THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 30, Issue of October 25. pp. 17868-17872,1989 Printed in lJ S.A.

A Novel ATP-requiring Protease from Skeletal Muscle That Hydrolyzes Non-ubiquitinated Proteins*

(Received for publication, May 31, 1989)

Julie M. Fagan and Lloyd Waxman$ From the Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08903

Previously, we isolated an ATP-dependent proteo- lytic pathway in muscle, liver, and reticulocytes that requires ubiquitin and the enzymes which conjugate ubiquitin to proteins. We report here that skeletal mus- cle contains another soluble alkaline energy-dependent (but ubiquitin-independent) proteolytic activity. The cleavage of non-ubiquitinated protein substrates by the partially purified protease requires ATP hydrolysis since ATP in the absence of Mg2+, nonhydrolyzable ATP analogs, and pyrophosphate all fail to stimulate proteolysis. Proteolytic activity is also stimulated by UTP, CTP, and GTP, although not as effectively as by ATP ( K r n ( * ~ p ) = 0.027 mM). The enzyme is inactivated by the serine protease inhibitors diisopropyl fluoro- phosphate and 3,4-dichloroisocoumarin, but not by specific inhibitors of aspartic, thiol, or metallopro- teases. It is maximally active at pH 8 and has a molec- ular weight of approximately 600,000. This new activ- ity differs from the 720-kDa multicatalytic proteinase, but resembles the soluble ATP-dependent proteolytic system that we previously isolated from murine eryth- roleukemia cells.

The breakdown of proteins in animal cells can occur by several different pathways, and some of these require energy. Certain abnormal and short-lived proteins are degraded via a soluble ATP- and ubiquitin-dependent proteolytic pathway. ATP hydrolysis is required both for the conjugation of ubiqui- tin to free amino groups on proteins and for the subsequent degradation of ubiquitinated proteins (1). Although best char- acterized in reticulocytes (l), the ATP- and ubiquitin-depend- ent system has been demonstrated in extracts of muscle and liver (2) and baby hamster kidney cells (3).

Mammalian cells also appear to contain nonlysosomal ATP-dependent proteases that hydrolyze proteins not conju- gated to ubiquitin. An ATP-dependent protease is present in mitochondria (4, 5 ) which has properties similar to protease La, an ATP-dependent enzyme in Escherichia coli (6, 7). A soluble high molecular weight ATP-dependent protease has also been isolated from murine erythroleukemia cells (S), and a particulate ATP-requiring enzyme has been described in human erythroleukemia cells (9). The 720-kDa multisubunit

-

* This work was supported by research grants from the National Institute of Arthritis, Musculoskeletal, and Skin Diseases, the United States Department of Agriculture, the Muscular Dystrophy Associa- tion of America, and the New Jersey Agricultural Experiment Station, which is supported by State funds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Dept. of Biological Chemistry, Merck Sharp and Dohme Research Laboratories, West Point, PA 19486.

proteinase purified from a variety of mammalian tissues (10- 16) has been reported 1) to be unaffected or slightly inhibited by ATP (E-lS), 2) to be stabilized by ATP (19-21), 3) to be stimulated by ATP without ATP hydrolysis (22-25), or 4) to hydrolyze proteins (3, 26) and peptides (26) in an ATP- requiring fashion. Antibodies to the multicatalytic proteinase were found to block ATP- and ubiquitin-dependent proteol- ysis in reticulocytes (22, 27) and baby hamster kidney cell extracts (3), suggesting that this enzyme may be involved in the ATP- and ubiquitin-dependent proteolytic pathway as well. In this report, we describe a new soluble high molecular weight ATP-dependent ubiquitin-independent protease from chicken skeletal muscle.

