytoplasmic volume and the inhibition of growth, of the electron transfer chain and of four different types of sugar transport system: the lactose proton symport, the glucose phosphotransferase system, the binding-protein dependent maltose transport system and the glycerol facilitator. In contrast to NaCl and sucrose, the permeant solute glycerol had no marked effect. These inhibitions could be partially relieved by glycine betaine. Despite these inhibitions, the internal pH, the protonmotive force and the ATP pool were maintained. It is concluded that inhibition of electron transfer and of sugar transport is the consequence of conformational changes caused by the deformation of the membrane. It is also concluded that the arrest of growth observed upon osmotic upshock is not due to energy limitations and that it cannot be explained by the inhibition of carbohydrate transport.     

Osmotic stress drastically inhibits active transport of carbohydrates by Escherichia coli

In intact Escherichia coli cells, severe osmotic stress almost totally inhibited active transport of carbohydrate by all of the systems known to transport carbohydrates in E. coli: group translocation (glucose), binding-protein mediated transport (maltose), proton symport (lactose), and sodium cotransport (melibiose). Detailed study of glucose transport showed that this inhibition of transport was not secondary to the inhibition of growth by osmotic stress, but rather that the inhibition of transport of a source of carbon and energy was sufficient to cause the complete inhibition of growth observed during severe osmotic upshock. Transport and growth inhibition did not result from cell death; upshocked cells were viable and metabolically active.     

Betaine and L-carnitine transport by Listeria monocytogenes Scott A in response to osmotic signals.

The naturally occurring compatible solutes betaine and L-carnitine allow the food-borne pathogen Listeria monocytogenes to adjust to environments of high osmotic strength. Previously, it was demonstrated that L. monocytogenes possesses an ATP-dependent L-carnitine transporter (A. Verheul, F. M. Rombouts, R. R. Beumer, and T. Abee, J. Bacteriol. 177:3205-3212, 1995). The present study reveals that betaine and L-carnitine are taken up by separate highly specific transport systems and support a secondary transport mechanism for betaine uptake in L. monocytogenes. The initial uptake rates of betaine and L-carnitine are not influenced by an osmotic upshock, but the duration of transport of both osmolytes is directly related to the osmotic strength of the medium. Regulation of uptake of both betaine and L-carnitine is subject to inhibition by preaccumulated solute. Internal betaine inhibits not only transport of external betaine but also that of L-carnitine and, similarly, internal L-carnitine inhibits transport of both betaine and L-carnitine. The inhibition is alleviated upon osmotic upshock, which suggests that alterations in membrane structure are transmitted to the allosteric binding sites for betaine and L-carnitine of both transporters at the inner surface of the membrane. Upon osmotic downshock, betaine and L-carnitine are rapidly released by L. monocytogenes as a consequence of activation of a channel-like activity. The osmolyte-sensing mechanism described is new and is consistent with various unexplained observations of osmoregulation in other bacteria.     

The biochemical and ultrastructural effects of tunicamycin and D-glucosamine in L1210 leukemic cells

Tunicamycin was found to specifically inhibit the incorporation of a number of sugars into L1210 leukemia cell glycoproteins. This inhibition of glyco-protein biosynthesis led to a cessation of cell growth which was reversible in a dose-dependent and time-dependent manner. After removal of the antibiotic from L1210 cell cultures resumption of sugar incorporation preceded that of thymidine incorporation and the recovery of cell growth. The treatment of cells with tunicamycin resulted in a significant increase in the intracellular pool of UDP-N-acetylglucosamine which occurred concurrently with alterations in cell ultrastructure including distentions of the endoplasmic reticulum and nuclear membranes. Similar ultrastructural changes and increases in the intracellular pools of UDP-sugars were observed in L1210 cells exposed to 5 mM D-glucosamine, which suggested that the antiproliferative effects of tunicamycin may be related to the accumulation in the endoplasmic reticulum of one or more nucleotide sugar precursors of asparagine-linked glycoprotein biosynthesis. However, the biological effects of tunicamycin could be distinguished from those caused by D-glucosamine. Exposure of L1210 cells to tunicamycin resulted in specific alterations in the biochemical composition of the plasma membrane and in the inhibition of cellular agglutination by wheat germ agglutinin which were not apparent following exposure to equitoxic concentrations of the aminosugar. These studies, together with those which demonstrated that recovery of the cellular capacity to synthesize glycoproteins was obligatory for the recovery of cellular proliferation in tunicamycin-treated cells, suggested that inhibition of the synthesis of glycoproteins was the major factor limiting L1210 leukemic cell proliferation.     

