BIOCHEMISTRY
A Peptidohydrolase from Mammalian Fibroblasts (Bovine Dental Pulp) * Chr. Schwabet and G. Kalnitsky
ABSTRACT: A procedure for the isolation of a connective tissue peptidohydrolase (cathepsin) in a state of high purity has been developed and tested for reproducibility. It involves the classical methods of acid and ammonium sulfate precipitation, autolysis of the extract, and fractionation on DEAE, hydroxylapatite, and Bio-Gel p-60 columns. The over-all yield of the main enzyme is approximately 20 %. A 15oO-18OO-fold purification of this fraction was achieved. By measurement of the specific activity of a column effluent as well as by disk electrophoresis a high degree of purity of one of the cathepsins has been demonstrated. The
T
he investigation of the biochemistry of mammalian fibroblasts is a prerequisite for the understanding of the disorders of collagen synthesis and degradation. The difficulties involved in obtaining these cells in sufficient quantities may well have stifled intense research in this field. Studies on mammalian tissues are therefore inconclusive with respect to the mechanism of collagen degradation and do not add to the known fact that collagen turnover does occur (Woessner 1962; Neuberger and Slack, 1953). Collagen degradation has been studied in amphibian connective tissue by Gross (1964) who demonstrated the presence of collagenolytic activity in the tadpole tail by the action of whole explants on reconstituted calfskin collagen. The tissue investigated in this study (bovine dental pulp) provides nearly homogeneous population of typical fibroblasts with bone-forming capacity. An attempt is made to actually isolate the cathepsins demonstrated earlier (Schwabe and Kalnitsky, 1965) and to characterize them as enzymes and as protein molecules. It seems reasonable to expect that this information will eventually help to decide whether the enzymes participate in the turnover of collagen or other endogenous connective tissue proteins.
__ From the Department of Biochemistry, College of Medicine, University of iowa, Iowa City, Iowa. Received August 24, 1965. We gratefully acknowledge support of this research by the College of Medicine, University of Iowa. t National Institutes of Health Research Career Development Awardee l-K3-DE-11, 128-01. Present address: Department of Biological Chemistry, Harvard School of Dental Medicine, Boston, Mass.
*
158
CHR.
SCHWABE A N D
ti.
KALNITSKY
pH optimum of the purified enzyme is between pH 3.0 and 3.5 when hydrolyzing acid-denatured hemoglobin. An activation energy of 10,800 cal at pH 3.0 has been calculated from reaction rates at different temperatures. The molecular size of the cathepsins was estimated from molecular filtration on Bio-Gel p-100. The maximum molecular weight of the main fraction is 41,000 and that of a minor fraction 35,000. Evidence presented suggests a relatively narrow specificity for the major enzyme fraction. The enzyme is an acidactivated peptide peptidohydrolase (endopeptidase).
Materials and Methods Tissue Source and Preparation. Bovine dental pulp (100 kg wet weight) was obtained in cooperation with the Wilson Packing Co. of Cedar Rapids, Iowa. The pulps were extracted, lyophilized, and degraded to a 60-mesh powder as previously described (Schwabe and Kalnitsky, 1965). This powder, which is stable for indefinite periods at -20" and constitutes a convenient starting material, is referred to as pulp powder. Materials for Column Chromatography. DEAEcellulose (Schleicher & Schiill) was suspended in 0.1 N NaOH, stirred for 30 min, washed free from alkali with distilled water, and suspended in the desired buffer. Fine particles were removed by four decantations after each 10-min settling period. Hydroxylapatite was prepared following the procedure of Jenkins (1962). The arrangement for the precipitation of calcium phosphate (Schwabe, 1965) has proved to be advantageous in our hands. The apparatus permits automatic control of the relative rates of addition of the two reagents as well as precipitation from a constant volume of water. Dowex 50-X2 (Dow Chemical Co.) was prepared as described by Schroeder et al. (1962). Bio-Gel (Calbiochem) was suspended in 0.1 N acetic acid and left to swell overnight at 4" in the eluting buffer. A dimethyldichlorosilane-coated column was filled with 0.1 N acetic acid. The top of the column was then fitted witn a wide-mouth funnel and enough BioGel suspension introduced to fill the column under flow. This took about 5-6 hr. After the bed had settled, 0.1 N acetic acid was pumped through the column at a rate of either 6 or 12 ml/hr from an LKB miniflow dual-syringe pump for several hours, or in the molecular weight experiment, for 2 days.
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TABLE I
: Bovine Dental Pulp Cathepsins. Initial Purification Steps.a
NO.
