Quantitative discrimination of carrier-mediated excretion of isoleucine from uptake and diffusion in Corynebacterium glutamicum.

The efflux of isoleucine in whole cells of Corynebacterium glutamicum was studied. The different amino acid fluxes across the plasma membrane were functionally discriminated into passive diffusion, carrier-mediated excretion, and carrier-mediated uptake. Detailed kinetic analysis was made possible by controlled variation of internal isoleucine from low concentrations to 100 mM by feeding with mixtures of isoleucine-containing peptides. Isoleucine diffusion was experimentally separated and proceeded with a first-order rate constant of 0.083 min-1 or 0.13 microliters.min-1.mg (dry mass)-1, which corresponds to a permeability of 2 x 10(-8) cm.s-1. Uptake of isoleucine was constant at a rate of 1.1 nmol.min-1.mg (dry mass)-1. Carrier-mediated isoleucine excretion was zero below a threshold of 8 mM cytosolic isoleucine. Above this level, a Michaelis-Menten-type kinetics was observed, with a Km of 21 mM (13 mM plus 8 mM threshold value) and a Vmax of 14.5 nmol.min-1.mg (dry mass)-1. The activity of the isoleucine excretion carrier depended on the presence of a membrane potential. Excretion was specific for L-isoleucine (and presumably L-leucine) and could be inhibited by SH reagents.     

Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum.

Osmoregulatory uptake of glycine betaine in whole cells of Corynebacterium glutamicum ATCC 13032 (wild type) was studied. The cells actively take up glycine betaine when they are osmotically shocked. The total accumulation and uptake rate were dependent on the osmotic strength of the medium. Kinetic analysis revealed a high-affinity transport system (Km, 8.6 +/- 0.4 microM) with high maximum velocity (110 nmol.min-1.mg [dry weight]-1). Glycine betaine functioned as a compatible solute when added to the medium and allowed growth at an otherwise inhibitory osmotic strength of 1.5 M NaCl. Proline and ectoine could also be used as osmoprotectants. Glycine betaine is neither synthesized nor metabolized by C. glutamicum. The glycine betaine transport system is constitutively expressed at a basal level of activity. It can be induced up to eightfold by osmotic stress and is strongly regulated at the level of activity. The transport system is highly specific and has its pH optimum in the slightly alkaline range at about pH 8. The uptake of the zwitterionic glycine betaine is mediated by a secondary symport system coupled to cotransport of at least two Na+ ions. It is thus driven both by the membrane potential and the Na+ gradient. An extremely high accumulation (internal/external) ratio of up to 4 x 10(6) was measured, which represents the highest accumulation ratio observed for any transport system.     

Permeability to cefsulodin of the outer membrane of Pseudomonas aeruginosa and discrimination between beta-lactamase-mediated trapping and hydrolysis as mechanisms of resistance

A pair of strains of Pseudomonas aeruginosa (3-Pre: cefsulodin-sensitive, inducible beta-lactamase; and 3-Post: cefsulodin-resistant, elevated beta-lactamase, derived from 3-Pre by subculture in the presence of cefsulodin) were taken as representative of the class of bacteria resistant to third-generation cephalosporins due to elevated synthesis of the normally inducible, chromosomally encoded beta-lactamase. These two strains were used to differentiate between 'trapping' and 'hydrolytic' mechanisms of cefsulodin resistance by (a) measuring the outer-membrane permeabilities to cefsulodin, (b) measuring the kinetics of cefsulodin hydrolysis and the stoichiometry of cefsulodin trapping by the periplasmic beta-lactamase, and (c) comparing the predictions of the trapping and hydrolysis hypotheses with the minimum inhibitory concentrations (MIC) of cefsulodin. The MIC of cefsulodin for strains 3-Pre and 3-Post were 2.35 microM (1.25 micrograms ml-1) and 37.6 microM (20.0 micrograms ml-1) respectively. The permeability parameter for cefsulodin of the outer membrane of the resistant strain was 0.0034 cm3 min-1 mg dry mass-1, so the flux of cefsulodin across its outer membrane at the MIC was calculated to be 0.120 nmol min-1 mg dry mass-1. Hydrolysis of cefsulodin by the beta-lactamase in the periplasm occurred at a rate of 0.118 nmol min-1 mg dry mass-1 which can thus account for resistance by matching the above rate of inflow. Trapping by the beta-lactamase, even with a 1:1 stoichiometry, would require the enzyme to be synthesized at 5.0 micrograms protein min-1 mg dry mass-1 or about 40% of the dry mass/generation. We conclude that hydrolysis, but not trapping, adequately explains the resistance to cefsulodin in P. aeruginosa 3-Post. A similar calculation for latamoxef resistance, using data taken from the literature, led to the same conclusion.     

