Biochimica et Biophysiczl /tcta. 1074(1991) 56-61 ~:' 1991 ElsevierSciencePublishersB.V.0304-4165/91/$03.50 ADONIS 0304416591001538
The influence of pH on cystine and dibasic amino acid transport by rat renal brushborder membrane vesicles R o b e r t A . R e y n o l d s 1, S t e p h e n G . M a h o n e y i, P a m e l a D . M c N a m a r a S e g a l 1.2
1 and Stanton
i Division of Biochemical Development and Molecular Diseases, The Children ~ Hospital of Philadelphia. Philadelphia. PA (U.S.A.) and the -" Department of Pediatric:~ and Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA (U.S.A.)
(Received 17 December1990) The uptake of cystine and lysine by rat renal brushborder membrane vesicles was examined at various intravesicular and extravesicular hydrogen ion concentrations to discern ~hether ionic species are determinants of specificity for the shared transport system and whether hydrogen ion gradients play a role in determining uptake values. When intravesicular and extravesicular pH are identical, the highest uptake of cystine occurred at pH 7.4, with lesser uptake at pH 6.0 and 8.3. Since cystine is electroneutral at pH 6.0 and ~ anionic at pH 8.3, it appears that neither form of the amino acid is a preferred species for transport. A similar relationship between pH and uptake occurs for lysine, which is cationic at pH below 8.5. This suggests that pH affects the functioning of the membrane carrier system independent of ionic species of the substrate. There is no apparent relationship of eystine uptake to hydrogen ion gradients across the membrane. Over the range of extravesicular pH studied, optimal cystine uptake occurred whenever the intravesicular pH was 7.4. Competitive interactions between unlabeled amino acids and labeled cystine were not effected by the extravesicular pH and, therefore, did not seem determined by the ionic species of cystine.
Introduction H u m a n cystinuria, which involves defective handling of cystine and dibasic amino acids by the kidney, is the prototype of an inherited membrane transport disorder [1]. The need to understand the nature of the underlying defect in this disorder has been the impetus for determining the characteristics of the shared transport system of cystine and dibasic amino acids in renal cells [2,3] and intestinal mucosa [4] from affected humans, as well as, normal kidney from rat [5,6] and dog [7] and rat jejunal mucosa [8]. The findings in intact cells have been amplified and extended by the use of isolated brushborder membrane vesicles from normal rat proximal tubules [9] and jejunum [10], as well as, cystinuric dog kidney [11]. The defect in cystine transport shown previously in mucosal ceils [12,13] has recently been demonstrated in brushborder membranes prepared from jejunum of cystinuric humans [14]. In renal and intestinal brushborder membrane vesicles, the presence of a low Km cystine transport system shared with dibasic amino acids has been demonstrated by mutual inhibi-
Correspondence: S. Segal, Divisionof BiochemicalDevelopmentand Molecular Diseases, Children's Hospital of Philadelphia, 34th Street & Civic Center Blvd., Philadelphia,PA 19104, U.S.A.
tion and participation in exchange diffusion [9,10,15]. All of these studies have been performed when the intravesicular pH was 7.1 and extravesicular pH was 7.4. Bannai, who has delineated the system for cystine transport in cultured fibroblasts [16-18], has emphasized the importance of hydrogen ion concentration [19]. This derives from the fact that cystine can exist in multiple ionic forms dependent on pH, being electroneutral at pH 6, 30~ anionic at pH 7.4 and 90~ anionic at pH 8.3 [19]. The ionization of cystine may be important in the interaction with the carrier as well as the relationship with other substrates such as lysine which maintains its cationic character at pH below 8.5. The single carrier thought to be shared by cystine and lysine mediates the transport of one substrate, cystine, which can be electroneutral or anionic at physiologic pH and the other, lysine, which is cationic. Most of our current knowledge of cystine-lysine interactions is based on measurements made at pH 7.1-7.4. The present studies were designed to determine which ionic form of cystine is the preferred substrate for the rat renal brushborder cystine carrier, how the cystine ionic species influences the carrier interaction with dibasic and other amino acids, and whether hydrogen ion gradients play a role in the uptake of cystine and lysine. The results form the basis of this report.
