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Calcium-dependent proteolytic activity in rat liver: Identification of two proteases with different calcium requirements
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 211, No. 1, October 1, pp. 253-257, 1981
Calcium-Dependent Proteolytic Activity in Rat Liver: Identification Proteases with Different Calcium Requirements GEORGE Department
of Two
N. DE MARTIN0
of Physiology, University of Texas Health Science Center at DaUas, 5323 Harry Hines Boulevard, Dallas, Tesas 75235 Received
February
19, 1981
Soluble extracts from rat liver cytoplasm contained substantial endoprotease activity which was totally dependent on calcium ions. This activity was accounted for by two distinct proteases which were separated by anion-exchange chromatography. One protease was half maximally activated by ‘7 PM calcium while the other protease required 150 MM calcium for half-maximal activation.
Intracellular protein degradation is a highly regulated physiologic process which influences the levels of specific proteins as well as the overall growth or atrophy of tissues (1, 2). In mammalian cells, there is good evidence that both lysosomal and nonlysosomal proteases catalyze the degradation of cell proteins. In contrast to lysosomal proteases, nonlysosomal proteases are poorly defined and the manner by which their activities are regulated is not clear. Recently, however, two cytoplasmic proteases have been identified which are regulated in vitro by potential physiologically important mechanisms. For example, we have demonstrated in rat liver, a high-molecular-weight protease whose activity is stimulated by ATP (3). This property may be important because protein degradation in vivo requires cellular energy. In the course of these studies we have identified an additional cytoplasmic proteolytic activity which is totally dependent on calcium. This finding was not suprising because calcium-activated proteases have been described previously in a variety of tissues (4-10). The calcium-dependent protease has received considerable attention because calcium appears to regulate protein degradation in some intact cells (11) and this enzyme offers an attractive mechanism for this regulation. However, because the high cal253
cium concentrations required for protease activation in vitro (>l mM) may never be achieved under normal conditions in vivo, the physiologic significance of these in vitro effects has been unclear. In this report, we demonstrate that calcium-dependent proteolytic activity in rat liver homogenates is accounted for by at least two distinct enzymes which differ from each other in their requirements for calcium. Importantly, one enzyme is completely activated by calcium concentrations which may be achieved in intact cells. MATERIALS
AND
METHODS
Preparation of liver extracts. Normal male rats (Sprague-Dawley, 400-500 g) were anesthetized lightly with ether. In order to remove as much contaminating blood as possible from the liver, the portal vein was cannulated and the liver was perfused with 100 ml of 0.15 M NaCl. Approximately 100 g of liver were chilled on ice, minced, and homogenized with a Dounce homogenizer in 5 mM potassium phosphate buffer, pH 7.6, containing 0.25 M sucrose and 1 mM EDTA. The homogenate (20%. w/v) was filtered through two layers of surgical gauze and the pH adjusted to pH 7.4. All of the following steps were carried out at 0-4’C. The homogenate was centrifuged for 10 min at 660g. The supernatant was centrifuged successively at 16,300g (20 min) and 105,OOOg (90 min). The resulting postmicrosomal supernatant was dialyzed for 3 h against 5 mM potassium phos0003-9861/81/110253-05$02.00/O Copyright All rights
0 1981 hy Academic Press, Inc. of reproduction in any form reserved.