The effect of azaserine on glutamine uptake by rat renal brush-border membranes.

Azaserine added directly to isolated rat renal brush-border membrane vesicles inhibits uptake of L-glutamine. Azaserine acts as a non-competitive inhibitor of the low-Km system for glutamine transport, but has no effect on the high-Km system. Preincubation of the vesicles with azaserine at 37 degrees C min is not required for transport inhibition to occur, although it is a requirement for gamma-glutamyl transpeptidase inhibition. Removal of azaserine from the vesicle preparation by repeated resuspensions in buffer results in a reversal of the transport inhibition but not in reversal of enzyme inhibition. Azaserine also inhibits vesicle uptake of L-proline and alpha-methyl D-glucoside, indicating a generalized effect on membrane transport systems. The data cast doubt on the postulate that gamma-glutamyl transpeptidase might act as the carrier mechanism for glutamine reabsorption by renal cortical cells.     

Growth regulatory activities of endothelial extracellular matrix: mediation by transforming growth factor-beta

The potential of transforming growth factor-beta (TGF-beta) to modulate the growth of endothelial cells via alterations in the cell's extracellular matrix (ECM) was examined. Rat brain endothelial cells were cultured in the presence or absence of TGF-beta, and subsequently ECM was prepared from the cell cultures by hypotonic lysis of the cells. Untreated endothelial cells were then cultured on the various matrices. Cells grown on TGF-beta-treated ECM showed a significant decrease in cell number (41 +/- 6% mean growth inhibition at 6 days, P less than 0.005 by paired T-test) compared with cells grown on untreated ECM. The growth inhibitory activity of the ECM was depleted by 9 days of culture, and resumption of exponential cell growth was observed. A similar phenomenon was observed if anti-TGF-beta neutralizing antibodies were incubated with the ECM. When the TGF-beta-treated matrix was exposed to a brief dithiothreitol treatment in order to inactivate residual TGF-beta, an approximately equal degree of growth inhibition was observed initially, but the reversal of inhibition occurred at an earlier time point than that with unreduced TGF-beta-treated matrix. Analyses of the composition of matrices synthesized in the presence or absence of TGF-beta revealed about a twofold increase in the accumulation of various radioactive metabolic precursors in the TGF-beta-treated matrices. However, no qualitative alterations in the matrix or cellular-associated proteins or glycoproteins were observed, as analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. An increase in cell-associated heparan sulfate, however, was observed in TGF-beta-treated cells. The results suggest that certain growth regulatory effects of TGF-beta may be mediated, at least in part, by alterations in the ECM.     

Involvement of histidine residues and sulfhydryl groups in the function of the biotin transport carrier of rabbit intestinal brush-border membrane.

