Tracer Studies with - American Chemical Society

24 Tracer Studies with NH +, K+, and Mg 13

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A Bug's Eye View of the Periodic Table

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 22, 2017 | http://pubs.acs.org Publication Date: March 1, 1982 | doi: 10.1021/ba-1981-0197.ch024

S I M O N S I L V E R and R O B E R T D. P E R R Y Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO 63130

Free-living cells must accumulate needed inorganic nutrients by uphill energy-dependent transport processes, exclude those normally found cations and anions that are not utilized intracellularly, and regulate the equilibrium ratios and osmotic pressures due to both. Studies with short half-lived K and Mg have allowed characterization of highly sub strate-specific and energy-dependent transport systems for these inorganic nutrients. Evidence supports the separate existence of one or more bacterial systems for both cations (and for other required intracellular cations and anions) and shows differences in uptake and retention of the "natural" substrates (in kinetic constants and other characteristics) from those that would be deduced primarily from studies with longer half-lived analogues Rb or Cs (for K ) and Mn or Co (for Mg ). Transport studies using CH NH + as an analogue of NH + suggest that free-living cells discriminate between this analogue and NH . A real istic understanding of the manner in which free-living cells regulate and govern their intracellular inorganic milieu re quires use of the radionuclides of the normal environmental ion. 42

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T n this volume, with several chapters devoted to the assimilation and metabolism of N-labeled ammonia and nitrate into amino acids, purines, and pyrimidines that are the building units of cellular macromolecules, it is perhaps worthwhile to consider the larger context of basic inorganic biochemistry, or how cells view and utilize the elements of the periodic table (1,2,3). 13

0065-2393/81/0197-0453$05.00/0

© 1981 American Chemical Society

Root and Krohn; Short-Lived Radionuclides in Chemistry and Biology Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

454

SHORT-LIVED RADIONUCLIDES


K+

Figure 1. Hypothetical model for a cation-specific channel through a membrane-spanning transport pro tein (3)

Marcel Dekker

Although this report concerns bacterial cells ("bugs," but not the six-legged kind), and most frequently experiments with that most-studied of microbes, Escherichia coli, it is our experience that conclusions reached with specific bacteria will generalize to all bacteria, to free-living cells of higher organisms, and often to the cells and intracellular mitochondria and chloroplasts of animal and plant cells. Clearly, such generalizations do not always hold and must be made with caution; however, the exist ence and general properties of many cation and anion transport systems are quite universal for cellular membranes. This has been the primary interest of our laboratory for the last decade and has been summarized in several recent reviews (3-6). Our general conclusion is that quite separate and specific membrane transport systems exist for each and every inorganic cation or anion that is needed for cellular metabolism. The specificity of these systems is indicative of membrane-embedded proteins (Figure 1 ) , which we picture as serving as channels or as pumps. In a few cases, such as the mammalian Na /K ATPase (7) and the sacroplasmic reticulum Ca * ATPase ( 5 , 8 , 9 ) , the basic properties of these systems are being investigated with purified membrane proteins reconstituted into artificial membranes. At least for charged molecules, nothing crosses the membrane by bulk diffusion. In addition to our general thesis that quite separate membrane trans port systems exist in free-living cells for each and every inorganic cation or anion required for growth, cations and anions that are not normally accumulated are excreted by similar transport systems of opposite polarity. Figure 2 summarizes many of these systems. K and M g are the major intracellular cations required for growth of all cells. These are actively accumulated by two separate K transport systems (10,11,12) and two separate M g transport systems (13,14,15) in E . coli, as will be described in greater detail below. A K efflux system (16) has been identified, which probably serves as a K / H exchange system that regulates intra+

