Inhibition of Nitrate Transport by Anti-Nitrate Reductase IgG Fragments ...

Plant Physiol. (1988) 88, 1141-1145 0032-0889/88/88/1 141/05/$O 1.00/0

Inhibition of Nitrate Transport by Anti-Nitrate Reductase IgG Fragments and the Identification of Plasma Membrane Associated Nitrate Reductase in Roots of Barley Seedlings' Received for publication March 25, 1988 and in revised form June 24, 1988

MICHAEL R. WARD2, RUDOLF TISCHNER3, AND RAY C. HUFFAKER*

Plant Growth Laboratory, University of California, Davis, California 95616 ABSTRACT Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited N03- uptake by more than 90% but had no effect on N02- uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited N03uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on N03- uptake. The results present the possibility that N03- uptake and N03- reduction in the PM of barley roots may be related.

The transport of N03- by roots is the first step in the process of NO3 assimilation in plants. Transport is induced by NO3 and requires both RNA and protein synthesis, leading Jackson et al. (12) to propose that a specific N03 transport protein is synthesized. Although much is known about the activity of the NO3- transporter (7 and references therein), a NO3 transport proteins has not been identified in higher plants. Since both NR4 and N03 transport are simultaneously induced by NO3-, inhibited by protein and RNA synthesis inhibitors and increased in activity by supplying glucose to roots, Butz and Jackson (3) proposed that membrane associated NR functions as a carrier for N03- transport. Although plant NR is

'Supported in part by a grant from the United States National Aeronautics and Space Administration (NASA NCC-2-99). 2Present address: Department of Genetics, Harvard Medical School and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114. 3Present address: Planzenphysiologisches Institut der Universitat Gottingen, Untere Karspule 2, D-3400, Gottingen, West Germany. 4Abbreviations: NR, nitrate reductase; PM, plasma membrane; IgG, immunoglobulin G; FAD, flavin adenine dinucleotide; PMSF, phenylmethanesulfonyl flouride; PVPP, polyvinylpolypyrrolidone; T:p, Triton X-100 to protein ratio; TPI, triose phosphate isomerase; ADH, alcohol dehydrogenase.

generally thought to be localized in the cytoplasm (23), there are several reports suggesting that a portion of the total plant NR is membrane associated. Miflin (20) initially found a small percentage of the total barley root NR in a mitochondria enriched fraction. Later, he reported that membrane associated NR was localized in an unidentified particulate fraction distinct from the mitochondria (21). Lips and Avissar (17) suggested that NR was localized on the membranes of cell microbodies in tobacco leaves and that NR detected in soluble fractions had been released from the membranes during tissue extraction. Utilizing immunogold labeling techniques, Kamachi et al. (14) recently proposed the presence of NR in chloroplasts. Lopez-Ruiz et al. (18) localized NR of green algae in the pyrenoid. Although NR has been localized in the cell wall-PM region and in the tonoplast membranes of Neurospora crassa (26) and is present in the cytoplasmic membrane of Escherichia coli (19) it has not been localized in the PM of higher plants. The localization of NR in the PM is dependent on the isolation of highly purified PM fractions. We utilized aqueous two-phase partitioning to show that up to 4% of the total barley root NR is associated with enriched PM fractions. We also show the specific inhibition of N03 uptake by barley seedlings by anti-NR IgG fragments. The same antiserum that inhibited N03 uptake also inactivated the PM associated NR. These results present the possibility that NO3- transport and PM associated NR may be related. MATERIALS AND METHODS Plant Material. Barley (Hordeum vulgare L. var CM 72) seedlings were grown hydroponically (32). Seeds were soaked in 0.1% (v/v) chlorox solution for 15 min, rinsed with distilled water, and germinated at room temperature in aerated distilled water. After 24 h, the germinated seeds were spread on a layer of cheesecloth supported on a stainless steel screen about 1 cm above the surface of 5 L of aerated 0.2 mM CaCl2 solution, covered with saran wrap, and placed in the dark at 25°C. The saran wrap was removed after 3 d. After 5 d the seedlings were transferred to aerated one-tenth strength Hoagland solution lacking N (9) and placed in a controlled environment growth chamber. The continuous light growth growth conditions were: 60 to 65% RH and 25°C. The photo flux density at the seedling canopy was 400 ,uE m-2 s-' and was supplied by incandescent and coolwhite fluorescent lamps. After 2 d in continuous light, the seedlings were transferred for 24 h to one-tenth strength Hoagland solution containing 1 mm KNO3 to induce the NO3- uptake system and NR or 1 mm KNO2 to induce the N02- uptake system. Plasma Membrane Isolation. Barley roots (25 g) from 8 d old seedlings were detached just below the seeds, snipped into 1 cm

