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Sites of cleavage of rabbit muscle aldolase by purified cathepsin M from rabbit liver
BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
35, 191-198
(1986)
Sites of Cleavage of Rabbit Muscle Aldolase by Purified Cathepsin M from Rabbit Liver SUSAN ERICKSON-VIITANEN,
ETTORE BALESTRERI, AND B. L. HORECKER
Roche
Institute
of Molecular
Biology, Received
Roche
Research
September
Center,
MARTIN
Nutley,
MCDERMOTT,
New
Jersey
07110
20, 1984
Cathepsin M is a lysosomal protease of the B-type cathepsin family exhibiting relatively poor reactivity toward synthetic naphthylamide derivatives of blocked or unblocked amino acids but more significant proteolytic activity towards native protein substrates such as muscle and liver aldolases (l-3). A significant proportion of cathepsin M activity is associated with the lysosomal membrane and is expressed by intact lysosomes at neutral pH (1, 2). For this reason, cathepsin M is a likely candidate for the modification of aldolase observed in fasted animals in viva (46).
We have used rabbit muscle aldolase to study the peptide bond specificity of cathepsin M. This protein is composed of four subunits each containing COOHterminal tyrosine and otherwise identical except for microheterogeneity introduced by spontaneous partial deamidation of an asparaginyl residue near the COOHterminus (7). Digestion of rabbit muscle aldolase with carboxypeptidase A results in loss of activity with fructose 1, 6-bisphosphate as the substrate at a rate that is correlated with the rate of loss of COOH-terminal tyrosines (8). Cathepsin B from human liver (9) or rabbit liver (10) removes dipeptides sequentially from the COOHterminus of muscle aldolase with resultant inactivation of the enzyme. However, the quantitative relationship between COOH-terminal modification and loss of catalytic activity has not been clear; complete inactivation has been correlated with the loss of a single COOH-terminal dipeptide during digestion with rabbit liver cathepsin B (10) or with the loss of three of the four tyrosines after digestion with carboxypeptidase A (8). We report here the sites of cleavage of muscle aldolase by purified rabbit liver cathepsin M. Unlike the peptidyl dipeptidase activity of cathepsin B (9,10), we find that cathepsin M attacks peptide bonds near the COOH-terminus in a less regular manner. In addition, quantitative analysis of release of tyrosine-containing peptides reveals that loss of aldolase activity is related to proteolytic modification of three of the four subunits of aldolase; modification of the fourth subunit is observed after inactivation is complete. 0885-4505186 ‘ill
$3.00
Copyright 0 1986 by Academic Press. Inc. rights of reproduction in any form reserved.
192
ERICKSON-VIITANEN
ET AL.
MATERIALS
Rabbit muscle fructose 1,6-bisphosphate aldolase (type IV), fructose 1,6-bisphosphate, and carboxypeptidase A were purchased from Sigma Chemical Company, and NADH and glycerol-3-phosphate dehydrogenase/triose phosphate isomerase (6DH/TIM) from Boehringer Mannheim. Reagents for SDS’-polyacrylamide gel electrophoresis were from Bio-Rad. Fluorescamine (Fluram) was provided by Dr. William E. Scott of Hoffmann-LaRoche and o-phthaldehyde was purchased from Pierce. All other chemicals and solvents were chromatography grade. Solvents were redistilled as required. METHODS
The purification of cathepsin M from rabbit liver crude lysosomal fraction by a modification of the earlier procedure (1) will be described elsewhere. After the last step of the purification on carboxymethly cellulose, the active fractions were pooled and concentrated to 50-100 pg protein/ml by ultrafiltration. The concentrated enzyme solution was stored at -20” C in small aliquots. The specific activity of the preparation was 20,000 units/mg protein, where one unit corresponds to the amount required to catalyze the inactivation of 1 nmole of rabbit muscle aldolase in 30 min under standard assay conditions (see below). For digestion of rabbit muscle aldolase, the incubation mixture contained 1.2 mg/ml of aldolase, 10 mM DTT, 50 mM Na acetate, pH 5.0, and 2 (ug/ml cathepsin M. Measurement of aldolase activity was carried out at 26” C using the spectrophotometric assay of Gracy et al. (11). The one-ml cuvette contained 50 mM Tris-HCl, pH 7.4, 0.15 mM NADH, 1.0 mM fructose 1,6-bisphosphate or 10 mM fructose l-phosphate, and 10 pg of the glycerophosphate dehydrogenase/triose phosphate isomerase mixture (10: 1 w/w). The reaction was begun by the addition of 20-~1 aliquots of the aldolase digestion mixtures (see above), that had been diluted 20-fold in cold 67 mM Tris-HCl, pH 7.3. Peptides released from aldolase upon digestion with cathepsin M were separated and quantitated by reverse-phase HPLC. An aliquot of the digestion mixture (1 ml) was cooled and acidified with perchloric acid to a final concentration of 5%. After centrifugation the precipitate was washed with the same volume of 5% HC104 and the first supernatant solution and washing combined and neutralized with 10% KOH. After centrifuging to remove precipitated KCIOl the clear supernatant solution was lyophilized. The powder was dissolved in 250 ~1 of 0.5 M HCOOH/O * 1 M pyridine and 150~~1 aliquots were analyzed on an Ultrasphere ODS Cl8 column (5 p, 4.6 x 250 mm, Altex Scientific) with the fluorescence detection system described by Stein and Moshera (12). Amino acid analyses were carried out after hydrolysis of peptides with redistilled 6.0 M HCl at 150” for 90 min, using a Glenco MM-70 amino acid analyzer adapted for use of o-phthaldehyde and fluorescence detection as described by Benson and Hare (13). To determine the number of COOH-terminal tyrosines remaining after various times of incubation with cathepsin M, the HClO,-precipitated protein was digested ’ Abbreviations
used: DTT, 1,4-dithiothreitol;
SDS, sodium dodecyl sulfate.
