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Endocytosis and degradation of native, cathepsin D-degraded, and glutathione-inactivated aldolase by perfused rat liver
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 227, No. 2, December, pp. 367-3’72, 1983
Endocytosis and Degradation Glutathione-Inactivated JUDITH
of Native, Cathepsin D-Degraded, Aldolase by Perfused Rat Liver’
S. BOND’ AND NATHAN
N. ARONSON.
and
JR.
Departmed of Biochemistry, Virginia Commonwealth University, Richmond, Virginia 23298, and The A&house Laboratory, Biochemistry Program, Pennsylvania State University, University Park, Pennsylvania 16802 Received March 21, 1983, and in revised form July 18, 1983
The uptake and degradation of ‘%I-labeled (a) native aldolase, (b) cathepsin D-inactivated aldolase, and (c) aldolase inactivated by oxidized glutathione were studied in perfused rat liver. All three forms of aldolase were removed from the perfusion medium and degraded by the liver, but the uptake of the glutathione-inactivated enzyme (half-life in perfusate = 10 min) was much faster than that of the native enzyme (halflife = 30 min) or the cathepsin-inactivated enzyme (half-life = 42 min). The degradation of the enzyme was almost totally inhibited by leupeptin, indicating that thiol proteinases in lysosomes play an important role in the digestion process. Degradation of native and cathepsin D-inactivated aldolase appeared to be slower than that of the glutathioneinactivated enzyme but studies in which liver was preloaded with aldolase by perfusion at 19°C and then warming to 3’7’C indicated that the rate of degradation of all three forms was similar. It is concluded that the liver is capable of distinguishing between the glutathione-altered aldolase and native or partially degraded aldolase with regard to endocytosis, but that all three forms are degraded at similar rates once within lysosomes. tide bonds in the C-terminus (up to 20 amino acid residues can be released). However, further hydrolysis of native aldolase by these proteinases has not been observed in vitro. By contrast, when aldolase is reacted with disulfides (such as cystine or glutathione), the enzyme becomes unstable and is extensively degraded by these same lysosomal or non-lysosomal enzymes in vitro ((2); M. K. Offermann, M. J. McKay, M. W. Marsh, and J. S. Bond, unpublished work). The conformation of the protein, therefore, is a major determinant of the action of proteinases on aldolase in vitro. The perfused liver is a system whereby the relation between structure and susceptibility to endocytosis can be studied. Furthermore, because proteins are generally incorporated into cells via pinocytic vesicles which later fuse with preexisting lysosomes, the lysosomal digestion of pro-
Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) isolated from rabbit muscle is quite stable in vitro and resistant to extensive degradation by proteinases (l-5). Several purified endoproteinases (cathepsins B and D, chymotrypsin, papain, subtilisin, Staphylococcus aureus protease, meprin) (l-6), mixtures of lysosomal enzymes (5), and a membrane-associated lysosomal proteinase (7) are capable of inactivating native aldolase by cleaving pepi This work was supported by NIH Grants AM 19691 (J.S.B.) and AM 15465 (N.N.A.). J.S.B. is a recipient of a Research Career Development Award from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases. We wish to thank K. McWilliams for technical assistance with liver perfusions. ’ To whom correspondence should be addressed at the Department of Biochemistry, Virginia Commonwealth University, Richmond, Va. 23298. 367
0003-9861/83 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
368
BOND
AND
teins in intact tissue can be investigated with this system. With temperature-controled perfusions, the processes of endocytosis and degradation can be investigated separately; degradation, but not endocytosis, is decreased markedly when the temperature is lowered from 37 to 19°C (8). The goals of the present work were to determine whether ‘251-labeled aldolase was taken up and degraded by liver and, if so, to determine whether the structural alterations induced in aldolase by limited proteolysis (produced by cathepsin D) or by reactions with disulfides (oxidized glutathione) alter the rate of endocytosis and/ or degradation of the aldolase molecule by the intact tissue. EXPERIMENTAL
PROCEDURES