1,
J A N U A R Y
Preparation Initial extract pH 4.0 precipitate Autolysis 30-80 ammonium sulfate
1966
Volume (ml) 900
900 780 275
Protein (mgiml) at 280 mp 28.5 17.0 14.2 16.0
Specific Nitrogen Activityb (mgiml) x IO2 2.7 1.5 1.5 -
Prepurution ojthe Hernoglobin Substrute. Hemoglobin substrate powder (Worthington Biochemicals) was dissolved in distilled water to make an 8 solution. The pH was adjusted to 2.0 with a few drops of 1 N hydrochloric acid and the solution was stirred gently for 30 min at room temperature. The pH was then readjusted to 4.0 with 1 N sodium hydroxide and the total volume was increased to make a 4% solution by the addition of distilled water. The resulting stock solution was further diluted 1 :1 with 0.1 M sodium acetate buffer, pH 4.0, to yield the substrate solution usually at pH 4.0. Extensive dialysis prior to the final dilution reduced the blank readings in the assay somewhat. Dialysis was omitted, since reproducible results were obtained with considerable saving in preparation time. The hemoglobin solution should be used within 3 weeks after preparation. Assay Method. The hemoglobin method of Anson (1939) was adapted for the specific needs in this study. A solution (200 pl) of lyophilized enzyme extract or of a column effluent was pipetted into a series of tubes and placed in a water bath at 40". To each tube, 800 pl of prewarmed hemoglobin substrate solution was added from an automatic pipet. From a similar pipet 3 ml of 10% trichloroacetic acid was added at zero time and various intervals thereafter. The increase in trichloroacetic acid soluble material was measured at 280 mp in a Zeiss spectrophotometer. Specific activity as referred to in this study is defined as the change in optical density (AOD) obtained in 30 min per milligram of enzyme protein as measured at 280 mp (1 mg of protein/ml -1.0 OD). The 30-min value was calculated from the linear portion of the reaction. An enzyme unit is defined as the amount of activity that produces a AOD of 1.0/30 min extrapolated from the initial velocity at 40" under standard assay conditions. The purification number is arbitrary, starting with 1 for the first extract. Thin Layer Chromatography. The thin layer equipment from Brinkman DESAGA was used throughout the experiments. The plates were coated with silica gel (E. Merk A. G., Darmstadt, Germany) and air dried overnight. For the separation of dinitrophenyl deriva-
z
A
Purifica-
Total Units
100 120 150 107
1. o 2.2 4.6 6.0
290 50 360 i. 50 440 i 50 310 i 30
(z) tion No:
1. o 2.4 4.6 6.0
a The data in this table have been computed from more than five extractions. section c Arbitrary number starting with 1 for the original extract.
Yield
*
Defined in the Material and Methods
tives of amino acids, the method described by Brenner et ul. (1961) was used, including the developing chamber for horizontal chromatography described by the first two authors (DESAGA B-N Chamber). The end-group analyses were performed according to the method described by Fraenkel-Conrat et al. (1955). Nitrogen determinations were performed by the modified Kjeldahl procedure of McKenzie and Wallace (1954). Experimental Section and Results Preparation of the Original Extract. Pulp powder (50 g) was extracted with 700 m l of distilled water in a 2-1. homogenizer jar (Lourdes high-speed homogenizer) at room temperature. After 0.5 hr the suspension was centrifuged in a refrigerated Sorvall high-speed centrifuge at 8000 rpm for 15 min. The supernatant was collected through a Pyrex wool plug and the pellet was resuspended in 300 ml of distilled water, followed by a 15-min reextraction in the Lourdes homogenizer. The centrifugation was repeated, the supernatants were combined, and the pellet was discarded. Approximately 900 ml of a reddish extract was obtained which contained about 28 mg of protein/ml, or 25 g of the total protein as measured at 280 mp. Further extraction did not increase the yield of proteolytic activity. Isoelectric Precipitation of Impurities. The extract obtained from 50 mg of pulp powder was acidified with 1 N HC1 to a pH of 4.0. The process was monitored continuously with a Zeromatic pH meter. The precipitate forming at pH 4.0 was removed by centrifugation and the supernatant was tested for protein, nitrogen content, and activity. As seen from Table I a twofold purification was obtained in this step. Autolysis of the Pulp Extract. The enzymes under investigation have an optimum very close to pH 4.0 when acting on hemoglobin as substrate, and between pH 3.0 and pH 4.0 when acting on endogenous substrate (Schwabe and Kalnitsky, 1965). Given the right conditions it could be expected that the enzymes would digest some of the inactive endogenous proteins. Aliquots of the supernatant from the previous step were incubated
P E P T I D O H Y D R O L A S E
F R O M
M A M M A L I A N
159
F I B R O B L A S T S
B I O C H E M I S T R Y
' I
'
I
I
I
LI I L-T
10 ' - 230 0
I
0
1
IO
I
I
I
20
30 4 0 TEMPERATURE .C
CUR.