254
GEORGE
N. DE MARTIN0
phate buffer, pH 7.6,0.5 mM D’IT,’ 1 mM EDTA. The dialysate was then added to the anion exchanger, diethylaminoethyl cellulose (DE52, Whatman, 1 g DE52/5 g liver) which was washed and equilibrated with the same buffer. After gentle agitation for 45 min, the mixture was filtered and the resin treated successively with potassium phosphate buffer containing 0.25 and 1.25 M KCl. Each treatment was for 20 min and was followed by filtration. The volume of the elution buffer was 1110th the volume of the original postmicrosomal supernatant. The filtrate from the 1.25 M KC1 elution was dialyzed for 14 h against 50 mM Tris-HCl buffer, pH 7.6, 8 m?d KCl, 0.5 mM DTT, 0.5 mM EGTA. This extract contained approximately 30% of the total protein originally present in the postmicrosomal supernatant. The dialyzed extract was centrifuged at 10,OOOg (10 min) and the supernatant chromatographed on Sephacryl S-300 as described below. Sephacryl S-NO gelJ2tration chromatography. Ascending gel filtration chromatography was performed with Sephacryl S-300 in a 100 X 5-cm column. The gel was equilibrated with 50 mM Tris-HCl buffer, pH 7.6, 8 mM KCl, 0.5 mM DTT, 0.5mM EGTA. The column was calibrated with purified proteins of known molecular weights (Fig. 1). Aliquots of the loml fractions were assayed for protease activity at pH 7.8, 36”C, using [methyl-“Clglobin as substrate (see below). DE52 Ion-exchange column chromatography. DE52 (10 g) was washed and equilibrated with 50 mM TrisHCl buffer, pH 7.6, 0.5 mM DTT, 0.5 mM EGTA. Column dimensions were 10 X 1.5 cm. After sample application, a 0.0-0.4 M linear gradient of KC1 in the Tris-HCl buffer was run at 0.5 ml/min. Aliquots (90 ~1) of the 5-ml fractions were assayed for protease activity at pH 7.5, 22°C in the presence and absence of 1.7 mM CaCl*. Protease activity. Protease activity was measured by the hydrolysis of [methyl-“Cjglobin as described previously (3). Conditions were fixed such that the assays were linear with respect to time and with respect to enzyme protein. Specific conditions for the assays are given in the legends to the figures. Chemicals and reagents. All chemicals were of the highest grade commercially available. All solutions were prepared in deionized, double-distilled water.
RESULTS
Postmicrosomal supernatants of rat liver homogenates were treated as described under Methods and chromato’ Abbreviations aminoethyl ether) threitol.
used: EGTA, N,w-teraacetic
ethylene acid,
glycol his@DlT, dithio-
graphed on Sephacryl S-300 equilibrated with 50 mM Tris-HCl buffer, pH 7.6, 8 mM KCl, 0.5 mM DTT, and 0.5 mM EGTA. When the column fractions were assayed for protease activity at pH 7.8 with no added calcium, two peaks of high-molecular-weight (i.e., M, = 750,000 and 550,000) protease activity were observed (Fig. 1). This activity has been described previously (3) and will be the subject of a subsequent report.2 When the column fractions were assayed in the presence of 1.7 mM CaC12,the activities of the high-molecular-weight proteases were not greatly affected (data not shown). However, assays with calcium revealed an additional peak of alkaline protease activity at a position corresponding to M, = 150,000. This activity had some properties similar to calcium-dependent proteases described previously in other tissues. For example, other divalent cations such as Me or Mn2+ could not activate proteolysis. The activity had an alkaline pH optimum from pH 7.0 to 8.5 and was sulfhydryl dependent. Protease activity was stimulated by compounds such as DTT, cysteine, and /3mercaptoethanol but inhibited by alkylating agents such as N-ethylmaleimide and iodoacetimide (data not shown). Fractions from the Sephacryl column which contained calcium-dependent protease activity were pooled and subjected to DE52 ion-exchange column chromatography as described under Methods. This resolved two peaks of calcium-dependent protease activity. The first (Peak I) eluted at a position corresponding to 0.10 M KC1 and second (Peak II) eluted at a position corresponding to 0.22 M KC1 (Fig. 2). The relative activities of these two peaks were approximately equal. Each of the calcium-dependent proteases was purified further by ammonium sulfate precipitation. The respective precipitates formed after the addition of ammonium sulfate to 50% saturation were collected by centrifugation, redissolved in a small volume (3-4 ml) of 5 mM Tris-HCl buffer, pH 7.2, 8 mM KCl, 0.5 mM DTT, 0.5 * G. N. De Martin0 in preparation.