Preparation of native and inuctivated forms of aldolase. Rabbit muscle aldolase (sp act 12 pmol fructose-1,6-bisphosphate cleaved min-’ mg-‘) was purchased from Sigma Chemical Company (St. Louis, MO.). Minor protein contaminants were removed by cellulose phosphate chromatography; a single protein band (M, 40,000) was observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of mercaptoethanol (1, 2, 5). The enzyme was dialyzed 18 h against 100 mM sodium acetate, pH 4.8, before incubation with cathepsin D or against 50 mM Tris-HCl, pH 8.0, before reaction with oxidized glutathione. The enzyme concentration was determined by Am with Ey:E = 0.91 (9) or, after precipitation with triehloroacetic acid by the method of Lowry et al (10). Aldolase activity was measured as described by Johnson and Velick (11) using fructosel,&bisphosphate as substrate. For the inactivation of aldolase by eathepsin D: aldolase (2.5 mg ml-‘) in 100 mM sodium acetate buffer, pH 4.8, containing 100 mM NaCl, was incubated with cathepsin D (33 pg ml-‘) at 23°C for 3 h. The cathepsintreated aldolase had a specific activity of 0.8 pmol substrate cleaved min-’ mg-‘, which was 93% inactivated compared to the native enzyme. For inactivation of aldolase by oxidized glutathione: aldolase (2.5 mg ml-‘) in 50 mM Tris-HCI, pH 8.0, was incubated with 18 mM oxidized glutathione at 37°C for 90 min. The disulfide-treated enzyme had a specific activity of 0.6 pmol substrate cleaved min-’ mg-‘, which was 95% inactive compared to native enzyme. The disulfide-inactivated aldolase was dialyzed, to remove glutathione, before iodination. Iodinatimof &!&.-se. Three milligrams of each form of aldolase was iodinated with ‘%I [15.4 mCi (5’70 MBq) per pg iodine, Amersham Corp., Arlington Heights,
ARONSON Ill.] by the chloramine-T method (12). The iodinated proteins were separated from the unreacted ‘“I by Sephadex G-25 chromatography using 50 mM sodium phosphate buffer, pH 7.5, containing 150 mM KCl. The final preparations of iodinated forms of aldolase contained approx 0.3 mg protein ml-’ and had specific radioactivities of 5-15 X lo6 dpm mg-‘. The specific activities of aldolase preparations after iodination were native, 5.9; cathepsin D-treated, 0.6; and glutathione-inactivated, 0.5. Radioactive ‘=I was measured in polystyrene test tubes (12 X ‘75 mm) on a Packard Auto-Gamma scintillation spectrometer. There was no indication that iodination caused subunit dissociation but all forms of aldolase showed some degree (approx 15%) of aggregation as demonstrated by Sephadex G-200 gel chromatography (data not shown). Perfusion of rat liver. Male Wistar rats weighing 240 to 340 g were anesthetized with sodium pentobarbital (6.5 mg/lOO g of body wt.) and livers were prepared for perfusion as previously described (13). The isolated liver was perfused cyclically at 37°C with 100 ml of perfusate. The perfusate medium contained 4.62 mM KCI, 2.33 mM KH,PO,, 3.5 mM CaCl*, 2.31 mM MgSO,, 4.9 pM EDTA, 120 mMNaC1, 19.9 mM 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 5 mM D-ghCOSe, 2% (w/v) polyvinylpyrolidone-40 (PVP40), and 6 mg/lOO ml of penicillin, and was adjusted to pH 7.4 with 1 N NaOH. The perfusion medium was stored frozen and filtered through a Whatman No. 42 filter immediately prior to use. This system has been used extensively to monitor the degradation of extracellular proteins such as lZI-asialofetuin (e.g. (8, 13)). Materials. Human liver cathepsin D, prepared by methods described by Barrett (14), was a gift from Dr. Alan J. Barrett (Strangeways Laboratory, Cambridge, U.K.). Leupeptin was obtained from the Protein Research Foundation (Minohshi, Osaka, Japan). All other chemicals were obtained from Sigma Chemical Company except where noted. RESULTS
Radioiodinated native aldolase and cathepsin D-inactivated aldolase were removed from the perfusate by the liver at similar rates (Fig. 1). The loss of trichloroacetic acid-precipitable radioactivity from the perfusate followed first-order exponential kinetics for the first 30 min and the calculated half-lives for removal of these two forms was 30 min for native and 42 min for cathepsin D-treated aldolase (see Fig. 3). The increase in acid-soluble lEI in the perfusate with time indicated that these forms of aldolase were degraded
DEGRADATION
0
30 Time
60 (min)
OF ALDOLASE
90
FIG. 1. Endocytosis and degradation of ‘%I-labeled native or cathepsin D-treated aldolase by perfused rat liver. Livers were perfused at 37°C for 60 min and then 100 pg of iodinated aldolase was added to the perfusate. Native aldolase contained 4.9 X lo5 dpm/lOO pgg;cathepsin D-degraded aldolase 1.3 X lo6 dpm/lOO pg. Acid-soluble and acid-precipitable radioactivity were measured using 4% phosphotungstic acid in 2 N HCI as the precipitating reagent. A 0.2ml sample of perfusate was combined with 1 ml of cold acid solution in polystyrene tubes. After centrifugation at 2500 rpm for 10 min, the resulting supernatant fluid was removed and the sediment was washed once with 0.5 ml of cold acid solution. The washed sediment was solubilized with 1.0 ml of 1 N NaOH. Radioactivity in these samples was measured directly in a y spectrometer. Values shown are acidsoluble (triangles) and acid-precipitable (circles) radioactivity in the perfusate from native aldolase (open symbols) and cathepsin D-degraded aldolase (solid symbols).