DIGESTION
OF RABBIT
MUSCLE
ALDOLASE
BY CATHEPSIN
M
193
with carboxypeptidase A (14). The protein was suspended in 1.0 ml of 50 mM Hepes buffer containing 0.1% SDS, pH 7.8, and dissolved by heating to 50” C. Carboxypeptidase A, 5 ~1 of a solution containing 2 mg/ml in 10% LiCl, was added to 100 ~1 of the cooled aldolase solution containing 2.6 nmole protein (ratio of aldolase to carboxypeptidase = 13 : 1) at zero time and again after 2 hr incubation at 25” C. Aliquots (2 ~1) were removed at 0, 1, 2, 3, and 5 hr and mixed with 10 ~1 0.2 M Na borate, pH 9.3, and frozen until analysis. Released amino acids were quantitated by reverse-phase HPLC after derivitization with o-phthaldehyde as described by Jones et al. (15). The aldolase content was determined by amino acid analysis of an aliquot after hydrolysis in 6 M HCl at 150” C for 1.5 hr using values of 3 methionines and 15 arginine residues per subunit (16) for the calculation. Slab gel electrophoresis at pH 8.8 in the presence of SDS and mercaptoethanol was carried out according to the procedure of Laemmli (17). Coomassie blue G was used for staining. RESULTS
Effect of cathepsin M on the catalytic activity and subunit structure of rabbit muscle aldolase. Catalytic activity was assessed with fructose 1 ,dbisphosphate as the substrate (Fig. 1). Cathepsin M caused a decrease in the activity of rabbit muscle aldolase from an intitial value of 14.0 units/mg to a final value of 1.2 units/mg; activity was not further decreased by longer incubation or the addition
TIME km)
FIG. 1. Inactivation of aldolase by cathepsin M and appearance of acid-soluble peptides containing tyrosine. Reaction conditions were as described under Methods; at the times indicated, aliquots of the reaction mixture were assayed spectrophotometrically for aldolase activity with fructose 1,6bisphosphate (0). Tyrosine release was calculated from the sum of tyrosine-containing peptides separated by HPLC (see Fig. 3), adjusted for losses during chromatography (0). An average recovery factor of 64% was calculated by comparing the total tyrosine and phenylalanine in HPLC fractions with the tyrosine and phenylalanine present in an aliquot of the neutralized acid extract. The total aldolase content determined by amino acid analysis of the acid-precipitated material (see Table 1) was used to calculate nmole tyrosine released per nmole aldolase tetramer.
ERICKSON-VIITANEN
194
ET AL.
of fresh cathepsin M (data not shown). With fructose l-phosphate as the substrate, the initial catalytic activity was 0.22 units/mg and was not significantly altered during incubation with cathepsin M (data not shown). These effects of cathepsin M are similar to those observed with carboxypeptidase A (8) and with cathepsin B (9), and suggested that digestion was confined to a limited region at the COOHterminus. This was supported by analysis of the modified enzyme by SDS-gel electrophoresis (Fig. 2), which indicated a change in subunit molecular weight from an initial value of 40,000 to a final value of approximately 38,500. Modification at the COOH-terminus was confirmed by the finding that the decrease in activity toward fructose 1,dbisphosphate was related to the loss of tyrosine residues from the COOH-terminus (Table l), evaluated by treating the aldolase remaining with carboxypeptidase A (see Methods). This was accompanied by the appearance of acid-soluble peptides containing tyrosine (Fig. 1). Modification of the NH,terminus was excluded by the absence of proline (16) in the peptides released (data not shown). kDa
c-i
*
“7%
66.2
45.0
31.0
21.5
14.4
0
6
10
30
60
180
lime of digestion (minutes) FIG. 2. SDS-polyacrylamide gel electrophoresis of native and cathepsin M-digested rabbit muscle aldolase. Rabbit muscle aldolase was digested with cathepsin M (see Methods) and at the times indicated 2.5~pg aliquots were analyzed on 12.5% acrylamide gels. Running conditions: 20 mV, 5hr. Lanes 1 and 8 represent the molecular weight markers bovine serum albumin (66.2 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa).
DIGESTION
OF RABBIT
MUSCLE
ALDOLASE
BY CATHEPSIN
195
M
TABLE 1 Tyrosine from Cathepsin M-Digested Aldolase
Recovery of COOH-Terminal Time of incubation with cathepsin M (mitt)
Tyrosine released by carboxypeptidase A
Subunits aldolase
@mole)
(pmole)
45.2 41.0 22.6 10.4
ND 51.8 52.4 54.2
0
3 30 90
Note. Carboxypeptidase A digestion was carried out as described under Methods using the HClO,-precipitated protein recovered after digestion with cathepsin M for the indicated times. Values represent pmole tyrosine present in 2 ~1 of the carboxypeptidase reaction mixture after 5 hr digestion. The total aldolase present in the reaction mixture was calculated from the amino acid composition obtained by total acid hydrolysis of a 2 ~1 aliquot of the reaction mixture. y ND, not determined.
