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Isolation and characterization of a membrane-bound proteinase from rat liver
ARCHIVES
OF
Isolation
BIOCHEMISTRY
AND
BIOPHYSICS
and Characterization
177,
3X-363
of a Membrane-Bound Rat Liver
MIRNA JUSIC, SILVIA RAINER HAAS, AND Biochemisches
Institut
(1976)
der Universittit,
from
SEIFERT, ERICH WEISS, PETER C. HEINRICH
D-78
Received
Proteinase
Freiburg, March
Hermann-Herder-Strap
7, Germany
31, 1976
The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 M phosphate buffer. After a 1330fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 2 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone Hl, which was split into a high molecular weight fragment (M, 13,000). The highly phosphorylated histone Hl isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as Hl from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-L-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-L-tyrosine ethyl ester is 5.0 x lo-:’ M. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, L-l-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.
In the course of our studies on the regulation of rat liver chromosomal protein degradation, a histone degrading enzyme was found in the mitochondrial fraction (1). We showed that this proteinase contaminates chromatin prepared from a total homogenate (1). The degradation of histones in isolated chromatin found by several authors (2,3) may be due to the action of this proteinase. In the present paper, we describe the purification and some properties of this enzyme. MATERIALS
AND
ginine, bovine serum albumin, egg albumin, N-benzoyl-L-arginine ethyl ester, and ATEE’ were products of Serva (Heidelberg, Germany); alcohol dehydrogenase, glucose 6-phosphate dehydrogenase, malate dehydrogenase, and cytochrome c were from Boehringer (Mannheim, Germany); dithioerythritol (Cleland’s reagent) was from Calbiochem (Zurich, Switzerland); sucrose (ultrapure) was from Schwarzi Mann (Orangeburg, N.Y.). [l~SI]Insulin (sp act 165 mCi/mg) and [‘2SI]glucagon (sp act 170 mCi/mg) were obtained from Behringwerke AG (Marburg, Germany). Hydroxylapatite was prepared as de1 Abbreviations used: ATEE, N-acetyl-L-tyrosine ethyl ester; DFP, diisopropyl phosphofluoridate; fluoride; PhMeSO,F, phenylmethanesulfonyl TLCK, 1-chloro-3-tosylamido-7-amino-L-2-heptanone; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; CM, carboxymethyl.
METHODS
Materials Digitonin, trypsin, were obtained from phenylmethanesulfonyl
and soybean trypsin inhibitor Merck (Darmstadt, Germany); fluoride, polylysine, polyar355
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
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356
scribed by Levin (4). The yeast inhibitors of proteinases A and B and carboxypeptidase Y were kindly supplied by Prof. Dr. H. Holzer. Pepstatin was a gift of Dr. Umezawa and kallikrein trypsin inhibitor (Trasylol) was kindly supplied by Dr. E. Truscheit. All the other chemicals were of the highest purity grade available.
Methods Preparation ofproteinase substrates. The preparation of [3H]lysine-labeled histones from Ehrlich ascites tumor cells has been described previously (1). The very-lysine-rich histone Hl was isolated from total rat liver histones according to Balhorn and Chalkley (5). The nonhistone chromosomal protein fraction used was prepared from rat liver nuclei as detailed by Kish and Kleinsmith (6). The preparation was used a&er Biorex 70 treatment. Protein determination. Protein was determined by the method of Lowry et al. (7) with bovine serum albumin as standard. AC&amide-gel electrophoresis. Polyacrylamide gel electrophoresis was carried out as described by Davis (8) with Tris/glycine buffer with a running pH of 8.9, or according to the technique of Panyim and Chalkley (9) with 0.9 N acetic acid, pH 2.6. SDS gel electrophoresis was performed according to King and Laemmli (10). Densitometry was conducted by use of a Gilford Model 2520 gel scanner. Estimation of molecular weight. The molecular weight was estimated by sucrose gradient centrifugation (11). The proteinase (0.04 mg, sp act 7.0 pmol ATEE split/min x mg) mixed with 200 fig of egg white lysozyme, 25 wg of rabbit muscle lactate dehydrogenase, and 20 pg of catalase in a total volume of 0.2 ml was layered on top of a linear gradient of 5 to 20% sucrose in 0.05 M Tris/HCl buffer, pH 7.5. After centrifugation at 280,OOOg for 15 h (SW41 rotor, 40,000 rpm), 51 fractions of 0.24 ml each were collected at a flow rate of 18 ml/h and assayed for enzymatic activities. The molecular weight of the proteinase was estimated from 81 -= S2 distance S, S2
distance
traveled traveled
M C-J M,,
2,s ’
from meniscus
by unknown
from meniscus
by standard
assuming the same partial specific volume for the proteinase and the standards (11). Proteinase assays. The proteinase assay contained in a total volume of 80 al: 10 pmol of Tris/ HCl, pH 8.0; 0.036 mg of total [3H]lysine histones from Ehrlich ascites tumor cells (sp act 7.8 x 10” dpmimg protein), and the enzyme to be tested. The
ET
AL.
incubation time was 60 min at 37°C. Before the reaction was stopped by addition of 20 ~1 of TCA (110 g/100 ml), 20 ~1 of bovine serum albumin (10 mg/ml) was added. The reaction mixture was centrifuged at 12,000g for 5 min, and 100 ~1 of supernatant was used for scintillation counting in 10 ml of toluenei Triton X-100 scintillator, 2:l (I). A K, of 0.4 mgiml is obtained for total histones from Ehrlich ascites tumor cells used in the proteinase assay. Because of the limited amount of 13H1Lys histones available, the assays were carried out with suboptimal substrate concentrations. The assay was linear with time of incubation and amount of enzyme. Alternatively, the proteinase activity was assayed with the synthetic substrate N-acetyl-L-tyrosine ethyl ester in a coupled assay. The enzymatic hydrolysis of ATEE results in the formation of ethanol. The ethanol was measured with alcohol dehydrogenase and NAD by the increase in absorbance at 334 nm in a spectrophotometer. The proteinase assay contained in a final volume of 1.5 ml the following components: 0.15 M sodium pyrophosphate, pH 8.0; semicarbazide, 0.23 mmol; glycine, 0.06 mmol; NAD, 1 mg; alcohol dehydrogenase, 0.3 mg; ATEE (20 ~1 of a solution, containing 200 mg/ml dimethylsulfoxide); and the enzyme to be assayed. The assay was linear with time of incubation and amount of enzyme (not shown). Purification of the enzyme. Preparation of mitochondria (step 1). The mitochondria were prepared according to Loewenstein et al. (12). In the following purification procedure 270 g of liver tissue of adult male Wistar rats (250-300 g) was used as starting material, leading to 270 ml of a mitochondrial suspension. After freezing and thawing twice, the mitochondrial suspension was centrifuged in aliquots of 54 ml (step 2). Sucrose gradient centrifugation (step Z,J. This step was carried out five times with 54 ml of the mitochondrial fraction each time. The mitochondrial fraction of step 1 was layered in six g-ml aliquots on top of 12 ml of 1.0 M sucrose above 15 ml of 1.7 M sucrose, 10 mM Tris, pH 7.5 and centrifuged in a Spinco SW27 rotor at 27,000 rpm (135,OOOg) for 2 h. The six pellets were suspended in a total of 1.8 ml of distilled water. The volume of enzyme suspension at this step was 5 x 1.8 ml = 9 ml. Solubilization of the enzyme (step 3). The enzyme suspension of step 2 was centrifuged at 12,000g for 5 min; the sediment was resuspended, centrifuged five times in 20 ml of 20 mM Tris/HCl, pH 7, and finally resuspended in 9.0 ml of 0.5 M potassium phosphate buffer, pH 8.5. After incubation at 4°C for at least 2 days, the mixture was again centrifuged. The supernatant contained the soluble enzyme. A higher yield of the soluble proteinase was obtained after longer or repeated incubation of the sediment with 0.5 M potassium phosphate buffer. Acetone fractionation (step 4). A volume of 13.5 ml
PROTEINASE
FROM
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357
LIVER
Purity
of cold (-30°C) acetone was added to 9 ml of the solubilized enzyme (step 3) to give a final concentration of 60% (v/v). The precipitate was sedimented by centrifugation at 48,OOOg for 3 min and suspended in 3.6 ml of 0.1 M potassium phosphate buffer, pH 7.5. After centrifugation at 12,000g for 5 min, the clear supernatant was used for hydroxylapatite adsorption. Hydroxylupatite adsorption (step 5). A volume of 3.6 ml of step 4 enzyme, 7.2 ml of distilled H20, and 2.1 g (wet weight) of hydroxylapatite were incubated at 4°C for 30 min under vigorous shaking. The suspension was centrifuged at 12,000g for 5 min. The sediment was washed three times with an excess of 20 mM Tris/HCl, pH 7.0 and suspended in 11 ml of 50 mM sodium phosphate buffer, pH 9.0. The enzyme was eluted by addition of 9.0 ml of 2 M NaCl. After incubation for 15 min at 4”C, the suspension was centrifuged at 12,OOOg for 5 min. The supernatant was dialyzed against 0.2 M potassium phosphate buffer, pH 8.0 and concentrated in an Amicon ultrafiltration cell (8 MC Micro-UF System).