Possible involvement of histidine residues and sulfhydryl groups in the function of the intestinal brush-border membrane (BBM) transporter of biotin was investigated. This was done by examining the effects of pretreatment of BBM vesicle (BBMV) isolated from rabbit intestine with the histidine-specific reagent diethyl pyrocarbonate (DEPC) and the sulfhydryl group-specific reagents p-chloromercuribenzenesulfonic acid (p-CMBS) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) on carrier-mediated biotin transport. Pretreatment of BBMV with DEPC caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with DEPC provided significant protection to biotin transport. Treatment of DEPC-pretreated vesicles with the reducing agents dithiothreitol and 2,3-dimercaptopropanol failed to reverse the inhibitory effect of DEPC on biotin transport. The inhibitory effect of DEPC was found to be mediated through a marked decrease in the number of the functional biotin transport carriers with no change in their affinity, as indicated by the severe inhibition in the Vmax but not the apparent Km of the biotin transport process, respectively. Pretreatment of BBMV with p-CMBS and NBD-Cl also caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with p-CMBS (or NBD-Cl) failed to protect biotin transport from inhibition. On the other hand, treatment of vesicles pretreated with p-CMBS (or NBD-Cl) with the reducing agents dithiothreitol and mercaptoethanol caused significant reversal in the inhibition of biotin transport. The inhibitory effects of p-CMBS (and NBD-Cl) on biotin transport was also found to be mediated through inhibition in the Vmax, but not the apparent Km, of biotin transport process. These results indicate the involvement of histidine residues and sulfhydryl groups in the normal function of the biotin transport system of rabbit intestinal BBM. Furthermore, the results also suggest that the histidine residues are probably located at (or near) the substrate-binding site while the sulfhydryl groups are located at a site other than the substrate binding region.     

Examination of sodium/glucose cotransport by using a visible glucose analogue

The glucose derivative, 2,2,6,6-tetramethylpiperidine-1-oxylglucose (TEMPO-glucose) was synthesized and examined for its ability to substitute for glucose as a substrate for the intestinal brush border membrane Na+/glucose cotransporter. TEMPO-glucose inhibited Na(+)-dependent phlorizin binding with an apparent KI of 18 microM and Na(+)-dependent glucose uptake with an apparent KI of 70 microM. The transport competence of TEMPO-glucose was examined by using two measures of transport. The first involved comparing the reversal of trans Na+ inhibition by D-glucose and TEMPO-glucose. The second directly examined Na(+)-dependent TEMPO-glucose uptake by using TEMPO-glucose quenching of intravesicular fluorescein sulfonate fluorescence. Tryptophan fluorescence was sensitive to TEMPO-glucose in a Na(+)-dependent, glucose-inhibitable manner. The bulk of these tryptophans appeared to be located in hydrophobic environments based on Cs(+)-insensitivity. With the reconstituted cotransporter, TEMPO-glucose, and tryptophan quench reagents, the cotransporter was compared in three transport modes: zero trans uptake, zero trans uptake in the presence of a shunt of membrane potential, and substrate exchange. The results suggest that the cotransporter conformation varies depending on its mode of operation and that TEMPO-glucose may be a useful probe for localizing amino acid residues involved in glucose transport.     

Reversal of quinone-induced chlorophyll fluorescence quenching

We have used two methods to investigate the reversibility of the interaction of substituted quinones with the thylakoid membrane of plant chloroplasts. Treatment of chloroplasts with added quinones lowers the room-temperature Photosystem II chlorophyll fluorescence intensity by variable amounts depending on the identity and concentration of the quinone. The extent of restoration of the chlorophyll fluorescence level is used as a measure of the effectiveness of the reversal technique. One reversal method involves the addition of thiols to quinone-treated chloroplasts to alter the quinone in a chemical way via a nucleophilic 1,4-Michael addition. In general, the modified quinones exhibit a lower affinity for the thylakoid membrane, as evidenced by an accompanying increase in chlorophyll fluorescence. The thiol concentrations necessary for quenching reversal are found to be in the order [dithiothreitol] less than [2-mercaptoethanol] less than [glutathione]. The second reversal method examines the extent to which added quinones can be removed from thylakoid membranes using a concentration gradient established by resuspension of quinone-treated chloroplasts in quinone-free media. The results further support the reversible nature of the quinone inhibition and indicate that the extent of recovery is dependent upon the degree of fluorescence inhibition originally induced by the added quinone.     