ZdentiJication of the peptides releasedfrom rabbit muscle aldolase. The COOH356
terminal
sequence of rabbit muscle aldolase has been established 360
as Ser-Leu-
364
Phe-Ile-Ser-Asn-His-AlaTyr OH by Edman degradation of the chymotryptic and tryptic peptides containing the COOH-terminus (16, 18) and partially confirmed by cloning of the cDNA (19). After separation by HPLC (Fig. 3), the acid soluble
TIME (mid
FIG. 3. Separation of acid-soluble peptides from muscle aldolase digested with cathepsin M. The reaction mixture (1 ml) contained 1.2 mg of rabbit muscle aldolase, 2 pg of cathepsin M, 10 mM DDT, 50 mM Na acetate buffer, pH 5.0. After incubation for 30 min at 37” C the mixture was acidified with 0.25 ml of 25% HClO, and the acid extract collected and neutralized as described under Methods. The lyophilized extract was dissolved in 0.25 ml of 0.5 M HCOOH/O.l M pyridine, pH 2.8, and a 150+1 aliquot was injected onto an Ultrasphere ODS Cl8 column preequilibrated with the same buffer. A delayed linear gradient of CH$N in 1.0 M HCOOH/0.2 M pyridine was generated with a Waters System Controller Model 720 (Waters Assoc.) as shown (---). The flow rate was 0.6 ml/min and fractions of 1 min were collected. At 6-set intervals, 5 ~1 of the effluent was diverted for analysis with fluorescamine and the fluorescence recorded (-). Six major peaks were detected. They were identified by amino acid analysis as Ile-Ser-Asn-His (peak 2); Ala-Tyr (peak 3), Ile-Ser-AsnHis-Ala-Tyr (peak 4), Phe-Ile (peak 5) and Leu-Phe (peak 6). Peak 1 contained a mixture of free amino acids (Ser,Gly) and small peptides that yielded ZSer, 3Glu, 2Gly, 4Ala, Asp, and His on acid hydrolysis. The short retention time observed with the reverse-phase chromatography system employed suggests that this peak contains the dipeptide Asn-His plus other di- and/or tripeptides.
196
ERICKSON-VIITANEN
ET AL.
peptides released by cathepsin M were identified by amino acid analysis (data not shown) and the time course of their release determined (Fig. 4). The first peptide detected was the COOH-terminal dipeptide Ala-Tyr, following by LeuPhe, and the COOH-terminal hexapeptide. Analysis of the results suggests that the most susceptible bond was that between His362 and A1a363followed by the Se?56-Leu357 and Phe358-Ile359 bonds. The hexapeptide formed by hydrolysis of the Phe358-Ile359 bond was subsequently cleaved to yield a tetrapeptide and the COOH-terminal dipeptide. If we include the small peptides in Peak 1 (Fig. 3) the total amino acids released would account for the last 19 residues at the COOH-terminus of rabbit muscle aldolase (16). Correlation of COOH-terminal modfication with loss of activity. For this purpose we monitored catalytic activity with fructose 1,6-bisphosphate as substrate and the release of tyrosine in acid-soluble peptides (Fig. 1). With fructose 1,6bisphosphate as the substrate the end-point (92% loss of activity) was reached tyrosines were released as acidwhen only three of the four COOH-terminal
16 -
IO
30
50
70
90
II0
130
150
170
TIME (mid
FIG. 4. Major peptides released from muscle aldolase upon digestion with cathepsin M. Quantities of peptides recovered from HPLC was based on amino acid analysis of fluorescamine positive peaks shown in Fig. 3. The peptides recovered in peaks 1 and 5 (see Fig. 3) were estimated to be present in quantities approaching 2 nmole after 180 min of incubation.
