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Uptake of Proline by the Scutellum of Germinating Barley
Grain' Received for publication September 30, 1985 and in revised form December 18, 1985
EILA VAISANEN AND TUOMAS SOPANEN*2
Biotechnical Laboratory, Technical Research Centre of Finland, Tietotie 2, SF-02150 Espoo 15, Finland (E.V., T.S.), Institute of Botany, University of Helsinki, Unioninkatu 44, SF-001 70 Helsinki 17, Finland
scutellum and proposed that these two amino acids are taken up
by two amino acid uptake systems having broad specificities. In Scutella separated from germinating grains of barley (Hordeum vul- the present communication we suggest on the basis of kinetic gare L. cv Himalaya) took up 1 millimolar L-1"Cjproline at an initial rate that in addition to these two nonspecific uptake experiments of about 6.5 micromoles gram-' fresh weight hour-' (pH 5, 30°C). The a systems, third, uptake had a pH optimum at 5. The bulk of the uptake (93%) was via involved in the apparently proline-specific uptake system is uptake of proline. carrier-mediated active transport. All of the 19 L-amino acids tested at 10 millimolar concentration inhibited the mediated uptake of 1 millimolar proline, the inhibitions varying from 18 to 76%. By studying how large a fraction of the mediated uptake was inhibitable by asparagine, alanine, glutamine, and leucine, the mediated uptake was shown to be due to three components. Two of these are most probably attributable to the two nonspecific uptake systems proposed earlier to act in the uptake of glutamine and leucine. The third component was not inhibited by glutamine, asparagine, or alanine, but was inhibited by unlabeled proline and leucine. The uptake by this system was apparently carrier-mediated active transport. D-Proline inhibited this system as strongly as L-proline. Nine of the 16 L-amino acids tested at 50 millimolar concentrations did not inhibit the uptake of 1 millimolar proline by this system. Valine, leucine, isoleucine, and the basic amino acids were inhibitory, but in spite of this, they did not appear to be taken up by this system. It seems therefore that in addition to two nonspecific amino acid uptake systems the scutella have an uptake system which is specific for proline. It is likely that this proline-specific system accounts for the bulk of proline uptake in a germinating grain.
Proline comprises about 14% of the total amino acids in the endosperm of Himalaya barley recovered after hydrolysis and is the second most abundant amino acid residue in this tissue (15). It is especially abundant in hordeins, the alcohol-soluble storage proteins of barley ( 18). During the germination, proline-like other amino acids in the starchy endosperm-is liberated from the storage proteins by the concerted action of proteinases and carboxypeptidases (1 1, 13). Interestingly, the liberation of proline involves, in addition to three nonspecific carboxypeptidases, two proline-specific carboxypeptidases, which act only on peptides of the type ... XPro-Y (12). Apparently this is due to the unique structure of proline, which has an imino group instead of an amino group. This structural property might naturally also affect the uptake of proline. We have previously studied some properties and the regulation of the uptake of leucine (14, 20) and glutamine (21) into the 1 Supported by the Research Council for the Natural Sciences.
2Junior Research Fellow of the Research Council for Natural Sciences. 902
MATERIALS AND METHODS Plant Material. Grains of barley (Hordeum vulgare L. cv Himalaya) were obtained from The Agronomy Club, Washington State University, Pullman, WA. They were allowed to germinate aseptically at 20°C for 3 d as described earlier (1 1). Uptake Assay. The scutella were dissected out and weighed, and samples of 4 scutella were incubated for 10 min in 3 ml of 1 mM ['4C]proline in 5 mm sodium 2,2-dimethylglutarate buffer (pH 5) in a shaking water bath at 30°C. The label taken up was measured by liquid scintillation spectrometry (20, 21). For inhibition experiments, stock solutions of inhibitory amino acids (25-625 mM) were made in the pH 5 buffer, and the pH of these solutions was readjusted to 5 with 0.5 M NaOH or HCI. The results are given as umol amino acid taken up by 1 g fresh weight in 1 h. All the values reported are means ± SE from 4 assays and all experiments have been carried out at least twice. Reagents. The L-[U-'4C]proline was purchased from The Radiochemical Centre, Amersham, England. Unlabeled amino acids and proline derivatives were from Merck AG., Sigma Chemical Co., or Bachem AG., and 2,2-dimethylglutaric acid from Fluka AG.