Reversible and irreversible inhibition of phosphate transport in human erythrocytes by a membrane impermeant carbodiimide

Phosphate entry into chloride-loaded human erythrocytes is inhibited by treatment of cells with the water-soluble carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) in the absence of added nucleophile. EAC does not penetrate the erythrocyte membrane or lead to significant intermolecular cross-linking of membrane proteins. At neutral extracellular pH in chloride-free medium, only about 50% of transport is rapidly and irreversibly inhibited, but at alkaline pH, inhibition is more rapid and complete. Inhibition by EAC was reversible in the presence of extracellular NaCl. Modification of membrane sulfhydryl groups does not prevent inhibition of phosphate transport by EAC but almost complete protection is afforded by 4,4-dinitrostilbene-2,2-disulfonic acid, a reversible competitive inhibitor of anion transport. N-(4-Azido-2-nitrophenyl)-2-aminoethylsulfonate, a reversible noncompetitive inhibitor of anion transport did not protect against EAC inhibition of transport but prevented reversal of inhibition in saline medium. Transport inhibition by [3H]EAC did not lead to specific incorporation of radioactivity into Band 3, the anion transport protein. These results suggest that inhibition of anion transport by EAC is due to modification of a carboxylic acid residue in or near the transport site accessible from the external face of the membrane. The subsequent fate of the modified carboxyl residue appears to be sensitive to the orientation of the anion transport site.     

Cholestasis-induced alterations of the trans- and paracellular pathways in rat hepatocytes

Bile secretion depends on the vectorial transport of solutes from blood to bile and involves three different pathways: transcellular pathways mediated by transport proteins distributed asymmetrically in the basolateral and canalicular plasma membrane and by transcytotic vesicles, and a paracellular pathway allowing selective diffusion through tight junctions. All three pathways are impaired differentially by extrahepatic (bile duct ligation) or intrahepatic (ethinyloestradiol) cholestasis. Ethinyloestradiol treatment leads to tight junctional defects that are less severe than those induced by bile duct ligation. Junctional impairment is reflected functionally in increased permeability for horseradish peroxidase and structurally by decreased strand numbers and increased junctional length, but not by alterations at the level of the individual strands. The parallelism of physiophical and morphological perturbations indicates a structure-function relationship in hepatocellular tight junctions. In addition, impaired functional integrity of tight junctions following bile duct ligation is reflected in a partial loss of hepatocellular surface polarity owing to redistribution of some, but not all, domain-specific plasma membrane antigens, which might mimic the behaviour of transport systems. After ethinyloestradiol treatment no alterations of surface polarity were observed. Thus, immunohistochemistry supports the view that ethinyloestradiol results in less severe impairment of the tight junctions than bile duct ligation. Finally, bile duct ligation, but not ethinyloestradiol, affects the transcytotic vesicular pathway; severe impairment of this is reflected in the absence of a late horseradish peroxidase peak in bile and also in the accumulation of pericanalicular vesicles that are immunopositive for canalicular membrane proteins and accessible for bulk phase endocytic markers. This view is supported by the disappearance of the pericanalicular vesicles simultaneously with the resumption of transcytotic horseradish peroxidase transport following release of ligation, a finding that indicates rapid restoration of this defect.This paper was presented at the symposium Metabolic Zonation of the Liver: New Answers to Old Questions, held in honour of Prof. Dr. D. Sasse's 60th birthday, 26 August 1994, in Basel     

Selective inhibition of carbohydrate transport by the local anesthetic procaine in Escherichia coli.

Maltose and lactose transport systems have been used to investigate the action of procaine on insertion and activity of membrane proteins and translocation of exported proteins in Escherichia coli. Procaine mildly inhibited growth on lactose. The level of inhibition was consistent with the small reduction observed in active and facilitated transport functions of the lac permease. However, procaine caused a severe reduction of growth rate on maltose, as well as an inhibition of induction of maltose regulon activities. In both constitutive and inducible strains, the synthesis of both maltose transport activity (malB operon) and amylomaltase activity (malA operon) was inhibited. Coordinate inhibition of soluble and membrane products was not observed with the lac operon. beta-Galactosidase synthesis proceeded normally during growth on procaine, whereas, the appearance of new transport activity was reduced. Regardless of carbon source, procaine specifically inhibited the appearance of ompF protein in the membrane fraction.     