and A. L. Goldberg,
manuscript
CALCIUM-DEPENDENT
PROTEASES
Fmction
IN RAT
LIVER
255
Number
FIG. 1. Sephacryl S-300 column chromatography. A postmicrosomal supernatant, prepared and treated as described under Methods, was chromatographed on Sephacryl S-300 equilibrated with 50 mM Tris-HCl buffer, pH 7.6, 8 mM KCl, 0.5 mM EGTA, 0.5 mM DTT. Aliquots (90 ~1) of the loml fractions were assayed for protease activity in 50 mM Tris-HCl buffer, pH 7.8, at 36”C, with and without 1.7 mM CaCle. Enzyme activities in fractions 50-89 were similar under both of these conditions and the values for assays with CaCl, have been omitted for clarity. Similar elution patterns were obtained with eight separate enzyme preparations. Insert: Estimation of molecular weight of protease activities. Blue Dextran, thyroglobulin (M, = SSO,OOO), ferritin (Af, = 440,000), catalase (kf, = 240,000), and hemoglobin (M, = 64,000) were dissolved in column buffer and chromatographed. The elution pattern for each substrate was determined and plotted versus the log of molecular weight.
mM EGTA, and dialyzed for 12 h against the same buffer. After dialysis, any undissolved material was removed by centrifugation and the resulting supernatant was dialyzed for an additional 12 h against two changes (6 1 each) of 5 mM Tris-HCl buffer, pH 7.2, 0.5 mM DTT. The dialyzed extracts were assayed for protease activity at various calcium concentrations (Fig. 3). Each enzyme was completely dependent on added calcium for activity. However, the concentrations of calcium required for the activation of each enzyme were remarkably different. Peak I required approximately 7 pM calcium for half-maximal activity and was maximally active at 25-50 pM calcium. Higher concentrations of calcium (i.e., >450 pM) inhibited this protease by 15-30s in various enzyme preparations. In contrast to Peak I, Peak II required approximately 150 PM calcium
for half-maximal activity and greater than 450 pM calcium for maximal activity. Results similar to those shown in Fig. 3 were obtained with enzymes which had not been precipitated with ammonium sulfate. Similar results were also obtained with [methyZ-‘4C]bovine serum albumin as substrate. In some experiments with the Peak I enzyme, we used a series of Ca”-EGTA buffers designed to achieve free calcium concentrations between 0.4 and 9.4 PM at the pH and temperature of the assays. Assays performed in this manner also demonstrated half-maximal activation of Peak I by 7-8 pM calcium (data not shown). DISCUSSION
A calcium-dependent protease was first described in skeletal muscle (4,5). Because this enzyme could degrade some specific
256
GEORGE
N. DE MARTIN0
FIG. 2. DE52 ion-exchange chromatography. Pooled samples of calcium-dependent protease activity from Fig. 1 were subjected to DE52 ion-exchange chromatography as described under Methods. Aliquots (90 ~1) of the 5-ml fractions were assayed for protease activity at pH 7.5 (50 mM Tris-HCl buffer), 22°C with 1.7 mM CaClz (30 min). Under these conditions, product formation was linear with respect to time. Similar results were obtained with six separate enzyme preparations.
isolated contractile proteins as well as Zline proteins of intact myofibrils, it was ascribed a role in the turnover of myofibrillar proteins in wivo (5, 12, 13). Subsequently, a similar protease has been identified in nonmuscle cells (6-8). Thus, it has been unclear whether this enzyme degrades specific but different proteins in different cell types or whether it plays a general role in the degradation of many cell proteins. In either case, a feature of this protease which has obscured its possible physiologic role is the high concentrations of calcium (i.e., usually >l mM) required for maximal enzyme activity. Recently, however, Mellgren isolated from canine heart, two forms of calcium-dependent proteases (14). One was like other described calcium-dependent proteases in that it required millimolar levels of calcium for maximal activity. The other enzyme, however, required only 40 PM calcium for half-maximal activity and was maximally activated by 400 PM calcium. These enzymes had similar molecular weights (135,000 daltons) and were separated by ion-exchange chromatography. Interestingly, the more calcium-sensitive enzyme represented only a small percentage of the total calcium-dependent protease activity. These important findings
raised the possibility that the protease which required lower calcium concentrations had been overlooked in previous studies and that this form of the enzyme might be of greater physiologic significance. In fact, evidence for two forms of calcium-dependent protease was obtained in several studies, but possible differences in calcium requirements between the enzymes were not examined (6, 7). The enzymes described in the present report appear to be related to those described by Mellgren. For example, the rat liver enzymes had molecular weights similar to each other (M, = 150,000) and to the canine heart enzymes (J4* = 135,000). Also, the enzymes were resolved by ion-exchange chromatography and the separated enzymes demonstrated different calcium requirements. Some specific features of the rat liver enzymes, however, were quite different from the canine heart enzymes. Importantly, the rat liver proteases were much more sensitive to calcium (7 and 150 pM calcium for halfmaximal activiation, respectively) than the respective canine heart proteases (40 and 800 PM calcium for half-maximal activation). Furthermore, in our rat liver extracts, each enzyme accounted for ap-
r 100
_
x. 2
--jT-Ii.i .