Lysine excretion by Corynebacterium glutamicum. 1. Identification of a specific secretion carrier system

Corynebacterium glutamicum effectively excretes lysine when the internal lysine concentration is elevated. Lysine efflux was investigated using selected mutants which are not able to regulate lysine biosynthesis by feedback inhibition. Secretion of lysine is not the consequence of unspecific permeability of the plasma membrane but is mediated by a secretion carrier which is specific for lysine. Lysine export is characterized by high activation energy and follows Michaelis-Menten type kinetics with an internal Km of 20 mM and a Vmax of 12 nmol.min-1.mg dry cells-1. Excretion can proceed against a preexisting chemical gradient and against the electrical potential, which rules out a previously suggested pore model. Lysine excretion can also be observed in the wild-type strain especially under conditions of peptide uptake. Its possible physiological function may be related to regulation of internal amino acid concentrations under special growth conditions.     

Identification and characterization of γ-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum

Corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as γ-aminobutyric acid (GABA). In this study, a C. glutamicum GABA-specific transporter (GabP(Cg)) encoded by ncgl0464 was identified and characterized. GabP(Cg) plays a major role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabP(Cg) was weakly competed by l-Asn and l-Gln and stimulated by sodium ion (Na(+)). The K(m) and V(max) values were determined to be 41.1 ± 4.5 μM and 36.8 ± 2.6 nmol min(-1) (mg dry weight [DW])(-1), respectively, at pH 6.5 and 34.2 ± 1.1 μM and 67.3 ± 1.0 nmol min(-1) (mg DW)(-1), respectively, at pH 7.5. GabP(Cg) has 29% amino acid sequence identity to a previously and functionally identified aromatic amino acid transporter (TyrP) of Escherichia coli but low identities to the currently known GABA transporters (17% and 15% to E. coli GabP and Bacillus subtilis GabP, respectively). The mutant RES167 Δncgl0464/pGXKZ9 with the GabP(Cg) deletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabP(Cg) represents a new type of GABA transporter and is potentially important for engineering GABA-producing C. glutamicum strains.     

Characterization of a secondary uptake system for l-glutamate in Corynebacterium glutamicum

Abstract A new transport system for the uptake of l-glutamate was characterized in Corynebacterium glutamicum strain Δ glu, in which the previously described binding protein-dependent glutamate uptake system is not present. Kinetic characterization revealed a highly specific secondary transport system, dependent on sodium ions. Glutamate uptake showed Michaelis-Menten kinetics, with a K _m of 0.6 mM and a V _max of 15 nmol min⁻¹ (mg dw)⁻¹. For the co-transported sodium ions, a relatively low K _m of 3.3 mM was determined.     

Physiological basis for the high salt tolerance of Debaryomyces hansenii.