DIGESTION
OF RABBIT
MUSCLE
ALDOLASE
BY CATHEPSIN
M
197
soluble peptides and no further change was observed with loss of the fourth tyrosine. This would suggest either cooperativity between the subunits or, more likely, the presence of one inactive subunit in the aldolase employed in these experiments (see below). DISCUSSION
The susceptibility of the COOH-terminal region of native rabbit muscle aldolase to a variety of exo- and endopeptidases, including carboxypeptidase A (8, 20, 21), cathepsin B (9, lo), and subtilisin (22) provides a useful tool for the study of the specificities of lysosomal and other cellular proteinases. The use of this substrate has helped to characterize cathepsin B as a peptidyl dipeptidase (9). In the present work we show that cathepsin M hydrolyzes not only the penultimate peptide bond, but also acts as an endopeptidase, yielding fragments as large as hexapeptides. Cathepsin M exhibits a preference for hydrolysis of aliphatic amino acids; all of the peptides recovered contained NH,-terminal alanine, leucine, or isoleucine. However, this specificity will require further studies with other susceptible protein substrates. The results also suggest that the usual preparations of rabbit muscle aldolase may contain one silent, or inactive, subunit. We have previously shown that approximately one residue of organic phosphorous is present in the tetrameric protein (23) and the presence of a phosphorylated subunit appears to account for the relative resistance of this subunit to digestion with carboxypeptidase A (24). The number of protein-bound phosphate groups can be increased in a suicide reaction with glyceraldehyde 3-phosphate (25,26) which also protects the COOHterminal tyrosyl residues against digestion with carboxypeptidase A (24). The conclusion that aldolase contains one inactive subunit is supported by the observation that removal of tyrosine-containing peptides from the first three subunits is accompanied by decreases of 33, 67, and lOO%, respectively, of the original catalytic activity. SUMMARY
Rabbit liver cathepsin M, a sulfhydryl proteinase similar in catalytic properties to cathepsin B, causes a decrease in the activity of rabbit muscle aldolase assayed with fructose 1,dbisphosphate but not with fructose l-phosphate. Proteolytic modification of aldolase by cathepsin M is limited to the removal of small peptides from the COOH-terminus, including the COOH-terminal hexapeptide NH,-IleSer-Asn-His-Ala-TyrOH. Correlation of loss of aldolase activity with COOHterminal modification indicates that only three of the four subunits of muscle aldolase contribute to the catalytic activity of the tetrameric enzyme. REFERENCES 1. Pontremoli, Arch.
S., Melloni,
Biochem.
Biophys.
E., Salamino, F., Sparatore, B., Michetti, M., and Horecker, B. L., 214, 376 (1982).
2. Horecker, B. L., Erickson-Viitanen, S., Melloni, E., and Pontremoli, S., in “Current Topics in Cellular Regulation” (B. L. Horecker and E. R. Stadtman, Eds.), Vol. 25, p. 77. Academic Press. New York. 1985.
ERICKSON-VIITANEN
198
ET AL.
3. Erickson-Viitanen, S., Balestreri, E., McDermott, M., and Horecker, B. L. Fed. Proc., 43, 1853 (1984). 4. Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., and Horecker, B. L., Proc. Natl. Acad. Sci. U.S.A. 76, 6323 (1979). 5. Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., and Horecker, B. L., Arch. Biochem. Biophys 203, 390 (1980). 6. Pontremoli, S., Melloni, E., Michetti, M., Salamino, F., Sparatore, B., and Horecker, B. L., Proc. Natl. Acad. Sci. U.S.A. 79, 5194 (1982). 7. Lai, C. Y., and Horecker, B. L., in “Essays in Biochemistry” (P. N. Campell and F. Dickens, Eds.). Academic Press, New York, 1972. 8. Drechsler, E. R., Boyer, P. D., and Kowalsky, A. G., J. Biol. Chem. 234, 2627 (1959). 9. Bond, J. S., and Barrett, A. J., Biochem. J. 189, 177 (1980). 10. Nakai, N., Wada, K., Kobashi, K., and Hase, I., Biochem. Biophys. Res. Commun. 83, 881 (1978).
11. Gracy, R. W., Lacko, A. G., and Horecker, B. L., J. Biol. Chem. 244, 3913 (1969). 12. Stein, S., and Moschera, J., in “Methods in Enzymology” (S. Pestka, Ed.), Vol. 79, p. 7. Academic Press, New York, 1981. 13. Benson, J. R., and Hare, P. E., Proc. Natl. Acad. Sci. U.S.A. 72, 619 (1975). 14. Ambler, R. P., in “Methods in Enzymology” (C. H. W. Hirs, Ed.), Vol. 1I, p. 155. Academic Press, New York, 1967. 15. Jones, B. N., Paabo, S., and Stein, S., J. Liq. Chromatog. 4, 565 (1981). 16. Lai, C. Y., Nakai, N., and Chang, D., Science (Washington, D.C.) 183, 1204 (1974). 17. Laemmli, U. K., Nature (London) 227, 680 (1970). 18. Lai, C. Y., Chen, C., and Horecker, B. L., Biochem. Biophys. Res. Commun. 40, 461 (1970). 19. Tolan, D. R., Amsden, A. B., Putney, S. D., Urdea, M. S., and Penhoet, E. E., J. Biol. Chem. 259, 1127 (1984). 20. Rutter, W. J., Richards, 0. C., and Woodfin, B. M., J. Biol. Chem. 236, 3193 (1961). 21. Morse, D. E., Chan, W., and Horecker, B. L., Proc. Natl. Acad. Sci. U.S.A. 58, 628 (1967). 22. Hannappel, E., MacGregor, J. S., Davoust, S., and Horecker, B. L., Arch. Biochem. Biophys. 214, 293 (1982).
23. Kobashi, K., Lai, C. Y., and Horecker, B. L., Arch. Biochem..&ophys. 117, 437 (1966). 24. Adelman, R. C., Morse, D. E., Chan, W. and Horecker, B. L:: Arch. Biochem. Biophys. 126, 343 (1968). 25. Lai, C. Y., Martinez-de Dretz, G., Bacila, M., Marinello, E., and Horecker, B. L., Biochem. Biophys. Res. Commun. 30, 665 (1968). 26. Wagner, J., Lai, C. Y., and Horecker, B. L., Arch. Biochem. Biophys. 152, 398 (1972).