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Tracer Studies with NHf,

SILVER AND PERRY

K\ and z*Mg + 455

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cellular p H . N a and C a are the major monovalent and divalent cations that are not accumulated or found i n significant intracellular concentra tions. These cations are excreted b y one or more outwardly oriented transport systems (17-20). M n (6,21,22) and Z n (23) are essential intracellular divalent cations required at levels much lower than that needed for M g . They are accumulated by separate and distinct trans port systems (Figure 1; Reference 3). A l l cells require F e for growth and metabolism; E. coli has at least four distinct iron transport systems, whose regulation and function have been extensively studied ( 3 ) . Inorganic anions are accumulated b y separate, carefully regulated transport systems. There are at least two such systems for phosphate i n E. coli (24) and a separate one for sulfate (25). Chloride, the major inorganic anion found i n growth environments but not generally used for metabolism, is probably governed b y a separate, outwardly oriented transport system, whose function is to regulate osmotic pressure. I n this volume the first direct evidence is presented for N 0 " transport systems in bacteria (26). W e hypothesized the existence of such systems three years ago (3). Bacteria contain two types of nitrate reductase enzymes: ( i ) a low level assimilatory reductase synthesized during nitrate-depend ent growth; and ( i i ) a dissimilatory nitrate reductase that functions as an electron sink i n anaerobic respiration. I n the first case, either the N 0 ~ must be transported across the membrane prior to reduction or, less likely, the product ( N 0 " ) must be transported subsequently to continue the enzymatic reduction to N H and incorporation into amino acids. Since +

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8 +

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Figure 2. Some known membrane transport systems for cations and anions Root and Krohn; Short-Lived Radionuclides in Chemistry and Biology Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

456


N 0 " is toxic, it is likely (27,28) that the dissimilatory nitrate reductase functions at the membrane surface (as diagrammed in Figure 2) so that the N 0 " never enters the cell. Eucaryotic microbes also have N C V and N 0 " transport systems (29,30) whose function is regulated by the availability of N H (31). That leaves the two more complicated situations in Figure 2: Proton movement outward across the membrane is carried out by the respiratory chain (where each of the classic coupling sites is now pictured as a site of proton translocation) and by the reversible proton-translocating ATPase. This is the coupling factor, essentially identical in bacterial cells, mitochondria, and chloroplasts (32). The protons that have been excreted pass back into the cell through many paths including the anion transport systems and NaVproton and Ca Vproton "antiport" exchange systems (17-20) shown in Figure 2, the reversible ATPase (resulting in "coupled" oxidative phosphorylation or photophosphorylation), or "symport" with neutral organic substrates (providing the driving force in these transport systems). The proton excretion sets up a transmembrane electrical potential (A^) and p H gradient (ApH) that constitute the "energized" membrane state available for osmotic and chemical work (32). Finally, there is the still-open question of ammonium transport pictured in the upper-left portion of Figure 2. Two likely routes for ammonium uptake are envisaged: (i) passive diffusion of the relatively lipid-soluble and uncharged N H and (ii) active carrier-mediated uptake of charged N H through a membrane protein. Both mechanisms are diagrammed in Figure 2 because, in spite of considerable efforts to distinguish between these mechanisms, we are unfortunately unable, at this time, to rule out either. The p K for the transition from N H to N H is 9.3; therefore at p H 7.0, 99.5% of the available ammonium will be in the N H form. The arguments in favor of an N H transport system and N H uptake experiments designed to test this hypothesis are described in detail below. This volume is concerned with the use of short-lived radioisotopes (half-lives of minutes). We will expand this "short" range to include (*i/2 — 12.4 h) and M g (t — 21.3 h), as well as N H \ since many biologists would consider these radioisotopes "short-lived." Although differences exist between the three systems, the general conclusion that studies on the accumulation of these cations must utilize the radioisotopes of "natural" substrates can be made. Analogues such as R b and C s for K , M n and C o for M g , and C H N H for N H * are imperfect and can lead to inaccurate conclusions. 2

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Potassium Transport Because procaryotic and eucaryotic K transport systems are the best understood of all cation transport systems, we shall start with an exami+