MATERIALS AND METHODS

Reagents-N-Ethylmaleimide, diisopropyl fluorophosphate, fluo- rometric peptide substrates, and nucleotides were purchased from Sigma. Phosphoramidon, elastatinal, and 3,4-dichloroisocoumarin were obtained from Boehringer Mannheim. Pepstatin, chymostatin, leupeptin, and E-64l were from Cambridge Research Biochemicals. Rabbit hemoglobin purified as described previously (28) and egg white lysozyme (Miles Laboratories Inc.) were labeled by reductive meth- ylation (29) with [3H]formaldehyde (Du Pont-New England Nuclear) and sodium cyanoborohydride (Sigma). Globin was prepared accord- ing to the method of Guidotti (30). Heparin-Sepharose, Sephacryl S- 300, and molecular weight markers for gel filtration were obtained from Pharmacia LKB Biotechnology Inc. DEAE-cellulose (DE52) was from Whatman.

Gel Electrophoresis-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out on slab gels containing 12.5% poly- acrylamide (31). Molecular weight markers were obtained from Sigma. Proteins were electrophoresed under nondenaturing condi- tions (32) on gels containing a linear gradient of acrylamide from 3 to 13%. Molecular weight markers were obtained from Pharmacia LKB Biotechnology Inc.

Preparation of Extracts-Male Peterson Arboracre chickens (6-12 months old) were killed by asphyxiation with CO,, and the pectoral muscles were rapidly excised and placed on ice. All subsequent steps were carried out at 4 "C. The muscles were trimmed of fat and connective tissue, minced, and then ground in a prechilled meat grinder. Muscle (200-500 g) was suspended in 3 volumes (ml/g) of ice-cold buffer containing 20 mM Tris-HC1 (pH 81, 1 mM 2-mercap- toethanol, 1% glycerol, 1 mM EDTA, 1 mM EGTA, 50 p M chymosta- tin, 50 FM E-64, and 10 p~ pepstatin and homogenized in a Waring blender for 1 min at top speed. The homogenates were centrifuged (30,000 X g for 30 min), and the supernatant was filtered through glass wool. The pH was then adjusted to 7.0 with 1 M Tris base, and glycerol was added with stirring (final concentration of 20%).

Assays-Protein content was measured by the procedure of Lowry et al. (33) with bovine serum albumin as the standard. The degrada-

The abbreviations used are: E-64, trans-epoxysuccinyl-L-leucyl- amido-(4-guanidino)butane; -AMC, -4-methylcoumaryl-7-amide; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; DTT, dithi- othreitol; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Z-, benzyloxycarbonyl-; MES, 2-(N-morpho- 1ino)ethanesulfonic acid; PIPES, piperazine-N,N'-bis(2-ethanesul- fonic acid); CAPS, 3-(cyclohexylamino)propanesulfonic acid.

17868

ATP-requiring Protease from Skeletal Muscle 17869

tion of radiolabeled proteins and the hydrolysis of fluorometric sub- strates were determined as described previously (2, 17).

RESULTS

Previously, we demonstrated the presence of a soluble ATP- and ubiquitin-dependent proteolytic system in skeletal muscle and liver (2). To examine further the energy requirement for proteolysis in muscle, crude extracts were fractionated by ion- exchange and gel filtration chromatography, and the high mass fraction >450 kDa (2) was applied to a column of heparin-Sepharose (Fig. 1). The ubiquitin-conjugate-degrad- ing enzyme (2) eluted as a broad peak (between 0.05 and 0.4 M NaCl), whereas the 720-kDa multicatalytic proteinase bound less well and eluted as a sharp peak at -0.15 M NaCl as monitored by the hydrolysis of Z-Ala-Arg-Arg-AMC (17) (data not shown). At a much higher salt concentration (0.4- 0.5 M NaCl), we also detected an ATP-stimulated proteolytic activity which did not require the addition of ubiquitin or the factors required to generate ubiquitin-protein conjugates (Fig. 1).