MEDICINE
AND
METABOLIC
BIOLOGY
35, 191-198
(1986)
Sites of Cleavage of Rabbit Muscle Aldolase by Purified Cathepsin M from Rabbit Liver SUSAN ERICKSON-VIITANEN,
ETTORE BALESTRERI, AND B. L. HORECKER
Roche
Institute
of Molecular
Biology, Received
Roche
Research
September
Center,
MARTIN
Nutley,
MCDERMOTT,
New
Jersey
07110
20, 1984
Cathepsin M is a lysosomal protease of the B-type cathepsin family exhibiting relatively poor reactivity toward synthetic naphthylamide derivatives of blocked or unblocked amino acids but more significant proteolytic activity towards native protein substrates such as muscle and liver aldolases (l-3). A significant proportion of cathepsin M activity is associated with the lysosomal membrane and is expressed by intact lysosomes at neutral pH (1, 2). For this reason, cathepsin M is a likely candidate for the modification of aldolase observed in fasted animals in viva (46).
We have used rabbit muscle aldolase to study the peptide bond specificity of cathepsin M. This protein is composed of four subunits each containing COOHterminal tyrosine and otherwise identical except for microheterogeneity introduced by spontaneous partial deamidation of an asparaginyl residue near the COOHterminus (7). Digestion of rabbit muscle aldolase with carboxypeptidase A results in loss of activity with fructose 1, 6-bisphosphate as the substrate at a rate that is correlated with the rate of loss of COOH-terminal tyrosines (8). Cathepsin B from human liver (9) or rabbit liver (10) removes dipeptides sequentially from the COOHterminus of muscle aldolase with resultant inactivation of the enzyme. However, the quantitative relationship between COOH-terminal modification and loss of catalytic activity has not been clear; complete inactivation has been correlated with the loss of a single COOH-terminal dipeptide during digestion with rabbit liver cathepsin B (10) or with the loss of three of the four tyrosines after digestion with carboxypeptidase A (8). We report here the sites of cleavage of muscle aldolase by purified rabbit liver cathepsin M. Unlike the peptidyl dipeptidase activity of cathepsin B (9,10), we find that cathepsin M attacks peptide bonds near the COOH-terminus in a less regular manner. In addition, quantitative analysis of release of tyrosine-containing peptides reveals that loss of aldolase activity is related to proteolytic modification of three of the four subunits of aldolase; modification of the fourth subunit is observed after inactivation is complete. 0885-4505186 ‘ill
$3.00
Copyright 0 1986 by Academic Press. Inc. rights of reproduction in any form reserved.
192
ERICKSON-VIITANEN
ET AL.
MATERIALS
Rabbit muscle fructose 1,6-bisphosphate aldolase (type IV), fructose 1,6-bisphosphate, and carboxypeptidase A were purchased from Sigma Chemical Company, and NADH and glycerol-3-phosphate dehydrogenase/triose phosphate isomerase (6DH/TIM) from Boehringer Mannheim. Reagents for SDS’-polyacrylamide gel electrophoresis were from Bio-Rad. Fluorescamine (Fluram) was provided by Dr. William E. Scott of Hoffmann-LaRoche and o-phthaldehyde was purchased from Pierce. All other chemicals and solvents were chromatography grade. Solvents were redistilled as required. METHODS
The purification of cathepsin M from rabbit liver crude lysosomal fraction by a modification of the earlier procedure (1) will be described elsewhere. After the last step of the purification on carboxymethly cellulose, the active fractions were pooled and concentrated to 50-100 pg protein/ml by ultrafiltration. The concentrated enzyme solution was stored at -20” C in small aliquots. The specific activity of the preparation was 20,000 units/mg protein, where one unit corresponds to the amount required to catalyze the inactivation of 1 nmole of rabbit muscle aldolase in 30 min under standard assay conditions (see below). For digestion of rabbit muscle aldolase, the incubation mixture contained 1.2 mg/ml of aldolase, 10 mM DTT, 50 mM Na acetate, pH 5.0, and 2 (ug/ml cathepsin M. Measurement of aldolase activity was carried out at 26” C using the spectrophotometric assay of Gracy et al. (11). The one-ml cuvette contained 50 mM Tris-HCl, pH 7.4, 0.15 mM NADH, 1.0 mM fructose 1,6-bisphosphate or 10 mM fructose l-phosphate, and 10 pg of the glycerophosphate dehydrogenase/triose phosphate isomerase mixture (10: 1 w/w). The reaction was begun by the addition of 20-~1 aliquots of the aldolase digestion mixtures (see above), that had been diluted 20-fold in cold 67 mM Tris-HCl, pH 7.3. Peptides released from aldolase upon digestion with cathepsin M were separated and quantitated by reverse-phase HPLC. An aliquot of the digestion mixture (1 ml) was cooled and acidified with perchloric acid to a final concentration of 5%. After centrifugation the precipitate was washed with the same volume of 5% HC104 and the first supernatant solution and washing combined and neutralized with 10% KOH. After centrifuging to remove precipitated KCIOl the clear supernatant solution was lyophilized. The powder was dissolved in 250 ~1 of 0.5 M HCOOH/O * 1 M pyridine and 150~~1 aliquots were analyzed on an Ultrasphere ODS Cl8 column (5 p, 4.6 x 250 mm, Altex Scientific) with the fluorescence detection system described by Stein and Moshera (12). Amino acid analyses were carried out after hydrolysis of peptides with redistilled 6.0 M HCl at 150” for 90 min, using a Glenco MM-70 amino acid analyzer adapted for use of o-phthaldehyde and fluorescence detection as described by Benson and Hare (13). To determine the number of COOH-terminal tyrosines remaining after various times of incubation with cathepsin M, the HClO,-precipitated protein was digested ’ Abbreviations
used: DTT, 1,4-dithiothreitol;
SDS, sodium dodecyl sulfate.