RESULTS Validity of the Uptake Assay. The accumulation of 14C into the scutella during incubation in 1 mM ['4C]proline was linear with time at least for the first 15 min and the line went through the origin (data not shown). This shows that initial uptake rates were measured during the standard 10 min incubation. When samples of 30 scutella were incubated in the standard assay conditions for 40 min, the decrease of 14C from the medium corresponded to the accumulation of 14C into the scutella. The measured rates of uptake were thus not diminished by a loss of 14C02 after uptake and possible metabolism. Estimation of Nonmediated Uptake. To estimate the nonmediated uptake, the uptake of 1 mM [14C]proline was assayed in the presence of increasing concentrations of unlabeled proline and the inhibitory effect was extrapolated to an infinitely high concentration of unlabeled proline (21 and references cited therein). The extrapolation showed that 7% of the uptake of 1 mM ['4C]proline was not inhibitable by unlabeled proline (Fig. IA). A mean value from two experiments indicates that the nonmediated uptake in the present assay conditions was about
FIG. 2. Effect of pH on the total uptake of I 2,2-dimethyl glutarate buffers. E
metabolic energy. The rate of total uptake increased with increasing substrate concentration and no saturation was observed. When the values were corrected for nonmediated uptake, the curve resembled more closely the Michaelis-Menten curve, but clear saturation 1.185 1.2 20° was not observed below 50 mm proline. Nor was the Hofsteeplot of the values linear (Fig. 3). This indicates that there are two or more components in the uptake of proline and that at least one of them has a high Km. An approximate estimation gives a 0.002 0.004 0.006 0.008 0.010 Km of the order of 4 to 6 mm and a Vm, of 25 to 30 ,umol g-' fresh weight h-' for the main component of uptake. 10 [ 1] Inhibition by Other Amino Acids. The mediated uptake of 1 mM proline was inhibited by all of the 19 L-amino acids tested at 10 mm concentrations, the inhibitions varying from 18 to 76% 01 ~0§ (Table I). D-Leucine was not inhibitory, but D-arginine caused an inhibition of about 30%. 400 300 200 To study whether some amino acids could inhibit the uptake 100 Proj (mM) of proline completely or only partially, the uptake of 1 mM FIG. 1. Estimation of nonmediated uptake of proline. The uptake of proline was assayed at increasing concentrations of glutamine, alanine, asparagine, and leucine. After correction for nonme1 mM (A) or 10 mm (B) ['4C]proline was assayed in the presence of 0 to 400 mm concentrations of unlabeled proline. Insets, Extrapolation of the diated uptake the values of v03/(v0 - vi) were plotted against 1/I inhibitory effect to an infinitely high concentration of unlabeled proline. (Fig. 4) and the intercept of the line with the y axis was used to calculate the mediated uptake in the presence of an infinitely 0.45 timol g-' fresh weight h-'. A similar experiment at 10 mM high concentration of the inhibitory amino acid (v,*) (21 and proline gave a value of 4.45 ,umol g-' fresh weight h-' for the references cited therein). With asparagine 31% (1.48 tsmol g-' nonmediated uptake (Fig. 1B). This confirms that the nonme- fresh weight h-') ofthe mediated uptake was not inhibitable (Fig. diated uptake is a linear function of the substrate concentration. 4). The corresponding value for glutamine was 20% (0.98 ,mol When a similar assay was carried out with another batch of g-' fresh weight h-'), and a similar value (18%) was obtained for ['4C]proline, the value for the nonmediated uptake at 1 mm alanine. The uptake was virtually completely inhibitable by proline was 0.83 ,umol g-' fresh weight h-'. This difference is leucine. These results suggest that there are three components in possibly due to the fact that the second batch of ['4C]proline the uptake of proline: (a) an asparagine-inhibitable component, contained, according to the manufacturer, about 1.5% of radio- which is also inhibited by glutamine, leucine, and alanine (about active impurities whereas the batch giving the lower value con- 70% of mediated uptake at 1 mM proline), (b) a component tained 0.7% of impurities. This demonstrates that the method which is not inhibited by asparagine but is inhibited by glutaused to estimate the nonmediated uptake has the advantage that mine, alanine, and leucine (10% of mediated uptake), and (c) a the uptake of any radiochemical impurity, either by diffusion or component which is not inhibited by asparagine, glutamine, or by a carrier having no affinity for the substrate, will be included alanine, but is inhibited by leucine (20% of