To resolve this new ATP-dependent activity from the other high molecular weight enzymes, the 0.3 M NaCl DEAE eluate was precipitated with 5-15% polyethylene glycol 8000 and applied to a column of heparin-Sepharose in buffer containing 0.2 M NaCl. Most of the activities responsible for hydrolyzing fluorometric peptides failed to bind under these conditions (Fig. 2, middle) and were not present in the region of the NaCl gradient (0.2-0.5 M) containing the new ATP-stimu- lated proteolytic activity (Fig. 2, top). SDS-PAGE of samples from fractions across the column (Fig. 2, bottom) shows that the six to eight bands (from 22 to 31 kDa) characteristic of the multicatalytic proteinase (11, 14, 16) are present only in

ATP+Ub-dependent

, - - - - - - r - - I"-"-T-" I

0 2 0 4 0 6 0 80 Fraction Number

FIG. 1. Fractionation of three high molecular weight pro- teases from muscle on heparin-Sepharose. Skeletal muscle (200 g) was homogenized and applied to a 50-ml column of DE52. Bound proteins were eluted with 0.6 M NaC1, concentrated by ultrafiltration, and applied to a column (1.5 X 62 cm) of Sephacryl S-300. Fractions with M, >450,000 were pooled (31 mg of total protein) and applied to a 15-ml heparin-Sepharose column equilibrated in 20 mM Tris-HC1 (pH 7.5), 0.5 mM DTT, and 20% glycerol. Bound proteins were eluted with a linear gradient (total of 120 ml) of 0.0-0.6 M NaC1. Fractions (1 ml) were collected, and 25 p1 was assayed for the presence of the multicatalytic proteinase with Z-Ala-Arg-Arg-AMC for 15 min (data not shown). 75 pl was assayed for the degradation of 3H-lysozyme (5 pg, 140,000 dpm/pg) in the presence or absence of 5 mM ATP, 300 pg of muscle conjugating fraction (2), and 7.5 pg of ubiquitin (Ub). After 1.5 h at 37 "C, the reaction was stopped by the addition of trichloro- acetic acid (final concentration of 7.2%), and the acid-soluble radio- activity was determined. CF, conjugating fraction.

the flow-through fraction (Fig. 2, bottom, lane I ) and are absent from the fractions which contain this new ATP-de- pendent proteolytic activity (Fig. 2, bottom, lanes 5-13).

We have been unable to purify this enzyme to homogeneity. We found this new ATP-dependent proteolytic activity to be extremely labile. It loses almost all of its activity on certain resins (e.g. hydroxylapatite, reactive dye-agarose) or within 1 week following extraction from the tissue.

Several experiments were carried out to define better the properties of the protease. On gel filtration, the enzyme has a molecular weight of -600,000 (Fig. 3, left). Gel electrophoresis under nondenaturing conditions of the peak fraction of pro- teolytic activity shows one major band between 600 and 700 kDa (Fig. 3, right). Henceforth, we will refer to this new activity as "600-kDa protease" based on its apparent size. The enzyme is maximally active at alkaline pH, with an optimum near pH 8 (Fig. 4, bottom). The 600-kDa protease has a K m ( ~ ~ ~ ) of 27 WM (Fig. 4, top). Other nucleotides and some ATP analogs also support proteolysis, but only in the presence of Mg2+ (Table I). Nonhydrolyzable ATP analogs and pyrophos- phate do not stimulate proteolysis (Table I). Thus, ATP hydrolysis is required for the cleavage of proteins.

To determine to which class of protease the 600-kDa en- zyme belongs, we examined the effect of a variety of protease inhibitors (Table 11). The ATP-dependent degradation of lysozyme was not affected by pepstatin (an inhibitor of as- partic proteases) (34), o-phenanthroline and dipicolinic acid (metal chelators), or phosphoramidon (an inhibitor of metal- loproteases) (35). Although the general sulfhydryl reagent N - ethylmaleimide blocks proteolytic activity, the more specific inhibitors of thiol-dependent proteases, E-64 (36) and leupep- tin (37), had no effect. Other peptide aldehydes including chymostatin, an inhibitor of chymotrypsin-like enzymes (37), or elastatinal, an inhibitor of elastases (37), also failed to inhibit this activity. In contrast, the serine protease inhibitors diisopropyl fluorophosphate (38) and 3,4-dichloroisocoumarin (39) blocked ATP-dependent proteolytic activity, suggesting that the 600-kDa protease is a serine protease. Proteolytic activity also was blocked completely by a variety of peptide chloromethyl ketones (at 100 WM) (data not shown). However, we cannot presently eliminate the possibility that these re- agents inhibit by modifying a key sulfhydryl group and not the histidine residue in the catalytic triad of the typical serine protease.