The final proteinase preparation was subjected to acrylamide-gel electrophoresis. The enzyme exhibited a low mobility in the Tris/glycine buffer system (pH 8.9) (Fig. la). When a pH 2.6 separation system was used, the enzyme gave essentially one band (Fig. lb), which was absent when the proteinase was incubated with trypsin inhibitor bound to CM-cellulose prior to acrylamide gel electrophoresis (Table II). When the enzyme preparation was subjected to SDS-gel electrophoresis, one main band was found. In addition to this band, representing the proteinase, a fur-
RESULTS
Purification The purification procedure detailed in Materials and Methods and summarized in Table I begins with the isolation of mitochondria from a rat liver homogenate. The mitochondria were treated with digitonin in order to destroy lysosomes (12). The proteinase obtained after centrifugation through 1.7 M sucrose, 10 mM Tris/ HCl, pH 7.5 is insoluble in buffers of low molarities. No solubilization is achieved by use of 1% Triton X-100 or 1% SDS or 2 M NaCl-5 M urea. The enzyme can be solubilized by incubation with 0.5 M potassium phosphate buffer, pH 8.5 at 4°C for several days. The final enzyme preparation showed a 1330-fold increase in specific activity over the mitochondrial preparation.
‘
-.i
FIG. 1. Acrylamide-gel electrophoresis of purified proteinase: (a) 7.5% acrylamide, pH 8.9, Tris/ glycine buffer, pH 8.3 (8); (b) 15% acrylamide, 6.25 M urea, pH 3.2, 0.9 N acetic acid as electrolyte (9). Electrophoresis was carried out at a constant current of 2 mA/tube for 2-3 h. Samples of 10 pg (a) and 30 pg (b) of protein were applied to the gels.
TABLE PURIFICATION
Purification
1. 2. 3. 4. 5.
step
Volume (ml)
Mitochondria Sucrose gradient Phosphate buffer extract Acetone fractionation Hydroxylapatite adsorption a 1 unit
= 1 Fmol
of ATEE
OF THE
270 9.0 9.0 3.6 2.0 split
per minutc?at
b
a
I
MITOCHONDRIAL
Total
protein (mg)
9180 175 17 0.8 0.24 30°C under
PROTEINASE
Total activity (units)” 103 18 15.0 6.1 3.2
assay
conditions
Specific activity (units/ mg)
Purification factor (-fold)
0.01
1
0.10 0.88 7.6 13.3
10 88 760 1330
(see Materials
Yield (%I
100
and Methods).
18 15 6 3
358
JUSIC TABLE EFFECT
II
OF PROTEINASE Inhibitor
Control Soybean trypsin Kallikrein trypsin Yeast proteinase Yeast proteinase Yeast proteinase Pepstatin DFP PhMeSO,F TLCK TPCK p-Chloromercuribenzoate 2-Mercaptoethanol Dithioerythritol
inhibitor inhibitor inhibitorI* inhibitor inhibitor
INHIBITORS” Proteinase activity (%)
IB Ic
100 9 87 88 89 84 102 0 11 53 3 93 84 64
u The data are given as a percentage of control = no inhibitor added. The preincubation mixture contained, in a volume of 70 yl, Tris/HCl (pH 81, 10 pmol; proteinase, 10 pg, and the following inhibitors: soybean trypsin inhibitor, 20 pg; kallikrein trypsin inhibitor, 20 pg; yeast proteinase inhibitor I*, 10 gg = 0.16 units (13); yeast proteinase inhibitor IB, 12 pg = 0.6 units (14); yeast proteinase inhibitor Ic, 10 pg = 0.45 units (15); pepstatin, 10 fig; DFP, 1.0 pmol; PhMeSO,F, 2 Fmol; TLCK, 1.0 pmol, and TPCK, 1.0 Fmol dissolved in 10 ~1 of DMSO; pchloromercuribenzoate, 0.05 pmol; 2-mercaptoethanol, 18 pmol; dithioerythritol, 1 pmol. After a preincubation time of 30 min at 25°C the reaction was started by the addition of 0.054 mg of [SH]lysine histones. After another 30 min of incubation at 37”C, the reaction was stopped by addition of 20 ~1 of bovine serum albumin (IO mg/ml) and 20 ~1 of TCA (110%). The assay tubes were centrifuged and 100 ~1 of supernatant was used for the determination of radioactivity.
ther protein band with a slightly higher electrophoretic mobility was found. Since the enzymatic activity was lost under the conditions of SDS-gel electrophoresis, the proteinase was identified as a radioactively labeled and enzymatically inactive enzyme after reaction with [1,3-3Hldiisopropyl phosphofluoridate. Whether the additional band is a result of autodigestion or still an impurity of the proteinase is not known. Stability, Effect of pH, and Temperature The enzyme is rather stable. Repeated freezing and thawing does not affect the
ET
AL.