Materials and Methods Rat renal brushborder membrane vesicles were prepared from the kidney of normal adult male SpragueDawley rat fed ad libitum on Purina Rat Chow. The method for isolating brushborder membranes from renal cortical sections was previously described [9] and involves a modification of Mg 2÷ aggregation method described by Booth and Kenny [20]. After the final centrifugation, the brushborder membrane vesicles were resuspended in buffers at pH 6.0, 7.4 or 8.3 for 30 rain prior to their use in ',ransport studies. The buffer at pH 6.0 was composed of 100 mM mannitol + 2 mM Mes with the pH adjusted to 6.0 with Tris. The pH 7.4 buffer contained 100 mM mannitol + 2 mM Hepes adjusted to pH with Tris, and the pH 8.3 buffer was 100 mM mannitol + 2 mM Tris adjusted to pH with Hepes. Vesicles were suspended at a concentration of 3-5 mg p r o t e i n / m l as determined by Bio°Rad assay using "/globulin as the standard. Lysine at pH 7.4 carries a positive charge and is readily soluble. Cystine, on the other hand, has only limited solubility at neutral pH. To insure that cystine is in solution, crystalline cystine is dissolved in 0.5 M HCI which is then diluted to the desired concentration in the appropriate buffer at the start of the experiment. The amount of 0.5 M HCI was 10 ~tl/ml of external buffer. Three external buffers were used: Mes at pH 6.0 (60 mM mannitol + 20 mM Mes and titrated with Tris such that 10 l a l / ml 0.5 M HCI brought the final pH to 6.0); Hepes at pH 7.4 (60 mM mannitol + 20 mM Hepes and titrated with Tris such that 10 t t l / m l 0.5 M HCI brought the final pH to 7.4); and Tris at pH 8.3 (60 mM mannitol + 20 mM Tris and titrated with Hepes such that 10 # l / m l 0.5 M HCI brought the final pH to 8.3).
All experiments are performed with 100 mM NaCI in the external buffer in order to have an inward sodium gradient imposed on membrane vesicles. Incubations are started by adding 50 p,I membrane vesicles and allowed to proceed for the stated time. Measurements of uptake at 22°C were determined by rapid filtration techniques described previously [15] using Sartorius cellulose nitrate filters (SM 113. 0.45 p,m). For determinations of cystine binding to membrane proteins, trichloroacetic acid (TCA) was added to the transport incubation. TCA-precipitated protein was collected on glass fiber filters using the technique described from this laboratory [21]. All filters were airdried overnight and counted in a Packard Tricarb Scintillation Spectrometer. Total uptake was measured as membrane associated radioactivity present on the filter per milligram of protein above the appropriate background. Linear regression analysis by least squares determination packaged in Lotus 123, version 2.2 was used to calculate the straight lines in the LineweaverBurk plot. The significance of the difference in the slopes between the lines was determined by the formula for t-test given by Goldstein [22]. The student's t-test was used to determine significance for all other data. The radioactive material used in these experiments was purchased from two companies: L-[3-~Slcystine from Amersham Corporation and L-[t4C]lysine from ICN Radiochemicals. A strict criteria of cystine purity as described by Schafer and Watkins [23] was used in which quantitative reduction to cysteine by dithiothreitol was determined by high voltage electrophoresis and thin layer chromatography [20,,25] for each batch of labeled cystine. Only batches which showed greater than 95% reduction to cysteine were used. Cystine solubilized in 0.5 M HCI and diluted with buffer is
0.7 0.6 0,5, 0.4. 0.5 : , ~
•
05
0.2.
_. . . .
~
~ 0
.
2
~ ~
O. "''-
2 4
6
B I0 12 14 16 18 20
TIME (min.)
Z
0
0
,
,
2 ,4 6 8 I0 12 14 16 18 20 TIME (rain
Fig. I. Influenceof pH on time course of [3SSlcystineuptake. Vesicleswith intravesicularbuffer (pH 7.4) were added to incubation buffers of varying extravesicularpH = 6.0 ([3,m), 8.3 (o, e), 7.4 (A, A); (see text for composition of all buffers). In A, the solid symbols are the total accumulationof 0.02 mM cystineby renal brushborder membrane vesiclesat 2Oo C. The TCA precipitablecomponent of uptake by brushborder vesiclesis shown by the open symbols. In B the net uptake (free cystine) is the arithmeuc difference between the pairs of total uptake and the respectiveTCA precipitableuptake (bound cystine)at each pH0. Each value is the mean of 8 to 12 determinationsper point. Standard error of the mean is shown by the verticalbar. In caseswhere there is no standard error bar, it fallswithin the sizeof the symbolused to designatethe mean.