DISCUSSION

We have resolved in extracts of chicken skeletal muscle a high molecular weight protease (-600,000) that requires ATP for activity. Unlike the 1,500-kDa ATP-dependent ubiquitin conjugate-degrading enzyme (2,17, 18), the 600-kDa protease seems to require no additional protein factors such as ubiqui- tin or the proteins that conjugate ubiquitin to potential sub- strates. This new ATP-dependent proteolytic activity isolated from skeletal muscle may be similar to the soluble 600-kDa ATP-dependent enzyme demonstrated in murine erythroleu- kemia cells which will hydrolyze proteins not conjugated to ubiquitin (8). It differs significantly from the 720-kDa multi- catalytic proteinase and the ATP-stimulated enzyme believed to be a labile form of the multicatalytic proteinase (26). Although the 600-kDa protease is not pure, its specific activi- ties are 10-30-fold greater (lysozyme, 26 ng/Ng/h; globin, 75 ng/Hg/h) than those reported for the purified multicatalytic proteinase activated by ATP (lysozyme, poorly degraded; casein, 2.4 ng/pg/h) (20). The ATP requirement for proteol- ysis ( K , = 27 pM) is 10-fold lower than the concentration of ATP required for half-maximal activation of the ATP-stim-

17870 ATP-requiring Protease from Skeletal Muscle

- 0.4 f

5 0.3

m 1 v

.- L m U 5 0.2 aJ

c 0

0 .- n 0.1 a - * 0.0 +

h - 1 .oo

- 0.75

i2

I -0.50 n

0

cu

b- y - 2 0 o 2 0 4 0 6 0 8 0

Fraction Number

66 -

45 - 37 -

29 - 25 -

20 -

14 -

1.00 -

0.75 -

0.50 -

0.25 -

0.00 r

* SLLVT -ATP

0.25

0.00

- 0.50 - z

- 0.25 p - f - 0.00

- 2 0 0 2 0 4 0 6 0 8 0 Fraction Number

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 14 - 205

- 116 - 97

- 66

. 45

. 29

FIG. 2. Fractionation of 600-kDa protease on heparin-Sepharose. Top, following DE52 column chromatography, 150 mg of protein precipitated by polyethylene glycol (5-15%) was solubilized in 15 mi of buffer containing 20 mM Tris (pH 7.6), 0.5 mM DTT, 0.2 M NaCI, 20% glycerol and applied to a 20-ml heparin-Sepharose column equilibrated in the same buffer. Bound proteins were eluted with a linear gradient (total of 120 ml) of 0.2-0.5 M NaCI. 1.5-ml fractions were collected, and selected column fractions (75 pl) were assayed for the degradation of globin (2.5 pg, 30,000 dpmlpg) in the presence or absence of 5 mM ATP and 5 mM M&12 in buffer containing 50 mM Tris-HCI (pH 8) and 1 mM DTT. Middle, 25 p1 was assayed for 15 min at 37 "C for the hydrolysis of Z-Ala-Arg-Arg-AMC ( Z A R R ) , a substrate for the multicatalytic proteinase ( l l ) , and for succinyl-Leu-Leu-Val-Tyr-AMC (SLLVT), a substrate both for the multicatalytic proteinase (18, 26) and for the ubiquitin conjugate-degrading enzyme (18), in the presence or absence of ATP (2 mM) and MgC12 (2 mM). Bottom, SDS-PAGE (12.5% acrylamide) was carried out on selected heparin-Sepharose column fractions. Lane I , flow-throcgh fraction (25 pi); lanes 2 and 4, molecular mass standards (bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (37 kDa), carbonic anhydrase (29 kDa), trypsinogen (25 kDa), soybean trypsin inhibitor (20 kDa), and a-lactalbumin (14 kDa)); lane 3, purified multicatalytic proteinase (20 pg); lanes 5-13, column fractions 20, 25,30,38,44, 52,60,66, and 72 (100 pl each); lone 14, molecular mass standards (myosin (205 kDa), 8-galactosidase (116 kDa), phosphorylase (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa)). Proteins were detected by staining with Coomassie Blue.