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nation of the use of vs. R b in such studies. With a 12.4-h half-life ^ K * is much less convenient for experimentation than is the frequently used K analogue R b with a 18.7-day half-life. Nevertheless, 12.4 h translates into about 1% loss in 10 min, and we have found that ^ K * can be handled and used in transport studies essentially as would C or H-labeled compounds. ^ K * emits strong ft particles that can be conveniently counted on a Geiger counter or in a liquid scintillation counter either in organic scintillation fluid or in water via Cerenkov radiation (33); we have used all three methods with essentially equal ease and high efficiencies. With a shipment of a few mCi of ^K* arriving at the airport in the evening from a commercial source, we can experi mentally process and count around 1000 samples (one minute each with no repeat counting) during the 5 half-fives or so of useful activity. Radioisotope decay corrections may be made by normalization either from control samples counted every 10 min and/or from recorded elapsed counting times, as explained below for N H . Much biological information has been obtained from studies of transport of K and its analogues. The general conclusion is that cells of higher animals (7,34) and plants (35) do not discriminate effectively between ^ K * and R b for transport. Therefore experiments with R b yield biologically interesting and valid results. Some microorganisms, of which Rhodopseudomonas capsulata is the best studied example (36), discriminate somewhat between K* and Rb (and even more so against Cs ) (Table I). Other microbes, notably Escherichia coli (Table I; Reference 12) discriminate so strongly against Rb that its use as a K analogue is precluded. Note from Table I that the two K transport systems of E. coli have different degrees of discrimination against Rb . 86

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Table I. Kinetic Parameters of Bacterial Potassium Transport Systems 0

Bacterium R. capsulata

Cation Trans ported K

0.20

+

Rb Cs E. coli Kdp TrkA

K

+

(mM)

+

+

0.52 3 0.002 1.5

Competitive Inhibitors or Discrimination Against Rb+

(fmol/min perg) 8.0 5.9 2 150 550

K K K K

{ i {

t

— 0.56mMRb — 2.8mMCs* = 0.2mMK — 2.6mMCs

+

+

+

>1000XK7Rb

10X K / R b +

+

+

'Kinetic parameters for JB. capsulata (for photosynthetically grown cells) and the K s and FmaxS for E. coli are from previously published reports (10,12,86). The Kis for R. capsulata are kinetically determined values from experiments such as Ref erence 86. The ratios for the E. coli systems are not accurately determined K% values but rather calculated discrimination ratios of Reference 12. m


458


The "trace-scavenging" Kdp system (which is responsible for accumulat ing K / K gradients of greater than 10 :1; Reference 10,12) has a discrimination ratio (K /Rb ) of 1000, while the major TrkA system (which functions under conditions of abundant extracellular K ) has a ratio of only 10. The high affinity Kdp system includes a transport ATPase (11,37), which is in fact the first example of a cation-specific transport ATPase in bacteria. Very recently, Epstein and Laimins (10) have reevaluated K transport in E . coli and concluded that there are two, rather than four, separate transport systems for K . The two deleted transport systems were actually the TrkA system with mutationally altered kinetic parameters. +

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6

o u t

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+


+

+

These results are as might be expected and represent another generalization from studies of the last 10 years: the specificity of a transport system is tailored to meet cellular needs with systems that function under conditions of nutrient starvation having the highest degree of specificity. For the monovalent cation systems, discrimination is greatest against the related abundant cation (Na ) and lesser for rarer analogues (Rb or Cs ). Organisms such as E . coli (which sometimes grows in very dilute fresh water) or Hahbacter halobtium (which grows in supersaturated NaCl ponds) require systems that have specificity characteristics that will sustain the universal need for very high internal K levels. Cells growing in better K -buffered environments do not require such great specificity (except against abundant N a ) , and therefore animal cells need not discriminate against Rb at all. Even among the free-living bacteria, R. capsulata can grow with intracellular K replaced with Rb (36), whereas E . coli cannot (38). As the major intracellular inorganic cation, K levels are carefully regulated (38). The regulation is primarily in response to extracellular osmolarity (10,12,38). However, K transport is also regulated during the cell cycle in exponentially growing cells (39) and during develop mental cycles such as bacterial sporulation and spore germination (40). Among the Group l a elements of the Periodic Table, living cells accumulate K and exclude Na —in fact, N a is universally excreted by a pump of reversed polarity from inside to out (3,19,32). While some organisms utilize N a entry in a symport mechanism to accumulate amino acids and carbohydrates, they also have N a efflux systems (41,42,43). L i is a N a analogue and does not function as a K substitute. Although ionic radius is undoubtedly the primary factor utilized by the membrane transport proteins to distinguish between cations of similar charge, there is no direct evidence as to how the presumedly negatively charged chemical groups lining the transmembrane channel (Figure 1) accom plish this task. With the lipid-soluble 1100 molecular weight depsipeptide valinomycin (44,45), discrimination between transmembrane movement +

