THE JOURNAL OF BIOLOGICAL CHE~IISTRY

Vol. 247, No. 23, Issue of December 10, pp. 7684-7894, 1972

Printed in U.S.A.

A Binding Protein Involved in the Transport of Cystine

and Diaminopimelic Acid in Escherichia coZi*

(Received for publication, May 22, 1072) EDWARD A. BERGER AND LEON A. HEPPEL

From the Section of Biochemistry and d~olecular Biology, Cornell Snivel-sity, Ithaca, New York l&fio

SUMMARY

Escherichin coli strain W is capable of actively transporting L-cystine as well as the structurally related compound OL,E- diaminopimelic acid. Cystine transport is mediated by two systems distinguishable on the basis of specificity and affinity for substrate. The cystine general system (K, = 3 x 10e7 M) also transports diaminopimelic acid and is inhibited by a variety of analogues, while the cystine specific system (K, = 2 X 1O-8 M) is much more selective. A mutant strain, D2W, has only the lower affinity general system (K, = 1 x 1OF M).

The two systems display differing sensitivities toward os- motic shock. When cells are grown in a minimal medium, the general system is entirely lost from each strain by shock, whereas the specific system of W is unaffected. The shock fluids from these cells contain a single protein capable of binding cystine (the cystine general binding protein), and the binding is completely inhibited by diaminopimelate. This binding activity is reduced by 90% in mutants of strain D2W lacking the general transport system. When strain W is grown in an enriched medium, the general system is absent, but the activity of the specific system is increased 3-fold and can now be reduced by osmotic shock. The cystine general binding protein is absent from the shock fluid, but we now find the appearance of a cystine binding activity with a speci- ficity similar to that of the specific transport system.

The cystine general binding protein has been purified to homogeneity and has properties similar to those reported for other shock-releasable binding proteins. Its specificity closely parallels that of the general transport system. The results support the concept that the cystine general binding protein is involved in the recognition step of the general transport system.

The permease model of active transport (1) predicts the exist- ence of a carrier which recognizes and binds a substrate molecule from the external medium, carries it across the membrane, and

* This work was supported by Grant GB-27396X from the Na- tional Science Foundation and Grant AM 11789-05 from the Na- tional Institutes of Health and Training grant GM-O 0824-10 from t,he National Institutes of Hea1t.h. A portion of this work was presented at the Fifty-fourth Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, 1970 (Abstract 542), and also at the Miami Winter Symposia on the Molecular Basis of Biological Transport, 1972.

releases it to the inside of the cell. The over-all process requires metabolic energy and permits the accumulation of the substrate against its concentration gradient. Numerous attempts have been made to isolate such carrier molecules from bacterial mem branes (Z-40), and much recent activity has focused on a group of proteins released from the cell by the osmotic shock procedure of Neu and Heppel (41). These “binding proteins” reversibl) bind certain substrates or groups of substrates without catalyzing my chemical reactions, and in general they have properties con- sistent with those expected of transport carriers.

Shock-releasable binding proteins have been implicated in the transport of a wide variety of compounds in gram-negative bac- teria, including amino acids (5-21), sugars (7-9, 22-30), inor- ganic ions (31-37), and vitamins (38-40). Generally, where the activity of a given system is reduced by osmotic shock, a corre- sponding binding protein is found in the shock fluid, while systems unaffected by shock do not yield corresponding binding activities

(4, 21). Transport systems for the amino acids exhibit varying degrees of complexity and specificity. Glutamine, for example, appears to enter by a single, highly specific system (ll-13), whereas leucine may be transported by at least two systems, one shared with the neutral branched chain amino acids isoleucine and valine (5) and the other specific for leucine (10). In the cases studied, the specificities of the transport systems reduced by shock correlate well with the specificities of binding proteins released into the shock fluid, suggesting a role for these proteins in the transport process.

Leive and Davis (42) have shown that Escherichia coli strain W has two systems for eystine uptake: one shared with and competi- tively inhibited by DSP’ and the other not. A mutant strain, DzW, has only the shared system. Evidence is presented here that the shared system is inhibited by a variety of cystine and DAP analogues, while the unshared system exhibits much greater specificity.2 From the shock fluids of these strains, we have iso- lated and purified a cystine-binding protein which appears to bc involved in the activity of the general transport system.

MATERIALS

Bacterial Strains-E. coli strain W (ATCC 9637) and it,s de- rivative D,W were gifts from Dr. L. Leive, National Institutes of He&h. DzWSelR1, 1)7WSclR~, and D2WSelR3 were mu- tants selected from DzW 011 the basis of their resistance to the toxic analogue selenocystine.

1 The abbreviation used is: DAP, (Y, c-diaminopimelic acid. r The shared system is called the cystine general transport

system, and the unshared system is referred to as the rystine specific transport system.

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Growth ~Vedia-Bacteria were grown in a synthetic minimal medium described by Tanaka et al. (43) supplemented with 1 y0 sodium succinate (Baker and -1damson) as a carbon source. For experiments on the effects of enriched media on transport, a mediu:n containing 3%; yeast extract and 4y0 tryptone (Medium A) was used. Cultures were grown at 37” with shaking. Optical density at 600 nm was measured on a Gilford spectrophotometer model 240 (optical density 1 = log cell per ml).

Clzelnicals-L-[U-l”C]Cystine (specific activity 270 mCi per mmole) was obtained from New England Nuclear Corp. For most experiments, this was diluted with nonradioactive L-cystine (Mann Research) to give a final specific activity of 10 mCi per mmole. cy, +[I (7)-~4C]Diaminopimelic acid (specific activity 9.8 mCi per mmole) was purchased from Cal-Atomic, Los Angeles, California. This material was a mixture of LL, DD, and meso isomers.

Dinminoglutaric acid, diaminoadipic acid, diaminosuberic acid, and diaminoazelaic acid were gifts from Dr. E. Work, Imperial College of Science and Technology, London. Di- aminopimelic acid (a mixture of LL, DD, and meso isomers) was purchased from Sigma. The four racemic mixtures (A, B, C, and D) of fl-hydroxy-DAP (44) were generously donated by Dr. C. Gilvarg, Department of Biochemical Sciences, Princeton Irniversity. y-Methyl-l)hP was a gift from L. E. Rhuland, The Upjohn Co., Kalamazoo, Mich. Meso-2,3-diaminosuccinic acid was obtained from Burdick and Jackson Laboratories. ,411 other nonradioactive amino acids, including analogues of cystine and DAP, were purchased from either Mann Research or Sigma.