ATP-requiring Protease from Skeletal Muscle 17871 500

0" Std 136

400 -I x 0 r- W

0.3

0.2 0 OD N c) 0

0.1

0.0

670kDa

w * I

CI

20 3 0 4 0 5 0 6 0

Fraction Number

FIG. 3. Left, gel filtration chromatography of BOO-kDa protease. Following chromatography on heparin-Sepharose, the peak of ATP- requiring proteolytic activity (containing 7 mg of protein) was pooled and applied to a column (1.5 X 60 cm) of Sephacryi S-300 equilibrated in 20 mM Tris-HC1 (pH 7.5), 0.5 mM DTT, 25 mM NaCI, 0.1 mM EDTA, and 20% glycerol. 1-ml fractions were collected, and AzW (-) was determined. 150 p1 was assayed for ['Hlglobin degradation in the presence (W) or absence (W) of 5 mM ATP for 1 h. The elution position of thyroglobulin (670 kDa) is shown. Right, gel electrophoresis of 600-kDa protease. 100 pl of the most active material (fraction 36 ( f 3 6 ) ) was electrophoresed on a gradient gel (3-13% acrylamide) under nondenaturing conditions at 200 V for 12 h at 4 "C. Protein was detected by staining with silver. The standards ( S t d ) used were thyroglobulin (670 kDa), ferritin (440 kDa), catalase (230 kDa), lactate dehydrogenase (140 kDa), and albumin (67 kDa).

TABLE I Effect of Mg2+ and nucleotides on globin degradation by the

600-KDa protease Following gel filtration on Sephacryl S-300, the effect of M F and

various nucleotides on proteolytic activity was determined by incu- bating partially purified enzyme (10 pg) in a total volume of 200 pl, containing 50 mM Tris-HC1 (pH 8.01, 0.5 mM DTT, 2.5 pg of [3H] globin (30,000 dpm/pg), and 5 mM nucleotide. EDTA (1 mM) or MgC1, (5 mM) was included where indicated. Assays were carried out for 1 h a t 37 "C, and acid-soluble radioactivity was determined in a liquid scintillation spectrometer. Release of acid-soluble radioactivity was linear for 1.5 h (data not shown).

Globin degradation

d h

EDTA (1 mM) 0.22 0.22

ATP + EDTA 0.18 ATP + Mg2+ 0.60 AMP-PNP" + M e 0.22

AMP-PCP + Mg2+ 0.20 PP, + M e 0.22

Experiment 1

M F

AMP-CPP + M e 0.20

Experiment 2 MgZ+ 0.25 +ATP +CTP

1.00 0.56

+UTP 0.39 +GTP 0.43 +Deoxy-ATP 0.89 +8-Azido-ATP 0.51 +l,N'-Etheno-ATP 0.25

' AMP-PNP, adenosine 5'-(P,y-imino)triphosphate; AMP-CPP, adenosine 5'-(a,@-methylene)triphosphate; AMP-PCP, adenosine 5'- (&y-methy1ene)triphosphate.

ulated multicatalytic proteinase (26). Our new activity ap- pears not to hydrolyze fluorometric peptides which are sub- strates for the multicatalytic proteinase including Z-Ala-Arg-

- 4 0 - 2 0 0 2 0 4 0 6 0 llATP (mu)