58 utilized within 5 mir, for transport studies. Unlabeled a m i n o acids, salts, and buffer c o m p o n e n t s were of the high purity commercial available.
".~.~0 2 8 0 ~.O.2 4, Cn
.E~0.20.
Results
~0.16 E
Effect of extrauesicular pH (pilot on the time course of [¢~S]£t'stine uptake
~ 0.12
The uptake of cystine by vesicles with an intravesicular pH ( p H , ) of 7.4 was determined at time intervals up to 20 rain under conditions where extravesicular p H was initially 6.0. 7.4 or 8.3. Fig. 1A shows that, for the first 3 min, cystine uptake was the same whether the extravesicular pH was 6.0 or 7.4. After 3 min, the u p t a k e from pH 0 7.4 continued to increase, while that from pH u 6 leveled off. The uptake when p H 0 = 8.3 was diminished for the first few minutes after which it increased linearly, resembling the uptake from p H o 7.4. When pH~ and pHu are 7.1 and 7.4, respectively, negligible cystine binding to the vesicle protein occurs within the first 15 s of incubation [21]. At later time points, however, there is considerable b i n d i n g of cystine to the vesicle. Therefore, binding of cystine was measured at each time point by the T C A precipitation method [21]. Binding was again found to be negligible at 15 s for each p H 0 examined. For incubation times of 1 rain or longer, binding to vesicular protein was significant. For pH 0 7.4 and 8.3, the a m o u n t of cystine b o u n d was similar ap.d increased with time of incubation. F o r p H u 6, the binding was considerably less than for the buffers with higher extravesicular p H and reach a plateau by 10 min. Fig. IB reveals the net uptake of u n b o u n d cystine with time calculated by subtracting the T C A precipitable radioactivity from the total u p t a k e (Fig. 1At. The 15 s uptake at pHu 8.3, when b i n d i n g is negligible, was about half of that at PHo 6 or PH(t 7.4. The net u p t a k e at p H 0 8.3 was low and reached an a p p a r e n t steady-state at 20 rain. The uptake at p H 0 6 appeared to have
~0.08 •
•~
ze
0..24
.~ .20 E
0
pH,= 74
cot,on,c
610 6'.4 60 7'.2 716 8'.o B4 pH
slightly higher values at 2 and 3 min than u p t a k e at p H 0 7.4. Since b i n d i n g to the m e m b r a n e was not a factor at 15 s, all s u b s e q u e n t d e t e r m i n a t i o n s were m a d e at that i n c u b a t i o n time in parallel with previous studies of cystine u p t a k e from this laboratory [9,13].
[35S]Cystine and lysine at the same intra- and extrat;esicular pH U p t a k e of cystine and lysine was assessed at 15 s when the pH~ = PHo at 6, 7.4 and 8.3. The results are shown in Fig. 2. C y s t i n e u p t a k e (closed symbols) was highest at p H 7.4, with significantly lower u p t a k e at p H zs
B
T
c~
~
0H:60 .,
20
~E
~
pH,:;'4
~--" 0 8
.08
pH,: 8~ t3-"
5
_1 0 4 o
too % coT,o~,c
Fig. 2. Cystine and lysinc uptake by brushborder membrane vesicles in relation to pH. The data points represent the 15 s uptake of 0.02 mM cystine (e) and 0.02 mM lysine (o) when both the intravesicular and extravesicular pH were equal at the pH indicated. The designation of ionic species for cystine was based on the calculations of Bannai [19]: 0% anionic indicates 100% electroneutral species (- OOC)2R(NH3~ )2; 30% anionic at pH 7.4 indicates 70% electroneutral species and 30% (-OOC)2R(NH2)NH~- and q0% anionic at pH 8.3 indicates a sum of 60% (-OOC)2R(NHz)NH~. 30% (-OOC)2R(NH2) 2 and 10% electroneutral form (see text for buffer composition). Each point is the average of at least seven determinations with the standard error of the mean shown by the vertical bar. The absence of the vertical bar indicates the standard error was within the size of the symbol designating the mean.