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pieces with scissors, and ground in 60 ml of 50 mM Tris (pH 8.0), 250 mm sucrose, 3 mM EDTA. 1 AM Na2MoO4(H20)2, 5 AM FAD, 3 mm PMSF, 1 mM DTT, and 0.05% (w/w roots) PVPP in a chilled mortar and pestle. The homogenate was filtered through 4 layers ofcheesecloth and centrifuged at 12,000g for 10 min. In some experiments a sample of the 12,000g supernatant was used for enzyme analysis. The remainder of the 12,000g supernatant was centrifuged at 1 20,000g for 20 min to obtain a microsome pellet and a soluble fraction. Microsomal pellets from 25 g of roots were resuspended in 2.2 mL of 250 mm sucrose, 5 mm K-phosphate (pH 7.8), and 1 mM PMSF and partitioned on an aqueous polymer two-phase system (1 1). All steps in the procedure were carried out at 4°C as detailed by Larsson (15). Briefly, 2 mL of the resuspended microsome fraction was added to an 8 mL two-phase system that contained 6.5% (w/v) Dextran T 500 (Pharmacia), 6.5% polyethylene glycol 3350 (Sigma), 0.33 M sucrose, 3 mM KCI, and 5 mm Kphosphate (pH 7.8). The tube was mixed by inversion 40 to 50 times and centrifuged in a swinging bucket rotor at 1,200g for 5 min. The upper phase was collected and partitioned on a fresh lower phase (28). The remaining microsome material, the first lower phase and the second upper phase fractions were collected, diluted with 20 mL of resuspension buffer (2 mm Tris-HCl [pH 8.0], 1 AM Na2MoO4(H20)2, 5 AM FAD, 3 mM PMSF, and 1 mM DTT) and pelleted at 120,000g for 20 min. The pellets were resuspended in resuspension buffer. The upper phase pellet was resuspended, diluted with 20 mL of resuspension buffer, and repelleted at 120,000g for 20 min. The pellets were diluted with resuspension buffer to a protein concentration equivalent to the soluble fraction and enzymes were assayed. Enzyme Analysis. Triton X-100-stimulated UDPase activity was assayed according to Nagahashi and Kane (22). Triose phosphate isomerase activity was determined according to Gibbs and Turner (4). Cyt c oxidase and Antimycin A insensitive NADH-Cyt c reductase activity were determined according to Hodges and Leonard (10). Vanadate sensitive ATPase activities were determined in the presence and absence of 0.0125% Triton X-100 (T:p = 25) according to Sandstrom et al. (28). Nitrate sensitive ATPase activity was determined acording to O'Neil et al. (24). Alcohol dehydrogenase activity, a marker for the cytoplasm (25), was determined according to Suzuki and Kyuwa (29). Nitrate reductase was assayed in the presence and absence of 0.1% Triton X-100 (T:p = 10) in an assay volume of 600,L (1). Protein was measured using a modification of the Bradford method (2) in which 60 AL of 0.2% Triton X- 100 was included in each assay tube to solubilize membrane proteins. Antiserum Preparation. Antiserum to Chlorella sorokiniana (strain 211-8k from Sammlung von Algenkulturen, Gottingen, West Germany) NR was prepared as described (31). IgG was purified from the serum by protein A-Sepharose chromatography (5), concentrated using Centricon 30 filters (Amicon, Danvers, MA) and hydrolyzed with papain (16). The papain was inactivated with iodoacetamide (16). The cleaved IgG fragments were separated from papain by gel filtration on a Sephadex G-1 50 column (13, 16). The fragments were concentrated and washed with 50 mm Mes (pH 6.5) on Centricon 30 filters and used in the uptake experiments. Preimmune serum was digested with papain and IgG fragments were purified as above. Uptake Experiments. Uptake studies were carried out with one group of three N03 induced (NO3- uptake experiments) or three NO2 induced (NO2- uptake experiments) 8 d old seedlings. A group of three induced seedlings was transferred to 4.5 ml of aerated one-quarter strength N-free Hoagland solution (9) with 1 mM KNO3 (nitrate induced) or KNO2 (nitrite induced) and1 mM Mes (pH 6.2) for a 3 h equilibration period. Solutions were changed every 30 min. After 3 h, an initial uptake rate was determined by following depletion of NO3- or N027 from the