enzymatic activity. Preincubation at pH values of 6.5-11.0 at room temperature for 60 min does not change the enzymatic activity. Preincubation at pH values of 4.5 5.5 results in a 50% decrease in activity. The enzyme, assayed with total histones as substrate, shows a pH optimum of around 8.0. The enzyme is essentially inactive below pH 5 and above pH 11.0. The extent of inactivation as a function of temperature was also studied. Eightyeight percent of the activity is lost by heating above 65°C for 5 min. Molecular
Weight
Sucrose density gradient centrifugation was used for the determination of the molecular weight. From the distance traveled by the proteinase in relation to the distance traveled by the standards (lysozyme, lactate dehydrogenase, and catalase), a molecular weight of 22,500 +- 10% was calculated (11). A molecular weight of 23,500 2 10% was estimated by SDS-acrylamide gel electrophoresis. Effect of Metal Ions and EDTA Neither monovalent metal ions (Na+, K+, NH,+) nor divalent metal ions (Mg2+, Ca2+ Mn2+, Zn2+, Hgz+) in final concentrations of 1.0 or 10 mM had any influence on the enzymatic activity. EDTA in a 1.0 or 10 mM concentration was also without effect on the proteinase activity. Effects
of Inhibitors
Table II summarizes the effects of various proteinase inhibitors. The strongest inhibition was obtained with soybean trypsin inhibitor. Kallikrein trypsin inhibitor, the yeast proteinase inhibitors I*, IB, and Ic, and the bacterial proteinase inhibitor pepstatin had little effect on the proteinase activity. p-Chloromercuribenzoate did not affect the enzymatic activity. Therefore, the involvement of a thiol group at the active site of the enzyme seems to be unlikely. 2Mercaptoethanol and dithioerythritol, however, were partially inhibitory. The proteinase was strongly inhibited by the seryl reagents DFP and PhMeOsF, indi-
PROTEINASE
FROM
eating a serine-type enzyme. Of the two chloromethyl ketones tested, TLCK and TPCK, the affinity reagent for chymotrypsin-like active sites, TPCK, strongly inactivated the enzyme. The presence of a histidine residue at the active site of the enzyme also could be shown by the methylene blue mediated photooxidation, which led to complete loss of proteinase activity. Substrate
Specificity
The studies on substrate specificity were carried out either with the purified enzyme preparation or with the insoluble enzyme obtained after centrifugation of mitochondria through 1.7 M sucrose. No qualitative or quantitative differences were found for the two enzyme preparations with respect to the substrate specificity. The advantage of using the insoluble enzyme for incubation with the various substrates summarized in Table III is that the proteolytic reaction can be terminated by removing the enzyme from the incubation mixture by centrifugation. In the case of the pure soluble proteinase, the degradation was stopped by addition of SDS and 2-mercaptoethanol and heat treatment (95”C, 3 min). After incubation of the different protein substrates with and without proteinase, the proteins from the incubation mixtures were applied to SDS-acrylamide gels, subjected to electrophoresis, stained, and scanned. All protein substrate concentrations were kept at 0.5 mg/ml of incubation mixture. The protein amounts added to the gels fell into the range where the intensities of the stained protein bands were linear with amount of protein (lo-50 pg). It can be seen from Table III that the proteinase shows a high specificity for chromosomal proteins. Histones as well as nonhistone chromosomal proteins are readily degraded. It has been shown previously that histone Hl is degraded in a very specific manner (16). The degradation pattern of histone Hl is shown in Figs. 2a-e. The formation of one major degradation product was observed, even if the time of incubation was extended or the amount of enzyme was increased. These results indicate a limited proteolysis of Hl. Wheh the
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359
LIVER TABLE
SUBSTRATE
SPECIFICITY
III OF THE
Substrate
Histone Hl Total histones Nonhistone chromosomal Ovalbumin Myoglobin Cytochrome c Bovine serum albumin Lysozyme Insulin Glucagon Azocasein Azocoll
PROTEINASE”
Degradation (% of control)
proteins
100 46 48 0 0 0 0 0 3 95 0 0
n The proteinase assay contained in a final volume of 0.1 ml Tris/HCl, pH 8.0, 5 pmol; protein substrate, 15-50 pg; and insoluble proteinase, 0.02 mg (sp act 0.2 kmol N-acetyl-L-tyrosine ethyl ester split per minute x milligram). After vigorous shaking for 60 min at 37”C, the incubation mixture was centrifuged at 12,000g for 5 min. Twenty microliters of supernatant was mixed with 20 ~1 of 2% SDS, 2% 2-mercaptoethanol, and 0.05 M Tris, pH 8.1, and kept at 95°C for 5 min. After the addition of 5 ~1 of bromophenol blue (0.01%) and 10 ~1 of glycerol (87%) the mixture was applied to SDS gels (IO). The protein bands were stained with Coomassie blue and scanned. The intensity of the band obtained from incubations without added proteinase was used as control (=lOO%l. In the case of experiments with azocasein and azocoll, the incubation mixture contained, in a total volume of 0.5 ml, TrisiHCl pH 8, 5 pmol; azocoll or azocasein, respectively, 12 mg; and proteinase, 0.12 mg (sp act 0.2 wmol N-acetyl-r.tyrosine ethyl ester split per minute x milligram). After 60 min at 37”C, 0.5 ml of TCA was added. After centrifugation, the clear supernatants were measured at 520 or 422 nm, respectively, against a blank without addition of enzyme.
degradation product in the fast-moving protein band was eluted and subjected to SDS-acrylamide gel (15%) electrophoresis, one main protein band and a diffuse zone were observed. From the electrophoretie mobility a molecular weight of 13,000 2 10% has been estimated for the main protein band. The diffuse zone of high electrophoretic mobility contains further degradation products. It was not possible to decide by SDS gel electrophoresis whether this zone consists of one or
JUSIC
ET
AL.
d
e
FIG. 2. Degradation pattern of the very-lysine-rich histone Hl: (a) histone HI; (b) histone Hl plus proteinase, incubated for 3 min, (c) 6 min, (d) 10 min, (e) 30 min. The incubation mixture contained, in a total volume of 0.1 ml: 5 wmol of Tris/HCl, pH 8,18 wmol of histone Hl, and 8 yg of proteinase (sp act 2.0 pmol N-acetyl-L-tyrosine ethyl ester split/min x mg protein) (b-e). After incubation at 37”C, the reaction was stopped by the addition of 20 ~1 of 4.5 N acetic acid. The a&amide-gel electrophoresis was carried out according to Panyim and Chalkley (9).
more proteins of molecular weights of
seen in Fig. 3 that the large polypeptides were more rapidly degraded than the small ones. Under conditions optimal for histone and nonhistone proteolysis, the only other substrate found to be degraded by the proteinase was glucagon. Glucagon degradation was also observed with [Y]glucagon. On the other hand, insulin and [12511insulin were resistant to the proteinase action (Fig. 4). From a Lineweaver-Burk plot, a K, of 0.7 x 10m3 M was obtained for [12511glucagon. No degradation of azocoll and azocasein could be observed. Furthermore, alcohol dehydrogenase (yeast), malate dehydrogenase (pig heart), and glucose 6-phosphate dehydrogenase (yeast) were used as
PROTEINASE
FROM
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361
LIVER
cal plot, an apparent Michaelis constant for the hydrolysis of ATEE of 5.0 x lo-” M was determined. 8 DISCUSSION
a
b
The proteinase described here has several properties similar to trypsin and chymotrypsin: the pH optimum around 8, the inhibition by soybean trypsin inhibitor, and the inactivation by the serine-proteinase reagents DFP and PhMeSO,F. The presence of a histidine at the active site was shown by reaction with TPCK and the methylene blue-mediated photooxidation. On the other hand, very few substrates were found, which indicates a high specificity of the enzyme. Among the substrates, chosen rather arbitrarily (Table III), only glucagon and chromosomal proteins were degraded. It has been shown recently (17) that glucagon has very little secondary structure, i.e., all peptide bonds are easily accessible to proteolytic attack. This is not the case with the highly ordered insulin molecule. It was the histone degradation which led us to the studies on this proteinase. Some interesting features in respect to the his-
FIG. 3. Degradation of nonhistone chromosomal proteins. The nonhistone proteins used as substrate were prepared according to (6). The incubation conditions are described in the legend to Table III: (a) incubation in the presence of proteinase; (b) control, no proteinase added. The SDS gel-acrylamide electrophoresis was carried out according to King and Laemmli (101; 50 pg of protein was applied to the gel (bl.