by the liver. By 90 min, for these two forms of aldolase, approx 36% of originally injected lz51was in the perfusate in an acidsoluble form, 36% was acid-precipitable in the perfusate, 28% was in the liver, and 1% was in the bile. When the perfusion was continued for longer periods, there was a slow increase in the acid-soluble ‘%I that appeared in the perfusate; by 180 min 50% of the originally injected ‘%I-aldolase had been degraded. In contrast to the slow uptake and degradation of native and cathepsin D-treated aldolase, disulfide-treated aldolase was re-
369
BY LIVER
moved from the perfusate and degraded rapidly by the liver (Fig. 2). The half-life for removal of this form of aldolase was 10 min (Fig. 3). By 90 min, 68% of the initially injected l%I-aldolase had been degraded to acid-soluble ‘%I present in the perfusate, 14% was acid-precipitable in the perfusate, 15% was in the liver, and 1% was in the bile. To determine whether leupeptin affected the rate of degradation of the endocytosed aldolase, livers were perfused with this inhibitor prior to injection of radiolabeled aldolase (Fig. 2). Leupeptin markedly inhibited degradation of aldolase and had little if any effect on the uptake of aldolase into the liver. Ninety minutes after the radiolabeled, disulfide-treated aldolase was injected into leupeptin-treated systems, only 16% had been degraded to acid-soluble lz51present in the perfusate, 16% was acid-
Time
(min)
FIG. 2. Endocytosis and degradation of ‘l-labeled, oxidized glutathione-treated aldolase and the effect of leupeptin. Aldolase was inactivated by oxidized glutathione as described under Experimental Procedures and then iodinated. The enzyme (100 rg) containing 1.5 X lo6 dpm was added to the perfusate and samples of the perfusate were analyzed for acid-soluble (open triangles) and acid-precipitable (open circles) radioactivity as described in Fig. 1. The solid symbols are values obtained for acid-soluble (triangles) and acid-precipitable (circles) radioactivity for the same disulfide-treated aldolase preparation when 1 mg leupeptin was added to the perfusate 1 h prior to the enzyme.
370
BOND
AND
ARONSON
was the same as from glutathione-treated aldolase. DISCUSSION
8
A
*O20 0
30
10 Timetmin)
FIG. 3. Acid-precipitable radioactivity in the perfusate. Livers were perfused, as described in Figs. 1 and 2, with native aldolase (open circles), cathepsin D-degraded aldolase (solid circles), or glutathioneinactivated aldolase (triangles). The acid-precipitable radioactivity in the perfusate is expressed as the percentage of initial precipitable activity in the perfusate.
precipitable in the perfusate, 68% was in the liver, and 0.2% was present in the bile. While the degradation of native and cathepsin D-degraded aldolase appeared slower than that of the glutathione-inactivated aldolase (Figs. 1 and Z), it seemed possible that the apparent relative rates of degradation of the different forms were determined by the rates of entry into the lysosomes rather than by their innate susceptibility to lysosomal proteolysis. Therefore, an experiment was devised whereby the perfused liver was loaded with radiolabeled cathepsin D-treated aldolase at 19°C (Fig. 4). At this temperature, proteins are endocytosed, but they remain in vesicles that do not fuse with lysosomes (8). Upon rapidly increasing liver temperature to 37”C, these substrate-filled structures fuse with lysosomes and degradation proceeds. Under these experimental conditions (perfusing first at 19°C followed by rapid warming to 37”C), the rate of ‘%I acid-soluble material that appeared in the perfusate from cathepsin D-treated aldolase
The present work demonstrates clearly that the perfused liver is capable of taking up and degrading ‘%I-labeled aldolase. Furthermore, the liver is capable of distinguishing between aldolase that has been modified by reaction with oxidized glutathione and native or cathepsin D-degraded aldolase. Although these three forms of aldolase do not bind to synthetic phospholipid vesicles (Z), the more complex membranes of cells obviously do interact with all of these forms and recognize differences in the conformation of aldolase induced by disulfide. Disulfides do not affect the subunit molecular weight (40,000) or quaternary structure of aldolase but they do alter the secondary and/or tertiary structure of the enzyme, causing a marked decrease in thermal stability and in vitro proteolytic susceptibility (2). The number of titratable sulfhydryl groups on aldolase is also decreased by the disulfide. The liver may be able to recognize critical factors on the surface of aldolase that are exposed by the disulfide-treatment; perhaps, for example, hydrophobic areas are more prevalent in the glutathione-treated enzyme. Little is known about the structural factors of non-glycoproteins that may determine the rate of endocytosis of these proteins. Evidence is accumulating to indicate that several cytosolic enzymes are taken up and degraded by the liver and spleen (15) and that endocytosis is accelerated in diabetes (16). Aldolase may provide a good model to explore the mechanism of endocytosis of carbohydrate-free proteins. The fact that cathepsin D-inactivated aldolase is handled by the liver similarly to native aldolase is not surprising. The limited proteolysis catalyzed by cathepsin D evokes no change in aldolase antigenicity, quaternary structure, resistance to urea, or thermal denaturation, and this treatment does not alter the resistance of the bulk of the aldolase molecule to further proteolytic attack in vitro (5). The loss of
DEGRADATION
0
60
OF ALDOLASE
120 Time
160
371
BY LIVER
240
300
(min)
FIG. 4. Degradation of cathepsin D-treated aldolase by liver perfused at 19 and then at 37°C. An isolated rat liver was equilibrated for 30 min at 3’7°C and the perfusate temperature was then lowered to 19°C. After 15 min at 19°C 100 pg of cathepsin D-treated aldolase was added. At 210 min (indicated by arrow), the perfusate temperature was rapidly brought up to 37°C. Symbols and procedures were otherwise as in Fig. 1
amino acids (up to 20) and consequent loss of aldolase activity towards the substrate fructose-1,6-bisphosphate apparently are not recognized as a significant change to the endocytotic system. There is no indication from the present work that the different forms of aldolase are degraded at different rates once in the intact tissue. The proteolytic system in intact cells, therefore, does not recognize the structural differences between the various forms of aldolase. The fact that leupeptin is so effective in inhibiting degradation of aldolase makes it likely that the thiol proteinases of the lysosomes are important for the process (1’7). It seems probable that the environment in the lysosome (perhaps the acidic pH or high concentration of proteinases) is such that the structure of most proteins is not a determinative factor in the rate of degradation. We suggest, as others have (B-20), that the binding of proteins to either autophagic precursor membranes or the lysosomal membrane itself regulates the rate of degradation of proteins in intact tissue and that in this manner the physicochemical properties of a protein are determinative in degradation.
