Cathepsin D: Rapid Isolation by Affinity Chromatography on Haemoglobin-Agarose Resin

Eur. J . Biochem. 48, 245 - 254 (1 974)

Cathepsin D : Rapid Isolation by Affinity Chromatography on Haemoglobin-Agarose Resin Ross SMITH and Vito TURK Department of Biochemistry, J. Stefan Institute, University of Ljubljana

(Received April 17/May 30, 1974)

The intracellular proteinase, cathepsin D, has been isolated from bovine spleen and thymus, in times as short as several hours, by affinity chromatography of partially purified and unpurified tissue extracts on haemoglobin-agarose resin. After subsequent separation from an inactive higher- molecular-weight protein by gel permeation chromatography, the enzyme from both tissues shows three dominant proteolytically active bands on gel electrophoresis at pH 4.3 and 9.5 : this proteolytic activity is completely inhibited by the acid-proteinase inhibitor, pepstatin. These enzyme electrophoretic patterns were approximately constant with variation in isolation time and with various preliminary purification procedures. The enzyme shows only traces of polypeptides other than that with an apparent molecular weight of 42 000 on dodecylsulphate electrophoresis, in contrast to the enzyme prepared by conventional methods, which contains considerable amounts of smaller polypeptides. This difference in polypeptide composition is shown to be the result of degradation of the enzyme in vitro during isolation by the previously published methods.

Although the intracellular proteinase, cathepsin D, has been studied intensively for more than a decade, no coherent picture of its properties has emerged. The enzyme isolated from a single tissue, for example bovine spleen, has been reported to exist in so-called isoenzymic forms ranging in number from one or two [1,2] to more than ten [3]. Similarly, the published molecular weight of the spleen enzyme has spanned the range from 37000 [4] to 58000 [l-3,5,6].

The difficulties in preparing other proteinases and peptidases such as bovine factor X [7,8], bovine carboxypeptidases and their zymogen [9- 121, pepsin (e.g. [13]) and trypsin [14] are well documented. Each of these enzymes must be isolated with due regard for concomitant autolysis or proteolysis by other enzymes present in the tissues. Surprisingly little attention has been paid to this problem in the isolation of cathepsin D. Yet recent studies have shown that the isolated enzyme sometimes contains a variety of polypeptide chains which is best explained by partial degradation of the enzyme [15,16]. Whether this degradation occurs in vitro or in vivo has not previously been clearly established.

Enzymes (CBN Recommendations 1972). Cathepsin D (EC 3.4.23.5); cathepsin E (EC 3.4.23.-).

Degradation of the other proteolytic enzymes mentioned above is usually avoided by rapid isolation or by the addition of inhibitors of other proteinases present. In the absence of a detailed knowledge of all of the other proteinases present in the tissues used here, the latter tactic is not possible: therefore it is desirable to achieve the purification rapidly. The classical preparative methods do not lend themselves to such a rapid purification.

A novel preparative method is therefore presented here, which has allowed purification of the enzyme from whole tissues in times as short as four hours. The enzyme prepared in this way shows little evidence of the degradation which is apparent in the material prepared by previously published methods, lending support to the hypothesis that degradation in vitro is significant.

The essential step in the purification is specific adsorption of the enzyme onto agarose resin to which its best protein substrate, haemoglobin, is covalently bound. Further purification is subsequently achieved by chromatography on a small column of Sephadex G-100.

A partial characterization of the enzyme is presented here: the structure of the enzyme is at present under detailed investigation.

Eur. J. Biochem. 48 (1974)

246 Affinity Chromatography of Cathepsin D

METHODS

Unless otherwise noted, chemicals were of reagent grade or better. All solutions were prepared with deionized water and had sodium azide (1 mM) or chloroform (saturated) added to prevent the growth of microorganisms.

Agarose was Sepharose 4B (Pharmacia, Uppsala) or Biogel A-5M (Bio-Rad, Richmond, California). Sephddex G-100 was also purchased from Pharmacia. Haemoglobin was prepared in this laboratory as previously described [17]. Pepstatin (a generous gift from Prof. Umezawa, Institute of Microbial Chem- istry, Tokyo) was used without further purification.

