Uptake of [14C]methylammonium by plankton communities: a comparative assay for ammonium transport systems in natural waters

A diverse range of freshwater plankton communities were tested for their ability to take up [14C]methylammonium. Uptake occurred at low substrate levels by high-affinity, energy-requiring, transport systems which were competitively inhibited by ammonium but not by L-amino acids or nicotinamide. A simple competitive inhibition model was used to examine the effects of increasing ammonium levels on uptake in a eutrophic lake. Apparent K1 values for the labelled substrate markedly increased with increasing ammonium. The transport systems had an approximately five-fold greater affinity for ammonium than for methylammonium. The Vmax for methylammonium uptake was relatively insensitive to large changes in ambient ammonium levels. This kinetic parameter may be a useful comparative measure of ammonium transport capacity in natural waters, particularly where low ambient ammonium concentrations preclude the use of 15N.     

Role of glutamine synthetase in the uptake and metabolism of methylammonium by Azotobacter vinelandii.

Methylammonium is a substrate for the ammonium transport system of Azotobacter vinelandii. During cellular uptake methylammonium is rapidly converted to a less polar metabolite (E. M. Barnes, Jr., and P. Zimniak, J. Bacteriol. 146:512-516, 1981). This metabolite has been isolated from A. vinelandii and identified as gamma-glutamylmethylamide by mass spectroscopy, 1H nuclear magnetic resonance spectroscopy, and cochromatography with the authentic compound. Escherichia coli also accumulated gamma-glutamylmethylamide during methylammonium uptake. The biosynthesis of gamma-glutamylmethylamide in vitro required methylammonium, ATP, L-glutamate, and a soluble cell extract from A. vinelandii. The enzyme responsible for gamma-glutamylmethylamide synthesis was glutamine synthetase. In a crude extract, L-methionine-DL-sulfoximine was equipotent in inhibiting the activities for gamma-glutamyltransferase and for the synthesis of glutamine and gamma-glutamylmethylamide. Likewise, an antiserum against the glutamine synthetase of E. coli precipitated the transferase and both synthetic activities at similar titers. During repression by growth of cells on ammonium medium, the synthesis of glutamine and gamma-glutamylmethylamide in vitro was also inhibited coordinately. A partially purified preparation of glutamine synthetase from A. vinelandii utilized methylammonium as substrate (Km = 78 mM, Vmax = 0.30 mumol/min per mg), although less efficiently than ammonium (Km = 0.089 mM, Vmax = 1.1 mumol/min per mg). The kinetic properties of glutamine synthetase with methylammonium as substrate as well as the insensitivity of this activity to inhibition by T1+ were strikingly different from methylammonium translocation. Thus, methylammonium (ammonium) translocation and intracellular trapping as glutamylamides are experimentally distinguishable processes.     

Methylammonium uptake by Rhizobium sp. strain 32H1

We present evidence that methylammonium is transported into cowpea Rhizobium sp. strain 32H1 cells by a membrane carrier whose natural substrate is ammonium. After growth in low (0.2%) oxygen, which is necessary for nitrogen fixation by these cells, respiring rhizobial cells took up [14C]methylammonium to high intracellular levels. Cells grown in atmospheric (21%) oxygen did not take up methylammonium. Uptake (transport plus metabolism) was maximal in cells harvested in the early stationary phase of batch culture and had a distinct pH optimum of 6.5 to 7.0. Uptake was inhibited by metabolic poisons that dissipate the proton motive force or inhibit ATP synthesis. Inhibition of uptake by ammonium and the counterflow phenomenon indicated that ammonium and methylammonium share a transport carrier. Of the methylammonium taken up, about 15% was accumulated to intracellular levels 20 times higher than those in the medium; most of the methylammonium was metabolized to gamma-N-methylglutamine.     

Methylammonium as a substrate for autotrophic growth by wild type Thiobacillus versutus, and mutant strains with lesions in chemolithotrophic metabolism.