The observation that the disulfide-inactivated enzyme was preferentially taken up by the liver may possibly have implications for degradation of intracellular proteins as well as extracellular proteins. The oxidation of sulfhydryl groups on proteins has been suggested as the initial event in the degradation of several soluble proteins to amino acids and peptides (21), and the redox state of cells has been suggested to be important in the regulation of intracellular proteolysis in general (22, 23). Thus the oxidation state of proteins in cells may play a determinative role in the degradation of proteins and it is conceivable that the molecular basis for this phenomenon involves the ability of cellular membranes to distinguish between native structures of proteins and those that have been modified by reactions with disulfides. REFERENCES 1. BOND, J. S., AND BARRETT, J. 189, 17-25.
A. J. (1980)
Biochem
2. BOND, J. S., AND OFFERMANN, M. K. (1981) Acta Biol Med Gem. 40, 1365-13’74. 3. MIDELFORT, C. F., AND MEHLER, A. H. (1972) J. Bid Chem. 247, 3618-3621.
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4. HANNAPPEL, E., MACGREGOR, J. S., DAVOUST, S., AND HORECKER, B. L. (1982) Arch Biochem Biophys. 214,293-298. 5. OFFERMANN, M. K., CHLEBOWSKI, J. F., AND BOND, J. S. (1983) Biochem. J. 211, 529-534. 6. BEYNON, R. J., SHANNON, J. D., AND BOND, J. S. (1981) Biochem J. 199, 591-598. 7. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE, B., MICHETX, M., AND HORECKER, B. L. (1982) Arch. B&hem Biophys. 214,376385. 8. DUNN, W. A., HUBBARD, A. L., AND ARONSON, N. N., JR. (1980) .I. Biol. Chem 255,5971-5978. 9. GRAZI, E., AND BLANZIER, M. (1977) Biochem J. 167,361-366. 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) .I Biol Chem 193, 265-275. 11. JOHNSON, L. W.,, AND VELICK, S. F. (1972) J. Biol Chem 247,4138-4143. 12. BOLTON, A. E. (1977) in Radioiodination Techniques, pp. 45-48, Printarium, Wembley, U.K.
ARONSON 13. DUNN, W. A., LA BADIE, J. H., AND ARONSON, N. N., JR. (1979) J. BioL Chem 254,4191-4196. 14. BARRE=, A. J. (1973) Biochem J. 131, 809-822. 15. SINKE, J., BOUMA, J. M. W., KOOISTRA, T., AND GRUBER, M. (1979) Biochxm. J. 180, l-9. 16. HUTSON, N. J., LLOYD, C. E., AND MORTIMORE, G. E. (1982) Pm Natl Ad Sci USA 79,17371741. 17. BARRETT, A. J., AND KIRSCHKE, H . (1981) in Methods in Enzymology (Lorand, L., ed.), Vol. 80, pp. 535-561, Academic Press, New York. 18. DEAN, R. T. (1975) Biochem Biophys. Res. Cmmun. 67, 604-609. 19. WARD, W. F., Cox, J. R., AND MORTIMORE, G. E. (1977) J. Biol. Chem 252, 6955-6961. 20. MOORE, A. T., WILLIAMS, K. E., AND LLOYD, J. B. (1977) Biochem J. 164, 607-616. 21. FRANCIS, G. L., AND BALLARD, F. J. (1980) Biochem J. 186, 581-590. 22. TISCHLER, M. E. (1980) Biochem .I 192, 963-966. 23. WILLIAMS, E. H., KAO, R. L., AND MORGAN, H. E. (1981) Amer. J. Physiol 240, E268-E273.