The standard proteins for dodecylsulphate gel electrophoresis were purchased from Serva (Heidel- berg) and were of chromatographic grade, except a-chymotrypsinogen (A) which was a 6 times crystal- lized sample from bovine pancreas (Sigma, St. Louis, Missouri). All columns were run at 4 "C.

Enzymatic Assay

The assay was basically the method of Anson [17]. The enzyme (12 pg or less) in 400 pl solution was mixed with 2.0 ml of 2% haemoglobin or bovine serum albumin in acetate buffer pH 3.5 and incubated at 37 "C for 10 min or more. The enzymatic reaction was stopped by adding 4.0 ml of 0.3 M trichloroacetic acid and after 10 min the precipitate was filtered off. 2.0 ml of the filtrate was mixed with 4.0 ml of 0.5 M sodium hydroxide and 1.2 ml of the Folin-Ciocalteau reagent (diluted 1 : 3). After 5 min the absorbance was measured at 750 nm. One unit of activity is defined as that amount of enzyme which, in the above assay, after a 10-min incubation at 37 "C, gives an absorbance of 1.00 for a 1 cm pathlength. This unit corresponds to approximately 0.59 of the units of Press et al. [3] and to about 3.6 of the units of Woessner and Sham- berger [18], and therefore represents the release of about 24 microequivalents of tyrosine/hour, using the latter authors conversion factor. The amount of enzyme, which was always such that the absorbance in the assay was 0.100 to 0.450, was read from a cali- bration curve (following Anson).

Although the assay with haemoglobin is not specific for cathepsin D, the total activity of other proteinases present in bovine spleen and thymus is much lower under these conditions.

Pepstatin Inhibition

0.88 k 0.10 pg of pepstatin (as a 2.2 pg/ml solution in water) was added to the normal assay mixture, incubating the enzyme plus inhibitor for a few minutes

at room temperature before the addition of the substrate. In the assay mixture the enzyme concentration was 0.016-0.03 pM (assuming a molecular weight of 42000) and the pepstatin concentration was 0.55 pM. The period of incubation at 37 "C was increased to allow greater accuracy in measuring the residual activity.

Haemoglohin-Agarose Resin

The resin was prepared following Cuatrecasas [19]. The resin used in the majority of the studies was prepared by coupling haemoglobin at pH 6.5 for 24 h, and contained approximately 13 mg of haemoglobin/ ml of packed wet resin as determined by amino-acid analysis and nitrogen analysis. In preliminary small- scale experiments resins prepared by coupling at pH values from 4.9 to 8.6 and containing 5-30 mg/ml packed resin were used. All resins were washed extensively before use with the pH-3.5 and pH-8.6 buffers used for affinity chromatography.

Protein Analyses

The haemoglobin content of the resin used in most of the studies was determined by hydrolyzing about 1 ml in an equal volume of 6 M HCI, in vacuo, at 106 "C, for 24 h. The hydrolysate was analyzed on a Joel JLC-3 (Japan Electron Optics, Japan) auto- matic amino-acid analyzer [20]. No corrections were made for destruction of the less-stable amino acids. The haemoglobin content of all other resins was determined only by measuring the total nitrogen content by the method of Kjeldahl [21] : 6.3 mg of the haemoglobin used for coupling to the resin contained 1 mg of nitrogen.

Enzyme specific activities were derived from protein concentration measurements by the method of Lowry et al. [22]; this method gave values, even for tissue homogenate supernatants, comparable to the concentrations from dry-weight measurements on aliquots of salt-free solutions.

Isoelectric Focussing

The pure enzyme was subjected to isoelectric focussing [23] (and references therein), in an LKB 8100 Ampholine apparatus (LKB, Sweden). The sample was initially evenly distributed throughout the 110-ml continuous sucrose density gradient, which contained ampholytes isoelectric in the range pH 5 - 7. Reasonable resolution was attained in 3 days at 600 V. The apparatus was kept at 2 "C, and the pH

Eur. J . Biochem. 48 (1974)

R. Smith and V. Turk 241

of the effluent fractions was measured at the same temperature.