Methylammonium consumption and ribulose 1-5-bisphosphate carboxylase (RuBisCO) activity were monitored in cultures of wild type Thiobacillus versutus and mutants deficient in autotrophic metabolism grown under various growth conditions. Only mutants 22, 72, 73 (deficient in ability to oxidize thiosulphate) could grow and develop RuBisCO activity on methylammonium, and assimilate 14CO2 generated as a result of methylammonium metabolism. Mutants 40 and 76, deficient in autotrophic CO2 fixation, showed no 14C methylammonium assimilation and did not oxidize it as a sole substrate within normal incubation periods. Relations of substrate metabolism and RuBisCO regulation in cultures grown on mixtures of thiosulphate or sucrose and methylammonium are described. Further genetic analysis of mutants with defects in autotrophic metabolism may allow localisation of genes responsible for the metabolic effects described.     

Characterization of the high and low affinity components of the renal Ca2(+)-Mg2+ ATPase

The purpose of this study was to characterize the interrelationship between free calcium (Ca2+) and magnesium (Mg2+) in the Ca2+ ATPase enzyme cycle of kidney membranes. Experiments were performed with basolateral membranes from rat renal cortex and microdissected proximal and distal tubules from mice. Results were similar in the three types of preparations. We first investigated the effect of ATP concentration on Ca2(+)- and Mg2(+)-dependent ATP hydrolysis. With 0.2 microM Ca2+, the enzyme activity, as a function of ATP concentration, showed two saturable components: a high affinity component with a Km of 33 microM ATP and a low affinity component with a Km of 0.63 mM ATP. These components may represent either two distinct sites of ATP binding or two forms of the same site. For the sake of simplicity, it was assumed that the two components correspond to a high affinity and a low affinity substrate site. At the high affinity site (ATP = 50 microM), the Ca2+ dependence of ATP hydrolysis followed a single Michaelis-Menten kinetics with Km for Ca2+ of 0.08 microM. The addition of 1 mM Mg2+ resulted in a relatively constant increase in ATP hydrolysis at all Ca2+ concentrations, indicating that the effects of the two cations were additive. With high ATP concentration (ATP = 3 mM), Ca2+ also induced an ATP hydrolysis according to a saturable process, with a Km for Ca2+ of 0.2 microM. In contrast with what occurred with low concentrations of ATP, addition of millimolar Mg2+ completely curtailed the sensitivity of the enzyme to Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Nitrate Transport in Citrus Rootstocks Depending on Nitrogen Availability

Previously, we reported that in Citrus plants, nitrate influx through the plasmalemma of roots cells follows a biphasic pattern, suggesting the existence of at least two different uptake systems, a high and low affinity transport system (HATS and LATS, respectively). Here, we describe a novel inducible high affinity transport system (iHATS). This new nitrate transport system has a high capacity to uptake nitrate in two different Citrus rootstocks (Cleopatra mandarin and Troyer citrange). The iHATS was saturable, showing higher affinity than constitutive high affinity transport system (cHATS) to the substrate NO₃⁻. The V_max for this saturable component iHATS was higher than cHATS, reaching similar values in both rootstocks.Additionally, we studied the regulation of root NO₃⁻ uptake mediated by both HATS (iHATS and cHATS) and LATS. In both rootstocks, cHATS is constitutive and independent of N-status. Concerning the regulation of iHATS, this system is upregulated by NO₃⁻ and down-regulated by the N status and by NO₃⁻ itself when plants are exposed to it for a longer period of time. LATS in Cleopatra mandarin and Troyer citrange rootstocks is repressed by the N-status.The use of various metabolic uncouplers or inhibitors indicated that NO₃⁻ net uptake mediated by iHATS and LATS was an active transport system in both rootstocks.Key Words: Citrus, inducible high affinity transport system (iHATS), constitutive high affinity transport system (cHATS), nitrate uptake, regulation     

Differential binding of antiestrogens by rat uterine and chick oviduct cytosol

Analysis of the interactions of two synthetic estrogen antagonists, tamoxifen and CI 628, with rat uterine and chick oviduct cytosol revealed significant differences in the antiestrogen binding properties of these tissues. In the rat uterus CI 628, tamoxifen and estradiol were bound to a similar number of saturable binding sites and estradiol could completely inhibit the binding of tritiated antiestrogens to these sites. In contrast, high affinity, saturable antiestrogen binding sites in chick oviduct were present at three times the concentration of estradiol binding sites and estradiol could only partially inhibit the binding of tritiated antiestrogens to these sites. It is concluded that antiestrogens bind to the estrogen receptor in both tissues and that chick oviduct has an additional saturable antiestrogen binding site distinct from the classical estrogen receptor site.     