Characterization of the thermotolerant cell. I. Effects on protein synthesis activity and the regulation of heat-shock protein 70 expression

Exposure of mammalian cells to a nonlethal heat-shock treatment, followed by a recovery period at 37 degrees C, results in increased cell survival after a subsequent and otherwise lethal heat-shock treatment. Here we characterize this phenomenon, termed acquired thermotolerance, at the level of translation. In a number of different mammalian cell lines given a severe 45 degrees C/30-min shock and then returned to 37 degrees C, protein synthesis was completely inhibited for as long as 5 h. Upon resumption of translational activity, there was a marked induction of heat-shock (or stress) protein synthesis, which continued for several hours. In contrast, cells first made thermotolerant (by a pretreatment consisting of a 43 degrees C/1.5-h shock and further recovery at 37 degrees C) and then presented with the 45 degrees C/30-min shock exhibited considerably less translational inhibition and an overall reduction in the amount of subsequent stress protein synthesis. The acquisition and duration of such "translational tolerance" was correlated with the expression, accumulation, and relative half-lives of the major stress proteins of 72 and 73 kD. Other agents that induce the synthesis of the stress proteins, such as sodium arsenite, similarly resulted in the acquisition of translational tolerance. The probable role of the stress proteins in the acquisition of translational tolerance was further indicated by the inability of the amino acid analogue, L-azetidine 2-carboxylic acid, an inducer of nonfunctional stress proteins, to render cells translationally tolerant. If, however, analogue-treated cells were allowed to recover in normal medium, and hence produce functional stress proteins, full translational tolerance was observed. Finally, we present data indicating that the 72- and 73-kD stress proteins, in contrast to the other major stress proteins (of 110, 90, and 28 kD), are subject to strict regulation in the stressed cell. Quantitation of 72- and 73-kD synthesis after heat-shock treatment under a number of conditions revealed that "titration" of 72/73-kD synthesis in response to stress may represent a mechanism by which the cell monitors its local growth environment.     

Amino acids are general growth inhibitors of Nicotiana silvestris in tissue culture

The growth of Nicotiana silvestris in suspension culture is inhibited by all of the common protein amino acids at the millimolar level, except for L-glutamine. A defined experimental system for growth/inhibition studies has been established, and growth studies were carried out with cells that had been maintained in the exponential growth phase for at least 10 generations (EE cells). The following results were obtained after particularly detailed studies with aromatic amino acids. The onset of inhibition was preceded by a duration of normal growth rate which varied within a range of 12 to 48 h. The degree of inhibition was directly proportional to amino acid concentration and inversely related to the initial cell density of the inoculum. A slowed, but still exponential rate of growth persisted during an early phase of inhibition. Under sufficiently severe conditions, this was followed by progressive diminution of growth rate and eventual lysis. The most drastic inhibitory effects caused by aromatic amino acids were in the order: phenylalanine, tryptophan and tyrosine. When EE cells cultivated under conditions of growth inhibition were diluted into fresh medium, immediate resumption of growth at the uninhibited rate occurred and persisted. On the other hand, when growth-inhibited EE cells were diluted into medium containing the same concentration of amino acid used in the first round of growth, an initial burst of uninhibited growth lasting about 24 h was followed by a drastic, progressively declining growth rate which deteriorated to cell death and lysis. When cells in stationary phase were used as an inoculum, as is done in typical growth characterizations with suspension cultures, the sensitivity to inhibition during the subsequent exponential growth phase was several-fold greater than was the case with EE cells. Hypotheses that growth inhibition might be caused by ammonia toxicity, keto-acid toxicity, or by inhibition of nitrate utilization were ruled out. Observations that provide new insight are: (i)growth-inhibited cells undergo drastic plasmolysis, (ii) L-glutamine is an effective antagonist of amino-acid inhibitors, and (iii) growth-inhibited cells exhibit a transient restoration of normal growth rate upon dilution into fresh growth medium. These results implicate a linkage of amino acids with osmotic regulation and nitrogen metabolism.     