Endocytosis and Degradation Glutathione-Inactivated JUDITH
of Native, Cathepsin D-Degraded, Aldolase by Perfused Rat Liver’
S. BOND’ AND NATHAN
N. ARONSON.
and
JR.
Departmed of Biochemistry, Virginia Commonwealth University, Richmond, Virginia 23298, and The A&house Laboratory, Biochemistry Program, Pennsylvania State University, University Park, Pennsylvania 16802 Received March 21, 1983, and in revised form July 18, 1983
The uptake and degradation of ‘%I-labeled (a) native aldolase, (b) cathepsin D-inactivated aldolase, and (c) aldolase inactivated by oxidized glutathione were studied in perfused rat liver. All three forms of aldolase were removed from the perfusion medium and degraded by the liver, but the uptake of the glutathione-inactivated enzyme (half-life in perfusate = 10 min) was much faster than that of the native enzyme (halflife = 30 min) or the cathepsin-inactivated enzyme (half-life = 42 min). The degradation of the enzyme was almost totally inhibited by leupeptin, indicating that thiol proteinases in lysosomes play an important role in the digestion process. Degradation of native and cathepsin D-inactivated aldolase appeared to be slower than that of the glutathioneinactivated enzyme but studies in which liver was preloaded with aldolase by perfusion at 19°C and then warming to 3’7’C indicated that the rate of degradation of all three forms was similar. It is concluded that the liver is capable of distinguishing between the glutathione-altered aldolase and native or partially degraded aldolase with regard to endocytosis, but that all three forms are degraded at similar rates once within lysosomes. tide bonds in the C-terminus (up to 20 amino acid residues can be released). However, further hydrolysis of native aldolase by these proteinases has not been observed in vitro. By contrast, when aldolase is reacted with disulfides (such as cystine or glutathione), the enzyme becomes unstable and is extensively degraded by these same lysosomal or non-lysosomal enzymes in vitro ((2); M. K. Offermann, M. J. McKay, M. W. Marsh, and J. S. Bond, unpublished work). The conformation of the protein, therefore, is a major determinant of the action of proteinases on aldolase in vitro. The perfused liver is a system whereby the relation between structure and susceptibility to endocytosis can be studied. Furthermore, because proteins are generally incorporated into cells via pinocytic vesicles which later fuse with preexisting lysosomes, the lysosomal digestion of pro-
Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) isolated from rabbit muscle is quite stable in vitro and resistant to extensive degradation by proteinases (l-5). Several purified endoproteinases (cathepsins B and D, chymotrypsin, papain, subtilisin, Staphylococcus aureus protease, meprin) (l-6), mixtures of lysosomal enzymes (5), and a membrane-associated lysosomal proteinase (7) are capable of inactivating native aldolase by cleaving pepi This work was supported by NIH Grants AM 19691 (J.S.B.) and AM 15465 (N.N.A.). J.S.B. is a recipient of a Research Career Development Award from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases. We wish to thank K. McWilliams for technical assistance with liver perfusions. ’ To whom correspondence should be addressed at the Department of Biochemistry, Virginia Commonwealth University, Richmond, Va. 23298. 367
0003-9861/83 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
368
BOND
AND
teins in intact tissue can be investigated with this system. With temperature-controled perfusions, the processes of endocytosis and degradation can be investigated separately; degradation, but not endocytosis, is decreased markedly when the temperature is lowered from 37 to 19°C (8). The goals of the present work were to determine whether ‘251-labeled aldolase was taken up and degraded by liver and, if so, to determine whether the structural alterations induced in aldolase by limited proteolysis (produced by cathepsin D) or by reactions with disulfides (oxidized glutathione) alter the rate of endocytosis and/ or degradation of the aldolase molecule by the intact tissue. EXPERIMENTAL
PROCEDURES