Mutants of Aspergillus nidulans altered in the transport of methylammonium and ammonium

Summary Mutations to methylammonium resistance occur in at least two loci in Aspergillus nidulans, meaA in linkage group IV and meaB in linkage group III. Transport studies using methylammonium-¹⁴C, at a concentration which inhibits protein synthesis in the wild type but not in resistant mutants, show that meaA mutants are defective in methylammonium (and hence ammonium) transport. The ability of meaA mutations to be expressed in the absence of a substrate of the ammonium-methylammonium transport system suggests that ammonium efflux may be involved, although it has not been established whether ammonium influx is also affected.     

The binding of low density lipoprotein by liver membranes in the pig.

Porcine liver membranes are capable of high affinity binding of homologous low density lipoproteins (LDL). Binding is time and temperature dependant and substrate saturable. High affinity binding sites are half saturated at 11 μg/ml lipoprotein-protein. The binding of ¹²⁵I-LDL is inhibited by unlabelled homologous LDL, very low density lipoproteins (VLDL) and high density lipoproteins (HDL) and also be human LDL and HDL, but not by unrelated proteins tested. The binding and displacement patterns with membranes from several other porcine tissues are similar to those of liver membranes. These results suggest the presence of “lipoprotein binding sites” in liver membranes which recognize structural features common to the lipoproteins and further indicate that liver membranes are not unique in their ability to bind LDL.     

Evidence that fungal MEP proteins mediate diffusion of the uncharged species NH(3) across the cytoplasmic membrane

Methylammonium and ammonium (MEP) permeases of Saccharomyces cerevisiae belong to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NH(4)(+) + NH(3)). Transport and accumulation of the ammonium analog [(14)C]methylammonium, a weak base, led to the proposal that members of this family were capable of energy-dependent concentration of the ammonium ion, NH(4)(+). In bacteria, however, ATP-dependent conversion of methylammonium to gamma-N-methylglutamine by glutamine synthetase precludes its use in assessing concentrative transport across the cytoplasmic membrane. We have confirmed that methylammonium is not metabolized in the yeast S. cerevisiae and have shown that it is little metabolized in the filamentous fungus Neurospora crassa. However, its accumulation depends on the energy-dependent acidification of vacuoles. A Deltavph1 mutant of S. cerevisiae and a Deltavma1 mutant, which lack vacuolar H(+)-ATPase activity, had large (fivefold or greater) defects in the accumulation of methylammonium, with little accompanying defect in the initial rate of transport. A vma-1 mutant of N. crassa largely metabolized methylammonium to methylglutamine. Thus, in fungi as in bacteria, subsequent energy-dependent utilization of methylammonium precludes its use in assessing active transport across the cytoplasmic membrane. The requirement for a proton gradient to sequester the charged species CH(3)NH(3)(+) in acidic vacuoles provides evidence that the substrate for MEP proteins is the uncharged species CH(3)NH(2). By inference, their natural substrate is NH(3), a gas. We postulate that MEP proteins facilitate diffusion of NH(3) across the cytoplasmic membrane and speculate that human Rhesus proteins, which lie in the same domain family as MEP proteins, facilitate diffusion of CO(2).     

High-Affinity Uptake of Spermine by Slices of Rat Cerebral Cortex

Abstract: The accumulation of the polyamine spermine into 0.1-mm prisms of rat cerebral cortex has been investigated at both 37°C and at 4°C. Kinetic analysis of the temperature-sensitive portion of uptake indicates two high-aftinity saturable components together with an unsaturable component at high concentrations. The 'very high'– affinity saturable system ( K _m= 3.8 nM) was temperature- and sodium-dependent, and significantly reduced by metabolic inhibitors, findings that are consistent with an active transport system for spermine into brain tissue. The 'high'– affinity saturable component ( K _m= 0.44 μM) was sodium-dependent and inhibited by ouabain, but only partially susceptible to inhibition by 2,4-dinitrophenol and sodium cyanide. The significance of these results with respect to the function of spermine in the central nervous system is discussed.     