A T
oo
Q.. o~ ~ 21)0.04" tO0% -J co,,on,c
The influence of pH on cystine and dibasic amino acid transport by rat renal brushborder membrane vesicles R o b e r t A . R e y n o l d s 1, S t e p h e n G . M a h o n e y i, P a m e l a D . M c N a m a r a S e g a l 1.2
1 and Stanton
i Division of Biochemical Development and Molecular Diseases, The Children ~ Hospital of Philadelphia. Philadelphia. PA (U.S.A.) and the -" Department of Pediatric:~ and Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA (U.S.A.)
(Received 17 December1990) The uptake of cystine and lysine by rat renal brushborder membrane vesicles was examined at various intravesicular and extravesicular hydrogen ion concentrations to discern ~hether ionic species are determinants of specificity for the shared transport system and whether hydrogen ion gradients play a role in determining uptake values. When intravesicular and extravesicular pH are identical, the highest uptake of cystine occurred at pH 7.4, with lesser uptake at pH 6.0 and 8.3. Since cystine is electroneutral at pH 6.0 and ~ anionic at pH 8.3, it appears that neither form of the amino acid is a preferred species for transport. A similar relationship between pH and uptake occurs for lysine, which is cationic at pH below 8.5. This suggests that pH affects the functioning of the membrane carrier system independent of ionic species of the substrate. There is no apparent relationship of eystine uptake to hydrogen ion gradients across the membrane. Over the range of extravesicular pH studied, optimal cystine uptake occurred whenever the intravesicular pH was 7.4. Competitive interactions between unlabeled amino acids and labeled cystine were not effected by the extravesicular pH and, therefore, did not seem determined by the ionic species of cystine.
Introduction H u m a n cystinuria, which involves defective handling of cystine and dibasic amino acids by the kidney, is the prototype of an inherited membrane transport disorder [1]. The need to understand the nature of the underlying defect in this disorder has been the impetus for determining the characteristics of the shared transport system of cystine and dibasic amino acids in renal cells [2,3] and intestinal mucosa [4] from affected humans, as well as, normal kidney from rat [5,6] and dog [7] and rat jejunal mucosa [8]. The findings in intact cells have been amplified and extended by the use of isolated brushborder membrane vesicles from normal rat proximal tubules [9] and jejunum [10], as well as, cystinuric dog kidney [11]. The defect in cystine transport shown previously in mucosal ceils [12,13] has recently been demonstrated in brushborder membranes prepared from jejunum of cystinuric humans [14]. In renal and intestinal brushborder membrane vesicles, the presence of a low Km cystine transport system shared with dibasic amino acids has been demonstrated by mutual inhibi-
Correspondence: S. Segal, Divisionof BiochemicalDevelopmentand Molecular Diseases, Children's Hospital of Philadelphia, 34th Street & Civic Center Blvd., Philadelphia,PA 19104, U.S.A.
tion and participation in exchange diffusion [9,10,15]. All of these studies have been performed when the intravesicular pH was 7.1 and extravesicular pH was 7.4. Bannai, who has delineated the system for cystine transport in cultured fibroblasts [16-18], has emphasized the importance of hydrogen ion concentration [19]. This derives from the fact that cystine can exist in multiple ionic forms dependent on pH, being electroneutral at pH 6, 30~ anionic at pH 7.4 and 90~ anionic at pH 8.3 [19]. The ionization of cystine may be important in the interaction with the carrier as well as the relationship with other substrates such as lysine which maintains its cationic character at pH below 8.5. The single carrier thought to be shared by cystine and lysine mediates the transport of one substrate, cystine, which can be electroneutral or anionic at physiologic pH and the other, lysine, which is cationic. Most of our current knowledge of cystine-lysine interactions is based on measurements made at pH 7.1-7.4. The present studies were designed to determine which ionic form of cystine is the preferred substrate for the rat renal brushborder cystine carrier, how the cystine ionic species influences the carrier interaction with dibasic and other amino acids, and whether hydrogen ion gradients play a role in the uptake of cystine and lysine. The results form the basis of this report.