substrates for the insoluble proteinase in concentrations similar to those indicated in Table III. After incubation at 37°C for 60 min, aliquots of the incubation mixture were assayed. In no case could a loss of enzymatic activity be demonstrated. Among various synthetic substrates only ATEE and N-benzoyl-L-arginine ethyl ester were readily hydrolyzed, whereas N-benzoyl-L-tyrosine-p-nitroanilide and a number of otherp-nitroanilides of various acylated and nonacylated amino acids were not attacked at all. From a Lineweaver-Burk double-r&ipro-
[““I]
GLUCAGON
[““lj
INSULIN
()g ( yg)
) 0 A
FIG. 4. Proteolytic degradation of [‘2511glucagon and [‘251]insulin. In a total volume ofO.1 ml, 15 wmol of Tris/HCl, pH 8.0, 0.02 mg of proteinase and [lzJI]glucagon (sp act 12,400 cmplmg), or [‘*“I]insulin (sp act 21,000 cpmimg), respectively, were incubated for 30 min at 37°C. The reaction was stopped by addition of 20 ~1 of bovine serum albumin (10 mgi ml) and 20 ~1 of TCA (110%). Supernatant (100 ~1) was used for the determination of radioactivity in 10 ml of a 2:l toluene/Triton X-100 scintillation liquid. The counting efficiency for ““I was 85% in the l-‘Cchannel.
362
JUSIC
tone degradation were found. A characteristic fragment of a molecular weight of 13,000 is obtained by limited proteolysis of the very-lysine-rich histone HI (Fig. 2). A similar digestion of histone Hl with (Ychymotrypsin has been described recently by Bradbury et al. (18). These authors were able to split the Hl molecule into an N-terminal (l-106) and a C-terminal fragment (107212). In contrast to the degradation by the proteinase described in this work, a limited proteolysis with a-chymotrypsin was not observed by Bradbury et al. After the rapid initial cleavage of the Hl molecule by a-chymotrypsin, both fragments are further degraded. Because of the tendency of nonhistone chromosomal proteins to aggregate, only that fraction of the nonhistone proteins which is solubilized in 0.4 M NaCl was tested as substrate. Our finding that the large polypeptides of this nonhistone cbromosomal protein fraction tend to be degraded more rapidly than small ones (Fig. 3) is consistent with the hypothesis of correlation between turnover rate and protein molecular size (19). The proteinase which we isolated has properties similar to the enzyme that was purified from calf thymus chromatin by Kurecki and Toczko (3): for example, molecular weight, pH optimum, and inhibition by PhMeS03F. The proteinase activity associated with chromatin isolated according to the procedure of Bonner et al. (20) very probably originates from particles in the mitochondrial fraction (1). There are a few discrepancies between the properties of the proteinase isolated from rat liver chromatin by the group of Bonner (2) and the proteinase described here. A higher molecular weight and inhibition by Hg’+ ions has been described for the enzyme obtained from chromatin. The substrate specificities and the inhibition by PhMeSOzF are the same. From the finding that 18% of the proteinase activity found in isolated mitochondria sediment through 1.7 M sucrose (Table I, step 2) -the same sucrose concentration as used by Bonner for the preparation of chromatin -it is evident that the mitochondrial proteinase cosediments with chromatin and thus contaminates it.
ET AL.
From our results, it is likely that the proteinase is associated with the mitochondrial membrane. The possibility that the proteinase is of lysosomal origin and found associated with the mitochondrial membrane as a result of homogenization of the liver tissue cannot be completely excluded. The following experiment, however, favors a mitochondrial localization. Highly purified lysosomes, frozen and thawed twice, were added to minced rat liver and homogenized together with the liver tissue. Mitochondria isolated with lysosomes added and without lysosomes were assayed for proteinase activity. No difference with respect to proteinase activities was found for the two preparations. This finding is regarded as strong evidence for the mitochondrial origin of the proteinase. There is evidence for proteolytic activity in rat liver mitochondria from several publications (21-24). The purification of a proteinase from the mitochondrial fraction was described recently by Katunuma et al. (25). Katunuma et al. also found a high degree of substrate specificity for their enzyme. They called it “group specific” since they found only apoenzymes of pyridoxaldependent enzymes to be degraded. The proteinase of Katunuma et al. and our proteinase are similar with respect to their pH optima, sensitivity to inhibition by serine proteinase inhibitors, and probable mitochondrial localization. On the other hand, the two proteinases appear to be different in several properties. In our experience it is impossible to do Sephadex G-75 gel filtration and lyophilization under the conditions given by Katunuma et al. without losing all the enzymatic activity. Sephadex chromatography of the proteinase is only possible if Triton X-100 is present in the buffer. From the work of Katunuma et al. it is not clear whether the molecular weight of the proteinase from rat liver is 17,000 (Ref. 25, p. 43) or 13,000 (Ref. 25, Fig. 4). We find molecular weights of 22,500 and 23,500 by two independent methods. It is not yet possible to decide conclusively whether these two enzymes are the same. Experiments are in progress to determine whether the proteinase described in this paper degrades apo-orni-
PROTEINASE
FROM
thintransaminase in addition to glucagon and chromosomal proteins. Further work will be required to determine whether the proteinase described has any physiological role in the degradation of chromosomal proteins. ACKNOWLEDGMENTS The authors thank Professor R. L. Switzer for a critical reading of this manuscript. We also thank Professor H. Holzer for his interest in and support of this work. REFERENCES 1. HEINRICH, P. C., RAYDT, G., PUSCHENDORF, B., AND JUSIC, M. (1976) Eur. J. Biochem. 62, 3743. 2. CHONG, M. T., GARRARD, W. T., AND BONNER, J. (1974) Biochemistry 13, 5128-5134. 3. KURECKI, T., AND TOCZKO, K. (1974) Acta Biochim. Polon. 21, 225-233. 4. LEVIN, 0. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.),Vol V, p. 27. Academic Press, New York/London. 5. BALHORN, R., AND CHALKLEY, R. (1975) in Methods in Enzymology (O’Malley, B. W., and Hardman, J. G., eds.), Vol. 40, p. 138, Academic Press, New York/London. 6. KISH, V. M., AND KLEINSMITH, L. J. (1974) J. Biol. Chem. 249, 750-760. 7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951)5. Biol. Chem. 193, 265-275. 8. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 9. PANYIM, S., AND CHALKLEY, R. (1969)Arch. Biothem. Biophys. 130, 337-346. 10. KING, J., AND LAEMMLI, U. K. (1971) J. Mol. Biol. 62, 465-477.