8
Q
75
100
I ‘i
75 50
EE
/---’
-
25.
25 50
i 8
I 0 0
50
100
0 0
loo
200
300
400
Kx)
SW
700
so0
900
FIG. 3. Effect of calcium on Peak I and Peak II. Peak I (Fig. 2, fraction numbers 32-36) and Peak II (Fig. 2, fraction numbers 47-52) were treated as described in the text and assayed for protease activity at pH 7.5 (50 mM Tris-HCl buffer), 22°C (30 min), at the calcium concentrations indicated. Under these conditions, product formation was linear with respect to time. For each enzyme the highest activity was designated 100% and all other activities were expressed as a percentage of that value. Similar results were obtained with five separate enzyme preparations. Peak I, closed circles. Peak II, open circles.
CALCIUM-DEPENDENT
PROTEASES
proximately 50% of the total calcium-dependent proteolytic activity. The reasons for the differences between this work and that of Mellgren are not clear. Although it is possible that these discrepancies result from differences in purification or assay procedures, it is also possible that the enzymes have different characteristics in various tissues and/or species. It is also unclear whether these same possibilities can explain why many other workers have failed to demonstrate either multiple forms of the proteases or any form which was activated by the low concentrations of calcium observed here. In any case, the complete purification and characterization of these various enzymes will be required to determine relationships among proteases from different cell types as well as between proteases within the same tissues. Calcium-dependent proteases may play an important role in the nonlysosomal degradation of intracellular proteins. In addition, these enzymes may provide the biochemical mechanism for the regulation of overall protein degradation by calcium in tissues such as skeletal muscle (11). Although it is not known whether calcium can also regulate proteolysis in other cells such as hepatocytes, this possibility does not seem unlikely because calcium has been implicated in the control of a wide variety of cellular processes in many different cells and tissues. Liver, like many other tissues, is composed of a mixture of cell types and we do not know from which cell type(s) these proteases originate. Also, the livers used in this study were probably contaminated with small amounts of blood even though we were very successful in removing most of the blood by perfusion. In separate experiments, we have demonstrated similar calcium-dependent proteases in blood cells. Based on these studies, however, our cal-
IN RAT
257
LIVER
culations indicate that enzymes from contaminating blood cells could account, at most, for only a very small percentage of the activity in the liver extracts. We have previously demonstrated the high-molecular-weight protease separated on Sephacryl S-300 (Fig. 1) in isolated liver parenchymal cells2 and we are presently performing similar experiments to determine whether the calcium-dependent proteases are also located in these cells. ACKNOWLEDGMENTS This work was supported American Heart Association.
by
a grant
from
the
REFERENCES 1. GOLDBERG,
A. L., AND
Annu. Rev. B&hem.
ST. JOHN,
A. C. (1976)
45.747-803.
2. SCHIMKE, R. T. (1970) in Mammalian Protein Metabolism (Munro, H. N., ed.), Vol. IV, pp. 17’7-228, Academic Press, New York. 3. DEMARTINO, G. N., AND GOLDBERG, A. L. (1979) J. Biol. Chem. 254, 3712-3715. 4. MEYER, W. L., FISCHER, E. H. AND KREBS, E. G. (1964) Biochemistry 3,1033-1039. 5. DAYTON, W. R., GOLL, D. E., ZEECE, M. G., ROBSON, R. M., AND REVUE, W. J. (1976) Biochemistry 15, 2150-2158. 6. INUOE, M., KISHIMOTO, A., TAKAI, Y., AND NISHIZUKA, Y. (1977) J. Biol. Chem. 252,7610-7616. 7. TAKAI, Y., YAMAMOTO, M., INOUE, M., KISHIMOTO, A., AND NISHIZUKA Y. (1977) B&hem. Bio-
phys. Res. Commun. 77, 542-550. 8. PHILLIPS, D. R. AND JAKABOVA, M. (1977) J. BioZ. Chem. 252, 5602-5605. 9. PUCA, G. A., NOLA, E., SICA, V., AND BRESCIANI, F. (1977) J. Biol. Chem. 252,1358-1366. 10. TOYO-OKA, T., SHIMIZU, T., AND MASAKI, T. (1978)
B&hem.