mediated uptake). The same three components could be detected when the in the nonmediated uptake. We have not studied in detail the nature of the nonmediated concentration of proline was 10 mM (Fig. 5). At this concentrauptake. It is likely that it is at least to some extent ascribable to tion, the asparagine-inhibited component accounted for about label remaining in the apoplastic space (21) and to uptake of 50% (10.6 Mmol g-' fresh weight h-') of the mediated uptake, labeled impurities. The value obtained for nonmediated uptake the asparagine-uninhibited, glutamine-inhibited component acis thus just a term needed for calculation of the mediated uptake. counted for about 20% (4.7 Amol g-' fresh weight h-'), and the It does not represent diffusion of the substrate through plasma- glutamine-uninhibited component accounted for about 30% (6.7 lemma, the rate of which is likely to be much smaller than the umol g-' fresh weight h-'). In this case the relative contributions ofthe two minor components were greater than at 1 mm proline. value given for total nonmediated uptake. Some General Properties of Proline Uptake. The pH optimum 3 Abbreviations: v,,, uptake in the absence of inhibitor, vi, uptake in of proline uptake in sodium 2,2-dimethylglutarate buffers was about 5 (Fig. 2). Dinitrophenol (0.25 mm, 5 min preincubation) the presence of inhibitor, vi., uptake in the presence of an infinitely high inhibited the total uptake by about 80%, indicating a dependence concentration of the inhibitor, I, inhibitor. on
FIG. 3. Effect of substrate concentration on the uptake of proline. The uptake of 0.5 to 50 mM proline was assayed and the values were corrected for nonmediated uptake (proline concentration in mM x 0.83 umol g-' fresh weight h-' [- - -]). Inset, v against v/S plot of the mediated uptake.
L. 9 40 2
0 30 .9
Table I. Effect of 10 mm L-Amino Acids on the Uptake of I mM Proline Inhibitory Inhibition of Amino Total Uptake of Proline Mediated Uptake Acid 10 mM jimol g-'fresh wth' % None 6.23 ± 0.32 2.49 ± 0.08 Gly 69 Ala 2.16±0.09 75 Val 4.47 ± 0.05 33 Leu 3.22 ±0.34 56 Ile 3.82±0.16 45 Pro 3.22 ± 0.13 56 2.10±0.12 76 Cys Met 2.19±0.20 75 47 Phe 3.71 ± 0.33 Try 3.20±0.19 56 70 Ser 2.45 ± 0.26 Thr 3.83 ± 0.22 55 27 Asn 4.77 ±0.43 Gln 2.71 ± 0.13 65 5.24 ± 0.48 18 Asp Glu 3.23 ± 0.14 56 54 3.31 ±0.15 Arg 44 Lys 3.86 ±0.40 32 His 4.49±0.15 a The nonmediated uptake (0.83 ;mol g-' fresh wt h-') was subtracted from the original values and the inhibition was calculated as a percentage of the value without any inhibitor.
FIG. 4. Effect of increasing concentrations of asparagine, glutamine, alanine, and leucine on the uptake of I mm proline. The uptake of I mM proline was assayed in the presence of 0 to 300 mm concentrations of the inhibitory amino acid. Insets, Values were corrected for nonmediated uptake (0.45 smol g-' fresh weight h-') and the values for the mediated uptake were used to extrapolate the inhibitory effect to an infinitely high concentration ofthe inhibitory amino acid. (- - -), Nonmediated uptake.
adjustment of the pH. Alanylalanine (50 mM) also inhibited the uptake of proline, the inhibition being about 25%. In the standard conditions of the peptide uptake assay (19) the corresponding inhibition was about 35%. DISCUSSION The results of our earlier studies on the uptake of glutamine indicated that there are at least two amino acid uptake systems in the scutellum (21). One of these systems appears to be inhibited by all protein amino acids whereas the other (the minor one) is probably inhibited by all other amino acids but asparagine. The inhibitions suggest that both systems have a broad specificity. The fact that proline can inhibit completely and competitively the uptake of glutamine (21) already suggests that proline might be taken up by both of the nonspecific systems. The present experiments show that the general properties of the uptake of proline are similar to those of glutamine. They also show that in the uptake of proline there is a component which is inhibited both by asparagine and glutamine and another, minor component which is inhibited by glutamine but not by asparagine. It is
[Inhibitory amino acid] (mM) FIG. 5. Effect of increasing concentrations of asparagine, glutamine, and alanine on the uptake of 10 mm proline. The uptake of 10 mm proline was assayed in the presence of 0 to 200 mm concentrations of the inhibitory amino acids. Inset, Original values were corrected for nonmediated uptake (4.5 gmol g'l fresh weight h-1) and the values for the mediated uptake were used to extrapolate the inhibitory effect to an infinitely high concentration of the inhibitory amino acid. --Nonmediated uptake.