Plant Physiol. Vol. 88, 1988

4.5 mL of aerated nutrient solution. Solutions were sampled every 5 min for 30 min. Roots were rinsed three times in fresh uptake solution and allowed to equilibrate for 10 min in the third rinse solution. The seedlings were then sequentially supplied fresh uptake solution with 2.6 mg (2) of cleaved preimm6ne

serum IgG fragments, intact anti-NR serum or cleaved anti-NR IgG fragments for 30 min uptake studies. The seedlings were rinsed as above after each uptake period and wash uptake rates were determined in fresh nutrient solution. Depletion of N03 and N02 were assayed by HPLC (30). Rates of net influx (uptake) were determined from the depletion curves (6). Salt and Chelate Wash. Twice pelleted upper phase fractions were diluted to 0.5 mg/mL protein concentration in resuspension buffer and brought to a final concentration of 500 mm NaCl and 1 mm EDTA in 0.5 mL or were diluted with an equal amount of resuspension buffer and placed on ice. Both fractions were vigorously vortexed for 30 s. The salt and chelate treated fractions were sonicated on ice in a sonicator (American Brand, [American Scientific Product]). After 15 min, the fractions were diluted with 8 mL of resuspension buffer and pelleted at 150,000g for 15 min. Nitrate reductase activity was determined in the resuspended pellets. Triton X-100 Washes. Twice pelleted upper phase fractions were diluted to 0.5 mg/mL protein with NR resuspension buffer, brought to 0 or 0.1% Triton X-100 (T:p = 2.5) in a total volume of 0.5 mL, mixed vigorously, and placed on ice for 15 min. The fractions were then pelleted at 150,000g for 15 min without dilution. Nitrate reductase activity was determined in both the soluble and resuspended membrane fractions. Inactivation of NR Activity by Chlorella NR Antiserum. Upper phase fractions were treated with 0.1 % Triton X-100 and pelleted to remove the insoluble material as above. One hundred,uL of water, preimmune serum, or anti-Chlorella NR antiserum was added to 200 gL of the Triton solubilized upper phase fractions. The fractions were vortexed vigorously for 30 s, placed on ice for 2 h, and then centrifuged for 5 min at 12,000 rpm in a microfuge. Nitrate reductase activity was assayed in the supernatant fractions.

RESULTS AND DISCUSSION Anti-NR IgG fragments purified from Chlorella NR antiserum inhibited N03 uptake by more than 90% but did not affect NO2- uptake by barley seedlings (Table I). (Chlorella NR was used as an antigen because it is more stable than barley NR and pure preparations were obtained [8, 31]. The antiserum crossreacted with soluble NR from barley roots as evidenced by inactivation of NR activity). The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after three rinses in fresh uptake solution and a 10 min equilibration period. IgG fragments isolated from preimmune serum inhibited nitrate uptake 36% but the effect was completely reversible by rinsing. Intact anti-NR molecules did not affect N03 uptake (Table I) presumably because they are much larger (150 kD) than the cleaved fragments (50 kD) and could not move as easily through the root cell wall to bind to N03 transport sites. Intact antibodies to the yeast PM Pi binding protein did not affect in vivo Pi uptake until the yeast cell wall was removed or the IgG molecules were cleaved with papain. This led Jeanjean et al. (13) to suggest that the intact IgG molecules were too large to penetrate the yeast cell wall to bind to the Pi transporter. The inhibition of N03 uptake by anti-NR IgG fragments suggested that NR or an antigenically related protein involved in N03 transport were present in the PM, the primary barrier of ions (and IgG fragments) to the cell cytosol. To determine if NR were present in the PM, it was important to isolate highly enriched PM fractions. Aqueous two-phase partitioning of plant