BioCert yeast extract and BioCert tryptone were obtained from Fisher Scientific.

All other compounds were analytical grade.

METHODS

Transport Assay-Cells were harvested during midexponential phase of growth (1 X log cells per ml), washed twice in minimal medium at 23”, and resuspended in 20 ml of minimal medium per g, wet weight, of cells. Glucose and chloramphenicol were added to portions of this suspension at final concentrations of 0.2y0 and 80 pg per ml, respectively. After a 5-min preincuba- tion, the transport assay was initiated by adding a portion of these cells to a reaction mixture containing 0.2% glucose, 80 pg per ml of chloramphenicol, isotopically labeled substrate, and minimal medium to a final volume of 0.5 ml. The mixture was kept at 23”; and 0.2-ml aliquots were withdrawn at various time intervals, filtered on 25.mm nitrocellulose filters (0.45 ~1, Mathe- son-Higgins), and washed with 10 ml of wash solution containing 0.01 &r Tris-HCl (pH 7.3), 0.15 hI NaCl, and 0.5 mM %fgClz (9).

The filters were dried and counted in a solution consisting of 15 g of 2,5-diphenyloxazole (PPO) and 0.2 g of 1,4-bis[2-(4.methyl- 5-phenyloxazolyl)]benzene (dimethylPOPOP) dissolved in 3.81 liters of toluene. To insure linearity of initial rate of uptake, 15- and 30-s time points were taken, and the 15-s points were used for calculations. Cystine transport studies were performed at 10 px while 20 /AM DAP was used for DAP uptake measura- merits. In all experiments, the amount of cells was adjusted so that less than lO7o of the total radioactivity was taken up during the first 30 s (generally 200 to 400 pg of cellular protein). Up- take values are expressed as nanomoles per nun per mg of cellular protein.

Identification of Transported Radioactivity-Cells were incu- bated with substrate for 15 s, filtered, and washed exactly as described for transport assays. Immediately after washing, the filter was flushed with 10 ml of cold deionized water; and the

filtrate was collected, lyophilized to dryness, and redissolved in a small volume (20 to 50 ~1) of water. Thin layer chromatog- raphy was performed on avicel plates (?lnaltech) with nonradio- active amino acids spotted as standards. Plates were scanned for radioactivity on a Packard model 7201 radiochromatogram scanner. Samples derived from cystine uptake experiments were chromatographcd in pyridine-fert-amyl alcohol-H?0 (35:35:30), while t,hose derived from DAP transport experiments were chromatographed in methanol-H&IO N HCI-pyridine (80: 17.5: 2.5:10) (45).

Osmotic shock-Osmotic shock was performed by a modifica- tion of the procedure of Neu and Heppel (41). Cells harvested in midexponential phase of growth were washed three times with 0.01 M Tris-HCI, pH 7.3, containing 0.03 M NaCl at 4”, then resuspended in 20 volumes (w/v) of 0.033 M Tris-HCl, pH 7.3, at 23”. An equal volume of this buffer containing 40$& sucrose and 0.4 mhr EDT.4 was added, and the suspension was gently swirled for 5 min. After centrifugation at 4”, the pellet was rapidly dispersed in 40 volumes of cold 0.5 mM MgCl2, and the suspension was swirled for 10 min followed by centrifugation. The pellet was resuspended in 20 volumes of minimal medium at 23” for transport assays, and the supernatant containing the shock-releasable proteins was concentrated in an Amicon model 401 ultrafiltration ceil equipped with a UM-10 membrane. The concentrated crude shock fluid was then dialyzed against 5 m&f Tris-HCl, pH 7.3, and assayed for cystine-binding activity.

Binding Assays-For quantitative measurements, equilibrium dialysis was carried out in Plexiglas chambers consisting of two wells separated by a layer of dialysis membrane (10). One side contained protein solution in a volume of 0.1 ml while the other side contained an equal volume of isotopically labeled substrate in 0.02 M potassium phosphate, pH 7.0, containing 0.1 M NaCl and a trace of chloroform. The chambers were rotated overnight at 4” to allow equilibration, and aliquots of 0.05 ml were removed from each side and counted in a solution consisting of 15 g of PPO, 0.188 g of dimethylPOPOP, 1 g of Triton X-100, and 2 liters of toluene. Specific activit,ies are expressed as unit)s per mg of protein, with 1 unit representing 1 nmole of substrate bound at saturation (10 PM cystine).

For assays of column fractions, a more rapid method was de- sired, and we employed a modification of the filter assay described by Schleif (28). Protein solution (0.030 ml) was mixed with 0.025 ml of labeled cystine (10 PM) at 23”. One-half milliliter of this mixture was filtered through a 25.mm nitrocellulose filter (B-6, Schleicher and Schuell) and washed with 5 ml of deionized water. The filters were dried and counted as for transport as- says.

Purijcation oj Cysline General Binding Protein from Sirain IV--- For the large scale preparation of cystine-binding protein, cul- tures were grown in 18.liter carboys aerated through glass spar- gers. To increase the yield of binding protein, cells were har- vested in late stationary phase,3 collected by means of a refrig- erated Sharples centrifuge, and shocked as described above except for the following modifications: the washed cells were suspended in only 10 volumes of 0.033 M Tris-HCl, pH 7.3, to which was added an equal volume of 0.033 M Tris-HCl, pH 7.3, in 40% sucrose containing 8 mM EDTA. After swirling for 10 min, followed by centrifugation at 4” for 20 min, the pellet was rapidly

a The initial rates of cystine and DAP uptake as well as the elution uattern of the crude shock fluid on DEAE-cellulose were nearly identical in cells harvested in logarithmic and stationary phase.

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suspended in 20 volumes of cold deionized water, swirled for 5 min at 4”, and centrifuged for 30 min at 4”.

The supernatant shock fluid was concentrated 200.fold by means of an Amicon model 401 ultrafiltration cell equipped with a UM-10 membrane and desalted on a Bio-Gel P-10 column (5 x 50 cm) equilibrated with deionized water at 4”. The frac- tions containing protein were pooled, concentrated by ultrafiltra- tion, and assayed for cystine-binding activity by equilibrium dialysis.