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11. MARTIN, R. G., AND AMES, B. N. (196115. Biol. Chem. 236, 1372-1379. 12. LOEWENSTEIN, J., SCHOLTE, H. R., AND WITPEETERS, E. M. (1970) Biochim. Biophys. Acta 223, 432-436. 13. SAHEKI, T., MATSUDA, Y., AND HOLZER, H. (1974) Eur. J. Biochem. 47, 325-332. 14. BETZ, H., HINZE, H., AND HOLZER, H. (1974) J. Biol. Chem. 249, 4515-4521. 15. MATERN, H., HOFFMANN, M., AND HOLZER, H. (1974) Proc. Nut. Acad. Sci. USA 71, 48744878. 16. HAAS, R., WEISS, E., JUSIC, M., AND HEINRICH, P. C. (1976) FEBS Lett., 63, 117-119. 17. CHOU, P. Y., AND FASMAN, G. D. (1975) Biochemistry 14, 2536-2541. 18. BRADBURY, E. M., CHAPMAN, G. E., DANBY, S. E., HARTMANN, P. G., AND RICHES, P. L. (1975) Eur. J. B&hem. 57, 521-528. 19. DICE, J. F., AND GOLDBERG, A. L. (1975) Arch. Biochem. Biophys. 170, 213-219. 20. BONNER, J., CHALKLEY, G. R., DAHMUS, M., FAMBROUGH, D., FUJIMURA, F., HUANG, R.C. C., HUBERMAN, J., JENSEN, R., MARUSHIGE, K., OHLENBUSCH, H., OLIVERA, B., AND WIDHOLM, J. (1968) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, pp. 3-65, Academic Press, New York. 21. ALBERTI, K. G. M. M., AND BARTLEY, W. (1965) Biochem. J. 95, 641-656. 22. BAIRD, G. D. (1964) Biochim. Biophys. Acta 93, 293-303. 23. FERDINAND, W., BARTLEY, W., AND BROOMHEAD, V. (1973) Biochem. J. 134, 431-436. 24. L~VAAS, E. (1974) FEBS Lett. 45, 244-247. 25. KATUNUMA, N., KOMINAMI, E., KOBAYASHI, K., BANNO, Y., SUZUKI, K., CHICHIBU, K., HAMAGUCHI, Y., AND KATSUNUMA, T. (1975)&u. J. Biochem. 52, 37-50.
OF
Isolation
BIOCHEMISTRY
AND
BIOPHYSICS
and Characterization
177,
3X-363
of a Membrane-Bound Rat Liver
MIRNA JUSIC, SILVIA RAINER HAAS, AND Biochemisches
Institut
(1976)
der Universittit,
from
SEIFERT, ERICH WEISS, PETER C. HEINRICH
D-78
Received
Proteinase
Freiburg, March
Hermann-Herder-Strap
7, Germany
31, 1976
The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 M phosphate buffer. After a 1330fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 2 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone Hl, which was split into a high molecular weight fragment (M, 13,000). The highly phosphorylated histone Hl isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as Hl from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-L-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-L-tyrosine ethyl ester is 5.0 x lo-:’ M. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, L-l-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.
In the course of our studies on the regulation of rat liver chromosomal protein degradation, a histone degrading enzyme was found in the mitochondrial fraction (1). We showed that this proteinase contaminates chromatin prepared from a total homogenate (1). The degradation of histones in isolated chromatin found by several authors (2,3) may be due to the action of this proteinase. In the present paper, we describe the purification and some properties of this enzyme. MATERIALS
AND
ginine, bovine serum albumin, egg albumin, N-benzoyl-L-arginine ethyl ester, and ATEE’ were products of Serva (Heidelberg, Germany); alcohol dehydrogenase, glucose 6-phosphate dehydrogenase, malate dehydrogenase, and cytochrome c were from Boehringer (Mannheim, Germany); dithioerythritol (Cleland’s reagent) was from Calbiochem (Zurich, Switzerland); sucrose (ultrapure) was from Schwarzi Mann (Orangeburg, N.Y.). [l~SI]Insulin (sp act 165 mCi/mg) and [‘2SI]glucagon (sp act 170 mCi/mg) were obtained from Behringwerke AG (Marburg, Germany). Hydroxylapatite was prepared as de1 Abbreviations used: ATEE, N-acetyl-L-tyrosine ethyl ester; DFP, diisopropyl phosphofluoridate; fluoride; PhMeSO,F, phenylmethanesulfonyl TLCK, 1-chloro-3-tosylamido-7-amino-L-2-heptanone; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; CM, carboxymethyl.
METHODS
Materials Digitonin, trypsin, were obtained from phenylmethanesulfonyl
and soybean trypsin inhibitor Merck (Darmstadt, Germany); fluoride, polylysine, polyar355
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
JUSIC
356
scribed by Levin (4). The yeast inhibitors of proteinases A and B and carboxypeptidase Y were kindly supplied by Prof. Dr. H. Holzer. Pepstatin was a gift of Dr. Umezawa and kallikrein trypsin inhibitor (Trasylol) was kindly supplied by Dr. E. Truscheit. All the other chemicals were of the highest purity grade available.
Methods Preparation ofproteinase substrates. The preparation of [3H]lysine-labeled histones from Ehrlich ascites tumor cells has been described previously (1). The very-lysine-rich histone Hl was isolated from total rat liver histones according to Balhorn and Chalkley (5). The nonhistone chromosomal protein fraction used was prepared from rat liver nuclei as detailed by Kish and Kleinsmith (6). The preparation was used a&er Biorex 70 treatment. Protein determination. Protein was determined by the method of Lowry et al. (7) with bovine serum albumin as standard. AC&amide-gel electrophoresis. Polyacrylamide gel electrophoresis was carried out as described by Davis (8) with Tris/glycine buffer with a running pH of 8.9, or according to the technique of Panyim and Chalkley (9) with 0.9 N acetic acid, pH 2.6. SDS gel electrophoresis was performed according to King and Laemmli (10). Densitometry was conducted by use of a Gilford Model 2520 gel scanner. Estimation of molecular weight. The molecular weight was estimated by sucrose gradient centrifugation (11). The proteinase (0.04 mg, sp act 7.0 pmol ATEE split/min x mg) mixed with 200 fig of egg white lysozyme, 25 wg of rabbit muscle lactate dehydrogenase, and 20 pg of catalase in a total volume of 0.2 ml was layered on top of a linear gradient of 5 to 20% sucrose in 0.05 M Tris/HCl buffer, pH 7.5. After centrifugation at 280,OOOg for 15 h (SW41 rotor, 40,000 rpm), 51 fractions of 0.24 ml each were collected at a flow rate of 18 ml/h and assayed for enzymatic activities. The molecular weight of the proteinase was estimated from 81 -= S2 distance S, S2
distance
traveled traveled
M C-J M,,
2,s ’
from meniscus
by unknown
from meniscus
by standard
assuming the same partial specific volume for the proteinase and the standards (11). Proteinase assays. The proteinase assay contained in a total volume of 80 al: 10 pmol of Tris/ HCl, pH 8.0; 0.036 mg of total [3H]lysine histones from Ehrlich ascites tumor cells (sp act 7.8 x 10” dpmimg protein), and the enzyme to be tested. The
ET
AL.