PLASMA MEMBRANE NITRATE REDUCTASE AND N03- TRANSPORT

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Table I. Effect of NR Antibodies on Nitrate and Nitrite Uptake Uptake experiments were carried out with NO3- induced (NO3- uptake experiments) or NO2- induced (NO2- uptake experiments) as described in "Materials and Methods." Average rates ± SE for the 0.5 h uptake studies are reported. Each experiment was repeated 2 times and representative data are shown. N03- Uptake Rate NO2- Uptake Rate Treatment Experiment Order ,umol/g x h 3.6 ± 0.4 1. 3.3 ± 0.1 Initial uptake rate 3.2 ± 0.5 2.1 ± 0.1 Preimmune IgG fragments 2. 3.1 ± 0.4 3.0 ± 0.09 Preimmune IgG fragments after wash 3. 3.0 ± 0.2 3.3 ± 0.2 Intact anti-NR serum 4. 3.3 ± 0.4 3.69 ± 0.1 Intact anti-NR serum after wash 5. 3.2 ± 0.9 0.25 ± 0.08 6. Anti-NR IgG fragments 2.9 ± 0.2 1.89 ± 0.1 Anti-NR IgG fragments after wash 7. Table II. Marker Enzyme Assessment ofSoluble, Microsome, and Phase Partitioned Membrane Fractions Root fractions were isolated and enzymes were assayed as described in "Materials and Methods." Vanadate sensitive ATPase in the presence and absence (±T) of 0.0125% Triton X-100 (T:P = 25), NO3- sensitive ATPase, and latent UDPase activities are in Mmol Pi/mg protein x h. NADH Cyt c reductase and Cyt c oxidase activities are in ,mol/mg protein x min. TPI and ADH activities are in nmol NADH oxidized or Mmol NAD reduced per mg protein x min, respectively. Each experiment was repeated at least three times and representative data are shown. Plant Fraction FractionSoluble

Marker Vanadate sensitive ATPase (-T) Vanadate sensitive ATPase (+T) NO3- sensitive ATPase Latent UDPase NADH Cyt c reductase Cyt c oxidase TPI ADH