Cyanide inhibition and pyruvate-induced recovery of cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

The mechanism of cyanide’s inhibitory effect on the mitochondrial cytochrome c oxidase (COX) as well as the conditions for its recovery have not yet been fully explained. We investigated three parameters of COX function, namely electron transport (oxygen consumption), proton transport (mitochondrial membrane potential Δψ _m) and the enzyme affinity to oxygen (p ₅₀ value) with regard to the inhibition by KCN and its reversal by pyruvate. 250 μM KCN completely inhibited both the electron and proton transport function of COX. The inhibition was reversible as demonstrated by washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60–80% of the original values. When using low KCN concentrations of up to 5 μM, we observed a profound, 30-fold decrease of COX affinity for oxygen. Again, this decrease was completely reversed by washing mitochondria while pyruvate induced only a partial, yet significant recovery of oxygen affinity. Our results demonstrate that the inhibition of COX by cyanide is reversible and that the potential of pyruvate as a cyanide poisoning antidote is limited. Importantly, we also showed that the COX affinity for oxygen is the most sensitive indicator of cyanide toxic effects.     

Muscarinic cholinergic inhibition of beta-adrenergic stimulation of phospholamban phosphorylation and Ca2+ transport in guinea pig ventricles

The effects of muscarinic cholinergic stimulation on beta-adrenergic induced increases in phospholamban phosphorylation and Ca2+ transport were studied in intact myocardium. Isolated guinea pig ventricles were perfused via the coronary arteries with 32Pi, after which membrane vesicles were isolated from individual hearts. Isoproterenol produced reversible increases in 32P incorporation into phospholamban. Associated with the increases in 32P incorporation were increases in the initial rate of phosphate-facilitated Ca2+ uptake measured in aliquots of the same membrane vesicles isolated from the perfused hearts. The increases in 32P incorporation and calcium transport were significantly attenuated by the simultaneous administration of acetylcholine. Acetylcholine also attenuated increases in phospholamban phosphorylation and Ca2+ uptake produced by the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin. The contractile effects of all agents which increased cAMP levels (increased contractility and a reduction in the t1/2 of relaxation) were also attenuated by acetylcholine. The inhibitory effects of acetylcholine were associated with attenuation of the increases in cAMP levels produced by isoproterenol and isobutylmethylxanthine but not by forskolin. Acetylcholine also increased the rate of reversal of the functional and biochemical effects of isoproterenol by propranolol without affecting cAMP levels. These results suggest that cholinergic agonists inhibit the functional effects of beta-adrenergic stimulation in part by inhibition of phospholamban phosphorylation. This inhibition may be mediated by two potential mechanisms: inhibition of beta-adrenergic activation of adenylate cyclase and stimulation of dephosphorylation.     

Discrete and reversible vacuole-like dilations induced by osmomechanical perturbation of neurons