DIGESTION
OF RABBIT
MUSCLE
ALDOLASE
BY CATHEPSIN
M
193
with carboxypeptidase A (14). The protein was suspended in 1.0 ml of 50 mM Hepes buffer containing 0.1% SDS, pH 7.8, and dissolved by heating to 50” C. Carboxypeptidase A, 5 ~1 of a solution containing 2 mg/ml in 10% LiCl, was added to 100 ~1 of the cooled aldolase solution containing 2.6 nmole protein (ratio of aldolase to carboxypeptidase = 13 : 1) at zero time and again after 2 hr incubation at 25” C. Aliquots (2 ~1) were removed at 0, 1, 2, 3, and 5 hr and mixed with 10 ~1 0.2 M Na borate, pH 9.3, and frozen until analysis. Released amino acids were quantitated by reverse-phase HPLC after derivitization with o-phthaldehyde as described by Jones et al. (15). The aldolase content was determined by amino acid analysis of an aliquot after hydrolysis in 6 M HCl at 150” C for 1.5 hr using values of 3 methionines and 15 arginine residues per subunit (16) for the calculation. Slab gel electrophoresis at pH 8.8 in the presence of SDS and mercaptoethanol was carried out according to the procedure of Laemmli (17). Coomassie blue G was used for staining. RESULTS
Effect of cathepsin M on the catalytic activity and subunit structure of rabbit muscle aldolase. Catalytic activity was assessed with fructose 1 ,dbisphosphate as the substrate (Fig. 1). Cathepsin M caused a decrease in the activity of rabbit muscle aldolase from an intitial value of 14.0 units/mg to a final value of 1.2 units/mg; activity was not further decreased by longer incubation or the addition
TIME km)
FIG. 1. Inactivation of aldolase by cathepsin M and appearance of acid-soluble peptides containing tyrosine. Reaction conditions were as described under Methods; at the times indicated, aliquots of the reaction mixture were assayed spectrophotometrically for aldolase activity with fructose 1,6bisphosphate (0). Tyrosine release was calculated from the sum of tyrosine-containing peptides separated by HPLC (see Fig. 3), adjusted for losses during chromatography (0). An average recovery factor of 64% was calculated by comparing the total tyrosine and phenylalanine in HPLC fractions with the tyrosine and phenylalanine present in an aliquot of the neutralized acid extract. The total aldolase content determined by amino acid analysis of the acid-precipitated material (see Table 1) was used to calculate nmole tyrosine released per nmole aldolase tetramer.
ERICKSON-VIITANEN
194
ET AL.
of fresh cathepsin M (data not shown). With fructose l-phosphate as the substrate, the initial catalytic activity was 0.22 units/mg and was not significantly altered during incubation with cathepsin M (data not shown). These effects of cathepsin M are similar to those observed with carboxypeptidase A (8) and with cathepsin B (9), and suggested that digestion was confined to a limited region at the COOHterminus. This was supported by analysis of the modified enzyme by SDS-gel electrophoresis (Fig. 2), which indicated a change in subunit molecular weight from an initial value of 40,000 to a final value of approximately 38,500. Modification at the COOH-terminus was confirmed by the finding that the decrease in activity toward fructose 1,dbisphosphate was related to the loss of tyrosine residues from the COOH-terminus (Table l), evaluated by treating the aldolase remaining with carboxypeptidase A (see Methods). This was accompanied by the appearance of acid-soluble peptides containing tyrosine (Fig. 1). Modification of the NH,terminus was excluded by the absence of proline (16) in the peptides released (data not shown). kDa
c-i
*
“7%
66.2
45.0
31.0
21.5
14.4
0
6
10
30
60
180
lime of digestion (minutes) FIG. 2. SDS-polyacrylamide gel electrophoresis of native and cathepsin M-digested rabbit muscle aldolase. Rabbit muscle aldolase was digested with cathepsin M (see Methods) and at the times indicated 2.5~pg aliquots were analyzed on 12.5% acrylamide gels. Running conditions: 20 mV, 5hr. Lanes 1 and 8 represent the molecular weight markers bovine serum albumin (66.2 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa).
DIGESTION
OF RABBIT
MUSCLE
ALDOLASE
BY CATHEPSIN
195
M
TABLE 1 Tyrosine from Cathepsin M-Digested Aldolase
Recovery of COOH-Terminal Time of incubation with cathepsin M (mitt)
Tyrosine released by carboxypeptidase A
Subunits aldolase
@mole)
(pmole)
45.2 41.0 22.6 10.4
ND 51.8 52.4 54.2
0
3 30 90
Note. Carboxypeptidase A digestion was carried out as described under Methods using the HClO,-precipitated protein recovered after digestion with cathepsin M for the indicated times. Values represent pmole tyrosine present in 2 ~1 of the carboxypeptidase reaction mixture after 5 hr digestion. The total aldolase present in the reaction mixture was calculated from the amino acid composition obtained by total acid hydrolysis of a 2 ~1 aliquot of the reaction mixture. y ND, not determined.