Preparation of native and inuctivated forms of aldolase. Rabbit muscle aldolase (sp act 12 pmol fructose-1,6-bisphosphate cleaved min-’ mg-‘) was purchased from Sigma Chemical Company (St. Louis, MO.). Minor protein contaminants were removed by cellulose phosphate chromatography; a single protein band (M, 40,000) was observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of mercaptoethanol (1, 2, 5). The enzyme was dialyzed 18 h against 100 mM sodium acetate, pH 4.8, before incubation with cathepsin D or against 50 mM Tris-HCl, pH 8.0, before reaction with oxidized glutathione. The enzyme concentration was determined by Am with Ey:E = 0.91 (9) or, after precipitation with triehloroacetic acid by the method of Lowry et al (10). Aldolase activity was measured as described by Johnson and Velick (11) using fructosel,&bisphosphate as substrate. For the inactivation of aldolase by eathepsin D: aldolase (2.5 mg ml-‘) in 100 mM sodium acetate buffer, pH 4.8, containing 100 mM NaCl, was incubated with cathepsin D (33 pg ml-‘) at 23°C for 3 h. The cathepsintreated aldolase had a specific activity of 0.8 pmol substrate cleaved min-’ mg-‘, which was 93% inactivated compared to the native enzyme. For inactivation of aldolase by oxidized glutathione: aldolase (2.5 mg ml-‘) in 50 mM Tris-HCI, pH 8.0, was incubated with 18 mM oxidized glutathione at 37°C for 90 min. The disulfide-treated enzyme had a specific activity of 0.6 pmol substrate cleaved min-’ mg-‘, which was 95% inactive compared to native enzyme. The disulfide-inactivated aldolase was dialyzed, to remove glutathione, before iodination. Iodinatimof &!&.-se. Three milligrams of each form of aldolase was iodinated with ‘%I [15.4 mCi (5’70 MBq) per pg iodine, Amersham Corp., Arlington Heights,
ARONSON Ill.] by the chloramine-T method (12). The iodinated proteins were separated from the unreacted ‘“I by Sephadex G-25 chromatography using 50 mM sodium phosphate buffer, pH 7.5, containing 150 mM KCl. The final preparations of iodinated forms of aldolase contained approx 0.3 mg protein ml-’ and had specific radioactivities of 5-15 X lo6 dpm mg-‘. The specific activities of aldolase preparations after iodination were native, 5.9; cathepsin D-treated, 0.6; and glutathione-inactivated, 0.5. Radioactive ‘=I was measured in polystyrene test tubes (12 X ‘75 mm) on a Packard Auto-Gamma scintillation spectrometer. There was no indication that iodination caused subunit dissociation but all forms of aldolase showed some degree (approx 15%) of aggregation as demonstrated by Sephadex G-200 gel chromatography (data not shown). Perfusion of rat liver. Male Wistar rats weighing 240 to 340 g were anesthetized with sodium pentobarbital (6.5 mg/lOO g of body wt.) and livers were prepared for perfusion as previously described (13). The isolated liver was perfused cyclically at 37°C with 100 ml of perfusate. The perfusate medium contained 4.62 mM KCI, 2.33 mM KH,PO,, 3.5 mM CaCl*, 2.31 mM MgSO,, 4.9 pM EDTA, 120 mMNaC1, 19.9 mM 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 5 mM D-ghCOSe, 2% (w/v) polyvinylpyrolidone-40 (PVP40), and 6 mg/lOO ml of penicillin, and was adjusted to pH 7.4 with 1 N NaOH. The perfusion medium was stored frozen and filtered through a Whatman No. 42 filter immediately prior to use. This system has been used extensively to monitor the degradation of extracellular proteins such as lZI-asialofetuin (e.g. (8, 13)). Materials. Human liver cathepsin D, prepared by methods described by Barrett (14), was a gift from Dr. Alan J. Barrett (Strangeways Laboratory, Cambridge, U.K.). Leupeptin was obtained from the Protein Research Foundation (Minohshi, Osaka, Japan). All other chemicals were obtained from Sigma Chemical Company except where noted. RESULTS
Radioiodinated native aldolase and cathepsin D-inactivated aldolase were removed from the perfusate by the liver at similar rates (Fig. 1). The loss of trichloroacetic acid-precipitable radioactivity from the perfusate followed first-order exponential kinetics for the first 30 min and the calculated half-lives for removal of these two forms was 30 min for native and 42 min for cathepsin D-treated aldolase (see Fig. 3). The increase in acid-soluble lEI in the perfusate with time indicated that these forms of aldolase were degraded
DEGRADATION
0
30 Time
60 (min)
OF ALDOLASE
90
FIG. 1. Endocytosis and degradation of ‘%I-labeled native or cathepsin D-treated aldolase by perfused rat liver. Livers were perfused at 37°C for 60 min and then 100 pg of iodinated aldolase was added to the perfusate. Native aldolase contained 4.9 X lo5 dpm/lOO pgg;cathepsin D-degraded aldolase 1.3 X lo6 dpm/lOO pg. Acid-soluble and acid-precipitable radioactivity were measured using 4% phosphotungstic acid in 2 N HCI as the precipitating reagent. A 0.2ml sample of perfusate was combined with 1 ml of cold acid solution in polystyrene tubes. After centrifugation at 2500 rpm for 10 min, the resulting supernatant fluid was removed and the sediment was washed once with 0.5 ml of cold acid solution. The washed sediment was solubilized with 1.0 ml of 1 N NaOH. Radioactivity in these samples was measured directly in a y spectrometer. Values shown are acidsoluble (triangles) and acid-precipitable (circles) radioactivity in the perfusate from native aldolase (open symbols) and cathepsin D-degraded aldolase (solid symbols).
