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Effect of papain treatment on phenylalanine transport in rabbit ileum
Life Sciences, Vol . 24, pp . 633-638 Printed in the U.S .A .
Pergamon Press
EFFECT OF PAPAW TREATMENT ON PHENYLAhANINE TRANSPORT IN RABBIT ILEUM Robert L . Preston Department of Biology, Illinois State University Normal, Illinois 61761 (Received in final form January 11, 1979) Summary The unidirectional influx of phenylalanine across the mucosal brush border of rabbit ileum is reduced by pretreatment with papain . Phenylalanine influx is reduced to 10-15x of the control value by 60-90 minutes of preincubation with papain . Pretreatment with protease from Streptamycea griseus has ao effect on phenylalanine influx . Kinetic analysis of the effect of papain indicates that the maximum velocity is reduced with little change in the apparent Michaelis-constant for phenylalanine . The data suggest that papain attacks a membrane protein required for transport . This protein is unaffected by protease which indicates the susceptible region of the protein is shielded by the membrane or inaccessible to protease . The neutral amino acid transport system in rabbit ileum has been extensively investigated by Schultz, Curran, and coworkers (1, 2) . They have shown that the unidirectional influx of neutral amino acids is strongly dependent oa sodium in the mucosal solution . The entry of amino acids is coupled to the simultaneous entry of sodium on the same carrier. A number of studies has delineated some of the molecular and functional properties of the carrier . Preston et al . described the apparent functional geometry of the transport site (3) . Other studies such se those of Schaeffer et al . (4) and Preatoa (5) utilized group specific reagents as membrane probes to describe functional groups for transport . A number of investigators have employed proteolytic enzyme digestion se a tool for studying membrane structure and transport in other tissues (6-9) . The accessibility of the carrier to proteolytic enzymes and kinetic changes asso ciated with enzyme treatment yield pertinent information about functional membrane architecture . The object of this study is to investigate the effects of proteolytic enzymes and in particular papain on the neutral amino acid transport system in rabbit ileum in order to assess the utility of this approach for further studies on the molecular aspecta .of transport in this system . Materials and Methods The methods employed for flux meaàuremeata have been described in detail previously (1) . In brief, male New Zealand rabbits were sacrificed by injection of nembutal or air . The ileum was removed, slit along the mesenteric border and washed in Ringer solution . Segments of ileum were mounted mucosal aide up in lucite flux chambers (1) . The tissue was then equilibrated for 30 minutes at 37oC with Ringer solution . The controls were then placed in Ringer solution 0300-9653/79/0212-063302 .00/0 Copyright (c) 1979 Pergamon Press Ltd .
63 4
Papain, gffect on Phenylalanine Transport
Vol,
24, No . 7,
1979
in which the sodium chloride was replaced with choline chloride . All controls also contained the same amount of enzymes ae the experimental cases but the enzymes were first boiled for 45 minutes . The papain and protease treated tissue were preincubated with choline Ringer which had 0 .25 mg/ml papain and 20 mM dithiothreitol (DTT) or 1 mg/ml protease . All enzyme treatments and preincuba tions were carried out on tissue mounted in the flux chambers . The reason for replacing the sodium in the preincubation solutions is that Chez et al . (10) have shown that treatment of ileum with metabolic inhibitors in sodium Ringer leads to a gain is intracellular sodium, cell swelling and a subsequent decrease in amino acid influx . These effects are minimized by sodium-free Ringer . After preincubation, the controls and enzyme treated tissue was washed and exposed briefly to 14 C-L-phenylalanine in sodium Ringer . L-phenylalanine was used because it has been shown that all neutral amino acids are transported by the same carrier in rabbit ileum and phenylalanine has relatively high affinity for the carrier. The incubation solution also contained 3H-inulin as a marker for the apparent extracellular space . After 60 seconds the labeled solution was removed and the tissue flushed with ice-cold 0 .3 M mannitol . The tissue was then removed with a tissue punch, rinsed briefly, blotted, and extracted for 2 hours in 0,1 N nitric acid . An aliquot of this extract was then counted in a two channel scintillation counter . All flux measurements were corrected for the adherent extracellular component as estimated by 3H-inulin . The enzymes used were obtained from Sigma Chemical Company, St . Louis, Mo . The papain was 2x crystallized and suspended in 0 .01 thymol and sodium acetate (Sigma #P3125, 16-40 BASE unite/mg) . The protease was Sigma Type V from Streptomycea griseus (Sigma #P5005, 1 .2 unite/mg) . The radioisotopes were purchased from New England Nuclear Corporation, Boston, Masa .