DEAE-cellulose Chromatography of Crude Desalted Shock Fluid-The concentrated, desalted shock fluid was applied to a 200-ml column (2.5 x 41 cm) of Whatman DEAE-cellulose DE-52 (microgranular, 1 meq per g), equilibrated with 5 mM Tris-HCl, pH 7.2, at 4”. The column was eluted by means of a peristaltic pump, first with 2 bed volumes of 5 mM Tris-HCl, pH 7.2, followed by a 7-liter linear gradient of 0 to 0.15 M NaCl in 5 mM Tris-HCl, pH 7.2. Optical density at 280 nm was moni- tored by means of a Uvicord II flow cell (LKB Products, Rock- ville, Md.). Fractions of 20 ml were collected, and measurement of cystine-binding activity revealed two distinct peaks, CBP I and CBP II. The fractions within each peak were pooled sep- arately, concentrated by ultrafiltration, and assayed by equilib- rium dialysis. The further purification of each peak was carried out independently. For CBP I, Sephadex G-75 chromatography followed by isoelectric focusing was used, while for CBP II, a combination of preparative disc gel electrophoresis and electro- focusing was employed.

Sephadel: G-75 Chromatography-The concentrated DEAE- cellulose fraction containing CBP I, in a volume of 2.5 ml, was loaded onto a Sephadex G-75 column (2.5 x 100 cm) equilibrated with 5 mM Tris-HCl, pH 7.2, at 4”. The column was eluted by reverse flow, and fractions containing binding activity were pooled, concentrated, and assayed.

Preparative Isoelectric Focusing-Electrofocusing was per- formed using an apparatus designed in this laboratory (20). A linear gradient of 0 to 50% sucrose containing 1 y0 of an ampho- line solution with a pH range of 4 to 6 (LKB, Produktor) was poured into a water-jacketed glass column of 150 ml capacity (2.5 x 40 cm). The gradient was supported by an 8% poly- acrylamide gel at the bottom of the column, and a 4-ml layer of 1 y. HzS04 in saturated sucrose was poured above the gel before the gradient was made. The lower anode buffer consisted of 1 y. sulfuric acid while the upper cathode buffer layered above the sucrose gradient contained 2 To ethanolamine. The ampholytes were prefocused for 24 hours at 550 volts to allow formation of the pH gradient, after which 3.2 ml were removed from a region about 2 inches below the gradient-ethanolamine interface and evaporated to dryness. The protein solution (3.0 ml) was added to the residue, and the sample was loaded at the point of initial removal. The current was reapplied, and the protein was focused for 24 hours. The acrylamide gel was then pierced with a 22- gauge syringe needle attached by means of plastic tubing to a peristaltic pump, and the contents of the column were emptied through a Uvicord II 280 nm flow cell. Fractions of 1.2 ml were collected and assayed for binding, and those containing activity were pooled and concentrated. The ampholytes and sucrose were removed by dialysis first against two changes of 0.1 M NaCl, and then against two changes of deionized water. Equilibrium dialysis was performed to determine cystine-binding activity.

Preparatzve Disc Gel Electrophoresis-Preparative disc gel elec- trophoresis was performed with a Tris-glycine buffer system (46) using an apparatus designed in this laboratory.4 The water-

jacketed gel column was filled to a height of 3 cm with the re- solving gel solution containing 67, acrylamide and 0.2% N, N’- methylenebisacrylamide, and a layer of water was gently applied above the gel solution to provide a level gel surface. After polymerization, the surface of the gel was rinsed two times with stacking gel solution; and 3.5 ml of this solution were added above the resolving gel, layered with water, and polymerized with the aid of a fluorescent lamp. The gel column was rinsed and filled with the upper buffer, and the lower portion of the col- umn was immersed in the lower buffer reservoir. The sample, in a final volume of 3.0 ml, contained protein, stacking gel buffer, 12.5% glycerol, and 0.15 ml of 0.01 y0 bromphenol blue as a track- ing dye. The sample was layered above the stacking gel and subjected to electrophoresis at 14 ma (250 volts). The space below the bottom of the resolving gel was continuously eluted with lower buffer by means of a peristaltic pump, and fractions were collected once the dye band approached the bottom of the resolving gel. Absorbance at 280 nm was monitored with a Uvicord II flow cell. Fractions containing cystine-binding xc- tivity were pooled, concentrated, and assayed.

Analytical Polyacrylamide Disc Gel Electrophoresis-Analytical polyacrylamide gels were run according to the method of Orn stein and Davis (46). Samples, in a total volume of 0.1 ml, con- tained 5 to 50 pg of protein, 12.5% glycerol, and stacking gel buffer. The upper electrode buffer contained 0.1 ml of 0.01% bromphenol blue per 100 ml as a tracking dye. After electro- phoresis, the gels were fixed in 12.5% trichloroacetic acid for 30 min and stained overnight with 0.05% Coomassie blue in 12.5% trichloroacetic acid (47). The gels were destained with several changes of 12.5y0 trichloroacetic acid and stored in 7.5% acetic acid.

In addition, a cationic buffer system in which separation occurs at pH 2.75 was employed. The upper buffer contained 0.1 ml of 0.01 y. methyl green per 100 ml as a tracking dye

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed according to the method of Weber and Osborn (48).

Preparation of Antisera to Cystine General Binding Protein- One milligram of purified cystine general binding protein in Freund’s adjuvant was injected into the foot pad of a New Zea- land white rabbit. After 30 days, 0.5 mg was injected intrave- nously. On the 11th day after boostering, the rabbit w-as bled, and the antisera were obtained. The antibody titer, as measured by serial dilutions in microcapillary tubes, was 1:32. Double diffusion plates were prepared as described by Ouchterlony (49). After application of sera and antigen, the plates were incubated for 24 hours at 4” in a closed humidity chamber.

Enzyme Assays-Cystine reductase activity was measured by a modification of the method of Roman0 and Nickerson (50). DAP decarboxylase was assayed as described by Hirschfield et cd. (51), and DAP transaminase was determined by the method of Meadow and Work (52).

Protein Assay-Protein was determined by a micromodification of the method of Lowry et al. (53) with bovine serum albumin as a standard.

RESULTS

Dependence of Cystine and DAP Uptake on

Metabolic Energy

The time course of cystine and DAP uptake is shown in Fig. I. As reported by Leive and Davis (42), the “D” mutation results

4 C. E. Furlong, personal commlmication. 5 W. L. Nelson, personal communication.

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TIME (MINUTES)

FIG. 1. Time course of cystine and DAP uptake. The initial external substrate concentrations were cystine, 10 pM, and DAP, 20 PM. Transport assays were performed as described under “Methods.” 0, strain W; 0, strain DzW.

in an increased transport rate for both compounds. In order to demonstrate that a system is capable of active transport, it is necessary to show either that the substrate is accumulated un- changed against its electrochemical gradient, or that metabolic energy is required. As indicated in Fig. 1, the accumulation of radioactivity failed to plateau within 10 min with either cystine or DAP as substrate, and analysis of the radioactive pools (see YMethods”) from strain W indicated that within 15 s the trans- ported cystine had been metabolized to yield several products, including cysteine, and the DAP had been predominantly con- verted to lysine. Such rapid metabolism makes the demonstra- tion of active accumulation difficult, yet Leive and Davis (42), by studying lysine auxotrophs unable to convert DAP to lysine, have shown that DAP could be actively concentrated by these strains and that the D mutation directly affects DAP transport rather than metabolism.