The effects of KCl, NaCl, and LiCl on the growth of Debaryomyces hansenii, usually considered a halotolerant yeast, and Saccharomyces cerevisiae were compared. KCl and NaCl had similar effects on D. hansenii, indicating that NaCl created only osmotic stress, while LiCl had a specific inhibitory effect, although relatively weaker than in S. cerevisiae. In media with low K+, Na+ was able to substitute for K+, restoring the specific growth rate and the final biomass of the culture. The intracellular concentration of Na+ reached values up to 800 mM, suggesting that metabolism is not affected by rather high concentrations of salt. The ability of D. hansenii to extrude Na+ and Li+ was similar to that described for S. cerevisiae, suggesting that this mechanism is not responsible for the increased halotolerance. Also, the kinetic parameters of Rb+ uptake in D. hansenii (Vmax, 4.2 nmol mg [dry weight]-1 min-1; K(m), 7.4 mM) indicate that the transport system was not more efficient than in S. cerevisiae. Sodium (50 mM) activated the transport of Rb+ by increasing the affinity for the substrate in D. hansenii, while the effect was opposite in S. cerevisiae. Lithium inhibited Rb+ uptake in D. hansenii. We propose that the metabolism of D. hansenii is less sensitive to intracellular Na+ than is that of S. cerevisiae, that Na+ substitutes for K+ when K+ is scarce, and that the transport of K+ is favored by the presence of Na+. In low K+ environments, D. hansenii behaved as a halophilic yeast.     

Adenosine 3'5'-monophosphate phosphodiesterase of buffalo spermatozoa.

The cyclic AMP-phosphodiesterase (EC 3.1.4.17) of buffalo spermatozoa is distributed in the head, mid-piece and tail fractions and has multiple forms, 70% of which is in the bound form. The bound enzyme was not solubilized by Triton X-100, lubrol or hyamine 2389. Kinetic measurements of the soluble enzyme showed two apparent Km values for low and high cAMP concentrations, i.e. 4.5 and 100 micro M with Vmax values of 0.25 and 2.0 nmol cAMP hydrolysed min-1 mg protein-1. The bound enzyme had an apparent Km of 66.6 microM with a Vmax of 0.75 nmol cAMP hydrolysed min-1 mg protein-1. The pH for optimum enzyme activity was 7.5 and Mg2+ was essential for the activity of the soluble and bound enzymes. Methylxanthines, ATP, ADP and ppi inhibited the soluble and bound enzymes, ATP being the most potent inhibitor.     

Active transport of alanine by the neutral amino-acid exchanger ASCT1

ASCT1 protein is a member of the glutamate transporter superfamily, which shows system ASC selectivity and properties and has been characterized as a Na+-dependent neutral amino-acid exchanger. Here, by using ASCT1-expressing oocytes, the uptake of alanine and glutamate was measured to investigate ASCT1's ability to mediate a concentrative transport of alanine, ASCT1's sodium dependence, and the influence of pH on the mutual inhibition between alanine and glutamate. Alanine uptake was measured after 30 min incubation. Kinetic analysis of the Na+ dependence of alanine uptake showed an apparent K0.5 (affinity constant) value for Na+ of 23.1 +/- 4.3 mM (mean +/- SE). Concentration dependence of alanine uptake was tested at 100 and 1 mM Na+, with apparent K0.5 values of 0.16 +/- 0.04 and 1.8 +/- 0.4 mM, respectively, at pH 7.5, and 0.21 +/- 0.06 and 1.9 +/- 0.3 mM at pH 6. Vmax was not modified between 100 and 1 mM Na+ at either pH. ASCT1 actively transports alanine and accumulates it in the cytosol even when the Na+ concentration in the medium was as low as 1-3 mM. 22Na uptake studies revealed that Na+ transport was stimulated by the presence of alanine in the medium. Our results demonstrate that ASCT1 is able to mediate a concentrative transport of alanine, which is Na+-dependent but not coupled to the Na+ gradient.     

Cell-bound peptidase activities of Treponema denticola ATCC 33520 in continuous culture.