~.o a~
a~ a: o I
0
1
v
N
120
i
1N
FIG . 1 The effect of papain (~) and protease (o) preincubation on phenylalanine transport in rabbit ileum . Rabbit ileum mounted in lucite influx chambers was preincubated for various times with enzymes in choline Ringer medium . The controls contained enzymes which were boiled for 45 minutes and which were identical in every other respect . All flux measurements were done in sodium Ringer for a duration of 60 seconds . The fluxes were corrected for adherent extracellular isotope using 3H-inulin .
Vol . 24, No . 7,
1979
Papain, Effect on Phenylalanine Transport
635
Results Preincubation of rabbit ileum with papain causes a large decrease in the influx of phenylalanine (Figure 1) . However, no significant reduction in flux was observed with protease . The decrease in flux leveled off at about 10-15X of control value after 60-90 minutes . The effect of papain preincubstion After treatment with on the kinetics of phenylalanine influx was also measured . papain for 60 minutes the flux was reduced but still followed saturation kinetics (Figure 2) . An Eadie-Hofstee plot of the same data indicates that the major effect of papain treatment is to decrease the maximum velocity (Jmax) of phenylalanine influx with little effect on the apparent Michaelie-constant (Kt) of phenylalanine for the carrier (Figure 3) . The Jmax for the control is-14 .0 _+ 1 .2 um/hr/cm2 compared with 8 .00 + 0 .64 um/hr/cm2 in treated tissue . These values are significantly different when compared with Students t-test (p < .O1) . The Kt is 8 .94 + 1 .19 mM and 10 .1 _+ l.2 mM for control and treated tissue respectively . These values are not statistically different using the t-test . The kinetic constants were calculated from the slopes and intercepts of regression lines in the Eadie-Hofstee plot using the mean values for influx (N ~ 4 for control and treated tissue) . Calculation of these constants using individual values of Ji and Ji/S instead of mean values (N ~ 12 for each condition) lead to exactly the same conclusions se above but with somewhat different values for the kinetic constants .
u
r. rr ..,~wi..
FIG . 2
The unidirectional influx of phenylalanine as a function of phenylalanine concentration in papain treated (p) and control (/) tissue . After preincubstion with papain in choline Ringer, the tissue was washed thoroughly and incubated for 60 seconds with 14 C-L-phénylalanine in sôdium Ringer . The flux was corrected for extracellular phenylalanine by using 3H-inulin as a marker of adherent extracellular isotope . The bare indicate + one standard error (N~3) .
Discussion The results indicate that papain causes a reduction in the unidirectional influa of phenylalanine . This presumably occurs because the enzyme attacks a membrane protein associated with transport . Interestingly enough, protease does sot cause reduction in influx . This supports the idea that reduction in influx
63 6
Papain, Effect on Phenylalanine Transport
Vol . 24, No . 7, 1979
rI ^ . 3 Aa Eadie-Hofstee plot of the data in Figure 2 . The parallel slopes of the curves indicate that the apparent Michaelis coastanta for phenylalanine transport in papain treated (O) and control ( " ) tissue are the same . The maximum velocity for phenylalanine transport in papain treated tissue is about one-half the maximum velocity in control tissues .
observed with papain is not a nonspecifis effect due, for example, to a generalized decrease in membrane permeability or modification of membrane potential . Since both protease and papain have relatively broad apecif icity, it would be But it is known, however, that expected that both might have similar effects . papain can degrade protein substrates more extensively than moat proteasea, which might account for the difference (11) . It is also possible that a portion of the carrier molecule is more susceptible to attack by papain than protease, possibly because it is shielded from the action protease by other membrane structures . This idea agrees with studies with inhibitors which show some amino and sulfhydryl groups appear to be partially shielded from these probe reagents (4, 5) . The fact that Jmax is reduced by papain treatment suggests that the carThe rier or some portion of it is being attacked and denatured by the enzyme . Changes number of carriers available for transport appears to be decreasing . in membrane potential or sodium dependency should result in changes in the Rt rather than the Jmax . For example, inhibitors such as fluoro-2,4~initrobenzene (5) and p-chloramercuriphenyl sulfonic acid (4) cause changea in the Kt . This can be shown to be due primarily to increase in the dissociation constant of sodium for the carrier . In conclusion, the data suggest that papain treatment reduces phenylalanine influx by attacking a portion of the carrier moiety or some other memThe fact that protease has no effect brane constituent essential for transport . suggests that a portion of the essential protein is shielded by the membrane or is at least inaccessible to the enzyme . Additional studies utilizing papain in combination with chemical membrane probes would be useful in clarifying these possibilities .