To determine the energy requirements for cystine transport in strain W, we studied the dependence of uptake on exogenous carbon source as well as the effects of energy poisons. As shown in Table I, uptake was stimulated by the addition of either glucose or succinate, with the most dramatic effects being obtained when the cells were incubated under conditions which would be ex- pected to deplete endogenous energy stores (13). Furthermore, sodium azide and 2,4-dinitrophenol severely inhibit cystine trans- port (Table I).

EJect of Diaminopimelate on Cystine Uptake

The initial rate of cystine transport in cells grown in minimal medium was studied as a function of external DAP concentration (Fig. 2). The inhibition by DAP plateaued at about 45% in strain W whereas in DZW, cystine uptake was completely in- hibited at a 200-fold excess of DAP. Furthermore, cystine, lanthionine, and cystathionine, all inhibitors of the general trans- port system (see below), inhibited DAP uptake in both strains (data not shown). These findings are consistent with the report by Leive and Davis (42) that W has two components of cystine

TABLE I

Energy requirements for cystine transport in strain W Transport assays were performed as described under “Meth-

ods.” The initial concentration of cystine was 10 j.hM. Glucose and succinate were used at 0.2%. In Treatment A, cells were washed, suspended in 20 volumes of minimal medium for 5 min, and preincubated with chloramphenicol and the appropriate car- bon source as described in “Methods.” In Treatment B, cells were washed, suspended in 20 volumes of minimal medium, and incubated for 2 hours at 37” with no carbon source. They were then incubated with chloramphenicol and carbon source at 23” for 5 min. In Treatment C, cells were washed, suspended in 20 volumes of minimal medium, and stored overnight at 4”, then treated as in B. In the experiments with metabolic inhibitors, the cells were incubated in the presence of the inhibitor, glucose, and chloramphenicol for 5 min prior to the initiation of the trans- port assay. One hundred per cent is defined as the initial rate of uptake for unstarved cells with glucose as the carbon source.

Treatment

A A A A A

B B B

C C c

- Relative rate of cystine uptake

- Glucose 100

+ Sodium azide (0.2%) 39 + 2,4-Dinitrophenol (2 mM) 23

Succinate 100 None 56

Glucose 87 Succinate 6G None 40

Glucose 95 Succinate 69 None 36

-- DIAMINOPIMELIG ACID (mM)

FIG. 2. Effect of DAP on cystine uptake. The initial rate of cystine transport at 10 PM cystine, initial concentration, was measured as a function of external unlabeled DAP concentration, as described under “Methods.” 0, strain W; l , strain DZW.

transport, a general system shared with DAP and a system specific for cystine, while DZW has only the general system.

Kinetics of Cystine Uptake

The dependence of cystine uptake on external cystine concen- tration (Fig. 3) further supports the presence of two systems in strain W grown on minimal medium (K, = 2 X 1O-8 M, K, = 3 x lop7 M). In the presence of DAP (2 mM) the K, apparent of the lower affinity system was raised to 9 X 1O-7 M but the smaller K, was unaffected, indicating that the lower affinity

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L -1.0

FIG. 3

TABLE II 13.0 Specijicity of cystine transport and binding

a

I

Transport assays were performed as described under “Meth-

ods.” After preincubation of the cells with glucose and chloram-

-I ’ g

phenicol, a portion of cells was added to the reaction flask con-

,2.0 iy taining glucose, chloramphenicol, [‘%]cystine (10 PM), and the

2 analogue. Binding assays were performed on the homogenous

$ cystine general binding p rotein by ecluilibrium dialysis (see “Methods”). Analogues were added to the same side with [“Cl-

1

E cystine (10 PM). One hundred per cent is defined as the rate of

1.0

2

transport or the amount of binding in the absence of added ana- logue.

I H2N NH, I

bH-R-&H

- 0.5 0 10

0.5 IO 15 2.0 HO& AOOH CYSTINE (,uM)

Kinetics of cystine transport. Initial rates u-ere deter- TT~T&. mined as described under “Methods.” S represents the initial cystine concentration (PM) whereas V represents the initial rate

of uptake (nanomoles per min per mg of cell protein). 0, strain W; A, strain W measured in the presence of unlabeled DAP (2 rnM) ; 0, strain l&W.

Analague “‘111s

in R

component represents the general system.0 DQW, which had

only the general system, had only a single K, of relatively low affinity (1 X 10’M).

The inhibition by DAP of cystine uptake in strain D,W was competitive with a K; of 14 pM (data not shown).

SpeciJcity of Cystine Transport in Strains TV and D,lV

A variety of cystine and DhP analogues were tested for their

ability to inhibit cystine transport. In strain W, the effects of a given compound on the general and the specific systems could be distinguished by testing the inhibition in both the presence and absence of 2 InM DL4P. Since DtW has only a single system, the effect of each analogue was measured only in the absence of DAP. The results shown in Table II indicate that the general system displayed considerably broader specificity than did the specific system. In each strain, the general system was inhibited by several analogues possessing the o1, a’-diaminodicarboxylic acid groups, as well as by the cystine hydroxamate and the cystine dimethyl ester, whereas only cystathionine, selenocystine, the cystine hydroxamate, and the cystine dimethyl ester inhibited

the specific system. Furthermore, none of the amino acids found in protein inhibited the activity of the general transport system

(data not shown).

Eflect of Osmotic Shock on Cystine Uptake

Osmotic shock has been shown to reduce the activity of some

amino acid transport systems such as glutamine (11-13) ; leucine,

isoleucine, and saline (5); leucine specific (10) ; and lysine, a.r- ginine, and ornithine (19, 20) ; but not of others such as lysine

specific (21) ; and proline (4). The two cystine uptake systems

showed markedly different sensitivities to shock (Table III). In

strain W grown in minimal medium, cystine transport was re-

duced by 4070 by shock, and the residual uptake was completely

6 A paradox emerges upon careful scrutiny of Fig. 3. At, low cystine concentrations, where almost all of the transport in strain W was dlle to the specific system, initial rates of uptake should have been unaffected by DAP. The inhibition of transport by DAP seen at low substrate concentrations (Fig. 3) is difficult to explain by the simple model of two noninteracting systems. We have as yet no explanation for this discrepancy.