The oral spirochaete Treponema denticola ATCC 33520 was grown at a mean generation time of 10 h in anaerobic continuous culture in a serum- and carbohydrate-free medium at pH 7.0. The extracellular proteolytic activities of this spirochaete were then investigated by incubating washed cells with 68 2-naphthylamide derivatives of the Extended API System. Chymotrypsin-like, trypsin-like, elastase-like and iminopeptidase activities were demonstrated. The phenylalanine peptidase or chymotrypsin-like activity of T. denticola ATCC 33520, estimated with N-succinyl-L-phenylalanyl-L-leucyl-L-phenylalanine-thiobenzyl ester (SPLP) had a pH optimum at pH 8.5, a specific activity of 36.6 nmol min-1 (mg dry wt)-1 and was inhibited only slightly by HgCl2. The trypsin-like activity, estimated with benzoyl-DL-arginine-7-amido-4-methylcoumarin (BAMC), had a pH optimum at pH9, and a specific activity of 0.3 nmol min-1 (mg dry wt)-1; inhibition by HgCl2 indicated the involvement of active thiol groups. The activity should preferably be termed arginine peptidase activity, according to the carboxy-terminal amino acid of the test substrate. The extracellular proline peptidase activity, estimated with L-proline-7-amido-4-methylcoumarin. HBr (PRAMC), had an activity of 1.5 nmol min-1 (mg dry wt)-1, an optimum at pH 8.5 and the properties of a thiol protease. The main cell-bound and extracellular active peptidase activities of fast-growing cells of T. denticola ATCC 33520 are phenylalanine peptidase, proline peptidase, arginine peptidase and an oligopeptide-dependent alanine peptidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake of glutamate in Corynebacterium glutamicum. 2. Evidence for a primary active transport system

The inducible glutamate uptake system in Corynebacterium glutamicum (Kr?mer, R., Lambert, C., Hoischen, C. & Ebbighausen, H., preceding paper in this journal) was characterized with respect to its mechanism and energy coupling. All possible secondary active uptake mechanisms can be excluded. Glutamate transport is not coupled to the translocation of H+, Na+ or K+ ions. Although changes in membrane potential and uptake activity cannot completely be separated, no correlation between these two parameters is observed. The uptake of glutamate resembles a primary active, ATP-dependent transport mechanism in several respects. (a) The substrate affinity is very high (1.3 microM). (b) Accumulation of glutamate reaches values of greater than 2.10(5), at least as high as those reported for binding-protein-dependent systems in Gram-negative bacteria. (c) The uptake is unidirectional. Even after complete deenergization, the accumulation ratio was not significantly reduced. (d) The rate of glutamate uptake is directly correlated to the cytosolic ATP content and also to the ATP/ADP ratio. This is shown by varying internal ATP by different procedures applying inhibitors (NaCN, dicyclohexyl carbodiimide), uncouplers (carbonyl m-chlorophenylhydrazone), ionophores (valinomycin), and even by shifting the cells to anaerobiosis. Uptake is not promoted by cytosolic ATP levels below 1.5 mM, the maximum uptake rate is reached at 4-5 mM ATP.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

Regulation of hepatic glutamate metabolism. Role of 2-oxoacids in glutamate release from isolated perfused rat liver

In isolated perfused rat liver, addition of the oxoanalogues of leucine, isoleucine, methionine and phenylalanine is followed by a rapid and reversible stimulation of glutamate release. This is not observed with the corresponding amino acids or 2-oxoisovalerate, 2-oxoglutarate or oxaloacetate. The increased glutamate release by the liver is accompanied by a decrease in the tissue contents of 2-oxoglutarate and glutamate by about 25% and 50%, respectively. During the metabolism of glutamine, i.e. conditions with elevated tissue glutamate concentrations, 2-oxoacid-induced glutamate release is stimulated. In the presence of glutamine (5 mM), 2-oxoisocaproate, 2-oxo-4-methylvalerate and 2-oxo-4-methylthiobutyrate were found to be most effective and glutamate release by the liver increased linearly from about 80 nmol g-1 min-1 to 600 nmol g-1 min-1 at increasing 2-oxoacid concentrations up to 1 mM. When glutamate tissue levels were decreased by phenylephrine, stimulation of glutamate release by 2-oxoisocaproate was markedly diminished. 2-Oxoacid-stimulated glutamate release is independent of oxoacid metabolism, indicating that the effect is probably not explained by a 2-oxoacid/glutamate exchange across the liver plasma membrane. 2-Oxoacid-induced glutamate export predominantly occurs in a sodium-independent way. At low concentrations of 2-oxoisocaproate (below 0.2 mM), the increased glutamate release was accompanied by a slight inhibition of 14CO2 production from added [14C]glutamate, indicating a simultaneous glutamate uptake and release also under these conditions. Stimulation of glutamate release by 2-oxoisocaproate is followed by a decreased rate of urea and glutamine synthesis from portal ammonia, as a consequence of an increased glutamate release.     