incubation time was 60 min at 37°C. Before the reaction was stopped by addition of 20 ~1 of TCA (110 g/100 ml), 20 ~1 of bovine serum albumin (10 mg/ml) was added. The reaction mixture was centrifuged at 12,000g for 5 min, and 100 ~1 of supernatant was used for scintillation counting in 10 ml of toluenei Triton X-100 scintillator, 2:l (I). A K, of 0.4 mgiml is obtained for total histones from Ehrlich ascites tumor cells used in the proteinase assay. Because of the limited amount of 13H1Lys histones available, the assays were carried out with suboptimal substrate concentrations. The assay was linear with time of incubation and amount of enzyme. Alternatively, the proteinase activity was assayed with the synthetic substrate N-acetyl-L-tyrosine ethyl ester in a coupled assay. The enzymatic hydrolysis of ATEE results in the formation of ethanol. The ethanol was measured with alcohol dehydrogenase and NAD by the increase in absorbance at 334 nm in a spectrophotometer. The proteinase assay contained in a final volume of 1.5 ml the following components: 0.15 M sodium pyrophosphate, pH 8.0; semicarbazide, 0.23 mmol; glycine, 0.06 mmol; NAD, 1 mg; alcohol dehydrogenase, 0.3 mg; ATEE (20 ~1 of a solution, containing 200 mg/ml dimethylsulfoxide); and the enzyme to be assayed. The assay was linear with time of incubation and amount of enzyme (not shown). Purification of the enzyme. Preparation of mitochondria (step 1). The mitochondria were prepared according to Loewenstein et al. (12). In the following purification procedure 270 g of liver tissue of adult male Wistar rats (250-300 g) was used as starting material, leading to 270 ml of a mitochondrial suspension. After freezing and thawing twice, the mitochondrial suspension was centrifuged in aliquots of 54 ml (step 2). Sucrose gradient centrifugation (step Z,J. This step was carried out five times with 54 ml of the mitochondrial fraction each time. The mitochondrial fraction of step 1 was layered in six g-ml aliquots on top of 12 ml of 1.0 M sucrose above 15 ml of 1.7 M sucrose, 10 mM Tris, pH 7.5 and centrifuged in a Spinco SW27 rotor at 27,000 rpm (135,OOOg) for 2 h. The six pellets were suspended in a total of 1.8 ml of distilled water. The volume of enzyme suspension at this step was 5 x 1.8 ml = 9 ml. Solubilization of the enzyme (step 3). The enzyme suspension of step 2 was centrifuged at 12,000g for 5 min; the sediment was resuspended, centrifuged five times in 20 ml of 20 mM Tris/HCl, pH 7, and finally resuspended in 9.0 ml of 0.5 M potassium phosphate buffer, pH 8.5. After incubation at 4°C for at least 2 days, the mixture was again centrifuged. The supernatant contained the soluble enzyme. A higher yield of the soluble proteinase was obtained after longer or repeated incubation of the sediment with 0.5 M potassium phosphate buffer. Acetone fractionation (step 4). A volume of 13.5 ml
PROTEINASE
FROM
RAT
357
LIVER
Purity
of cold (-30°C) acetone was added to 9 ml of the solubilized enzyme (step 3) to give a final concentration of 60% (v/v). The precipitate was sedimented by centrifugation at 48,OOOg for 3 min and suspended in 3.6 ml of 0.1 M potassium phosphate buffer, pH 7.5. After centrifugation at 12,000g for 5 min, the clear supernatant was used for hydroxylapatite adsorption. Hydroxylupatite adsorption (step 5). A volume of 3.6 ml of step 4 enzyme, 7.2 ml of distilled H20, and 2.1 g (wet weight) of hydroxylapatite were incubated at 4°C for 30 min under vigorous shaking. The suspension was centrifuged at 12,000g for 5 min. The sediment was washed three times with an excess of 20 mM Tris/HCl, pH 7.0 and suspended in 11 ml of 50 mM sodium phosphate buffer, pH 9.0. The enzyme was eluted by addition of 9.0 ml of 2 M NaCl. After incubation for 15 min at 4”C, the suspension was centrifuged at 12,OOOg for 5 min. The supernatant was dialyzed against 0.2 M potassium phosphate buffer, pH 8.0 and concentrated in an Amicon ultrafiltration cell (8 MC Micro-UF System).
The final proteinase preparation was subjected to acrylamide-gel electrophoresis. The enzyme exhibited a low mobility in the Tris/glycine buffer system (pH 8.9) (Fig. la). When a pH 2.6 separation system was used, the enzyme gave essentially one band (Fig. lb), which was absent when the proteinase was incubated with trypsin inhibitor bound to CM-cellulose prior to acrylamide gel electrophoresis (Table II). When the enzyme preparation was subjected to SDS-gel electrophoresis, one main band was found. In addition to this band, representing the proteinase, a fur-
RESULTS
Purification The purification procedure detailed in Materials and Methods and summarized in Table I begins with the isolation of mitochondria from a rat liver homogenate. The mitochondria were treated with digitonin in order to destroy lysosomes (12). The proteinase obtained after centrifugation through 1.7 M sucrose, 10 mM Tris/ HCl, pH 7.5 is insoluble in buffers of low molarities. No solubilization is achieved by use of 1% Triton X-100 or 1% SDS or 2 M NaCl-5 M urea. The enzyme can be solubilized by incubation with 0.5 M potassium phosphate buffer, pH 8.5 at 4°C for several days. The final enzyme preparation showed a 1330-fold increase in specific activity over the mitochondrial preparation.
‘
-.i
FIG. 1. Acrylamide-gel electrophoresis of purified proteinase: (a) 7.5% acrylamide, pH 8.9, Tris/ glycine buffer, pH 8.3 (8); (b) 15% acrylamide, 6.25 M urea, pH 3.2, 0.9 N acetic acid as electrolyte (9). Electrophoresis was carried out at a constant current of 2 mA/tube for 2-3 h. Samples of 10 pg (a) and 30 pg (b) of protein were applied to the gels.
TABLE PURIFICATION
Purification
1. 2. 3. 4. 5.
step
Volume (ml)
Mitochondria Sucrose gradient Phosphate buffer extract Acetone fractionation Hydroxylapatite adsorption a 1 unit
= 1 Fmol
of ATEE
OF THE
270 9.0 9.0 3.6 2.0 split
per minutc?at
b
a
I
MITOCHONDRIAL
Total
protein (mg)
9180 175 17 0.8 0.24 30°C under
PROTEINASE
Total activity (units)” 103 18 15.0 6.1 3.2
assay
conditions
Specific activity (units/ mg)
Purification factor (-fold)
0.01
1
0.10 0.88 7.6 13.3
10 88 760 1330
(see Materials
Yield (%I
100
and Methods).
18 15 6 3
358
JUSIC TABLE EFFECT
II
OF PROTEINASE Inhibitor
Control Soybean trypsin Kallikrein trypsin Yeast proteinase Yeast proteinase Yeast proteinase Pepstatin DFP PhMeSO,F TLCK TPCK p-Chloromercuribenzoate 2-Mercaptoethanol Dithioerythritol
inhibitor inhibitor inhibitorI* inhibitor inhibitor
INHIBITORS” Proteinase activity (%)
IB Ic
100 9 87 88 89 84 102 0 11 53 3 93 84 64
u The data are given as a percentage of control = no inhibitor added. The preincubation mixture contained, in a volume of 70 yl, Tris/HCl (pH 81, 10 pmol; proteinase, 10 pg, and the following inhibitors: soybean trypsin inhibitor, 20 pg; kallikrein trypsin inhibitor, 20 pg; yeast proteinase inhibitor I*, 10 gg = 0.16 units (13); yeast proteinase inhibitor IB, 12 pg = 0.6 units (14); yeast proteinase inhibitor Ic, 10 pg = 0.45 units (15); pepstatin, 10 fig; DFP, 1.0 pmol; PhMeSO,F, 2 Fmol; TLCK, 1.0 pmol, and TPCK, 1.0 Fmol dissolved in 10 ~1 of DMSO; pchloromercuribenzoate, 0.05 pmol; 2-mercaptoethanol, 18 pmol; dithioerythritol, 1 pmol. After a preincubation time of 30 min at 25°C the reaction was started by the addition of 0.054 mg of [SH]lysine histones. After another 30 min of incubation at 37”C, the reaction was stopped by addition of 20 ~1 of bovine serum albumin (IO mg/ml) and 20 ~1 of TCA (110%). The assay tubes were centrifuged and 100 ~1 of supernatant was used for the determination of radioactivity.
ther protein band with a slightly higher electrophoretic mobility was found. Since the enzymatic activity was lost under the conditions of SDS-gel electrophoresis, the proteinase was identified as a radioactively labeled and enzymatically inactive enzyme after reaction with [1,3-3Hldiisopropyl phosphofluoridate. Whether the additional band is a result of autodigestion or still an impurity of the proteinase is not known. Stability, Effect of pH, and Temperature The enzyme is rather stable. Repeated freezing and thawing does not affect the
ET
AL.