Meso-2,3-diaminosuccinic acid (200 PM).

Diaminoglutaric acid (2

rnM).....................

Diaminoadipic acid (2 mM) DL-Meso-DAP (2 mi%)

p-Hydroxy-DAP (Mixture A) (1 mM)

P-Hydroxy-DAP (Mixture

B) (1 mM). p-Hydroxy-DAP (Mixture

C) (1 rnM).

P-Hydroxy-DAP (Mixture D) (1 mM).

DL-Lanthionine (200 .uM)

r-Methyl-DAP (2 m?a) DL-Allocystathionine (200

&M) .

DL-8elenOCJc3tine (500 PM).

Diaminosuberic acid (2 rnM)

n-Cystine (200 PM). Diaminoazelaic acid (2 mM) L-Djenkolic acid (ZOO PM)

L-Homocystine (200 PM).

L-Cystine hydroxamate (5OC PM)

L-Cystine dimethyl ester (200 ,.‘M)

.I

)I

0

1 2

3

3

3

3

3

3 3

4

4

4

4 5 5

G

4

4

1 Relative rate of cystine transport

Specific system of strain W

%

GWled system of strain Wa

Relative amount of

binding

84

92 118

100

-I- % %

98 110

80 100 103

0 105

15

9G 50 56

91 20 27

101 77 84

80

87 98

93 20 77

9

0

4

0

101

95 102 77

86

27 70

92 93 87

0

23

5

21

100 6:

101

0 20

43 100

92 99

120

18

47

a Similar results were obtained with cystine transport in strain

DZW.

insensitive to DAP, suggesting that osmotic shock affected only the general system. (Some variations in the extent of reduction of the general system occurred in several experiments, but in all cases, the general system was affected much more than the specific system.) Cystine transport in DtW, which proceeds entirely via the general system, was decreased by 83% after osmotic shock. As expected, DAP uptake, which is a direct measure of the general transport system, was sererely reduced by shock in both strains. Hence, it appears that’, of the two cystine uptake sgs- t,ems, osmotic shock reduced only the general one.

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Cystine Binding in Crude Shock Fluids

The loss of the cystine general transport system after osmotic shock encouraged us to look for a DAP-inhibitable cystine-bind- ing activity as well as for DAP binding in the crude shock fluid of cells grown in minimal medium. As shown in Table IV, the shock fluid from strain W contained these activities. The cystine binding was completely inhibited by a ZOO-fold excess of cold DAP, and the protein is therefore referred to as the rystine gen- eral binding protein. The shock fluid from strain D2W also contained this protein at approximately the same specific ac- tivity (data not shown).

Cystine Binding in Mutants Lacking Cystine General Transport System

Mutants lacking the cystine general transport system were se- lected on the basis of their resistance to the toxic analogue selenocystine. Strain D2W, which has only the general system, was used for the selection, and, of the mutants isolated which could grow in the presence of 3 X 1O-5 M selenocystine, most had little detectable cystine or DAP transport (Table V). Sev-

TABLE III Effect of osmotic shock on cystine and DAP uptake in cells grown

in minimal medkm and in Medium A Cells were harvested during the midexponential phase of

growth. A portion of these (control cells) was washed two times with minimal medium and suspended in 20 volumes of t’his me-

dium. The remaining cells were osmotically shocked (see “Meth- ods”) and suspended in 20 volumes of minimal medium. The shocked cells w-ere incubated at 37” with 0.2% glucose for 10 min,

and then both control and shocked cells were incubated with glu- cose and chloramphenicol for 5 min at room temperature. Trans- port assags were performed as described under “Methods.”

Groath medium Strain Treatment

-

Minimal

Minimal Minimal.

h!linimal A. A.

A. A..

w w DzW DzW W w

DzW

DzW

Initial rate of uptake centrated to a final volume of 180 ml. The shock fluid contained

cuyp;i;t Cyst&

I I

1640 mg of protein and had a specific activity for cystine binding

uptake + DAP of 0.79 unit per mg of protein (Table VI). 2 ITIM DAP uptake DEAE-cellulose Chronzafography-Fractionation of the con-

Control 1.25 Shocked 0.77 Control 2.55 Shocked 0.43 Control 2.23 Shocked 0.35 Control 0.16 Shocked 0.00

era1 of these were osmotically shocked and yielded greatly reduced levels of cystine binding in the crude shock fluids (Ta- ble V) .

Cystine Transport and Binding dctivity in Strain

W Grown in Jledium A

Strain W was grown under a, variety of conditions to see whether cystine transport activity could be influenced by the nature of the medium. Growth in rich media has been shown to alter the activity levels of some uptake systems as well as t,o cause certain systems which are normally resistant to shock to become shock-sensitive (4, 21, 54). The results with cells grown in Medium A (Table III) indicate that the cystine general transport system, as measured either by DAP-inhibitable cgstine uptake or by total DAP transport, was nearly absent from both W and DQW, while the activity of the specific system of W was increased almost 3-fold. Furthermore, when strain W grown under these conditions was osmot,ically shocked, cystine transport was reduced by more than 807;. We therefore expected to find greatly lowered levels of the cgstine general binding protein in the shock fluid, along with the a.ppearance of a cystinc-binding activity which was not inhibited by DAI’. The results &own in Table IV confirm these expectations. The cystine binding in the Medium A shock fluid was completely insensitive to l)AP,

and there was no detecta.ble D-Q binding in t,he shock fluid.

Puri$cation oj” Cystine General Binding Protein from Strain W

Growth and Osmotic Xhock of Cells-Forty-five liters of strain W were harvested in late stationary phase to yield 270 g (wet weight) of cells. Osmotic shock was performed as described under “Methods,” and the shock fluid was concentrated by ultrafiltration, desalted on a Bio-Gel P-10 column, and recon-

-

~7Id~S/WZi?S/?~g centrated desalted shock fluid on DEAE-cellulose yielded two

0.81 neighboring but distinctly separated peaks of cystine binding, n nr: the first (CBP I) eluting at 0.026 nr XaCl and the second (C’BP

0.79 0.78 0.03

0.00 2.17 0.18 0.00 0.00

V.“”

2.80 II) at 0.032 M NaCl (Fig. 4). Fractions containing cystine- 1.10 binding activity in each of t.hese peaks were pooled and conceIl- 0.18 trated by ultrafiltration, and the purification of each peak was 0.00 carried out separately. The specific activity of CBP I was 7.7 0.19 units per mg of protein, while that of CRP II was 9.4 units per 0.00 mg of protein (Table VI).