Biophys. Res. Commun. 82,484-491.
11. KAMEYAMA, T., AND ETLINGER, J. D. (1979) Nature (London) 279, 344-346. 12. REDDY, M. K., ETLINGER, J. D., RABINOWITZ, M., FISCHMAN, D. A., AND ZAK, R. (1975) J. Biol.
Chem. 250.4278-4284. 13. DAYTON, W. R., REVUE, W. J., GOLL, D. E., AND STROMER, M. H. (1976) Biochemistry 15,21592167. 14. MELLGREN, R. L. (1980) FEBS L&t. 109.129-133.
Calcium-Dependent Proteolytic Activity in Rat Liver: Identification Proteases with Different Calcium Requirements GEORGE Department
of Two
N. DE MARTIN0
of Physiology, University of Texas Health Science Center at DaUas, 5323 Harry Hines Boulevard, Dallas, Tesas 75235 Received
February
19, 1981
Soluble extracts from rat liver cytoplasm contained substantial endoprotease activity which was totally dependent on calcium ions. This activity was accounted for by two distinct proteases which were separated by anion-exchange chromatography. One protease was half maximally activated by ‘7 PM calcium while the other protease required 150 MM calcium for half-maximal activation.
Intracellular protein degradation is a highly regulated physiologic process which influences the levels of specific proteins as well as the overall growth or atrophy of tissues (1, 2). In mammalian cells, there is good evidence that both lysosomal and nonlysosomal proteases catalyze the degradation of cell proteins. In contrast to lysosomal proteases, nonlysosomal proteases are poorly defined and the manner by which their activities are regulated is not clear. Recently, however, two cytoplasmic proteases have been identified which are regulated in vitro by potential physiologically important mechanisms. For example, we have demonstrated in rat liver, a high-molecular-weight protease whose activity is stimulated by ATP (3). This property may be important because protein degradation in vivo requires cellular energy. In the course of these studies we have identified an additional cytoplasmic proteolytic activity which is totally dependent on calcium. This finding was not suprising because calcium-activated proteases have been described previously in a variety of tissues (4-10). The calcium-dependent protease has received considerable attention because calcium appears to regulate protein degradation in some intact cells (11) and this enzyme offers an attractive mechanism for this regulation. However, because the high cal253
cium concentrations required for protease activation in vitro (>l mM) may never be achieved under normal conditions in vivo, the physiologic significance of these in vitro effects has been unclear. In this report, we demonstrate that calcium-dependent proteolytic activity in rat liver homogenates is accounted for by at least two distinct enzymes which differ from each other in their requirements for calcium. Importantly, one enzyme is completely activated by calcium concentrations which may be achieved in intact cells. MATERIALS
AND
METHODS
Preparation of liver extracts. Normal male rats (Sprague-Dawley, 400-500 g) were anesthetized lightly with ether. In order to remove as much contaminating blood as possible from the liver, the portal vein was cannulated and the liver was perfused with 100 ml of 0.15 M NaCl. Approximately 100 g of liver were chilled on ice, minced, and homogenized with a Dounce homogenizer in 5 mM potassium phosphate buffer, pH 7.6, containing 0.25 M sucrose and 1 mM EDTA. The homogenate (20%. w/v) was filtered through two layers of surgical gauze and the pH adjusted to pH 7.4. All of the following steps were carried out at 0-4’C. The homogenate was centrifuged for 10 min at 660g. The supernatant was centrifuged successively at 16,300g (20 min) and 105,OOOg (90 min). The resulting postmicrosomal supernatant was dialyzed for 3 h against 5 mM potassium phos0003-9861/81/110253-05$02.00/O Copyright All rights
0 1981 hy Academic Press, Inc. of reproduction in any form reserved.