enzymatic activity. Preincubation at pH values of 6.5-11.0 at room temperature for 60 min does not change the enzymatic activity. Preincubation at pH values of 4.5 5.5 results in a 50% decrease in activity. The enzyme, assayed with total histones as substrate, shows a pH optimum of around 8.0. The enzyme is essentially inactive below pH 5 and above pH 11.0. The extent of inactivation as a function of temperature was also studied. Eightyeight percent of the activity is lost by heating above 65°C for 5 min. Molecular
Weight
Sucrose density gradient centrifugation was used for the determination of the molecular weight. From the distance traveled by the proteinase in relation to the distance traveled by the standards (lysozyme, lactate dehydrogenase, and catalase), a molecular weight of 22,500 +- 10% was calculated (11). A molecular weight of 23,500 2 10% was estimated by SDS-acrylamide gel electrophoresis. Effect of Metal Ions and EDTA Neither monovalent metal ions (Na+, K+, NH,+) nor divalent metal ions (Mg2+, Ca2+ Mn2+, Zn2+, Hgz+) in final concentrations of 1.0 or 10 mM had any influence on the enzymatic activity. EDTA in a 1.0 or 10 mM concentration was also without effect on the proteinase activity. Effects
of Inhibitors
Table II summarizes the effects of various proteinase inhibitors. The strongest inhibition was obtained with soybean trypsin inhibitor. Kallikrein trypsin inhibitor, the yeast proteinase inhibitors I*, IB, and Ic, and the bacterial proteinase inhibitor pepstatin had little effect on the proteinase activity. p-Chloromercuribenzoate did not affect the enzymatic activity. Therefore, the involvement of a thiol group at the active site of the enzyme seems to be unlikely. 2Mercaptoethanol and dithioerythritol, however, were partially inhibitory. The proteinase was strongly inhibited by the seryl reagents DFP and PhMeOsF, indi-
PROTEINASE
FROM
eating a serine-type enzyme. Of the two chloromethyl ketones tested, TLCK and TPCK, the affinity reagent for chymotrypsin-like active sites, TPCK, strongly inactivated the enzyme. The presence of a histidine residue at the active site of the enzyme also could be shown by the methylene blue mediated photooxidation, which led to complete loss of proteinase activity. Substrate
Specificity
The studies on substrate specificity were carried out either with the purified enzyme preparation or with the insoluble enzyme obtained after centrifugation of mitochondria through 1.7 M sucrose. No qualitative or quantitative differences were found for the two enzyme preparations with respect to the substrate specificity. The advantage of using the insoluble enzyme for incubation with the various substrates summarized in Table III is that the proteolytic reaction can be terminated by removing the enzyme from the incubation mixture by centrifugation. In the case of the pure soluble proteinase, the degradation was stopped by addition of SDS and 2-mercaptoethanol and heat treatment (95”C, 3 min). After incubation of the different protein substrates with and without proteinase, the proteins from the incubation mixtures were applied to SDS-acrylamide gels, subjected to electrophoresis, stained, and scanned. All protein substrate concentrations were kept at 0.5 mg/ml of incubation mixture. The protein amounts added to the gels fell into the range where the intensities of the stained protein bands were linear with amount of protein (lo-50 pg). It can be seen from Table III that the proteinase shows a high specificity for chromosomal proteins. Histones as well as nonhistone chromosomal proteins are readily degraded. It has been shown previously that histone Hl is degraded in a very specific manner (16). The degradation pattern of histone Hl is shown in Figs. 2a-e. The formation of one major degradation product was observed, even if the time of incubation was extended or the amount of enzyme was increased. These results indicate a limited proteolysis of Hl. Wheh the
RAT
359
LIVER TABLE
SUBSTRATE
SPECIFICITY
III OF THE
Substrate
Histone Hl Total histones Nonhistone chromosomal Ovalbumin Myoglobin Cytochrome c Bovine serum albumin Lysozyme Insulin Glucagon Azocasein Azocoll
PROTEINASE”
Degradation (% of control)
proteins
100 46 48 0 0 0 0 0 3 95 0 0
n The proteinase assay contained in a final volume of 0.1 ml Tris/HCl, pH 8.0, 5 pmol; protein substrate, 15-50 pg; and insoluble proteinase, 0.02 mg (sp act 0.2 kmol N-acetyl-L-tyrosine ethyl ester split per minute x milligram). After vigorous shaking for 60 min at 37”C, the incubation mixture was centrifuged at 12,000g for 5 min. Twenty microliters of supernatant was mixed with 20 ~1 of 2% SDS, 2% 2-mercaptoethanol, and 0.05 M Tris, pH 8.1, and kept at 95°C for 5 min. After the addition of 5 ~1 of bromophenol blue (0.01%) and 10 ~1 of glycerol (87%) the mixture was applied to SDS gels (IO). The protein bands were stained with Coomassie blue and scanned. The intensity of the band obtained from incubations without added proteinase was used as control (=lOO%l. In the case of experiments with azocasein and azocoll, the incubation mixture contained, in a total volume of 0.5 ml, TrisiHCl pH 8, 5 pmol; azocoll or azocasein, respectively, 12 mg; and proteinase, 0.12 mg (sp act 0.2 wmol N-acetyl-r.tyrosine ethyl ester split per minute x milligram). After 60 min at 37”C, 0.5 ml of TCA was added. After centrifugation, the clear supernatants were measured at 520 or 422 nm, respectively, against a blank without addition of enzyme.
degradation product in the fast-moving protein band was eluted and subjected to SDS-acrylamide gel (15%) electrophoresis, one main protein band and a diffuse zone were observed. From the electrophoretie mobility a molecular weight of 13,000 2 10% has been estimated for the main protein band. The diffuse zone of high electrophoretic mobility contains further degradation products. It was not possible to decide by SDS gel electrophoresis whether this zone consists of one or
JUSIC
ET
AL.
d
e
FIG. 2. Degradation pattern of the very-lysine-rich histone Hl: (a) histone HI; (b) histone Hl plus proteinase, incubated for 3 min, (c) 6 min, (d) 10 min, (e) 30 min. The incubation mixture contained, in a total volume of 0.1 ml: 5 wmol of Tris/HCl, pH 8,18 wmol of histone Hl, and 8 yg of proteinase (sp act 2.0 pmol N-acetyl-L-tyrosine ethyl ester split/min x mg protein) (b-e). After incubation at 37”C, the reaction was stopped by the addition of 20 ~1 of 4.5 N acetic acid. The a&amide-gel electrophoresis was carried out according to Panyim and Chalkley (9).