Mechanism of glutamate uptake in Zymomonas mobilis.

The energetics of the anaerobic gram-negative bacterium Zymomonas mobilis, a well-known ethanol-producing organism, is based solely on synthesis of 1 mol of ATP per mol of glucose by the Entner-Doudoroff pathway. When grown in the presence of glucose as a carbon and energy source, Z. mobilis had a cytosolic ATP content of 3.5 to 4 mM. Because of effective pH homeostasis, the components of the proton motive force strongly depended on the external pH. At pH 5.5, i.e., around the optimal pH for growth, the proton motive force was about -135 mV and was composed of a pH gradient of 0.6 pH units (internal pH 6.1) and a membrane potential of about -100 mV. Measurement of these parameters was complicated since ionophores and lipophilic probes were ineffective in this organism. So far, only glucose transport by facilitated diffusion is well characterized for Z. mobilis. We investigated a constitutive secondary glutamate uptake system. Glutamate can be used as a nitrogen source for Z. mobilis. Transport of glutamate at pH 5.5 shows a relatively high Vmax of 40 mumol.min-1.g (dry mass) of cells-1 and a low affinity (Km = 1.05 mM). Glutamate is taken up by a symport with two H+ ions, leading to substantial accumulation in the cytosol at low pH values.     

Structure of the gluABCD cluster encoding the glutamate uptake system of Corynebacterium glutamicum.

To assess the mechanism and function of the glutamate uptake system of gram-positive Corynebacterium glutamicum, a mutant deficient in glutamate uptake was isolated and was then used to isolate a DNA fragment restoring this deficiency. In a low-copy-number vector, this fragment resulted in an increased glutamate uptake rate of 4.9 nmol/min/mg (wild type, 1.5 nmol/min/mg). In addition, carbon source-dependent regulation of the glutamate uptake system was determined with the fragment, showing that the entire structures required for expression and control reside on the fragment isolated. Sequencing of 3,977 bp revealed the presence of a four-gene cluster (gluABCD) with deduced polypeptide sequences characteristic of a nucleotide-binding protein (GluA), a periplasmic binding protein (GluB), and integral membrane proteins (GluC and GluD), identifying the glutamate transporter as a binding protein-dependent system (ABC transporter). This identification was confirmed by the kinetic characteristics obtained for cells grown in the presence of globomycin, which exhibited an increased Km of 1,400 microM (without globomycin, the Km was 1.5 microM) but a nearly unaltered maximum velocity. By applying gene-directed mutagenesis, a strain with the entire cluster deleted was constructed. With this mutant, the glutamate uptake rate was reduced from 1.4 to less than 0.1 nmol/min/mg, which is proof that this system is the only relevant one for glutamate uptake. With this strain, the glutamate excretion rate was unaffected (18 nmol/min/mg), showing that no component of gluABCD is involved in export but rather that a specific machinery functions for the latter purpose.     

Investigations into enzymes of nitrogen metabolism of the ectomycorrhizal basidiomycete, Suillus bovinus