Materials and Methods Rat renal brushborder membrane vesicles were prepared from the kidney of normal adult male SpragueDawley rat fed ad libitum on Purina Rat Chow. The method for isolating brushborder membranes from renal cortical sections was previously described [9] and involves a modification of Mg 2÷ aggregation method described by Booth and Kenny [20]. After the final centrifugation, the brushborder membrane vesicles were resuspended in buffers at pH 6.0, 7.4 or 8.3 for 30 rain prior to their use in ',ransport studies. The buffer at pH 6.0 was composed of 100 mM mannitol + 2 mM Mes with the pH adjusted to 6.0 with Tris. The pH 7.4 buffer contained 100 mM mannitol + 2 mM Hepes adjusted to pH with Tris, and the pH 8.3 buffer was 100 mM mannitol + 2 mM Tris adjusted to pH with Hepes. Vesicles were suspended at a concentration of 3-5 mg p r o t e i n / m l as determined by Bio°Rad assay using "/globulin as the standard. Lysine at pH 7.4 carries a positive charge and is readily soluble. Cystine, on the other hand, has only limited solubility at neutral pH. To insure that cystine is in solution, crystalline cystine is dissolved in 0.5 M HCI which is then diluted to the desired concentration in the appropriate buffer at the start of the experiment. The amount of 0.5 M HCI was 10 ~tl/ml of external buffer. Three external buffers were used: Mes at pH 6.0 (60 mM mannitol + 20 mM Mes and titrated with Tris such that 10 l a l / ml 0.5 M HCI brought the final pH to 6.0); Hepes at pH 7.4 (60 mM mannitol + 20 mM Hepes and titrated with Tris such that 10 t t l / m l 0.5 M HCI brought the final pH to 7.4); and Tris at pH 8.3 (60 mM mannitol + 20 mM Tris and titrated with Hepes such that 10 # l / m l 0.5 M HCI brought the final pH to 8.3).
All experiments are performed with 100 mM NaCI in the external buffer in order to have an inward sodium gradient imposed on membrane vesicles. Incubations are started by adding 50 p,I membrane vesicles and allowed to proceed for the stated time. Measurements of uptake at 22°C were determined by rapid filtration techniques described previously [15] using Sartorius cellulose nitrate filters (SM 113. 0.45 p,m). For determinations of cystine binding to membrane proteins, trichloroacetic acid (TCA) was added to the transport incubation. TCA-precipitated protein was collected on glass fiber filters using the technique described from this laboratory [21]. All filters were airdried overnight and counted in a Packard Tricarb Scintillation Spectrometer. Total uptake was measured as membrane associated radioactivity present on the filter per milligram of protein above the appropriate background. Linear regression analysis by least squares determination packaged in Lotus 123, version 2.2 was used to calculate the straight lines in the LineweaverBurk plot. The significance of the difference in the slopes between the lines was determined by the formula for t-test given by Goldstein [22]. The student's t-test was used to determine significance for all other data. The radioactive material used in these experiments was purchased from two companies: L-[3-~Slcystine from Amersham Corporation and L-[t4C]lysine from ICN Radiochemicals. A strict criteria of cystine purity as described by Schafer and Watkins [23] was used in which quantitative reduction to cysteine by dithiothreitol was determined by high voltage electrophoresis and thin layer chromatography [20,,25] for each batch of labeled cystine. Only batches which showed greater than 95% reduction to cysteine were used. Cystine solubilized in 0.5 M HCI and diluted with buffer is
0.7 0.6 0,5, 0.4. 0.5 : , ~
•
05
0.2.
_. . . .
~
~ 0
.
2
~ ~
O. "''-
2 4
6
B I0 12 14 16 18 20
TIME (min.)
Z
0
0
,
,
2 ,4 6 8 I0 12 14 16 18 20 TIME (rain
Fig. I. Influenceof pH on time course of [3SSlcystineuptake. Vesicleswith intravesicularbuffer (pH 7.4) were added to incubation buffers of varying extravesicularpH = 6.0 ([3,m), 8.3 (o, e), 7.4 (A, A); (see text for composition of all buffers). In A, the solid symbols are the total accumulationof 0.02 mM cystineby renal brushborder membrane vesiclesat 2Oo C. The TCA precipitablecomponent of uptake by brushborder vesiclesis shown by the open symbols. In B the net uptake (free cystine) is the arithmeuc difference between the pairs of total uptake and the respectiveTCA precipitableuptake (bound cystine)at each pH0. Each value is the mean of 8 to 12 determinationsper point. Standard error of the mean is shown by the verticalbar. In caseswhere there is no standard error bar, it fallswithin the sizeof the symbolused to designatethe mean.