TABLE IV

Binding activity in crude shock &id from strain Ii- Cells were harvested in midexponential phase of growth and

osmotically shocked (see “Methods”). The shock fluid was con- centrated by ultrafiltration, dialyzed, and assayed for binding by

equilibrium dialysis as described under “Methods.” Cystine binding assays were performed at 10 PM and DAP assays at 20 pM.

Binding activity in shock fluid

Growth medium Cystine binding c~$n~~i~d~Pg DAP binding

unit/mg

Minimal medium.. 0.79 0.00 I 0.56 Medium A............... 0.80 0.78 1 0.00

TABLE v

Cystine trunsport and bi,nding in mu/ants resistant to selenocystine

Mutants were selected as single colony isolates on agar plates containing minimal medium, 15; sodium succinate, and 3 X 10m5

M selenocystine. Transport assays and preparation and assay of shock fluids were performed as described under “Methods.”

Initial rate of uptake Strain Cystine-binding activity in

shock fluid Cystine DAP

nmoles/min/nzg unilslg se1 weight of cells

D,W. 2.2 2.7 3.2 DzWSelR, 0.0 0.4 0.3

DzWSelRz. 0.0 0.3 0.3

D2WSelR3.. 0.0 0.4 0.4

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Further Purification of CBP I-The concentrated DEAE- only a single band when subjected to analytical polyacrylamide cellulose fraction (2.5 ml) was subjected to gel filtration disc gel clectrophoresis at pH 10.1 over a broad range of acryl- on Sephadex G-75 to yield 34 mg of protein with a specific ac- amide concentrations. However, analytical disc gel electro- tivity of 13 units per mg of protein (Table VI). The material phoresis at pH 2.7 as well as polyacrylamide gels in the presence from the Sephadex column was then divided into two equal por- of sodium dodecyl sulfate revealed the presence of a contam- tions, and each was further fractionated by electrofocusing as inating protein band (Fig. 6). The material from the pre- described under “Methods.” Appropriate fractions were pooled, parative disc gel electrophoresis step was therefore subjected to concentrated, and dialyzed to remove ampholytes and sucrose, electrofocusing in a pH gradient of 4 to 6 (Fig. 5). Cystine- and the final specific activity for cystine binding was 21 units binding protein with a specific activity of 25 units per mg of per mg of protein with a recovery of 40% compared to the DEAE- protein was obtained in 34y0 yield relative to the DEAE-cellulose cellulose fraction (Table VI). fraction (Table VI).

Further Purification of CBP II-The concentrated material from the DEAE-cellulose column was divided into two equal portions, and each was subjected to preparative disc gel elec- trophorcsis to yield cystine-binding protein with a specific ac- tivity of 14 units per mg (Table VI). This material produced

TABLE VI

Purifcation of cystine general binding protein

The purification steps are described under “Methods.” Cys- tine binding was determined by equilibrium dialysis.

Tests for Homogeneity-Both CUP I and CBP II were judged to be homogeneous after their respective purifiea.tions by the following criteria: (a) analytical polyacrylamide disc gel elec- trophoresis at pH 10.1 and pH 2.7 over a range of acrylamide concentrations from 4 to 10% revealed only a single band of protein (Fig. 6) ; (b) the proteins each migrated as a single band in polyacrylamide gels in the presence of sodium dodecyl sulfate; (c) sedimentation equilibrium ultracentrifugation produced a straight line in the log C versus r* plot for each protein (Fig. 7); (d) amino acid analysis indicated nearly identical composition for the two protein fractions; (e) antisera prepared against CBP I gave a single precipitin band with both CBP I and CUP II in Ouchterlony double diffusion tests.

Purification step Total units

Crude shock fluid. 1300 DEAE-cellulose

CBP I. . . 440 CBP II................. 490

Further purification of

CBP I

Total protein

m

1640

57 53

0.79

7.7 9.4

Sephadex G-75. 430 Electrofocusinga 176

Further purification of CBP

II

34 8.4

13 21

Preparative disc gel elec-

trophoresisa Electrofocusing..

490 34 14

168 6.7 25

-

-

-

-

Specific activity

-

R

-

ecwery

%

100 100

98 40

100 34

a Yields are based on the entire amount of starting material,

but only one-half of the material from the previous step was used at a time.

FIG. 4. DEAE-chromatography of the crude shock fluid from strain W. Desalted shock fluid protein (1640 mg) was applied to a column of DEAE-cellulose equilibrated with 5 nlM Tris-HCl, pH 7.2. The column was washed with this buffer and eluted with a linear gradient of 0 to 0.15 M NaCl in 5 mM Tris-HCI, pH 7.2. Arrow 1 indicates the beginning of the gradient. ATTOW 2 indi- cates where the column was washed with 1 M NaCl. Cystine- binding activity ( l - - -0) was measured by equilibrium dialysis (see “Methods”). Absorbance was followed at 280 nm (---).

Identity of CBP I and CBP II-Experiments with purified CBP I and CBP II have revealed no differences in their physical, chemical, immunochernical, or binding properties. By every criterion examined, including mobility on gels, molecular weight, amino acid composition, dissociation constant, and binding specificity, CUP I and CBP II are identical. In addition, an- tisera against CBP I gave a line of identity with both protein fractions in Ouchterlony double diffusion plates. We, therefore, believe it unnecessary to distinguish between CBP I and CBP II, and we refer to each as the cystine general binding protein. It should be emphasized that each of the experiments described below has been performed with material from both peaks with essentially identical results. We have as yet no explanation for the consistent appearance of two peaks on DEAE-cellulose, though similar phenomena have been noted in this laboratory with other shock-releasable binding proteins.

T”“““‘l”“““/ i I

L /*ANODE : •~

‘\ uw CATHODE-, I I I I I I I.l I-I I I1 I I I I I I

20 40 60 80

FRACTION NUMBER

FIG. 5. Isoelectric focusing of t,he cystine general binding protein. The protein was focused in a pH gradient of 4 to 6 and then eluted as described under “1Methods.” Cystine binding ( l - - -0) was assayed by equilibrium dialysis (see “Methods”). O---O represents the pH of individual fractions. The iso- electric point of t.he cystine-binding protein was 4.8.