[PROJ (mM) FIG. 6. Effect of substrate concentration on the uptake of proline by the proline-specific system. The uptake of 10 to 100 mm proline was assayed in the presence of 150 mM alanine. The original values (0) were corrected for nonmediated uptake (proline concentration in mm x 0.45 Mmol g-' fresh weight h-') and the values for the mediated uptake were used for the v/(v/S) plot (inset). most likely that these two components correspond to the two carriers proposed to be involved in the uptake of glutamine and leucine. The fact that the relative strength of inhibition caused by other amino acids is similar whether the substrate is glutamine (21, Table I) or proline (Table I) gives strong support to the idea
Table II. Inhibition ofthe Uptake of1 mM Proline in the Presence of 150 mM Glutamine by Various Amino Acids at 50 mM Concentration Amino Acid
Total Uptake of Proline
50 mM gmol g-'fresh wt h' % None 1.56 ± 0.06 Gly 1.42 ± 0.08 13 Ala 1.62 ± 0.09 -5 Val 1.35±0.06 19 24 Leu 1.29 ± 0.04 le 1.39 ± 0.04 15 Pro 0.78 ± 0.04 70 Met 1.53±0.10 3 1.49 ± 0.06 Ser 6 Thr -4 1.60 ± 0.08 Asn -11 1.69 ± 0.06 Gln 1.57±0.10 -1 Asp 1.02 ± 0.07 49 Glu 1.03 ± 0.05 48 Arg 1.03 ± 0.05 48 Lys 0.97 ± 0.04 53 His 1.05 ± 0.06 46 a The nonmediated uptake (0.45 umol g-' fresh wt h-') was subtracted from the original values and the inhibition was calculated as a percentage of the value without any inhibitor.
that the bulk of the uptake of these amino acids is due to common carriers. Some properties of these two carriers have been discussed earlier (21). The presence of a third uptake system for proline was revealed by the fact that a component of the mediated uptake of proline was not inhibited by glutamine or alanine, which inhibit completely the two nonspecific systems (21). Saturation at high proline concentrations and inhibition by dinitrophenol indicate that the uptake by this system is due to carrier-mediated active transport. The specificity of this system differs completely from that of the two apparently nonspecific systems. Although tested at a high concentration (50 mM), most of the protein amino acids were not inhibitory and therefore they cannot be taken up by this system. Two groups of inhibitory amino acids were observed, however: the branched-chained aliphatic and the basic amino acids. We have earlier shown that the uptake of leucine is completely inhibitable by glutamine (21). Therefore, this third, glutamine-uninhibited uptake system is apparently not involved in the uptake ofleucine. This is probably also true for the uptake of isoleucine and valine. On the other hand, the uptake of the
Plant Physiol. Vol. 80, 1986
basic amino acids does involve a component which is not inhibited by glutamine (T Sopanen, unpublished results). Proline does not, however, inhibit this component, which is probably due to a fourth amino acid uptake system, specific for basic amino acids. It is possible that the basic and branched-chain amino acids bind loosely to the carrier but are not transported. These results indicate that the only protein amino acid taken up by this glutamine-uninhibited system is proline and therefore this system will hereafter be called the proline-specific system. Naturally, the uptake of other types of possible substrates than amino acids cannot be ruled out. The facts that D-proline inhibits the proline-specific system as strongly as L-proline and that methylation or amidation of the carboxyl group increases the affinity indicate that the carboxyl group does not play an important role in the binding of the substrate to the carrier. In this respect the proline-specific system differs clearly from the two unspecific amino acid uptake systems. Apparently, carbon 4 and the imino group have a role in the binding because hydroxylation of carbon 4 and acetylation of the imino group decrease the affinity strongly. This also seems to be true for the two unspecific uptake systems. The inhibition of the proline-specific system by alanylalanine raises the question of whether this system could be identical with the peptide uptake system(s) found in barley scutellum (19 and references cited therein). At pH 4.5 the Km for the uptake of alanylalanine is 1 mM (T Sinervo, T Sopanen, unpublished data). If the proline-specific system were identical with the peptide uptake system(s), alanylalanine should give a much stronger inhibition (more than 95%) than it actually does (35%). This discrepancy and the inability of p-chloromercuribenzene sulfonate to inhibit the uptake of proline as strongly as it inhibits the uptake of glycylsarcosine (T Sopanen, unpublished results) indicate