by the liver. By 90 min, for these two forms of aldolase, approx 36% of originally injected lz51was in the perfusate in an acidsoluble form, 36% was acid-precipitable in the perfusate, 28% was in the liver, and 1% was in the bile. When the perfusion was continued for longer periods, there was a slow increase in the acid-soluble ‘%I that appeared in the perfusate; by 180 min 50% of the originally injected ‘%I-aldolase had been degraded. In contrast to the slow uptake and degradation of native and cathepsin D-treated aldolase, disulfide-treated aldolase was re-
369
BY LIVER
moved from the perfusate and degraded rapidly by the liver (Fig. 2). The half-life for removal of this form of aldolase was 10 min (Fig. 3). By 90 min, 68% of the initially injected l%I-aldolase had been degraded to acid-soluble ‘%I present in the perfusate, 14% was acid-precipitable in the perfusate, 15% was in the liver, and 1% was in the bile. To determine whether leupeptin affected the rate of degradation of the endocytosed aldolase, livers were perfused with this inhibitor prior to injection of radiolabeled aldolase (Fig. 2). Leupeptin markedly inhibited degradation of aldolase and had little if any effect on the uptake of aldolase into the liver. Ninety minutes after the radiolabeled, disulfide-treated aldolase was injected into leupeptin-treated systems, only 16% had been degraded to acid-soluble lz51present in the perfusate, 16% was acid-
Time
(min)
FIG. 2. Endocytosis and degradation of ‘l-labeled, oxidized glutathione-treated aldolase and the effect of leupeptin. Aldolase was inactivated by oxidized glutathione as described under Experimental Procedures and then iodinated. The enzyme (100 rg) containing 1.5 X lo6 dpm was added to the perfusate and samples of the perfusate were analyzed for acid-soluble (open triangles) and acid-precipitable (open circles) radioactivity as described in Fig. 1. The solid symbols are values obtained for acid-soluble (triangles) and acid-precipitable (circles) radioactivity for the same disulfide-treated aldolase preparation when 1 mg leupeptin was added to the perfusate 1 h prior to the enzyme.
370
BOND
AND
ARONSON
was the same as from glutathione-treated aldolase. DISCUSSION
8
A
*O20 0
30
10 Timetmin)
FIG. 3. Acid-precipitable radioactivity in the perfusate. Livers were perfused, as described in Figs. 1 and 2, with native aldolase (open circles), cathepsin D-degraded aldolase (solid circles), or glutathioneinactivated aldolase (triangles). The acid-precipitable radioactivity in the perfusate is expressed as the percentage of initial precipitable activity in the perfusate.
precipitable in the perfusate, 68% was in the liver, and 0.2% was present in the bile. While the degradation of native and cathepsin D-degraded aldolase appeared slower than that of the glutathione-inactivated aldolase (Figs. 1 and Z), it seemed possible that the apparent relative rates of degradation of the different forms were determined by the rates of entry into the lysosomes rather than by their innate susceptibility to lysosomal proteolysis. Therefore, an experiment was devised whereby the perfused liver was loaded with radiolabeled cathepsin D-treated aldolase at 19°C (Fig. 4). At this temperature, proteins are endocytosed, but they remain in vesicles that do not fuse with lysosomes (8). Upon rapidly increasing liver temperature to 37”C, these substrate-filled structures fuse with lysosomes and degradation proceeds. Under these experimental conditions (perfusing first at 19°C followed by rapid warming to 37”C), the rate of ‘%I acid-soluble material that appeared in the perfusate from cathepsin D-treated aldolase
The present work demonstrates clearly that the perfused liver is capable of taking up and degrading ‘%I-labeled aldolase. Furthermore, the liver is capable of distinguishing between aldolase that has been modified by reaction with oxidized glutathione and native or cathepsin D-degraded aldolase. Although these three forms of aldolase do not bind to synthetic phospholipid vesicles (Z), the more complex membranes of cells obviously do interact with all of these forms and recognize differences in the conformation of aldolase induced by disulfide. Disulfides do not affect the subunit molecular weight (40,000) or quaternary structure of aldolase but they do alter the secondary and/or tertiary structure of the enzyme, causing a marked decrease in thermal stability and in vitro proteolytic susceptibility (2). The number of titratable sulfhydryl groups on aldolase is also decreased by the disulfide. The liver may be able to recognize critical factors on the surface of aldolase that are exposed by the disulfide-treatment; perhaps, for example, hydrophobic areas are more prevalent in the glutathione-treated enzyme. Little is known about the structural factors of non-glycoproteins that may determine the rate of endocytosis of these proteins. Evidence is accumulating to indicate that several cytosolic enzymes are taken up and degraded by the liver and spleen (15) and that endocytosis is accelerated in diabetes (16). Aldolase may provide a good model to explore the mechanism of endocytosis of carbohydrate-free proteins. The fact that cathepsin D-inactivated aldolase is handled by the liver similarly to native aldolase is not surprising. The limited proteolysis catalyzed by cathepsin D evokes no change in aldolase antigenicity, quaternary structure, resistance to urea, or thermal denaturation, and this treatment does not alter the resistance of the bulk of the aldolase molecule to further proteolytic attack in vitro (5). The loss of
DEGRADATION
0
60
OF ALDOLASE
120 Time
160
371
BY LIVER
240
300
(min)
FIG. 4. Degradation of cathepsin D-treated aldolase by liver perfused at 19 and then at 37°C. An isolated rat liver was equilibrated for 30 min at 3’7°C and the perfusate temperature was then lowered to 19°C. After 15 min at 19°C 100 pg of cathepsin D-treated aldolase was added. At 210 min (indicated by arrow), the perfusate temperature was rapidly brought up to 37°C. Symbols and procedures were otherwise as in Fig. 1
amino acids (up to 20) and consequent loss of aldolase activity towards the substrate fructose-1,6-bisphosphate apparently are not recognized as a significant change to the endocytotic system. There is no indication from the present work that the different forms of aldolase are degraded at different rates once in the intact tissue. The proteolytic system in intact cells, therefore, does not recognize the structural differences between the various forms of aldolase. The fact that leupeptin is so effective in inhibiting degradation of aldolase makes it likely that the thiol proteinases of the lysosomes are important for the process (1’7). It seems probable that the environment in the lysosome (perhaps the acidic pH or high concentration of proteinases) is such that the structure of most proteins is not a determinative factor in the rate of degradation. We suggest, as others have (B-20), that the binding of proteins to either autophagic precursor membranes or the lysosomal membrane itself regulates the rate of degradation of proteins in intact tissue and that in this manner the physicochemical properties of a protein are determinative in degradation.