58 utilized within 5 mir, for transport studies. Unlabeled a m i n o acids, salts, and buffer c o m p o n e n t s were of the high purity commercial available.
".~.~0 2 8 0 ~.O.2 4, Cn
.E~0.20.
Results
~0.16 E
Effect of extrauesicular pH (pilot on the time course of [¢~S]£t'stine uptake
~ 0.12
The uptake of cystine by vesicles with an intravesicular pH ( p H , ) of 7.4 was determined at time intervals up to 20 rain under conditions where extravesicular p H was initially 6.0. 7.4 or 8.3. Fig. 1A shows that, for the first 3 min, cystine uptake was the same whether the extravesicular pH was 6.0 or 7.4. After 3 min, the u p t a k e from pH 0 7.4 continued to increase, while that from pH u 6 leveled off. The uptake when p H 0 = 8.3 was diminished for the first few minutes after which it increased linearly, resembling the uptake from p H o 7.4. When pH~ and pHu are 7.1 and 7.4, respectively, negligible cystine binding to the vesicle protein occurs within the first 15 s of incubation [21]. At later time points, however, there is considerable b i n d i n g of cystine to the vesicle. Therefore, binding of cystine was measured at each time point by the T C A precipitation method [21]. Binding was again found to be negligible at 15 s for each p H 0 examined. For incubation times of 1 rain or longer, binding to vesicular protein was significant. For pH 0 7.4 and 8.3, the a m o u n t of cystine b o u n d was similar ap.d increased with time of incubation. F o r p H u 6, the binding was considerably less than for the buffers with higher extravesicular p H and reach a plateau by 10 min. Fig. IB reveals the net uptake of u n b o u n d cystine with time calculated by subtracting the T C A precipitable radioactivity from the total u p t a k e (Fig. 1At. The 15 s uptake at pHu 8.3, when b i n d i n g is negligible, was about half of that at PHo 6 or PH(t 7.4. The net u p t a k e at p H 0 8.3 was low and reached an a p p a r e n t steady-state at 20 rain. The uptake at p H 0 6 appeared to have
~0.08 •
•~
ze
0..24
.~ .20 E
0
pH,= 74
cot,on,c
610 6'.4 60 7'.2 716 8'.o B4 pH
slightly higher values at 2 and 3 min than u p t a k e at p H 0 7.4. Since b i n d i n g to the m e m b r a n e was not a factor at 15 s, all s u b s e q u e n t d e t e r m i n a t i o n s were m a d e at that i n c u b a t i o n time in parallel with previous studies of cystine u p t a k e from this laboratory [9,13].
[35S]Cystine and lysine at the same intra- and extrat;esicular pH U p t a k e of cystine and lysine was assessed at 15 s when the pH~ = PHo at 6, 7.4 and 8.3. The results are shown in Fig. 2. C y s t i n e u p t a k e (closed symbols) was highest at p H 7.4, with significantly lower u p t a k e at p H zs
B
T
c~
~
0H:60 .,
20
~E
~
pH,:;'4
~--" 0 8
.08
pH,: 8~ t3-"
5
_1 0 4 o
too % coT,o~,c
Fig. 2. Cystine and lysinc uptake by brushborder membrane vesicles in relation to pH. The data points represent the 15 s uptake of 0.02 mM cystine (e) and 0.02 mM lysine (o) when both the intravesicular and extravesicular pH were equal at the pH indicated. The designation of ionic species for cystine was based on the calculations of Bannai [19]: 0% anionic indicates 100% electroneutral species (- OOC)2R(NH3~ )2; 30% anionic at pH 7.4 indicates 70% electroneutral species and 30% (-OOC)2R(NH2)NH~- and q0% anionic at pH 8.3 indicates a sum of 60% (-OOC)2R(NHz)NH~. 30% (-OOC)2R(NH2) 2 and 10% electroneutral form (see text for buffer composition). Each point is the average of at least seven determinations with the standard error of the mean shown by the vertical bar. The absence of the vertical bar indicates the standard error was within the size of the symbol designating the mean.
A T
oo
Q.. o~ ~ 21)0.04" tO0% -J co,,on,c