Axenic mycelia of the ectomycorrhizal basidiomycete, Suillus bovinus, were grown in liquid media under continuous aeration with compressed air at 25 degrees C in darkness. Provided with glucose as the only carbohydrate source, they produced similar amounts of dry weight with ammonia, with nitrate or with alanine, 60-80% more with glutamate or glutamine, but about 35% less with urea as the respectively only exogenous nitrogen source. In crude extracts of cells from NH4(+)-cultures, NADH-dependent glutamate dehydrogenase exhibited high aminating (688 nmol x mg protein(-1) x min(-1)) and low deaminating (21 nmol x mg protein(-1) x min(-1)) activities. Its Km-values for 2-oxoglutarate and for glutamate were 1.43 mM and 23.99 mM, respectively. pH-optimum for amination was about 7.2, that for deamination about 9.3. Glutamine synthetase activity was comparatively low (59 nmol x mg protein(-1) x min(-1)). Its affinity for glutamate was poor (Km = 23.7 mM), while that for the NH4+ replacing NH2OH was high (Km = 0.19 mM). pH-optimum was found at 7.0. Glutamate synthase (= GOGAT) revealed similar low activity (62 nmol x mg protein(-1) x min(-1)), Km-values for glutamine and for 2-oxoglutarate of 2.82 mM and 0.28 mM, respectively, and pH-optimum around 8.0. Aspartate transaminase (= GOT) exhibited similar affinities for aspartate (Km = 2.55 mM) and for glutamate (Km = 3.13 mM), but clearly different Km-values for 2-oxoglutarate (1.46 mM) and for oxaloacetate (0.13 mM). Activity at optimum pH of about 8.0 was 506 nmol x mg protein(-1) x min(-1) for aspartate conversion, but only 39 nmol x mg protein(-1) x min(-1) at optimum pH of about 7.0 for glutamate conversion. Activity (599 nmol x mg protein(-1) x min(-1)), substrate affinities (Km for alanine = 6.30 mM, for 2-oxoglutarate = 0.45 mM) and pH-optimum (6.5-7.5) proved alanine transaminase (= GPT) also important in distribution of intracellular nitrogen. There was comparatively low activity of the obviously constitutive enzyme, urease, (42 nmol x mg protein(-1) x min(-1)) whose substrate affinity was rather high (Km = 0.56 mM). Nitrate reductase proved substrate induced; activity could only be measured after exposure of the mycelia to exogenous nitrate. Routes of entry of exogenous nitrogen and tentative significance of the various enzymes in cell metabolism are discussed.     

Calcium accumulation and release by the sarcoplasmic reticulum of digitonin-lysed adult mammalian ventricular cardiomyocytes.

We have developed a model for characterizing calcium handling by the intact cardiac sarcoplasmic reticulum (SR) that yields data consistent with both mathematical simulations of in situ SR Ca2+ uptake and deduced behavior of the Ca2(+)-induced Ca2+ efflux channels in mechanically skinned single cardiac cells. In Na(+)-based media (37 degrees C, pH 7.2, 50 mM Pi, 10 mM MgATP, pMg 3.3, 10 mM phosphocreatine), SR 45Ca2+ uptake by digitonin-lysed rat myocytes as a function of free [Ca2+] peaked at pCa 6.2, declined until pCa 5.6 and increased again at lower pCa. When Ca2(+)-induced Ca2+ efflux was inhibited with 30 microM ruthenium red and 10 mM procaine, uptake was saturable with a Vmax of 160 +/- 5 nmol.min-1.mg-1, K0.5 of 500 nM free [Ca2+] and slope factor of 1.6. In K(+)-based media, maximum Pi- and oxalate-supported uptake increased to 220 and 260 nmol.min-1.mg-1, respectively. Without phosphocreatine, 45Ca2+ uptake declined under all conditions; this was correlated with a decrease in ATP/ADP. Vmax for 45Ca2+ uptake was increased 20% in hyperthyroid myocytes but depressed 30% in myocytes from heart failure-prone rats. In canine myocytes, Vmax was the same as in normal rat cells, but K0.5 was 830 nM. Without efflux inhibitors, ryanodine caused a concentration-dependent decline in net Pi-supported 45Ca2+ uptake at pCa 6.3 (K0.5 = 1 microM), while 10 microM ryanodine depressed uptake at all pCa between 7.2 and 5.6. Ruthenium red/procaine fully reversed this effect.     

Energy-dependent uptake of 4-chlorobenzoate in the coryneform bacterium NTB-1.