Ultrafiltration

Solutions were concentrated by ultrafiltration, under nitrogen pressure, through Amicon UM-10 or PM-10 membranes (Amicon, Lexington, Mass.). The filtrates were without enzymatic activity.

Polyacrylamide-Gel Electrophoresis

Disc electrophoresis in gels containing 7 - 10 acrylamide was performed as outlined by Davis (at pH 9.5) [24] and by Reisfeld et al. (at pH 4.3) [25]. The gels were stained by heating in a 1 % solution of Coomassie brilliant blue G-250 (Serva, Heidelberg) in 7 % acetic acid-10% methanol, and they were destained electrophoretically, after being cut into two approximately equal pieces if necessary.

The polypeptide chain composition of the enzyme was investigated using electrophoresis in 10 or 5 % acrylamide gels containing dodecylsulphate [26,27]. The protein was dissolved, with or without heating, in buffer (pH 7.4) containing 1 % sodium dodecylsulphate and 0.1 M 2-mercaptoethanol before mixing with a bromophenol-blue - sucrose solution for layer- ing on the gel tops. Samples have also had 6 M urea added for electrophoresis, the enzyme has stood for times varying from several hours to several weeks in the gel buffer, and it has also initially been denatured in 6 M guanidine hydrochloride before dialysis against water then gel buffer: none of these treatments gave different electrophoretic patterns. Proteins used as molecular weight standards included bovine serum albumin (molecular weight assumed : 69 000), ovalbu- min (43 000), pepsinogen (43 000), a-chymotrypsinogen (25700) and lysozyme (14300).

When the molecular weights were measured the dye had travelled at least 11 cm; for qualitative exa- mination shorter gels were used. The molecular weights given in this paper are based solely on gel electrophoresis and are therefore only apparent values which should be accepted with caution until confirmed by more direct methods.

To determine which electrophoretic bands contained active enzyme, longer gels were run (gel length 17 cm, CJ usual 6 cm) and afterwards some were sliced transversely into 1.5-mm sections and others stained. Corrections for elongation during staining and destaining were made as in dodecylsulphate- polyacrylamide gel electrophoresis [27]. The gel slices were incubated in 0.50 ml water at 4 "C overnight and 25 - 200 pl of each solution taken for enzymatic assay.

PREPARATION OF CATHEPSIN D Initial Concentration and Partial Purfication by Precipitation

Bovine spleen and thymus, taken within 1 h of slaughtering, were transported to the laboratory on ice and all subsequent operations were carried out at 4 "C or below. The tissues were freed of large blood vessels and fat (thymus) then 0.5 - 2 kg homogenized in an equal weight of water. Cellular debris was removed from the homogenate by centrifugation (requiring approx. 500000 x g,, x min) in a Sorvall GSA rotor (Ivan Sorvall, Norwalk, Connecticut) and the supernatant filtered through gauze to remove fat particles.

Several methods were then used to reduce the protein solution to a volume suitable for application to the affinity column. The protein in the homogenate supernatant was precipitated by the addition of ammonium sulphate (to 70 % saturation) or acetone (to 70 v/v). The precipitate was collected by centrifugation (up to 200000 x g,, x min) and resuspended in a small volume of water. In some experiments fractions precipitating in the narrower ranges of 30- 65 saturated ammonium sulphate or 40 - 65 % (v/v) acetone were taken without change in the composition of the isolated enzyme.

The resuspended precipitate was dialyzed overnight against water then re-centrifuged to remove un- dissolved material. The supernatant was adjusted to pH 3.5 (0.1 M sodium acetate, 1 M sodium chloride) by the addition, with vigorous stirring, of one third the volume of concentrated buffer and the small amount of precipitate which formed was centrifuged Off.

In some experiments with thymus a more rapid isolation was achieved by omitting the salt or acetone precipitation. The homogenate supernatant was adjusted immediately to pH 3.5 by the addition of concentrated buffer, then centrifuged before adding the haemoglobin-agarose resin.