0.8 I

3 5 7 9 1 1 1 3 PH

FIG. 4. Catalytic properties of 600-kDa protease. Top, &(ATP). The K,(ATP) was determined by measuring the amount of [3H]globin (2.5 pg/assay) hydrolyzed in 1 h in the presence of increas- ing concentrations of ATP (0.02-0.2 mM). The assay mixture also included 50 mM Tris-HC1 (pH 8), 10 mM MgC12, 1 mM DTT, and 10 pg of partially purified enzyme. After subtracting the activity meas- ured in the absence of ATP, the K, was determined from a double reciprocal plot of the data. Bottom, pH dependence of lysozyme hydrolysis. The pH optimum for proteolysis was determined by measuring the amount of 'H-lysozyme hydrolyzed in 1 h over the pH range 3.5-12. Included in each assay mixture was 5 pg of 3H-lysozyme, 5 mM ATP, 1 mM DTT, 10 mM MgClZ, 10 pg of partially purified enzyme, and buffer a t a concentration of 50 mM. The buffers used were: sodium citrate (pH 3.5 and 3.8) (m), sodium/MES (pH 5.6)(+), sodium/PIPES (pH 6.25) (+), Tris-HC1 (pH 7.2, 7.5, 7.85, and 8.1) (a), sodium glycine (pH 8.0 and 9.2) (A), and sodium/CAPS (pH 10.6 and 12) (A). Each pH was measured at 37 "C and included all components of the assay mixture.

Arg-AMC and succinyl-Leu-Leu-Val-Tyr-AMC (11, 18, 26) or for the ubiquitin conjugate-degrading enzyme which hydro- lyzes succinyl-Leu-Leu-Val-Tyr-AMC in an ATP-stimulated fashion (18). Furthermore, SDS-PAGE of the ATP-depend- ent proteolytic activity identified in our study shows none of the subunits characteristic of the multicatalytic proteinase.

The enzyme described in this report is one of a growing class of ATP-dependent proteases which are present in bac- teria (6, 7, 40, 41), mitochondria (4, 5), and the cytoplasm of mammalian cells (2, 3, 8, 17, 18, 26). The role of ATP hydrolysis in the function of these enzymes is still unclear. Due to the low K m ( * ~ p ) (27 PM), it is unlikely that cellular concentrations of ATP per se regulate the activity of this enzyme. Thus, ATP probably serves another function, per- haps in recognizing specific structural features of potential substrates. Alternatively, ATP hydrolysis may enable the protease to cleave proteins more efficiently by enabling it to function in a processive mode. Our efforts are now directed toward purifying sufficient amounts of this enzyme to be able to answer some of these important mechanistic questions.

17872 ATP-requiring Protea

TABLE I1 Effect of inhibitors on lysozyme degradation by the 600-kDa protease

Following gel filtration, partially purified enzyme (10 pg) was preincubated a t 37 "C in the presence of inhibitor at the concentration indicated. All assays contained dimethyl sulfoxide a t a concentration of 2.5%. After 15 min, the assays were put on ice, and DTT (final concentration of 1 mM) and MgC12 (final concentration of 5 mM) were added. "H-Lysozyme (5 pg, 140,000 dpmlpg) was added, without or with ATP (5 mM); and the mixtures were incubated a t 37 "C. After 1 h, assays were terminated, and the percent inhibition of the ATP- dependent degradation of lysozyme (265 ng of lysozyme was degraded Der h in the absence of inhibitor) was calculated.

-

Pepstatin (50 pM) N-Ethylmaleimide (2 mM)

Leupeptin (100 p ~ ) Phosphoramidon (100 p ~ ) o-Phenanthroline (1 mM) Dipicolinic acid (5 mM) Dichloroisocoumarin (0.1 mM) Diisopropyl fluorophosphate (5 mM) Chymostatin (100 pM) Elastatinal (100 pM)

E-64 (100 p M )

Lysozyme degradation

% inhibition 0

96 0 0 0 0 0

88 76 0 8

Acknowledgment-We are grateful to Carole Hannan for her as- sistance in preparing this manuscript.

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