254
GEORGE
N. DE MARTIN0
phate buffer, pH 7.6,0.5 mM D’IT,’ 1 mM EDTA. The dialysate was then added to the anion exchanger, diethylaminoethyl cellulose (DE52, Whatman, 1 g DE52/5 g liver) which was washed and equilibrated with the same buffer. After gentle agitation for 45 min, the mixture was filtered and the resin treated successively with potassium phosphate buffer containing 0.25 and 1.25 M KCl. Each treatment was for 20 min and was followed by filtration. The volume of the elution buffer was 1110th the volume of the original postmicrosomal supernatant. The filtrate from the 1.25 M KC1 elution was dialyzed for 14 h against 50 mM Tris-HCl buffer, pH 7.6, 8 m?d KCl, 0.5 mM DTT, 0.5 mM EGTA. This extract contained approximately 30% of the total protein originally present in the postmicrosomal supernatant. The dialyzed extract was centrifuged at 10,OOOg (10 min) and the supernatant chromatographed on Sephacryl S-300 as described below. Sephacryl S-NO gelJ2tration chromatography. Ascending gel filtration chromatography was performed with Sephacryl S-300 in a 100 X 5-cm column. The gel was equilibrated with 50 mM Tris-HCl buffer, pH 7.6, 8 mM KCl, 0.5 mM DTT, 0.5mM EGTA. The column was calibrated with purified proteins of known molecular weights (Fig. 1). Aliquots of the loml fractions were assayed for protease activity at pH 7.8, 36”C, using [methyl-“Clglobin as substrate (see below). DE52 Ion-exchange column chromatography. DE52 (10 g) was washed and equilibrated with 50 mM TrisHCl buffer, pH 7.6, 0.5 mM DTT, 0.5 mM EGTA. Column dimensions were 10 X 1.5 cm. After sample application, a 0.0-0.4 M linear gradient of KC1 in the Tris-HCl buffer was run at 0.5 ml/min. Aliquots (90 ~1) of the 5-ml fractions were assayed for protease activity at pH 7.5, 22°C in the presence and absence of 1.7 mM CaCl*. Protease activity. Protease activity was measured by the hydrolysis of [methyl-“Cjglobin as described previously (3). Conditions were fixed such that the assays were linear with respect to time and with respect to enzyme protein. Specific conditions for the assays are given in the legends to the figures. Chemicals and reagents. All chemicals were of the highest grade commercially available. All solutions were prepared in deionized, double-distilled water.
RESULTS
Postmicrosomal supernatants of rat liver homogenates were treated as described under Methods and chromato’ Abbreviations aminoethyl ether) threitol.
used: EGTA, N,w-teraacetic
ethylene acid,
glycol his@DlT, dithio-
graphed on Sephacryl S-300 equilibrated with 50 mM Tris-HCl buffer, pH 7.6, 8 mM KCl, 0.5 mM DTT, and 0.5 mM EGTA. When the column fractions were assayed for protease activity at pH 7.8 with no added calcium, two peaks of high-molecular-weight (i.e., M, = 750,000 and 550,000) protease activity were observed (Fig. 1). This activity has been described previously (3) and will be the subject of a subsequent report.2 When the column fractions were assayed in the presence of 1.7 mM CaC12,the activities of the high-molecular-weight proteases were not greatly affected (data not shown). However, assays with calcium revealed an additional peak of alkaline protease activity at a position corresponding to M, = 150,000. This activity had some properties similar to calcium-dependent proteases described previously in other tissues. For example, other divalent cations such as Me or Mn2+ could not activate proteolysis. The activity had an alkaline pH optimum from pH 7.0 to 8.5 and was sulfhydryl dependent. Protease activity was stimulated by compounds such as DTT, cysteine, and /3mercaptoethanol but inhibited by alkylating agents such as N-ethylmaleimide and iodoacetimide (data not shown). Fractions from the Sephacryl column which contained calcium-dependent protease activity were pooled and subjected to DE52 ion-exchange column chromatography as described under Methods. This resolved two peaks of calcium-dependent protease activity. The first (Peak I) eluted at a position corresponding to 0.10 M KC1 and second (Peak II) eluted at a position corresponding to 0.22 M KC1 (Fig. 2). The relative activities of these two peaks were approximately equal. Each of the calcium-dependent proteases was purified further by ammonium sulfate precipitation. The respective precipitates formed after the addition of ammonium sulfate to 50% saturation were collected by centrifugation, redissolved in a small volume (3-4 ml) of 5 mM Tris-HCl buffer, pH 7.2, 8 mM KCl, 0.5 mM DTT, 0.5 * G. N. De Martin0 in preparation.