Vol,
24, No . 7,
1979
Papain, Effect on Phenylalanine Transport
637
Acknowledgments This research was supported in part by Illinois State University Research Grants ISU-76-22 and ISU-77-18 . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
S . G . SCHULTZ, P. F . CURRAN, R. _50, 1241-1260 (1967) . P . F, CURRAN, S . G . SCHULTZ, R. _50, 1261-1281 (1967) . R. L. PRESTON, J . F . SCHAEFFER, 467 (1974) . J. F. SCHAEFFER, R. L. PRESTON, 131-146 (1973) .
A. CHEZ, and R. E. FUISZ .
J . Gen . Physiol .
A. CHEZ, and R. E . FUISZ .
J . Gen . Physiol .
and P. F . CURRAN,
J . Gen . Physiol . _64, 443-
and P. F . CURRAN .
J . Gen. Physiol . _62,
R. L. PRESTON . Riochim . Biophys . Acta (submitted for publication) . W. W. BENDER, H . GARAN, and H . BERG . J . Mol . Biol . _58, 783-797 (1971) . C . M. ARMSTRONG, F . BEZANILLA, a~ E . ROJAS . J. Gen . Physiol . _62, 375391 (1973) . D. LOUVARD, S . MAROUX, CH . VANNIER, and D . DESNUELLE . Biochim. Biophys . Acaa _375, 236-248 (1975) . R. B. TRIPLET and R . L . CARRAWAY . Biochem . _11, 2897-2903 (1972) . R. A. CHEZ, R. R . PALMER, S . G . SCHULTZ, and P. F. CURRAN . J . Gen . Physiol . _50, 2357-2375 (1967) . R. ARNON. Methode in Enzymology , Vol . RIR (Penman, G . E . and Lorand, L . eda .) p . 226, Academic Press, New York (1970) .
Pergamon Press
EFFECT OF PAPAW TREATMENT ON PHENYLAhANINE TRANSPORT IN RABBIT ILEUM Robert L . Preston Department of Biology, Illinois State University Normal, Illinois 61761 (Received in final form January 11, 1979) Summary The unidirectional influx of phenylalanine across the mucosal brush border of rabbit ileum is reduced by pretreatment with papain . Phenylalanine influx is reduced to 10-15x of the control value by 60-90 minutes of preincubation with papain . Pretreatment with protease from Streptamycea griseus has ao effect on phenylalanine influx . Kinetic analysis of the effect of papain indicates that the maximum velocity is reduced with little change in the apparent Michaelis-constant for phenylalanine . The data suggest that papain attacks a membrane protein required for transport . This protein is unaffected by protease which indicates the susceptible region of the protein is shielded by the membrane or inaccessible to protease . The neutral amino acid transport system in rabbit ileum has been extensively investigated by Schultz, Curran, and coworkers (1, 2) . They have shown that the unidirectional influx of neutral amino acids is strongly dependent oa sodium in the mucosal solution . The entry of amino acids is coupled to the simultaneous entry of sodium on the same carrier. A number of studies has delineated some of the molecular and functional properties of the carrier . Preston et al . described the apparent functional geometry of the transport site (3) . Other studies such se those of Schaeffer et al . (4) and Preatoa (5) utilized group specific reagents as membrane probes to describe functional groups for transport . A number of investigators have employed proteolytic enzyme digestion se a tool for studying membrane structure and transport in other tissues (6-9) . The accessibility of the carrier to proteolytic enzymes and kinetic changes asso ciated with enzyme treatment yield pertinent information about functional membrane architecture . The object of this study is to investigate the effects of proteolytic enzymes and in particular papain on the neutral amino acid transport system in rabbit ileum in order to assess the utility of this approach for further studies on the molecular aspecta .of transport in this system . Materials and Methods The methods employed for flux meaàuremeata have been described in detail previously (1) . In brief, male New Zealand rabbits were sacrificed by injection of nembutal or air . The ileum was removed, slit along the mesenteric border and washed in Ringer solution . Segments of ileum were mounted mucosal aide up in lucite flux chambers (1) . The tissue was then equilibrated for 30 minutes at 37oC with Ringer solution . The controls were then placed in Ringer solution 0300-9653/79/0212-063302 .00/0 Copyright (c) 1979 Pergamon Press Ltd .