Vol

Prot

11.8

22.1

21.2

51.8

PM

14.3

30.3

24.5

84.5

Tonoplast Golgi

0.70 3.2 66 0 0.27 50.1

0.95 10.4 195 97.5 2 0

0 0 55.2 0.64 0 0

ER

Mitochondria Plastid/cytoplasm Cytoplasm

Specific

Total

-Triton +Triton -Triton +Triton mL mg/mL

nmol/mgx h protein

nmol/h

12 K

Supernatanta 65 59 Solubleb Microsomec 13

0.56 0.5 0.41

Lower Phase I

0.48

2.2

21,300 19,200

13.9

840

284 394 2.6

7.1

116

6.7

10,300 11,600

~~~~~~~~~Phase

Uppe ~~~~~~~LowerUpper

PM

Table III. Nitrate Reductase Activity Distribution in Root Fractions Root fractions were isolated as described in "Materials and Methods." Nitrate reductase was assayed in the presence and absence of 0.1 % Triton X-100 (T:P = 10). The experiment was repeated at least four times and data from a representative experiment are shown. Nitrate Reductase Activity

Sample

Microsome

586 651 157 110

Upper 284 14 5.4 109 1.1 0.35 Phase II b a From 12,000g centrifugation. From 120,000g centrifugac Pellet from 120,000g centrifugation. tion.

membrane fractions is a convenient technique for isolating PM fractions of high purity (1 1, 15). Marker enzyme assessment of two-phase partitioned barley root microsomal fractions is shown in Table II. A 2.3- to 2.8fold enrichment of the PM ATPase activity was detected in the upper phase over the microsome fraction in the presence and

0.31 4

203 37.9 2.44 0

Table IV. Triton Solubilization of PM Associated NR Upper phase (PM) fractions were isolated and treated with Triton X100 as described "Materials and Methods." NR was assayed in the soluble and pellet fractions in the presence of 0.1% Triton X-100. The experiment was repeated five times; data from a representative experiment are shown. Nitrate Reductase Activity Treatment Soluble Pellet nmol/mg protein x h 211 0 -Triton X-100 301 26 +Triton X-100 Table V. Salt and Chelate Wash of PM Fractions Plasma membrane fractions were isolated and washed with or without 500 mm NaCl, 1 mm EDTA and sonicated as described in "Materials and Methods." After treatment, the PM fractions were diluted with resuspension buffer and pelleted. The resulting pellets were assayed for NR activity in the presence of Triton X-100 (T:p = 10). The experiment was repeated three times and data from a representative experiment are shown. Nitrate Reductase Activity Treatment nmol/mg protein x h 211 -NaCl 224 +NaCl, EDTA

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Plant Physiol. Vol. 88, 1988

Table VI. Inactivation of PM NR Solubilized with Triton X-100 Nitrate reductase was solubilized from the PM fractions as described in "Materials and Methods." One hundred jsL of water, preimmune serum, or anti-NR antiserum was added to 200 ,uL of the Triton solubilized PM fractions. The fractions were kept on ice for 2 h and then centrifuged for 5 min at 12,000 rpm in a microfuge. Nitrate reductase was assayed in the supernatant fractions. Each experiment was repeated at least four times and representative data are shown. Treatment Nitrate Reductase Activity nmol/ml x h -Antiserum 15.8 +Preimmune serum 16.0 +Anti-NR serum 0.0

NR from the PM fraction suggesting that the association was not simply ionic but that NR was tightly associated with the PM lipid bilayer. To further characterize the PM associated NR activity, PM vesicles were isolated, treated with Triton X- 100 (T:P = 2.5) to remove NR from the membrane pellet (Table IV) and then incubated with either preimmune or anti-NR serum (Table VI). The PM associated NR was inactivated by NR antiserum but was not affected by the preimmune serum. A single spot on Western blots of Triton solubilized PM fractions separated by two-dimensional electrophoresis was detected with the NR antiserum (MR Ward, PR Lafayette, RL Travis, RC Huffaker,

PM. The NR activity distribution in root soluble and phase partitioned membrane fractions is shown in Table III. Most of the NR activity was soluble as is well established (23); however, approximately 4% of the total NR was associated with the membrane fraction when assayed in the presence of Triton. A 1.8- to 5.4-fold enrichment of the NR activity in the upper phase (PM) fraction over the microsome in the presence and absence of Triton, respectively, (Table III) was detected. Taken together, the results from the marker enzymes and from the NR activity show that a significant portion of barley root NR was present in highly enriched PM fractions. The addition of 0.1% Triton X- 100 (T:P = 10) to the reaction mixture stimulated membrane associated NR specific activity 60-fold in the microsome fraction, 16-fold in the lower, and 20fold in the upper phase, but increased soluble NR activity by only a factor of 1.7 (Table III). In contrast to NR, upper phase PM ATPase activity was stimulated by Triton whereas lower phase PM ATPase activity was not affected (Table II). These results agree with others who found that inside-out PM vesicles partition in the lower phase of aqueous polymer two-phase systems (15). Triton X-100 does not stimulate the PM ATPase of lower phase vesicles because the hydrolytic site of the enzyme is on the outside and is accessible to ATP (15, 28). The lack of significant NR activity in the absence of Triton in both the lower and upper phase PM fractions indicated that the membrane associated NR NO3- reducing and/or NADH oxidizing sites were not directly exposed to either side of the PM. Most of the PM associated NR was solubilized by 0.1% Triton X-100 (T:P = 2.5) (Table IV). In contrast, similar concentrations of Triton did not solubilize the PM ATPase (data not shown). This suggests that the latency of the membrane associated NR was a function of the solubilization of NR from the membrane. To determine the extent of the association of NR and the PM, PM vesicles were vigorously vortexed in 500 mM NaCl and 1 mM EDTA, sonicated for 15 min, and then pelleted after dilution (Table V). The salt/chelate/sonication treatment did not remove