The uptake of 4-chlorobenzoate (4-CBA) in intact cells of the coryneform bacterium NTB-1 was investigated. Uptake and metabolism of 4-CBA were observed in cells grown in 4-CBA but not in glucose-grown cells. Under aerobic conditions, uptake of 4-CBA occurred with a high apparent affinity (apparent Kt, 1.7 microM) and a maximal velocity (Vmax) of 5.1 nmol min-1 mg of protein-1. At pH values below 7, the rate of 4-CBA uptake was greatly reduced by nigericin, an ionophore which dissipates the pH gradient across the membrane (delta pH). At higher pH values, inhibition was observed only with valinomycin, an ionophore which collapses the electrical potential across the membrane (delta psi). Under anaerobic conditions, no uptake of 4-CBA was observed unless an alternative electron acceptor was present. With nitrate as the terminal electron acceptor, 4-CBA was rapidly accumulated by the cells to a steady-state level, at which uptake of 4-CBA was balanced by excretion of 4-hydroxybenzoate. The mechanism of energy coupling to 4-CBA transport under anaerobic conditions was further examined by the imposition of an artificial delta psi, delta pH, or both. Uptake of 4-CBA was shown to be coupled to the proton motive force, suggesting a proton symport mechanism. Competition studies with various substrate analogs revealed a very narrow specificity of the 4-CBA uptake system. This is the first report of carrier-mediated transport of halogenated aromatic compounds in bacteria.     

Energy-driven uptake of humic acids by Aspergillus niger.

The uptake of humic acids by mycelia of Aspergillus niger was demonstrated to be energy-dependent with a sensitivity to sodium azide and to 2,4-dinitrophenol. Greater uptake of humic acids by submerged mycelium occurred at pH 3.0 and at 32 degrees C. The rate of uptake was influenced by the concentration of humic acids with an apparent Km of 0.2 grams/ml and with a Vmax of 0.13 mg humic acids per gram mycelial dry weight.10 min-1. In the absence of added energy source, Vmax of 0.05 mg humic acids per gram mycelial dry wt.10 min-1 was obtained; however, the affinity for humic acids by this uptake system was the same as for the energy-driven process. Apparent binding of humic acids to cell structures was indicated because only 41.8% of the humic acids taken up by the energy-dependent system could be recovered.     

(Na,K)-ATPase-mediated cation pumping in cultured rat hepatocytes. Rapid modulation by alanine and taurocholate transport and characterization of its relationship to intracellular sodium concentration

(Na,K)-ATPase is thought to maintain the transmembrane electrochemical sodium gradient which powers secondary active sodium-coupled transport of a variety of solutes including amino acids and bile acids. However, little is known regarding the effect of sodium-coupled solute transport on intracellular sodium concentration ( [Na]ic) and on (Na,K)-ATPase-mediated cation pumping in the intact cell. In order to address this question, we have measured 22Na uptake rate, steady state 22Na content, and ouabain-suppressible 86Rb uptake rate in primary cultures of adult rat hepatocytes under a variety of conditions. Compared with control conditions (sodium uptake rate = 6.00 +/- 0.40 nmol X min-1 X mg-1; [Na]ic = 11.96 +/- 0.54 mM; cation pumping = 2.53 +/- 0.18 nmol X min-1 X mg-1), cation pumping was increased by taurocholate (less than or equal to 158%), alanine (less than or equal to 246%), monensin (less than or equal to 400%), and cold exposure (less than or equal to 525%), and this increase was accompanied by increases in Na uptake and [Na]ic. In contrast, preincubation in low sodium medium decreased all three variables. These changes in cation pumping were blocked in the absence of extracellular sodium and were not accompanied by changes in ouabain-suppressible ATP hydrolysis measured in cell homogenate. An overall plot of cation pumping versus [Na]ic yielded a sigmoid-shaped curve. Values for KNa (17.8 +/- 1.4 mM) and Vmax (8.98 +/- 0.62 nmol X min-1 X mg-1) for cation pumping were estimated assuming three sodium sites per pump unit. These findings indicate that: 1) uptake of alanine and taurocholate is associated with a rapid increase in (Na,K)-ATPase cation pumping; 2) this increase probably results from an increase in pumping per pump unit rather than an increase in the total number of pump units, and it appears to be mediated via an increase in sodium influx and [Na]ic; 3) [Na]ic under control conditions is close to the apparent KNa of cation pumping, implying that substrate availability may be the mechanism whereby sodium uptake is tightly linked to (Na,K)-ATPase cation pumping in intact hepatocytes.