that the proline-specific system is separate from the peptide uptake system(s). At present we can only make tentative calculations of the relative roles of the three uptake systems in the uptake of proline in the whole germinating grain. The average concentration of proline in the starchy endosperm after germination for 4 d in our conditions is about 8 mm (preliminary results). At 10 mm concentration the mediated uptake in vitro is about 23 jmol g-' fresh weight h-' and of this about 11 mol g-' fresh weight h-' (50%) is by the asparagine-inhibited system, 5 ,umol g-' fresh weight h-' (20%) by the asparagine-uninhibited, glutamine-inhibited system and 7 Mmol g-' fresh weight h-' (30%) by the proline-specific system. In vivo, the uptake of proline by the two nonspecific systems is slowed down because of competition with other amino acids. There are nine amino acids which inhibit
Table III. Effect of L-Proline and Some of its Analogues and Derivatives on the Uptake of Proline The uptake of 1 mm proline alone was assayed in the presence of 10 mm concentrations of the inhibitory compounds. To assay the uptake by the proline-specific system, the uptake of 1 mM proline was assayed in the presence of 150 mM alanine; the concentration of the inhibitory compounds in this experiment was 50 mM.
Uptake of I nmm
Uptake of 1 mM
Proline with 150 Inhibition of ofh1imr Addit'ionUPtake mt Alanine; Inhibitory ioproine;at10mm rati Mediated Uptakea A
(10 or 50 mM) None
L-Proline D-Proline Proline methyl ester Proline amine N-Acetyl proline Hydroxyproline a See footnote in Table II.
UPTAKE OF PROLINE BY BARLEY SCUTELLUM more strongly than proline the uptake of glutamine (21) and the sum of their average concentrations in the starchy endosperm is
(T Sopanen, unpublished results). If they average affinity as glutamine, they would inhibit the uptake of proline by the nonspecific systems by about 65% (calculated from Fig. 5). Because the weakly inhibitory amino acids also compete with proline, it is likely that the uptake of proline by the nonspecific systems is not high in a whole grain. Although 50 mm concentrations of some amino acids inhibit the uptake of 1 mm proline by the proline specific system, these inhibitions are probably negligible in vivo, because in vivo the concentrations of the inhibitory amino acids are much lower and the concentration of proline is much higher than in the experiment (Table II). Therefore, it appears that in vivo the prolinespecific system could work uninhibited at about the same rate as in vitro (7 umol g-' fresh weight h-') and that in vivo the bulk of proline is probably taken up by this system. Because of the high amount of proline in the starchy endosperm, the proline-specific system appears therefore to play an important role in the nutrition of the growing seedling. In most higher plant tissues studied, proline inhibits the uptake of other amino acids and is therefore probably taken up by a general amino acid uptake system(s) (e.g., 1-4, 8, 9, 16), which resembles the nonspecific systems in barley scutellum. The uptake of proline has not generally been directly assayed and it is not possible to reach any conclusions about the presence in other plants of a proline-specific system similar to that in the scutellum. In Lemna, however, it has been shown that when the uptake of alanine, valine, and leucine occurs at a saturating concerftration, addition of proline does not cause a transient membrane depolarization (6). This indicates that in Lemna there is no cotransport of proline and H+ by a proline-specific system. In Chlorella, glucose or depletion of nitrogen induce an amino acid uptake system which transports proline, but this system also transports glycine, alanine, and serine (5, 17) and therefore differs from the proline-specific system in barley scutellum. Although proline-specific uptake systems have not earlier been detected in plants, systems which seem to prefer proline to other amino acids have been found at least in yeasts (10), animal cells (22, 24), and bacteria (7, 23). These systems have Km values in the range of 0.6 to 300 ,uM. Thus, the proline-specific transport system in barley scutellum differs from the proline-specific systems of other organisms in having a much higher Km (about 65 mM
are assumed to have the same
Acknowledgments-We thank Dr. J. Mikola for useful discussions and suggestions during the preparation of the manuscript, Mr. Pasi Sintonen and Ms. Ritva Linna for technical assistance, and Mr. M. J. Bailey, M.Sc., for checking the language of the text.
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