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Tracer Studies with NH \ K\ and Mg *

SILVER AND PERRY

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of K and of Na is governed by the stability of the complex, which is in turn dependent on ionic radius [K (r — 0.169 nm); N a (r — 0.095 nm) ]. Whether membrane transport proteins (Figure 1) utilize a mechanism similar to this model compound (46,47) to discriminate between K and Rb is an open question. +

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Magnesium Transport It is precisely because of the absence of a long half-lived radioisotope of M g that there have been few studies of M g transport in biological systems. M g has a 21.3-h half-life and emits a 0.45-meV p particle as well as y radiation (33). It is not available from commercial sources, but it has been made on a fixed schedule of every two or three weeks from the Hot Laboratory Division, Brookhaven National Laboratory in the United States and was available on request from the Radiochemical Centre, Amersham, Great Britain. Jasper and Silver (4) summarized all known single-cell and subcellular studies on M g and biological mate rials (animal, plant, and microbial) in 1977. We are not aware of newer studies with M g at the cellular or subcellular levels. Transport of M g by a variety of bacteria has been studied in our laboratory (15,48,49,50). The most thoroughly studied example for M g transport is E. coli, where M g transport was first reported in 1969 (50,51). The eucaryotic microbe Euglena and human K B cells in tissue culture also have M g transport systems (4,52). In fact, all tested cells have active transport systems for M g (4) with the one exception of mature human red blood cells (unpublished data). It is possible that this transport process is lost during red blood cell maturation just as Na /K ATPase activity decreases during red blood cell maturation (53). E. coli has two M g transport systems (13,14). Competitive inhibitors and alternative substrates for E . coli M g transport include C o and M n , two cations for which longer-lived, more convenient radioisotopes exist. Indeed C o , N i , and M n have been used in these studies (4,14). Nevertheless, the existence of mutants with altered discrimination against C o and M n (14,15) and the ratio of the K s for M g and for other divalent cations (often 10:1 in favor of M g ) point out the essential requirement that studies of magnesium transport and metabolism utilize the short half-life radioisotope M g . Faulty conclusions can be drawn from overreliance on imperfect analogues. 2+

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Ammonium (a) Uptake There are several alternative mechanisms for movement of N H / N H across biological membranes (Table II). Indeed, more than one mecha nism may occur in nature, and different organisms may utilize different 3


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Table II.

Possible Mechanisms for Ammonia Uptake

0

Mechanisms Likely Passive diffusion of N H Carrier-mediated transport of NH

Predicted Characteristics A p H dependent and unsaturable A p H and A ^ dependent; saturable

3

4

+

Unlikely Passive diffusion of N H Carrier-mediated diffusion of


4

A p H and A ^ dependent; unsaturable A p H dependent and saturable

+

NH3

•All mechanisms would be followed by the "pull" from ATP-dependent saturable assimilation into amino acids.