The observation that the disulfide-inactivated enzyme was preferentially taken up by the liver may possibly have implications for degradation of intracellular proteins as well as extracellular proteins. The oxidation of sulfhydryl groups on proteins has been suggested as the initial event in the degradation of several soluble proteins to amino acids and peptides (21), and the redox state of cells has been suggested to be important in the regulation of intracellular proteolysis in general (22, 23). Thus the oxidation state of proteins in cells may play a determinative role in the degradation of proteins and it is conceivable that the molecular basis for this phenomenon involves the ability of cellular membranes to distinguish between native structures of proteins and those that have been modified by reactions with disulfides. REFERENCES 1. BOND, J. S., AND BARRETT, J. 189, 17-25.
A. J. (1980)
Biochem
2. BOND, J. S., AND OFFERMANN, M. K. (1981) Acta Biol Med Gem. 40, 1365-13’74. 3. MIDELFORT, C. F., AND MEHLER, A. H. (1972) J. Bid Chem. 247, 3618-3621.
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4. HANNAPPEL, E., MACGREGOR, J. S., DAVOUST, S., AND HORECKER, B. L. (1982) Arch Biochem Biophys. 214,293-298. 5. OFFERMANN, M. K., CHLEBOWSKI, J. F., AND BOND, J. S. (1983) Biochem. J. 211, 529-534. 6. BEYNON, R. J., SHANNON, J. D., AND BOND, J. S. (1981) Biochem J. 199, 591-598. 7. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE, B., MICHETX, M., AND HORECKER, B. L. (1982) Arch. B&hem Biophys. 214,376385. 8. DUNN, W. A., HUBBARD, A. L., AND ARONSON, N. N., JR. (1980) .I. Biol. Chem 255,5971-5978. 9. GRAZI, E., AND BLANZIER, M. (1977) Biochem J. 167,361-366. 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) .I Biol Chem 193, 265-275. 11. JOHNSON, L. W.,, AND VELICK, S. F. (1972) J. Biol Chem 247,4138-4143. 12. BOLTON, A. E. (1977) in Radioiodination Techniques, pp. 45-48, Printarium, Wembley, U.K.
ARONSON 13. DUNN, W. A., LA BADIE, J. H., AND ARONSON, N. N., JR. (1979) J. BioL Chem 254,4191-4196. 14. BARRE=, A. J. (1973) Biochem J. 131, 809-822. 15. SINKE, J., BOUMA, J. M. W., KOOISTRA, T., AND GRUBER, M. (1979) Biochxm. J. 180, l-9. 16. HUTSON, N. J., LLOYD, C. E., AND MORTIMORE, G. E. (1982) Pm Natl Ad Sci USA 79,17371741. 17. BARRETT, A. J., AND KIRSCHKE, H . (1981) in Methods in Enzymology (Lorand, L., ed.), Vol. 80, pp. 535-561, Academic Press, New York. 18. DEAN, R. T. (1975) Biochem Biophys. Res. Cmmun. 67, 604-609. 19. WARD, W. F., Cox, J. R., AND MORTIMORE, G. E. (1977) J. Biol. Chem 252, 6955-6961. 20. MOORE, A. T., WILLIAMS, K. E., AND LLOYD, J. B. (1977) Biochem J. 164, 607-616. 21. FRANCIS, G. L., AND BALLARD, F. J. (1980) Biochem J. 186, 581-590. 22. TISCHLER, M. E. (1980) Biochem .I 192, 963-966. 23. WILLIAMS, E. H., KAO, R. L., AND MORGAN, H. E. (1981) Amer. J. Physiol 240, E268-E273.