and A. L. Goldberg,
manuscript
CALCIUM-DEPENDENT
PROTEASES
Fmction
IN RAT
LIVER
255
Number
FIG. 1. Sephacryl S-300 column chromatography. A postmicrosomal supernatant, prepared and treated as described under Methods, was chromatographed on Sephacryl S-300 equilibrated with 50 mM Tris-HCl buffer, pH 7.6, 8 mM KCl, 0.5 mM EGTA, 0.5 mM DTT. Aliquots (90 ~1) of the loml fractions were assayed for protease activity in 50 mM Tris-HCl buffer, pH 7.8, at 36”C, with and without 1.7 mM CaCle. Enzyme activities in fractions 50-89 were similar under both of these conditions and the values for assays with CaCl, have been omitted for clarity. Similar elution patterns were obtained with eight separate enzyme preparations. Insert: Estimation of molecular weight of protease activities. Blue Dextran, thyroglobulin (M, = SSO,OOO), ferritin (Af, = 440,000), catalase (kf, = 240,000), and hemoglobin (M, = 64,000) were dissolved in column buffer and chromatographed. The elution pattern for each substrate was determined and plotted versus the log of molecular weight.
mM EGTA, and dialyzed for 12 h against the same buffer. After dialysis, any undissolved material was removed by centrifugation and the resulting supernatant was dialyzed for an additional 12 h against two changes (6 1 each) of 5 mM Tris-HCl buffer, pH 7.2, 0.5 mM DTT. The dialyzed extracts were assayed for protease activity at various calcium concentrations (Fig. 3). Each enzyme was completely dependent on added calcium for activity. However, the concentrations of calcium required for the activation of each enzyme were remarkably different. Peak I required approximately 7 pM calcium for half-maximal activity and was maximally active at 25-50 pM calcium. Higher concentrations of calcium (i.e., >450 pM) inhibited this protease by 15-30s in various enzyme preparations. In contrast to Peak I, Peak II required approximately 150 PM calcium
for half-maximal activity and greater than 450 pM calcium for maximal activity. Results similar to those shown in Fig. 3 were obtained with enzymes which had not been precipitated with ammonium sulfate. Similar results were also obtained with [methyZ-‘4C]bovine serum albumin as substrate. In some experiments with the Peak I enzyme, we used a series of Ca”-EGTA buffers designed to achieve free calcium concentrations between 0.4 and 9.4 PM at the pH and temperature of the assays. Assays performed in this manner also demonstrated half-maximal activation of Peak I by 7-8 pM calcium (data not shown). DISCUSSION
A calcium-dependent protease was first described in skeletal muscle (4,5). Because this enzyme could degrade some specific
256
GEORGE
N. DE MARTIN0
FIG. 2. DE52 ion-exchange chromatography. Pooled samples of calcium-dependent protease activity from Fig. 1 were subjected to DE52 ion-exchange chromatography as described under Methods. Aliquots (90 ~1) of the 5-ml fractions were assayed for protease activity at pH 7.5 (50 mM Tris-HCl buffer), 22°C with 1.7 mM CaClz (30 min). Under these conditions, product formation was linear with respect to time. Similar results were obtained with six separate enzyme preparations.