mechanisms. The two mechanisms that are considered most likely to occur are (i) the passive diffusion of lipid-soluble un-ionized N H across the lipid bilayer regions of membranes, and (ii) the protein-carrier mediated energy-dependent transport of ionized N H . The primary characteristics of the former mechanism is an absence of saturation kinetics—bulk solubility parameters of lipid surfaces would govern the rate of uptake, rather than a small number of specific sites—and a dependence on the p H gradient across the membrane for the final equilibrium ratio. With a p K of 9.3, essentially all the ammonia will occur as N H at physiological p H , and a difference in p H (internal alkaline) will result in an equflibrium concentration of a factor of 1 0 X less ammonia internally for every p H difference across the membrane. An internal p H more acidic than the external p H would be required for net accumulation of ammonia by this mechanism. Yet, the Mitchell chemiosmotic theory for chloroplasts, mitochondria, and bacterial mem branes predicts an internal p H alkaline relative to the outside p H ; this has indeed been found repeatedly by direct experiment (e.g., see Refer ence 32 for review). Nevertheless, passive diffusion of N H appears to be the actual mechanism of ammonia uptake in mammalian cells and across membranes such as the blood-brain barrier (54,55,67). Perhaps with the stable low N H levels in blood serum (about 5 yM; Reference 55) and the use of organic rather than inorganic compounds as nitrogen sources within animals, there is no need for an ammonium transport system in animal cells comparable to that required for growth by freeliving cells dependent on inorganic nitrogen for nutrient supply. There have been several reports of passive diffusion of N H in freeliving microbial cells as well: Suzuki et al. (56) measured the effect of p H on whole-cell oxidation rates of N H to N 0 " by Nitrosomonas europaea and concluded that N H was the form that moved across the cellular membrane to the intracellular oxidation site. This conclusion was based on the questionable assumptions of equal concentrations across 3

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SILVER AND PERRY

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the cell membrane and no p H gradient. More recently, Bhandari and Nicholas (57), using the same organism, came to the opposite conclusion, that is, that N. europaea utilizes a specific N H translocase for ammonia uptake. Subsequent extrusion of protons leads to a net N H uptake, but the initial N H uptake process was inhibited more completely by energy inhibitors than were subsequent oxygen-dependent stages (57). Zarlengo and Abrams (58) measured the entry of N H to aged (for days at 4°C) stationary phase Streptococcus fecalis. With cells starting with an internal p H of near 5, entry of un-ionized N H was rapid. Under these physiologically nonactive conditions, bacterial cells were indeed rapidly penetrable to N H , with a decrease in the p H gradient across the membrane an immediate result of the process. Uptake led to a net accumulation of ammonia, starting with cells that were acidic inside relative to the incubation medium. 4

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Stevenson and Silver (59) used the radioactive N H analogue CH NH in transport studies with E. coli. Methylamine had been previously used as an N H analogue in studies with nonbacterial microbes (see below). Energy-dependent uptake of methylamine was demon strated. The p H dependence of this uptake was complex, but optimum uptake was obtained at pH 9, well below the p K of C H N H to C H N H of 10.9. Stevenson and Silver (59) postulated the existence of one or possibly two specific N H transport systems in E. coli. Subsequently, Clostridium pasteurianum cells (60) were shown to accumulate N H (and C H N H ) by physiologically active processes that were interpreted to occur across a membrane essentially impermeable to un-ionized N H . C. pasteurianum cells maintained a gradient of N H + N H of 100:1 inside to outside while growing on N as nitrogen source (60). This result is consistent with a basically impermeable membrane. Valinomycin released cellular N H by functioning as a membrane carrier of N H . C. pasteurianum reduces N to N H only when there is no alternative nitrogen source available; N H in the environment is scavenged down to traces below lfiM. The transport system utilized for scavenging was studied with C H N H as an analogue substrate for the N H transport system;, concentration gradients of 70:1 (internal:external C H N H ) were established. This gradient was almost completely eliminated by addition of low levels of N H , indicating a preference for the natural substrate over the analogue. +

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Laane et al. (61) demonstrated energy-dependent N H uptake in another N -fixing bacterial species, Azotobacter vinelandii. This uptake was in response to the cellular membrane potential (internal negative). C H N H was used again as a radioactive analogue of N H . Whereas intact cells of free-living N -fixing A. vinelandii accumulated C H N H , bacteroids of Rhizobium leguminosarum (which is symbiotic in plant 4