ZdentiJication of the peptides releasedfrom rabbit muscle aldolase. The COOH356
terminal
sequence of rabbit muscle aldolase has been established 360
as Ser-Leu-
364
Phe-Ile-Ser-Asn-His-AlaTyr OH by Edman degradation of the chymotryptic and tryptic peptides containing the COOH-terminus (16, 18) and partially confirmed by cloning of the cDNA (19). After separation by HPLC (Fig. 3), the acid soluble
TIME (mid
FIG. 3. Separation of acid-soluble peptides from muscle aldolase digested with cathepsin M. The reaction mixture (1 ml) contained 1.2 mg of rabbit muscle aldolase, 2 pg of cathepsin M, 10 mM DDT, 50 mM Na acetate buffer, pH 5.0. After incubation for 30 min at 37” C the mixture was acidified with 0.25 ml of 25% HClO, and the acid extract collected and neutralized as described under Methods. The lyophilized extract was dissolved in 0.25 ml of 0.5 M HCOOH/O.l M pyridine, pH 2.8, and a 150+1 aliquot was injected onto an Ultrasphere ODS Cl8 column preequilibrated with the same buffer. A delayed linear gradient of CH$N in 1.0 M HCOOH/0.2 M pyridine was generated with a Waters System Controller Model 720 (Waters Assoc.) as shown (---). The flow rate was 0.6 ml/min and fractions of 1 min were collected. At 6-set intervals, 5 ~1 of the effluent was diverted for analysis with fluorescamine and the fluorescence recorded (-). Six major peaks were detected. They were identified by amino acid analysis as Ile-Ser-Asn-His (peak 2); Ala-Tyr (peak 3), Ile-Ser-AsnHis-Ala-Tyr (peak 4), Phe-Ile (peak 5) and Leu-Phe (peak 6). Peak 1 contained a mixture of free amino acids (Ser,Gly) and small peptides that yielded ZSer, 3Glu, 2Gly, 4Ala, Asp, and His on acid hydrolysis. The short retention time observed with the reverse-phase chromatography system employed suggests that this peak contains the dipeptide Asn-His plus other di- and/or tripeptides.
196
ERICKSON-VIITANEN
ET AL.
peptides released by cathepsin M were identified by amino acid analysis (data not shown) and the time course of their release determined (Fig. 4). The first peptide detected was the COOH-terminal dipeptide Ala-Tyr, following by LeuPhe, and the COOH-terminal hexapeptide. Analysis of the results suggests that the most susceptible bond was that between His362 and A1a363followed by the Se?56-Leu357 and Phe358-Ile359 bonds. The hexapeptide formed by hydrolysis of the Phe358-Ile359 bond was subsequently cleaved to yield a tetrapeptide and the COOH-terminal dipeptide. If we include the small peptides in Peak 1 (Fig. 3) the total amino acids released would account for the last 19 residues at the COOH-terminus of rabbit muscle aldolase (16). Correlation of COOH-terminal modfication with loss of activity. For this purpose we monitored catalytic activity with fructose 1,6-bisphosphate as substrate and the release of tyrosine in acid-soluble peptides (Fig. 1). With fructose 1,6bisphosphate as the substrate the end-point (92% loss of activity) was reached tyrosines were released as acidwhen only three of the four COOH-terminal
16 -
IO
30
50
70
90
II0
130
150
170
TIME (mid
FIG. 4. Major peptides released from muscle aldolase upon digestion with cathepsin M. Quantities of peptides recovered from HPLC was based on amino acid analysis of fluorescamine positive peaks shown in Fig. 3. The peptides recovered in peaks 1 and 5 (see Fig. 3) were estimated to be present in quantities approaching 2 nmole after 180 min of incubation.