in preparing the antiserum and P. R. Lafayette, R. L. Travis, and M. Aslam for helpful discussions.

unpublished data).

The inactivation of the PM associated NR and the inhibition of NO3- transport by the NR antiserum present the possibility that NO3 transport and the PM associated NR may be related. absence of Triton X-100, respectively. The Triton stimulation of The PM associated NR may be a part of an enzyme complex the ATPase of phase partitioned PM fractions is well established that both transports and reduces N03 as proposed by Butz and (Table II; Refs. 15, 28). The latency indicates that the vesicles Jackson (3). Alternatively, the NO3- transporter and the PM are sealed and right side out and is attributed to increased associated NR may be completely separate but antigenically accessibility of ATP to the hydrolytic site of the PM ATPase. related systems. Rufty et al. (27) recently proposed the possibility Recently, it has been proposed that Triton X- 100 also activates that the receptor for the NR induction system and the functional the PM ATPase possibly by altering the lipid environment near NR protein may both be associated with the PM of root cells. the ATPase or by removing an inhibitory component (28). The results demonstrated that anti-NR lgG fragments specifiEvaluation of markers for the tonoplast, Golgi apparatus, mito- cally inhibited NO3- uptake. We have localized membrane aschondria, ER, plastid and cytoplasm demonstrated that each of sociated NR in the PM and have shown that the same antibodies these activities was significantly reduced by the two-phase pro- that inhibited NO3- uptake also inactivated PM NR. These cedure. Of particular significance was the absence of cytoplasmic results indicate a possible relationship between N03- transport contamination in the upper phase fractions since NR is generally and NO3 reduction in the PM of barley roots. considered to be a cytosolic enzyme (23). The marker enzyme data indicate that the upper phase fraction consisted mainly of Acknowledgments-The authors would like to thank P. Velasco for assistance LITERATURE CITED 1. ASLAM M, JL ROSICHAN, RC HUFFAKER 1987 Comparative induction of nitrate reductase by nitrate and nitrite in barley leaves. Plant Physiol 83: 579-584 2. BRADFORD MM 1976 A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principles of proteindye binding. Anal Biochem 72: 248-254 3. BUTZ RG, WA JACKSON 1977 A mechanism for nitrate transport and reduction. Phytochemistry 16: 409-417 4. GIBBS M, MJ TURNER 1964 Enzymes of glycolysis. In HF Linskens, BD Sanwal, M Tracey, eds, Modern Methods of Plant Analysis, Vol 7. Springer Verlag, Berlin, p 520 5. GODING JW 1976 Conjugation of antibodies with fluorochromes: modifications to the standard methods. J Immunol Methods 13: 215-226 6. GOYAL SS, RC HUFFAKER 1986 A novel approach and fully automated, microcomputer based system to study kinetics of NO3 , NO2- and NH4' transport simultaneously by intact wheat seedlings. Plant Cell Environ 9: 209-2 12 7. GOYAL SS, RC HUFFAKER 1986 The uptake of NO3-, NO2- and NH4' by intact wheat (Triticum aestivum) seedlings. I. Induction and kinetics of transport systems. Plant Physiol 82: 1051-1056 8. HAGEMAN RH, AJ REED 1980 Nitrate reductase from higher plants. Methods Enzymol 69: 270-280 9. HOAGLAND DR, DI ARNON 1950 The water culture method for growing plants without soil. Calif Agric Exp Stn Bull 347 10. HoDGES TK, RT LEONARD 1974 Purification of a plasma membrane bound adenosine triphosphatase from plant roots. Methods Enzymol 32: 392-406 11. HoDGEs TK, D MILLS 1986 Isolation of the plasma membrane. Methods Enzymol 118: 41-54 12. JACKSON WA, D FLESHER, RH HAGEMAN 1973 Nitrate uptake by dark grown corn seedlings. Plant Physiol 52: 120-127 13. JEANJEAN R, S BEDU, J ROCCA-SERARA, C FOUCAULT 1984 Phosphate uptake in the yeast Candida tropicalis: purification of phosphate-binding protein and investigations about its role in phosphate uptake. Arch Microbiol 137: 215-219 14. KAMACHI K, Y AMEMIYA, N OGURA, H NAKAGAWA 1987 Immuno-gold localization of nitrate reductase in spinach (Spinacia oleracea) leaves. Plant Cell Physiol 28: 333-338 15. LARSSON C 1985 Plasma membranes. In HF Linskens, JF Jackson, eds, Modern Methods of Plant Analysis. New Series Vol 1, Cell Components. SpringerVerlag, Berlin, pp 85-104 16. LIFTER J, YS CHOI 1978 Separation of IgG Fab and Fc fragments by isoeCectnc focusing. J Immunol Methods 23: 297-302

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