isolated contractile proteins as well as Zline proteins of intact myofibrils, it was ascribed a role in the turnover of myofibrillar proteins in wivo (5, 12, 13). Subsequently, a similar protease has been identified in nonmuscle cells (6-8). Thus, it has been unclear whether this enzyme degrades specific but different proteins in different cell types or whether it plays a general role in the degradation of many cell proteins. In either case, a feature of this protease which has obscured its possible physiologic role is the high concentrations of calcium (i.e., usually >l mM) required for maximal enzyme activity. Recently, however, Mellgren isolated from canine heart, two forms of calcium-dependent proteases (14). One was like other described calcium-dependent proteases in that it required millimolar levels of calcium for maximal activity. The other enzyme, however, required only 40 PM calcium for half-maximal activity and was maximally activated by 400 PM calcium. These enzymes had similar molecular weights (135,000 daltons) and were separated by ion-exchange chromatography. Interestingly, the more calcium-sensitive enzyme represented only a small percentage of the total calcium-dependent protease activity. These important findings
raised the possibility that the protease which required lower calcium concentrations had been overlooked in previous studies and that this form of the enzyme might be of greater physiologic significance. In fact, evidence for two forms of calcium-dependent protease was obtained in several studies, but possible differences in calcium requirements between the enzymes were not examined (6, 7). The enzymes described in the present report appear to be related to those described by Mellgren. For example, the rat liver enzymes had molecular weights similar to each other (M, = 150,000) and to the canine heart enzymes (J4* = 135,000). Also, the enzymes were resolved by ion-exchange chromatography and the separated enzymes demonstrated different calcium requirements. Some specific features of the rat liver enzymes, however, were quite different from the canine heart enzymes. Importantly, the rat liver proteases were much more sensitive to calcium (7 and 150 pM calcium for halfmaximal activiation, respectively) than the respective canine heart proteases (40 and 800 PM calcium for half-maximal activation). Furthermore, in our rat liver extracts, each enzyme accounted for ap-
r 100
_
x. 2
--jT-Ii.i .
8
Q
75
100
I ‘i
75 50
EE
/---’
-
25.
25 50
i 8
I 0 0
50
100
0 0
loo
200
300
400
Kx)
SW
700
so0
900
FIG. 3. Effect of calcium on Peak I and Peak II. Peak I (Fig. 2, fraction numbers 32-36) and Peak II (Fig. 2, fraction numbers 47-52) were treated as described in the text and assayed for protease activity at pH 7.5 (50 mM Tris-HCl buffer), 22°C (30 min), at the calcium concentrations indicated. Under these conditions, product formation was linear with respect to time. For each enzyme the highest activity was designated 100% and all other activities were expressed as a percentage of that value. Similar results were obtained with five separate enzyme preparations. Peak I, closed circles. Peak II, open circles.
CALCIUM-DEPENDENT
PROTEASES
proximately 50% of the total calcium-dependent proteolytic activity. The reasons for the differences between this work and that of Mellgren are not clear. Although it is possible that these discrepancies result from differences in purification or assay procedures, it is also possible that the enzymes have different characteristics in various tissues and/or species. It is also unclear whether these same possibilities can explain why many other workers have failed to demonstrate either multiple forms of the proteases or any form which was activated by the low concentrations of calcium observed here. In any case, the complete purification and characterization of these various enzymes will be required to determine relationships among proteases from different cell types as well as between proteases within the same tissues. Calcium-dependent proteases may play an important role in the nonlysosomal degradation of intracellular proteins. In addition, these enzymes may provide the biochemical mechanism for the regulation of overall protein degradation by calcium in tissues such as skeletal muscle (11). Although it is not known whether calcium can also regulate proteolysis in other cells such as hepatocytes, this possibility does not seem unlikely because calcium has been implicated in the control of a wide variety of cellular processes in many different cells and tissues. Liver, like many other tissues, is composed of a mixture of cell types and we do not know from which cell type(s) these proteases originate. Also, the livers used in this study were probably contaminated with small amounts of blood even though we were very successful in removing most of the blood by perfusion. In separate experiments, we have demonstrated similar calcium-dependent proteases in blood cells. Based on these studies, however, our cal-
IN RAT
257
LIVER
culations indicate that enzymes from contaminating blood cells could account, at most, for only a very small percentage of the activity in the liver extracts. We have previously demonstrated the high-molecular-weight protease separated on Sephacryl S-300 (Fig. 1) in isolated liver parenchymal cells2 and we are presently performing similar experiments to determine whether the calcium-dependent proteases are also located in these cells. ACKNOWLEDGMENTS This work was supported American Heart Association.
by
a grant
from
the
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