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Properties of Cyst&e General Binding Protein

Physical Properties-The isoelectric point of the cystine general binding protein determined by isoelectric focusing in a pH gra- dient of 4 to 6 is 4.8 (Fig. 5). The molecular weight has been measured by three methods. (a) Sedimentation equilibrium ultracentrifugation was performed by the method of Yphantis (55). A speed of 36,000 rpm was sufficient to deplete the me- niscus, and the molecular weight determined from the slope of the log absorbance (280 nm) versus r* plot (Fig. 7) was 27,000, assum- ing a v of 0.74. (5) Electrophoresis in the presence of sodium do- decyl sulfate was performed with the purified cystine general bind- ing protein as well as with a series of standard proteins of known molecular weight. From the relationship of log molecular weight versus mobility (48)) a molecular weight of 28,000 was calculated. (c) The cystine general binding protein and a series of standards were individually chromatographed on a Sephadex G-150 col- umn (1.5 x 33 cm) in the presence of a blue dextran dye marker. All the binding activity migrated as a single peak corresponding to a molecular weight of 27,000.

Amino Acid Composition-Amino acid analysis was performed on a Beckman model 102 C analyzer according to the method of Moore and Stein (56). The results are shown in Table VII.

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Binding Parameters-The dissociation constant for cystine was determined by equilibrium dialysis. The conditions were adjusted so that at each concentration of cystine, not more than 5% of the isotope added was bound. Hence, it became nec- essary, especially at low cystine concentrations, to dialyze against large volumes containing the labeled amino acid. Dialysis bags were therefore used instead of the Plexiglas chambers. The KD determined from a plot of S/V versus S was 1 x 10e8 M (Fig.

8). The cystine general binding protein was not inhibited by any

of the amino acids naturally found in protein but showed a rel- atively broad specificity for other diamino-dicarboxylic acids. The ability of various cystine and DAP analogues to inhibit cystine binding is shown in Table II, and it can be seen that the specificity of the binding protein closely paralleled that of the general transport system. The nature of the DAP inhibition of cystine binding was competitive with a Ki of 17 PM (data not shown). The purified protein also bound DAP.

The binding of cystine to the cystine general binding protein measured at saturating substrate concentration displayed a broad pH optimum between pH 3 and 10 and was unaffected by alterations in ionic strength up to 0.5 M NaCl and by alterations in temperature between 4 and 37”.

Absence of Enzymatic Activities-The purified cystine general binding protein displayed no observable enzymatic activity toward either cystine or DAP. After incubation with the pro- tein, [14C]cystine chromatographed identically with the starting

FIG. 6. Polyacrylamide disc gel electrophoresis of the cystine general binding protein. Gels were run at pH 2.7. The resolving gel contained 6% acrylamide. A, crude shock fluid (50 rg): B, CBP I, DEAE-cellulose fraction (30 fig) ; C, CBP I, Sephadex G-75 fraction (20 pg); D, CBP I, electrofocusing fraction (10 pg); E, CBP II, DEAE-cellulose fraction (35 pg); P, CBP II, preparative disc gel electrophoresis fraction (35 pg); and G, CBP II, electro- focusing fraction (10 pg).

FIG. 7. Sedimentation equilibrium ultracentrifugation. High speed sedimentation equilibrium ultracentrifugation was per- formed according to the method of Yphantis (55). A sample (0.1 ml), containing 0.02 mg of cystine-binding protein in 0.1 M

NaCl in 0.01 M potassium phosphate buffer, pH 7.5, was added to one sector of a 12-mm double sector aluminum epoxy cell with sapphire windows. The reference sector contained 0.1 M NaCl in 0.01 M potassium phosphate buffer, pH 7.5. Cent,rifugat,ian was performed in a Spinco model E analytical ultracentrifuge at 20” at 36,000 rpm until equilibrium was reached. The cells were scanned along their length for absorbance at 280 nm. T represents _ . . the distance from the center of rotation.

I---

3-

j-

1-

2-

I-

/ /

d

/

P /

P /

I I I I I I 43.6 43.8 44.0 44.2 44.4 44.6 a

r* (cm2) 8

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T.ln~r; VII Amino acid composition of cystine general binding protein

Samples, 0.1 mg, of purified cystine-binding protein were sealed

in evacuated glass vials in 6 N HCl and hydrolyzed at 110”. One sample was hydrolyzed for 20 hours and another for 44 hours. In addition, a third sample was subjected to performic acid oxida- tion by the method of Moore (57) and then to a 20.hour HCl hy- drolysis. The hydrolyzed samples Tyere evaporated to dryness, suspended in 0.25 ml of deionized water, and subjected to amino acid analysis on a Beckman model 120 C analyzer according to the method of Moore and Stein (56). Analyses were performed in duplicate. Tryptophan was determined by the absorbance at

280 and 288 nm in the presence of 6 M guanidine hydrochloride as described by Edelhoch (58). Residues per molecule were calcu- lated assuming a molecular weight. of 27,000.

Amino acid Residues per molecule

Lysine.................................... Histidine Arginine.................................. Aspartic acid, Threonine. . . . . . . . . . . . Serine Glutamic acid. . Proline Glycine................................... Alanine.......................

Half-cystine Valine Methionine . . .

Isoleucine................................. Leucine................................... Tyrosine..................................

Phenylalanine............................. Tryptophan.

23.2

2.3 10.2 31.4 17.2 11.7 27.6

5.7 24.3 20.2

3.0 18.5

1.7

9.2 25.7

5.4

7.8 2.7

material. Furthermore, direct enzymatic assay for cystine reductase, DAP decarboxylase, and DAP transaminase revealed none of these activities for the purified binding protein, with supernatants from cell lysates prepared by freeze-thawing used as controls.

Stability-The cystine general binding protein was nearly fully active after exposure to a boiling water bath for 10 min, and the denaturation caused by 8 M urea could be completely reversed by dialysis. The protein was stable to storage at 3” and at -80” for at least 6 months.

DISCUSSION

117hm E. coli st.rain W is incubated in the presence of chlor- amphenicol and labeled L-cyst.&, radioactivity is accumulated by the cells by a process which is dependent upon exogenous carbon source and inhibited by metabolic energy poisons (Table I). Demonstration of active concentration against a chemical gradient is difficult, however, owing to the rapid me- tabolism of the transported compound. Hence the question arises as to whether the initial rate measured truly reflects the transport process or instead the rate of metabolism. Leive and Davis (42) have shown that lysine auxotrophs derived from W or D2W, unable to convert DAP to lysine, can actively ac- cumulate DAP, and that the initial rate of entry in these mutants is identical to that in the respective parents. Thus, they con- cluded that the initial rate of incorporation into the cell truly reflects transport and not metabolism, and that the D mutation directly affects the transport process.