DIGESTION
OF RABBIT
MUSCLE
ALDOLASE
BY CATHEPSIN
M
197
soluble peptides and no further change was observed with loss of the fourth tyrosine. This would suggest either cooperativity between the subunits or, more likely, the presence of one inactive subunit in the aldolase employed in these experiments (see below). DISCUSSION
The susceptibility of the COOH-terminal region of native rabbit muscle aldolase to a variety of exo- and endopeptidases, including carboxypeptidase A (8, 20, 21), cathepsin B (9, lo), and subtilisin (22) provides a useful tool for the study of the specificities of lysosomal and other cellular proteinases. The use of this substrate has helped to characterize cathepsin B as a peptidyl dipeptidase (9). In the present work we show that cathepsin M hydrolyzes not only the penultimate peptide bond, but also acts as an endopeptidase, yielding fragments as large as hexapeptides. Cathepsin M exhibits a preference for hydrolysis of aliphatic amino acids; all of the peptides recovered contained NH,-terminal alanine, leucine, or isoleucine. However, this specificity will require further studies with other susceptible protein substrates. The results also suggest that the usual preparations of rabbit muscle aldolase may contain one silent, or inactive, subunit. We have previously shown that approximately one residue of organic phosphorous is present in the tetrameric protein (23) and the presence of a phosphorylated subunit appears to account for the relative resistance of this subunit to digestion with carboxypeptidase A (24). The number of protein-bound phosphate groups can be increased in a suicide reaction with glyceraldehyde 3-phosphate (25,26) which also protects the COOHterminal tyrosyl residues against digestion with carboxypeptidase A (24). The conclusion that aldolase contains one inactive subunit is supported by the observation that removal of tyrosine-containing peptides from the first three subunits is accompanied by decreases of 33, 67, and lOO%, respectively, of the original catalytic activity. SUMMARY
Rabbit liver cathepsin M, a sulfhydryl proteinase similar in catalytic properties to cathepsin B, causes a decrease in the activity of rabbit muscle aldolase assayed with fructose 1,dbisphosphate but not with fructose l-phosphate. Proteolytic modification of aldolase by cathepsin M is limited to the removal of small peptides from the COOH-terminus, including the COOH-terminal hexapeptide NH,-IleSer-Asn-His-Ala-TyrOH. Correlation of loss of aldolase activity with COOHterminal modification indicates that only three of the four subunits of muscle aldolase contribute to the catalytic activity of the tetrameric enzyme. REFERENCES 1. Pontremoli, Arch.
S., Melloni,
Biochem.
Biophys.
E., Salamino, F., Sparatore, B., Michetti, M., and Horecker, B. L., 214, 376 (1982).
2. Horecker, B. L., Erickson-Viitanen, S., Melloni, E., and Pontremoli, S., in “Current Topics in Cellular Regulation” (B. L. Horecker and E. R. Stadtman, Eds.), Vol. 25, p. 77. Academic Press. New York. 1985.
ERICKSON-VIITANEN
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ET AL.
3. Erickson-Viitanen, S., Balestreri, E., McDermott, M., and Horecker, B. L. Fed. Proc., 43, 1853 (1984). 4. Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., and Horecker, B. L., Proc. Natl. Acad. Sci. U.S.A. 76, 6323 (1979). 5. Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., and Horecker, B. L., Arch. Biochem. Biophys 203, 390 (1980). 6. Pontremoli, S., Melloni, E., Michetti, M., Salamino, F., Sparatore, B., and Horecker, B. L., Proc. Natl. Acad. Sci. U.S.A. 79, 5194 (1982). 7. Lai, C. Y., and Horecker, B. L., in “Essays in Biochemistry” (P. N. Campell and F. Dickens, Eds.). Academic Press, New York, 1972. 8. Drechsler, E. R., Boyer, P. D., and Kowalsky, A. G., J. Biol. Chem. 234, 2627 (1959). 9. Bond, J. S., and Barrett, A. J., Biochem. J. 189, 177 (1980). 10. Nakai, N., Wada, K., Kobashi, K., and Hase, I., Biochem. Biophys. Res. Commun. 83, 881 (1978).
11. Gracy, R. W., Lacko, A. G., and Horecker, B. L., J. Biol. Chem. 244, 3913 (1969). 12. Stein, S., and Moschera, J., in “Methods in Enzymology” (S. Pestka, Ed.), Vol. 79, p. 7. Academic Press, New York, 1981. 13. Benson, J. R., and Hare, P. E., Proc. Natl. Acad. Sci. U.S.A. 72, 619 (1975). 14. Ambler, R. P., in “Methods in Enzymology” (C. H. W. Hirs, Ed.), Vol. 1I, p. 155. Academic Press, New York, 1967. 15. Jones, B. N., Paabo, S., and Stein, S., J. Liq. Chromatog. 4, 565 (1981). 16. Lai, C. Y., Nakai, N., and Chang, D., Science (Washington, D.C.) 183, 1204 (1974). 17. Laemmli, U. K., Nature (London) 227, 680 (1970). 18. Lai, C. Y., Chen, C., and Horecker, B. L., Biochem. Biophys. Res. Commun. 40, 461 (1970). 19. Tolan, D. R., Amsden, A. B., Putney, S. D., Urdea, M. S., and Penhoet, E. E., J. Biol. Chem. 259, 1127 (1984). 20. Rutter, W. J., Richards, 0. C., and Woodfin, B. M., J. Biol. Chem. 236, 3193 (1961). 21. Morse, D. E., Chan, W., and Horecker, B. L., Proc. Natl. Acad. Sci. U.S.A. 58, 628 (1967). 22. Hannappel, E., MacGregor, J. S., Davoust, S., and Horecker, B. L., Arch. Biochem. Biophys. 214, 293 (1982).
23. Kobashi, K., Lai, C. Y., and Horecker, B. L., Arch. Biochem..&ophys. 117, 437 (1966). 24. Adelman, R. C., Morse, D. E., Chan, W. and Horecker, B. L:: Arch. Biochem. Biophys. 126, 343 (1968). 25. Lai, C. Y., Martinez-de Dretz, G., Bacila, M., Marinello, E., and Horecker, B. L., Biochem. Biophys. Res. Commun. 30, 665 (1968). 26. Wagner, J., Lai, C. Y., and Horecker, B. L., Arch. Biochem. Biophys. 152, 398 (1972).