more proteins of molecular weights of
seen in Fig. 3 that the large polypeptides were more rapidly degraded than the small ones. Under conditions optimal for histone and nonhistone proteolysis, the only other substrate found to be degraded by the proteinase was glucagon. Glucagon degradation was also observed with [Y]glucagon. On the other hand, insulin and [12511insulin were resistant to the proteinase action (Fig. 4). From a Lineweaver-Burk plot, a K, of 0.7 x 10m3 M was obtained for [12511glucagon. No degradation of azocoll and azocasein could be observed. Furthermore, alcohol dehydrogenase (yeast), malate dehydrogenase (pig heart), and glucose 6-phosphate dehydrogenase (yeast) were used as
PROTEINASE
FROM
RAT
361
LIVER
cal plot, an apparent Michaelis constant for the hydrolysis of ATEE of 5.0 x lo-” M was determined. 8 DISCUSSION
a
b
The proteinase described here has several properties similar to trypsin and chymotrypsin: the pH optimum around 8, the inhibition by soybean trypsin inhibitor, and the inactivation by the serine-proteinase reagents DFP and PhMeSO,F. The presence of a histidine at the active site was shown by reaction with TPCK and the methylene blue-mediated photooxidation. On the other hand, very few substrates were found, which indicates a high specificity of the enzyme. Among the substrates, chosen rather arbitrarily (Table III), only glucagon and chromosomal proteins were degraded. It has been shown recently (17) that glucagon has very little secondary structure, i.e., all peptide bonds are easily accessible to proteolytic attack. This is not the case with the highly ordered insulin molecule. It was the histone degradation which led us to the studies on this proteinase. Some interesting features in respect to the his-
FIG. 3. Degradation of nonhistone chromosomal proteins. The nonhistone proteins used as substrate were prepared according to (6). The incubation conditions are described in the legend to Table III: (a) incubation in the presence of proteinase; (b) control, no proteinase added. The SDS gel-acrylamide electrophoresis was carried out according to King and Laemmli (101; 50 pg of protein was applied to the gel (bl.
substrates for the insoluble proteinase in concentrations similar to those indicated in Table III. After incubation at 37°C for 60 min, aliquots of the incubation mixture were assayed. In no case could a loss of enzymatic activity be demonstrated. Among various synthetic substrates only ATEE and N-benzoyl-L-arginine ethyl ester were readily hydrolyzed, whereas N-benzoyl-L-tyrosine-p-nitroanilide and a number of otherp-nitroanilides of various acylated and nonacylated amino acids were not attacked at all. From a Lineweaver-Burk double-r&ipro-
[““I]
GLUCAGON
[““lj
INSULIN
()g ( yg)
) 0 A
FIG. 4. Proteolytic degradation of [‘2511glucagon and [‘251]insulin. In a total volume ofO.1 ml, 15 wmol of Tris/HCl, pH 8.0, 0.02 mg of proteinase and [lzJI]glucagon (sp act 12,400 cmplmg), or [‘*“I]insulin (sp act 21,000 cpmimg), respectively, were incubated for 30 min at 37°C. The reaction was stopped by addition of 20 ~1 of bovine serum albumin (10 mgi ml) and 20 ~1 of TCA (110%). Supernatant (100 ~1) was used for the determination of radioactivity in 10 ml of a 2:l toluene/Triton X-100 scintillation liquid. The counting efficiency for ““I was 85% in the l-‘Cchannel.
362
JUSIC
tone degradation were found. A characteristic fragment of a molecular weight of 13,000 is obtained by limited proteolysis of the very-lysine-rich histone HI (Fig. 2). A similar digestion of histone Hl with (Ychymotrypsin has been described recently by Bradbury et al. (18). These authors were able to split the Hl molecule into an N-terminal (l-106) and a C-terminal fragment (107212). In contrast to the degradation by the proteinase described in this work, a limited proteolysis with a-chymotrypsin was not observed by Bradbury et al. After the rapid initial cleavage of the Hl molecule by a-chymotrypsin, both fragments are further degraded. Because of the tendency of nonhistone chromosomal proteins to aggregate, only that fraction of the nonhistone proteins which is solubilized in 0.4 M NaCl was tested as substrate. Our finding that the large polypeptides of this nonhistone cbromosomal protein fraction tend to be degraded more rapidly than small ones (Fig. 3) is consistent with the hypothesis of correlation between turnover rate and protein molecular size (19). The proteinase which we isolated has properties similar to the enzyme that was purified from calf thymus chromatin by Kurecki and Toczko (3): for example, molecular weight, pH optimum, and inhibition by PhMeS03F. The proteinase activity associated with chromatin isolated according to the procedure of Bonner et al. (20) very probably originates from particles in the mitochondrial fraction (1). There are a few discrepancies between the properties of the proteinase isolated from rat liver chromatin by the group of Bonner (2) and the proteinase described here. A higher molecular weight and inhibition by Hg’+ ions has been described for the enzyme obtained from chromatin. The substrate specificities and the inhibition by PhMeSOzF are the same. From the finding that 18% of the proteinase activity found in isolated mitochondria sediment through 1.7 M sucrose (Table I, step 2) -the same sucrose concentration as used by Bonner for the preparation of chromatin -it is evident that the mitochondrial proteinase cosediments with chromatin and thus contaminates it.
ET AL.
From our results, it is likely that the proteinase is associated with the mitochondrial membrane. The possibility that the proteinase is of lysosomal origin and found associated with the mitochondrial membrane as a result of homogenization of the liver tissue cannot be completely excluded. The following experiment, however, favors a mitochondrial localization. Highly purified lysosomes, frozen and thawed twice, were added to minced rat liver and homogenized together with the liver tissue. Mitochondria isolated with lysosomes added and without lysosomes were assayed for proteinase activity. No difference with respect to proteinase activities was found for the two preparations. This finding is regarded as strong evidence for the mitochondrial origin of the proteinase. There is evidence for proteolytic activity in rat liver mitochondria from several publications (21-24). The purification of a proteinase from the mitochondrial fraction was described recently by Katunuma et al. (25). Katunuma et al. also found a high degree of substrate specificity for their enzyme. They called it “group specific” since they found only apoenzymes of pyridoxaldependent enzymes to be degraded. The proteinase of Katunuma et al. and our proteinase are similar with respect to their pH optima, sensitivity to inhibition by serine proteinase inhibitors, and probable mitochondrial localization. On the other hand, the two proteinases appear to be different in several properties. In our experience it is impossible to do Sephadex G-75 gel filtration and lyophilization under the conditions given by Katunuma et al. without losing all the enzymatic activity. Sephadex chromatography of the proteinase is only possible if Triton X-100 is present in the buffer. From the work of Katunuma et al. it is not clear whether the molecular weight of the proteinase from rat liver is 17,000 (Ref. 25, p. 43) or 13,000 (Ref. 25, Fig. 4). We find molecular weights of 22,500 and 23,500 by two independent methods. It is not yet possible to decide conclusively whether these two enzymes are the same. Experiments are in progress to determine whether the proteinase described in this paper degrades apo-orni-
PROTEINASE
FROM
thintransaminase in addition to glucagon and chromosomal proteins. Further work will be required to determine whether the proteinase described has any physiological role in the degradation of chromosomal proteins. ACKNOWLEDGMENTS The authors thank Professor R. L. Switzer for a critical reading of this manuscript. We also thank Professor H. Holzer for his interest in and support of this work. REFERENCES 1. HEINRICH, P. C., RAYDT, G., PUSCHENDORF, B., AND JUSIC, M. (1976) Eur. J. Biochem. 62, 3743. 2. CHONG, M. T., GARRARD, W. T., AND BONNER, J. (1974) Biochemistry 13, 5128-5134. 3. KURECKI, T., AND TOCZKO, K. (1974) Acta Biochim. Polon. 21, 225-233. 4. LEVIN, 0. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.),Vol V, p. 27. Academic Press, New York/London. 5. BALHORN, R., AND CHALKLEY, R. (1975) in Methods in Enzymology (O’Malley, B. W., and Hardman, J. G., eds.), Vol. 40, p. 138, Academic Press, New York/London. 6. KISH, V. M., AND KLEINSMITH, L. J. (1974) J. Biol. Chem. 249, 750-760. 7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951)5. Biol. Chem. 193, 265-275. 8. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 9. PANYIM, S., AND CHALKLEY, R. (1969)Arch. Biothem. Biophys. 130, 337-346. 10. KING, J., AND LAEMMLI, U. K. (1971) J. Mol. Biol. 62, 465-477.
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