I I I I I I I I 3.0-

I -20 0 20 40 60 60

S (nM)

FIG. 8. Kinetics of binding to the cystine general binding pro- tein. Equilibrium dialysis was performed in dialysis bags, each containing 0.5 ml of protein solution (0.3 unit per ml). The external solution cont.ained 1 to 2 nmoies of [‘G]cystine (271 mCi per mmole) in volumes between 20 ml and 1 liter. After equilibra- tion, 0.25 ml was removed from both the bag and the external solution and counted as described under “Methods.” S repre- sents the cystine concentration (nM) and V represents the counts per min bound.

We have not yet isolated mutants unable to metabolize cgstine, yet studies of other amino acid transport systems in our lab- oratory indicate that the initial rate of entry into the cell is not driven by metabolism. Transported L-arginine is rapidly me- tabolized by E. co& yet the initial uptake rate is the same in the presence of aminooxyacetic acid, an inhibitor of arginine me- tabolism (20). Similarly, azaserine blocks the conversion of L-glutamine to glutamate but does not alter the initial rate of glutamine uptake (13). By analogy to these other amino acid transport systems, we feel justified in assuming that the initial rate of entry of cystine is a direct measure of the activity of a

transport system. Leive and Davis (42) have reported that E. coli strain W 113s

two components of cystine uptake, one shared with DAP (which we refer to as the cystine general system) and the other not (the cystine specific system). We have presented several lines of evidence to support this view: (a) DAP inhibits only a portion of cystine uptake in strain W, whereas all of the uptake in strain DaW is competed for by DAP (Fig. 2); (b) the two cystine trans- port systems are kinetically distinct (Fig. 3) and display dif- ferent specificities (Table II) ; (c) osmotic shock of cells grown in minimal medium selectively reduces cystine general transport activity (and DAP uptake) in both W and D2W, while the spe- cific system of W is unaffected; (d) cells grown in Medium A are deficient in both cystine general and DAP uptake, whereas the activity of the specific system is enhanced (Table III); (e) strain DSW, which was derived from a strain selected for increased DAP uptake, also has a higher level of cystine transport activity. Similarly, both cystine and DAP uptake are reduced in seleno- cystine-resistant mutants derived from D2W (Table V). We have also derived fresh mutants from strain W deficient in the specific system by selecting for selenocystine-resistant colonies growing in Medium A. Such mutants exhibit no cystine spe- cific uptake when grown in either minimal or Medium A though both cystine general transport activity and DAP uptake are normal.

In conjunction with the reduction of the general system by shock of cells grown in minimal medium, a binding protein of

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similar specificity is released into the shock fluid (Table IV). Since the activity of the general system is 5 times as great in D,W as in W and most is released by shock, we might expect DZW to yield significantly greater binding activity. Such is not the case, for the specific activities are similar in each shock fluid. Thus, the mutation in D,W producing enhanced general system transport must be due to an alteration in some compo- nent of the transport system other than the total number of binding protein molecules. It might be argued that the increased general transport in D,W is due simply to the absence of the specific system which in W competed for some subsequent step in the over-all transport process. This interpretation seems unlikely in view of the observation that a DAP auxotroph, 173- 25, from which DzW was ultimately derived, also has only the general system but at the low activity characteristic of W (42). The lesion responsible for the 1) mutation is still under investi- gation.

The purified cystine general binding protein has properties similar to those reported for other binding proteins believed to be involved in transport, and it exhibits a specificity which closely parallels that of the cystine general transport system (Table II). A variety of cystine and DAP analogues possessing the a,(~‘- diaminodicarboxylic acid functions were tested for their ability to inhibit both cystine uptake and binding, and it was found that compounds containing a bridge between the a and a! car- bons of 3 or 4 units are effective as competitors, with the ex- ception of y-methyl-DAP. Compounds with shorter or longer bridges do not inhibit. In addition, the cystine hydroxamate and the cystine dimethyl ester are potent inhibitors of both transport and binding. A possible explanation for the failure of y-methyl-DAP to inhibit transport or binding is that the ma- terial used was a mixture of eight stereoisomers (four racemic pairs) and it is possible that one or more of these species do in- hibit the general system but were present in such low quantity at the concentration used that IIO inhibition could be detected. This interpretation becomes plausible in view of the stereoselec- tivity of both transport and binding: n-cystine is a poor inhibitor, and of the four racemic mixtures of /3-hydroxy-DAP only Mix- tures A and B inhibit significantly. DAP competitively inhibits both t,he general transport system and the binding protein with nearly identical Ki values (14 and 17 PM, respectively).

We have studied transport mutants as well as growth COII-

ditions which alter the activity of transport to obtain correlations between uptake and the level of binding activity in the crude shock fluid. Mutants of strain D2W lacking the cystine general transport system release much smaller amounts of cystine general binding protein into the shock fluid (Table V). Furthermore, when strain W is grown in Medium A, the activity of the cystine general transport system is lost (Table III), and 110 cystine gen- eral binding protein is found in the shock fluid (Table IV).

An additional effect of growth in Medium A is to enhance the activity of the cystine specific transport system and to render it sensitive to osmotic shock (Table III). The shock fluid does contain cystine-binding activity (Table IV), and further studies in our laboratory indicate that this binding, though insensitive to DAP, is inhibited by cystathionine, selenocystine, the cystine hydrosamate, and the cystine dimethyl ester, all potent inhib- itors of the cystine specific system. We are presently attempting to isolate this binding factor from the yeast extract-tryptone shock fluid with the aim of demonstrating its involvement in the cystine-specific transport system.

Acknowledgments-The authors are indebted to Dr. L. Leive

7693

and Dr. B. Davis, whose initial studies on diaminopimelate and cystine transport served as the impetus for this work. Dr. Leive kindly donated the bacterial strains and provided much valuable advice during the early phases of this study. We wish to thank Dr. Richard Crepeau for his assistance with the analytical ultra- centrifuge. We are also grateful to Miss Kathleen Quinn and to Miss -&ny Shandell for their technical assistance.

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Edward A. Berger and Leon A. HeppelEscherichia coliin

A Binding Protein Involved in the Transport of Cystine and Diaminopimelic Acid

1972, 247:7684-7694.J. Biol. Chem. 

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