Purine Nucleoside and Nucleobase Cell Membrane Transport in Giardia lamblia

ABSTRACT. Giardia lamblia is dependent on the salvage of preformed purines and pyrimidines. This study investigated purine nucleoside and nucleobase transport utilizing rapid uptake determinations. Nucleoside substrate/velocity curves exhibited the hyperbolic kinetics of a saturable carrier-mediated system. Deoxynucleosides exhibited a much lower affinity for the transporter. Inhibition studies confirmed the relative camer affinities of these ribonucleosides and deoxyribonucleosides. The nucleobase adenine did not exhibit saturation lunetics at a comparable substrate range, and did not inhibit nucleoside transport. Dipyridamole markedly inhibited nucleoside but not nucleobase transport, confirming the separate entry pathways. When cells were depleted of ATP, the velocity of nucleoside and nucleobase transport was unchanged, indicating that it is a non-energy-dependent process. Three nucleoside analogs, formycin A, adenine arabinoside and 7–deazaadenosine, were studied. Transport kinetics ranged widely among this group and could not completely account for their cytotoxic effect. When the apparent Km and Vmax of the nucleosides were compared, an approximately linear relationship (r²= 0.95) was noted. This suggests that a high affinity of the nucleoside permease for the substrate retards disassociation of the substrate-carrier complex, slowing net influx.     

Interaction between phloretin and the red blood cell membrane

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane.     

Two different carriers transport both ammonium and methylammonium in Chlamydomonas reinhardtii

A new methylammonium-resistant mutant strain from Chlamydomonas reinhardtii, henceforth termed 2172 (ma-2), has been isolated. This mutant is affected in a single mendelian gene different from and linked to the ma-1 locus which is defective in the methylammonium-resistant mutant 2170. Both mutations in ma-1 (2170) and ma-2 (2172) are linked to the nit-1 gene coding for the nitrate reductase apoenzyme. Mutant 2172 is affected in methylammonium but not in ammonium uptake capacity and shows derepressed nitrate and nitrite reductase activities in media containing nitrate plus methylammonium but not in nitrate plus ammonium media. The following two enzymatic components for the transport of both ammonium and methylammonium in wild-type cells have been identified: component 1, with high Vmax and K values, which is constitutive, and component 2, with low Vmax and K values, which is ammonium-repressible. Mutant 2170 lacks component 1 whereas mutant 2172 lacks component 2 for both methylammonium and ammonium transport. From genetic and kinetic evidences we conclude that in C. reinhardtii two different carriers are responsible for the transport of both ammonium and methylammonium and that methylammonium (ammonium) transport is a reversible process probably inhibited by the intracellular ammonium which, in turn, regulates nitrate and nitrite reductase levels.     

Feedback inhibition of ammonium (methylammonium) ion transport in Escherichia coli by glutamine and glutamine analogs.

When cultured with glutamate or glutamine as the nitrogen source, Escherichia coli expresses a specific ammonium (methylammonium) transport system. Over 95% of the methylammonium transport activity in washed cells was blocked by incubation with 100 microM L-glutamine in the presence of chloramphenicol (100 micrograms/ml). The time course for the onset of this glutamine inhibition followed a first-order rate expression with a t1/2 of 2.8 min. The inhibition of transport by L-glutamine was noncompetitive (Ki = 18 microM) with respect to the [14C]methylammonium substrate. D-Glutamine had no significant effect. The glutamine analogs gamma-L-glutamyl hydroxamate (Ki = 360 microM) and gamma-L-glutamyl hydrazide (Ki = 800 microM) were also noncompetitive inhibitors of methylammonium transport, suggesting that glutamine metabolism is not required. The role of the intracellular glutamine pool in the regulation of ammonium transport was investigated by using mutants carrying defects in the operon of glnP, the gene for the glutamine transporter. The glnP mutants had normal rates of methylammonium transport but were refractory to glutamine inhibition. Glycylglycine, a noncompetitive inhibitor of methylammonium uptake in wild-type cells (Ki = 43 microM), was equipotent in blocking transport in glnP mutants. Although ammonium transport is also subject to repression by growth of E. coli in the presence of ammonia, this phenomenon is unrelated to glutamine inhibition. A GlnL RegC mutant which constitutively expressed ammonium transport activity exhibited a sensitivity to glutamine inhibition similar to that of wild-type cells. These findings indicate that ammonium transport in E. coli is regulated by the internal glutamine pool via feedback inhibition.     