In cultured Lymnaea stagnalis neurons, osmolarity increases (upshocks) rapidly elicited large membranous dilations that could be dislodged and pushed around inside the cell with a microprobe. Subsequent osmolarity decreases (downshocks) caused these vacuole-like dilations (VLDs) to disappear. Additional upshock/downshock perturbations resulted in repeated appearance/disappearance (formation/reversal) of VLDs at discrete sites. Confocal microscopy indicated that VLDs formed as invaginations of the substrate-adherent surface of the neuron: extracellular rhodamine-dextran entered VLDs as they formed and was expelled during reversal. Our standard VLD-inducing perturbation was: 2–4 min downshock to distilled water, upshock to normal saline. However, a wide range of other osmotic perturbations (involving osmolarities up to 3.5x normal, perturbations with or without Ca²⁺, replacement of ions by sucrose) were also used. We concluded that mechanical, not chemical, aspects of the osmo-mechanical shocks drove the VLD formation and reversal dynamics and that extracellular Ca²⁺ was not required.Following a standard perturbation, VLDs grew from invisible to their full diameter (>10 m) in just over a minute. Over the next 0.5–3 hr in normal saline, neurons recovered. Recovery eliminated any visible VLDs and was accompanied by cytoplasmic turmoil around the VLDs. Recovery was prevented by cytochalasin B, brefeldin A and N-ethylmaleimide but not by nocodazole. In striking contrast, these drugs did not prevent repeated VLD formation and reversal in response to standard osmo-mechanical perturbations; VLD disappearance during reversal and during recovery are different.The osmo-mechanical changes that elicited VLDs may, in an exaggerated fashion, mimic tension changes in extending and retracting neuntes. In this context we postulate: (a) the trafficking or disposition of membrane between internal stores and plasma membrane is mechano sensitive, (b) normally, this mechanosensitivity provides an on demand system by which neurons can accommodate stretch/release perturbations and control cell shape but, (c) given sudden extreme mechanical stimuli, it yields VLDs. Present address: Biology Department, Washington University, St Louis, MO 63130-4899This work was supported by NSERC of Canada research grants to CEM and to LRM. CR was the recipient of a postdoctoral fellowship from the Ministere francais de la Recherche et de l'Espace. We thank J-M. Trifaro for the use of his image processing equipment.     

Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain

Effects of long-term, subtotal inhibition of Na⁺-K⁺ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K⁺ medium, are described for HeLa cells. After prolonged growth in 2 × 10^?8 M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing amodest induction of Na⁺, K⁺-ATPase. In contrast, after long-term growth in low K⁺ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na⁺] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na⁺]_i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding, V_max for transport, and specific phosphorylation. Parallel exposure of cryptic Na⁺, K⁺-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na⁺, K⁺-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.     

Direct observation of the transmembrane recruitment of band 3 transport sites by competitive inhibitors. A 35Cl NMR study

Numerous models describing anion exchange across the red cell membrane by band 3 have been discussed in literature. These models are readily distinguished from one another by an experiment which tests the ability of band 3 transport sites to be recruited to one side of the membrane. In order to observe directly the transmembrane recruitment of transport sites, we have developed 35Cl NMR techniques that resolve the two transport site populations on opposite sides of the membrane. Using these techniques, we show that the inhibitors 4,4'- dinitrostilbene -2,2'-disulfonate and p- nitrobenzensulfonate each recruit all of the transport sites on both sides of the membrane to the extracellular facing conformation. This result indicates that band 3 has an alternating site transport mechanism: each band 3 transport unit possesses a single functional transport site which is alternately exposed first to one side of the membrane then to the other.     

Glucose transport in Streptococcus agalactiae and its inhibition by lactoperoxidase-thiocyanate-hydrogen peroxide.

Transport of 2-deoxyglucose or glucose in Streptococcus agalactiae was strongly inhibited if the cells were first exposed to a combination of lactoperoxidase-thiocyanate-hydrogen peroxide (LP-complex). The inhibition was completely reversible with dithiothreitol. N-ethylmaleimide and p-chloromercuribenzoate inhibited sugar transport, and the inhibition was also reversible with dithiothreitol. Sodium fluoride also inhibited sugar transport. Glucolysis was completely inhibited, and dithiothreitol completely reversed the inhibition. Phosphoenolpyruvate-dependent phosphotransferase activity in S. agalactiae was not strongly inhibited by the LP-complex. Interference of the entry of glucose into cells of S. agalactiae by the LP-complex could well account for its growth inhibitory properties with this organism. The inhibition of glucose transport by the LP-complex and its reversibility with dithiothreitol suggest the modification of functional sulfhydryl groups in the cell membrane as a cause of transport inhibition.