50
at 4, 31, 40, and 46", all at pH 4.0, for 18 hr (Figure 1 ) . Table I shows that the increase in specific activity is accompanied by an increase in total activity. These results may be interpreted in terms of an activatable enzyme precursor or the breakage of lysosomes with subsequent enzyme release. Interference of bacterial contamination has been excluded by the addition of Zephiran chloride (Schwabe, 1965). The preparation is enriched nearly fourfold with respect to cathepsins after autolysis. In subsequent preparatory work, autolysis was carried out at 37". Ammonium Sulfate Precipitation. The autolyzed extract was centrifuged briefly in the Sorvall high-speed centrifuge (10,000 rpm) for 15 min and the supernatant was collected and 3 0 z saturated with ammonium sulfate at room temperature. A light precipitate was centrifuged off after 1 hr under the above conditions. The supernatant was brought to 8 0 z ammonium sulfate saturation by the addition of the solid salt. Precipitation was allowed to occur overnight at 4", followed by centrifugation, this time collecting the pellet and discarding the supernatant. The specific activity after dialysis was increased 1.4 times compared to the autolysate, while the total activity decreased only slightly (Table I). Chromatography on Diethylaminoethylcellulose. Further purification of the dental pulp cathepsins was achieved through column chromatography. The adsorbent was equilibrated with 5 X 10-4 M potassium buffer at pH 6.8 at 3 '. The 3&80z ammonium sulfate fraction (10 g) was dissolved in 70 ml of 5 X M phosphate at pH 6.8 and applied to the column. The material was permitted to enter the cellulose followed by 10 ml of the same buffer. The column was then connected to the reservoir consisting of two 4-1. bottles in series containing 2 1. of 5 X M phosphate and 2 1. of 1 X IOF2 M phosphate, both at pH 6.8. The effluent was monitored by a flow scanner (Gilson Medical Electronics). Fractions (12 ml, 1 m1,imin) were collected
S C H W A B E
A N D
G.
KALNITSKY
'
70 90 90 110 TUBE NUMSER
I130 iO
150 ; ; 1170 I50
NoOH
wash
2: Effluent record of the DEAE-cellulose column. The activity represents the AOD produced by 100 p1 of the effluent in 30 min under the usual assay conditions; 12-ml aliquots were collected in each tube. FIGURE
>
FIGURE 1 : Effect of temperature during autolysis on the specific activity of bovine dental pulp cathepsins.
160
' '.io 50 '
A
in a Buechler refrigerated fraction collector at 3". The conditions of pH and ionic strength as described appear to be rather critical with the type of DEAE-cellulose used in these experiments (Schleicher & Schiill Type 70). Initial buffer concentrations higher than 5 X IO-4 M led to poor separation while lower concentrations did not improve separation but caused partial or total irreversible inactivation. Figure 2 shows a typical elution pattern of 10 g of the ammonium sulfate fraction from such a DEAE-cellulose column. After the first peak had emerged, 4 M sodium chloride solution was added to the second reservoir to make its concentration approximately 5 X M M in salt, neglecting the small increase in volume as far as the gradient is concerned. Both activities shown in Figure 2 were dialyzed and lyophilized; the main activity was then further purified while peak 2 was stored for later examination. Chromatography on Hydroxylapatite-Cellulose Columns. Approximately 45 g of a hydroxylapatite slurry (settled from a suspension) was placed in 0.02 M phosphate buffer (all at pH 6.8) together with 15 g of the cellulose slurry and stirred for 15 min and then packed into a 2 x 25 cm column. The adsorbent was permitted to settle under buffer flow until all of the 60 g of adsorbent was introduced, yielding a 9-10-cm adsorbent bed. During these operations and all subsequent steps, the column was cooled to 3" by pumping the coolant of the refrigerated fraction collector through the jacket of the column. About 400 mg of the DEAE-cellulose fraction 1 was dissolved in 2.5 ml of 2 X 10-2 M phosphate buffer, placed onto the column, and forced into the adsorbent with 10 psi air pressure. At this time cracks may form in the top part of the apatite-cellulose or the sample may be forced along the wall between the adsorbent and the column. In this case the top layer of the column may be stirred to about 2-3-cm depth and permitted to settle, while the air pressure is reapplied. The material was washed into the column with another milliliter of
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NO.
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J A N U A R Y
TABLE 11 : Purification
of Bovine Dental Pulp Cathepsins by Column Chromatography.
Prepn Ammonium sulfate DEAE Fraction 1 Fraction 2 Hydroxylapatitecellulose Fraction 1 Fraction 2 HydroxylapatiteAmberlite Fraction 1 Fraction 2 Bio-Gel p-60 Above Fraction 1. Above Fraction 2. a
1966
Units of Activity Placed on the Column 600 120
Units Recovered
=t50
Specific Activity
Purification No.
0.06
100
100
6
390 40 100 f 20
0.40 0.07
65 17
65
40
54 f 5 26 f 5
4.50 0.30
45 22
30
450
65 i 5 5 44
10-12 5-8
55 38
18
1000- 1200 500-800
14-18 12-15
100 100
18
1400- 1800 1200-1500
*
* 10
Yield Yield (over-all (%)of %)of Previous Main Step Fraction
120 1. 10
*
From hydroxylapatite-Amberlite column.
-100 -50 7'5
,.
,'-a\