Effect of temperature and chloride on steady-state inhibition of angiotensin I-converting enzyme by enalaprilat and ramiprilat.

Recent work has provided new evidence that ATP is the major constituent of the low-Mr iron pool in the reticulocyte. The interaction of the iron complex of ATP with mitochondria was investigated in the present experiments. When ATP-Fe3+ was incubated with mitochondria, Fe3+, free of ATP, bound with high affinity to Fe3+ receptors on the mitochondria. The binding was saturable and reversible. Iron which was complexed to PPi, nitrilotriacetate, citrate, ADP and GTP also showed saturable binding to mitochondria; Fe3+ complexed to AMP bound non-specifically, as did Fe2+/ascorbate complexed to AMP bound non-specifically, as did Fe2+/ascorbate and Fe2+/dithionite.     

Specific binding of oestradiol to guinea-pig prostate cytosol and nuclear fractions

A high affinity (Kd approximately 0.15 nM), saturable oestradiol binding site, which is specific for natural and synthetic oestrogens has been identified in guinea-pig prostate cytosol fractions. The binding site is protein in nature (heat- and protease-sensitive) and has a sedimentation coefficient of approx. 8S on glycerol gradients. A high affinity (Kd approximately 0.16 nM), saturable oestradiol binding site was also identified in salt-extracted (0.5 M KC1) nuclear fractions. The optimum incubation conditions for measuring the cytosolic and nuclear oestradiol binding sites were determined to be 20 h at 4 degrees C. Saturation analysis studies revealed that following oestrogen treatment of intact animals, approx. 80% of the specific oestradiol binding sites in prostatic cytosol fractions were transferred into the nucleus. The presence of a specific oestradiol binding protein with characteristics of an oestrogen receptor in the guinea-pig prostate, is consistent with oestrogen having biological activity in this tissue. In view of the abundance of stroma in the prostate of this species, and the consistent finding that the stroma of male accessory sex tissues is oestrogen sensitive, the guinea-pig may be an appropriate experimental animal for further investigating the role of oestrogen in the growth and development of the prostate.     

Receptor of prolactin and its effect on binding of corticotropin in human and guinea pig adrenocortical cells

Specific binding of prolactin (PRL) by human and guinea pig isolated adrenocortical cells is saturable and reach equilibrium during 60 min. Characteristics of PRL binding by adrenocortical microsomes were determined. A single receptor species related to the high affinity and low binding capacity receptor class was revealed. The PRL-receptor affinities in adrenal cortex and liver are similar (approximately 10(9) M-1). Human adrenals have a higher affinity than that in guinea pigs. PRL increased corticotropin binding by adrenocortical cells of both species. Corticotropin binding rise may be due to the increase in the number of available receptors of low affinity and high capacity.     

Developmental and other characteristics of lysine uptake by rat brain synaptosomes.

Synaptosomes isolated from adult or newborn rat cerebrum take up L-lysine by two saturable systems, one with a high affinity low capacity and the other with a low affinity high capacity. Initial rate of uptake for low lysine concentrations is mort tissue. Analysis of kinetic data indicates that synaptosomes of the newborn have a higher Vmax than those of the adult for high affinity system but adult for high affinity system but adult synaptosomes have a higher Vmax than newborn for low affinity system. At a physiological lysine concentration of 0.5 mM, the calculated contributions of two systems indicate that the adult uptake occurs for about 71% by low affinity system but the newborn utilizes both systems to the same extent. The uptake is sodium independent but pH dependent. Lysine uptake is inhibited by other dibasic amino acids, arginine and ornithine but not cystine. Kinetic analysis indicates that arginine specifically inhibits the high affinity, low Km system for lysine uptake.     

Widespread occurrence of specific, high affinity binding sites for amino acids.

The high affinity binding of several amino acids to various membrane and protein preparations has been measured. Binding of radioactive amino acids suspected of being neurotransmitters and also of leucine and tyrosine to brain, liver and heart muscle membranes was saturable, reversible and stereospecific. Similar characteristics were found using chloroform-methanol extracted brain tissue and heat denatured albumin. Compounds thought to act as blockers of postsynaptic binding such as strychnine, bicuculline and kainic acid did not inhibit binding. Thus, specific high affinity interactions between amino acids and proteins are widespread and largely unrelated to neurotransmission.