63 4
Papain, gffect on Phenylalanine Transport
Vol,
24, No . 7,
1979
in which the sodium chloride was replaced with choline chloride . All controls also contained the same amount of enzymes ae the experimental cases but the enzymes were first boiled for 45 minutes . The papain and protease treated tissue were preincubated with choline Ringer which had 0 .25 mg/ml papain and 20 mM dithiothreitol (DTT) or 1 mg/ml protease . All enzyme treatments and preincuba tions were carried out on tissue mounted in the flux chambers . The reason for replacing the sodium in the preincubation solutions is that Chez et al . (10) have shown that treatment of ileum with metabolic inhibitors in sodium Ringer leads to a gain is intracellular sodium, cell swelling and a subsequent decrease in amino acid influx . These effects are minimized by sodium-free Ringer . After preincubation, the controls and enzyme treated tissue was washed and exposed briefly to 14 C-L-phenylalanine in sodium Ringer . L-phenylalanine was used because it has been shown that all neutral amino acids are transported by the same carrier in rabbit ileum and phenylalanine has relatively high affinity for the carrier. The incubation solution also contained 3H-inulin as a marker for the apparent extracellular space . After 60 seconds the labeled solution was removed and the tissue flushed with ice-cold 0 .3 M mannitol . The tissue was then removed with a tissue punch, rinsed briefly, blotted, and extracted for 2 hours in 0,1 N nitric acid . An aliquot of this extract was then counted in a two channel scintillation counter . All flux measurements were corrected for the adherent extracellular component as estimated by 3H-inulin . The enzymes used were obtained from Sigma Chemical Company, St . Louis, Mo . The papain was 2x crystallized and suspended in 0 .01 thymol and sodium acetate (Sigma #P3125, 16-40 BASE unite/mg) . The protease was Sigma Type V from Streptomycea griseus (Sigma #P5005, 1 .2 unite/mg) . The radioisotopes were purchased from New England Nuclear Corporation, Boston, Masa .
~.o a~
a~ a: o I
0
1
v
N
120
i
1N
FIG . 1 The effect of papain (~) and protease (o) preincubation on phenylalanine transport in rabbit ileum . Rabbit ileum mounted in lucite influx chambers was preincubated for various times with enzymes in choline Ringer medium . The controls contained enzymes which were boiled for 45 minutes and which were identical in every other respect . All flux measurements were done in sodium Ringer for a duration of 60 seconds . The fluxes were corrected for adherent extracellular isotope using 3H-inulin .
Vol . 24, No . 7,
1979
Papain, Effect on Phenylalanine Transport
635
Results Preincubation of rabbit ileum with papain causes a large decrease in the influx of phenylalanine (Figure 1) . However, no significant reduction in flux was observed with protease . The decrease in flux leveled off at about 10-15X of control value after 60-90 minutes . The effect of papain preincubstion After treatment with on the kinetics of phenylalanine influx was also measured . papain for 60 minutes the flux was reduced but still followed saturation kinetics (Figure 2) . An Eadie-Hofstee plot of the same data indicates that the major effect of papain treatment is to decrease the maximum velocity (Jmax) of phenylalanine influx with little effect on the apparent Michaelie-constant (Kt) of phenylalanine for the carrier (Figure 3) . The Jmax for the control is-14 .0 _+ 1 .2 um/hr/cm2 compared with 8 .00 + 0 .64 um/hr/cm2 in treated tissue . These values are significantly different when compared with Students t-test (p < .O1) . The Kt is 8 .94 + 1 .19 mM and 10 .1 _+ l.2 mM for control and treated tissue respectively . These values are not statistically different using the t-test . The kinetic constants were calculated from the slopes and intercepts of regression lines in the Eadie-Hofstee plot using the mean values for influx (N ~ 4 for control and treated tissue) . Calculation of these constants using individual values of Ji and Ji/S instead of mean values (N ~ 12 for each condition) lead to exactly the same conclusions se above but with somewhat different values for the kinetic constants .
u
r. rr ..,~wi..