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root nodules and which fixes nitrogen only in association with the legumous host plant) did not accumulate C H N H , as if they lacked an N H uptake system. Rhizobium bacteroids in fact excreted C H N H in response to the p H gradient (0.45 p H units internal alkaline; Refer ence 61). Pseudomonas sp. M A actively transports C H N H . However, this transport system was inducible only under conditions where methyl amine served as carbon source. That and the absence of inhibition of methylamine uptake by ammonia indicate that this methylamine transport system was not an ammonia transport system (62). In summary, there appears to be evidence for both mechanisms of N H / N H transport in bacterial cells. Passive diffusion of N H would not generally lead to net accumulation by cells with an alkaline interior p H relative to the medium pH. Passive diffusion would be rate-limited by the low level of N H available at physiological p H and by the solubility properties of the cell membrane. N H active transport would be required for net ammonium uptake. However, net nitrogen accumulation could be accomplished by rapid assimilation of internal ammonia into amino acids. Net accumulation might be limited by the balance of the two processes: uptake by active transport vs. loss by passive diffusion. More extensive studies characterizing N H accumulation have been carried out in free-living eucaryotic organisms using C H N H as an analogue for N H . In Aspergillus nidulans, an active methylamine trans port system (K = 20^M), which was repressed by growth on high N H , has been characterized (63). Competitive inhibition of C H N H uptake by N H was not tested, but mutants were identified with phenotypic characteristics, which include poor growth on N H and altered methylamine transport activities. A repressible methylamine active transport system (K = 220/AM with ammonia as a strong com petitive inhibitor) was identified in Saccharomyces cerevisiae (64,65). Penicillium chrysogenum also has a repressible active C H N H trans port system ( K = lOpM). Both N H and C H N H were competitive inhibitors of the system, with K s of 0.25/AM and 100/AM respectively (66). 1 4

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N-Ammonium

13

Accumulation

Although the use of methylamine as an N H * analogue has been moderately successful with the above organisms, its similar application to many bacterial systems may lead to erroneous conclusions. Accord ingly, we have started N H uptake experiments designed to charac terize bacterial N H accumulation. The Mallinckrodt Institute of Radiology at Washington University School of Medicine produces N H • NH can be conveniently counted in a standard commercial liquid scintillation spectrophotometer, 4

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SILVER AND PERRY

Tracer Studies with

,3

NH , ^K* and 4

+

Mg *

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463

obviating the need for special counting equipment. Approximately 100 samples can be processed and counted (0.5-1.0 min each with no repeat counting) before decay precludes further analysis. With a half-life of 9.96 min, it is necessary periodically to record the "clock time" on the counter printout. Decay corrections for each sample may then be calcu lated by the equation: Decay correction factor = antilog (0.0302 X elapsed time in minutes) either by computer or by hand calculator. Our experiments have used primarily S. typhimurium strains with wild-type and mutant phenotypes for ammonia assimilation. Figure 3 illustrates the p H profiles for [ N]ammonia accumulation of S. typhimurium strains JL907 (wild-type) and JB801, which lacks glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT). L-MethionineDL-sulfoximine is an inhibitor of the first steps of the ammonia assimilatory process depicted in Figure 2. The p H profile for strain JB801 showed a 13

Figure 3.

pH profile of NHf/NH accumulation strains JL907 (wild-type) andJBSOl (GDH' 18

s

9

by S. typhimurium GOGAT').

The cells were grown to mid-log phase in a minimal medium (M9) with 2mM NHiCl (JL907) or lOmM L-glutamate (JB801). The cells were then washed and resuspended in M9 medium without a N-source. After 4 h of incubation at 42°C, the cells were washed and resuspended in 50mM Tris-72.5mM NaCU0.4% glucose of the appropriate pH. After equilibration at 37°C, 71\M N H + NH was added; 0.5-mL samples were filtered after 30 s of incubation and washed twice with 5-mL volumes of appropriate pH buffer (without NHfil). 200/M. i

Tracer Studies with - American Chemical Society

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