FIG . 2
The unidirectional influx of phenylalanine as a function of phenylalanine concentration in papain treated (p) and control (/) tissue . After preincubstion with papain in choline Ringer, the tissue was washed thoroughly and incubated for 60 seconds with 14 C-L-phénylalanine in sôdium Ringer . The flux was corrected for extracellular phenylalanine by using 3H-inulin as a marker of adherent extracellular isotope . The bare indicate + one standard error (N~3) .
Discussion The results indicate that papain causes a reduction in the unidirectional influa of phenylalanine . This presumably occurs because the enzyme attacks a membrane protein associated with transport . Interestingly enough, protease does sot cause reduction in influx . This supports the idea that reduction in influx
63 6
Papain, Effect on Phenylalanine Transport
Vol . 24, No . 7, 1979
rI ^ . 3 Aa Eadie-Hofstee plot of the data in Figure 2 . The parallel slopes of the curves indicate that the apparent Michaelis coastanta for phenylalanine transport in papain treated (O) and control ( " ) tissue are the same . The maximum velocity for phenylalanine transport in papain treated tissue is about one-half the maximum velocity in control tissues .
observed with papain is not a nonspecifis effect due, for example, to a generalized decrease in membrane permeability or modification of membrane potential . Since both protease and papain have relatively broad apecif icity, it would be But it is known, however, that expected that both might have similar effects . papain can degrade protein substrates more extensively than moat proteasea, which might account for the difference (11) . It is also possible that a portion of the carrier molecule is more susceptible to attack by papain than protease, possibly because it is shielded from the action protease by other membrane structures . This idea agrees with studies with inhibitors which show some amino and sulfhydryl groups appear to be partially shielded from these probe reagents (4, 5) . The fact that Jmax is reduced by papain treatment suggests that the carThe rier or some portion of it is being attacked and denatured by the enzyme . Changes number of carriers available for transport appears to be decreasing . in membrane potential or sodium dependency should result in changes in the Rt rather than the Jmax . For example, inhibitors such as fluoro-2,4~initrobenzene (5) and p-chloramercuriphenyl sulfonic acid (4) cause changea in the Kt . This can be shown to be due primarily to increase in the dissociation constant of sodium for the carrier . In conclusion, the data suggest that papain treatment reduces phenylalanine influx by attacking a portion of the carrier moiety or some other memThe fact that protease has no effect brane constituent essential for transport . suggests that a portion of the essential protein is shielded by the membrane or is at least inaccessible to the enzyme . Additional studies utilizing papain in combination with chemical membrane probes would be useful in clarifying these possibilities .
Vol,
24, No . 7,
1979
Papain, Effect on Phenylalanine Transport
637
Acknowledgments This research was supported in part by Illinois State University Research Grants ISU-76-22 and ISU-77-18 . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
S . G . SCHULTZ, P. F . CURRAN, R. _50, 1241-1260 (1967) . P . F, CURRAN, S . G . SCHULTZ, R. _50, 1261-1281 (1967) . R. L. PRESTON, J . F . SCHAEFFER, 467 (1974) . J. F. SCHAEFFER, R. L. PRESTON, 131-146 (1973) .
A. CHEZ, and R. E. FUISZ .
J . Gen . Physiol .
A. CHEZ, and R. E . FUISZ .
J . Gen . Physiol .
and P. F . CURRAN,
J . Gen . Physiol . _64, 443-
and P. F . CURRAN .
J . Gen. Physiol . _62,
R. L. PRESTON . Riochim . Biophys . Acta (submitted for publication) . W. W. BENDER, H . GARAN, and H . BERG . J . Mol . Biol . _58, 783-797 (1971) . C . M. ARMSTRONG, F . BEZANILLA, a~ E . ROJAS . J. Gen . Physiol . _62, 375391 (1973) . D. LOUVARD, S . MAROUX, CH . VANNIER, and D . DESNUELLE . Biochim. Biophys . Acaa _375, 236-248 (1975) . R. B. TRIPLET and R . L . CARRAWAY . Biochem . _11, 2897-2903 (1972) . R. A. CHEZ, R. R . PALMER, S . G . SCHULTZ, and P. F. CURRAN . J . Gen . Physiol . _50, 2357-2375 (1967) . R. ARNON. Methode in Enzymology , Vol . RIR (Penman, G . E . and Lorand, L . eda .) p . 226, Academic Press, New York (1970) .