Affinity Chromatography

5 - 25 ml of protein solution at pH 3.5, containing 50- 150 mg protein/ml, was applied to a 1-cm diameter x 19-cm column of the haemoglobin-agarose resin which had been equilibrated with the same buffer. After application of the sample, the column was washed with 0.1 M acetate buffer, pH 5.0, 1 M NaCl at 12 ml/h until the effluent had a constant absorbance at 280nm of less than 0.05. The column was then eluted with 0.1 M Tris buffer pH 8.6-1 M NaC1. 4-ml fractions were collected.

In the rapid experiments 8-60 ml of the resin was added to 300-2000 ml of the acidified thymus



! 25 2 5 1

0 5 10 15 20 25 30 50 55 60 65 Fraction number

Fig. 1 . Haemoglobin-agarose chromatography of a 40- 70 % (v/v) acetone-precipitated fraction from thymus. Immediately following sample application the buffer was changed to pH 5 from pH 3.5. Later, at the point marked with an arrow, the

homogenate supernatant and stirred for 30- 60 min before filtering, washing the resin, and eluting the enzyme, as above.

Further Purification

The affinity column enzyme peak was concentrated by ultrafiltration to 2- 3 ml and applied to a Sephadex G-100 column (3 cm diameter x 40 cm) equilibrated with the pH-8.6 buffer. The flow rate was 12ml/h and 3 - 6-ml fractions were collected.

For electrophoretic analysis the peak from this column was concentrated and dialyzed against a large volume of water.

RESULTS

The precipitation steps with ammonium sulphate and acetone are commonly used (e.g. [3,28,29]) and will not be described at length here. These steps were used primarily for concentration of the enzyme fractions to a volume suitable for application to the small haemoglobin-agarose column. The enzyme losses in these stages were appreciable and often little purification resulted : if some preliminary purification with higher yield is desired then another method, such as precipitation with dioxane [18], may be more salis- factory.

Resins prepared at pH between 4.9 and 8.6 with coupling haemoglobin solutions of 10 - 200 mg/ml, when separately mixed with equal aliquots of a crude thymus fraction containing cathepsin D at pH 3.5,

pH was changed to 8.6. The absorbance at 280nm of the effluent was measured with 0.1-cm pathlength cells. Proteo- lytic activity (----); absorbance at 280 nm (-)

all extracted about the same amount of the proteolytic activity (about 70%) per unit volume of resin. In similar small-scale experiments it was found that the proportion bound increased sharply below pH 5.0 (where 35% was bound), and was a little greater at pH 3.0 (79%) than at pH 3.5 (70%); however, the last pH was routinely used. The optimal pH for binding to the resin is, not unexpectedly, close to the pH for digestion of haemoglobin by this enzyme.

The pH at which the enzyme is released from the resin was established by applying a pH gradient from pH 5.0- 8.6, at constant ionic strength. Elution of the enzyme began near pH 6.2 but a single change from pH 5.0 to pH 8.6 (at which the enzyme activity to- wards haemoglobin is negligible) was routinely used for elution from the column. A typical affinity column chromatogram is given in Fig. 1, and the result of a representative thymus preparation is given in Table 1. Spleen enzyme behaved similarly.

It can be seen that the recovery of activity from the Sephadex G-100 column appears to exceed that eluted from the affinity resin; this was noted several times in varying degrees. Possibly the enzyme is eluted from the affinity column together with an inhibitor which is subsequently removed on gel permeation chromatography.

The presence of a high salt concentration during affinity chromatography is essential : in experiments in 0.1 M buffer without added NaCl none of the proteolytic activity was retained on the resin at pH 3.5. However, in the presence of 1 M salt almost all of the inactive protein, and a small amount of haemoglobin- digesting enzyme, passes through the column at



Table 1. Two typical extractions of cathepsin D from thymus homogenates usingaffinity chromatography, with and withoutpreliminary purification The activity units are explained under Methods

Step Total Specific Volume Purification Yield activity activity

units units/mg ml -fold % Homogenate supernatant from

1900 g tissue 2240 0.050 1175 Ammonium sulphate to 65 % saturation 1370 0.033 840

Ammonium sulphate to 70 % and Acetone to 75 % 1150 0.160 553

trating 488 0.107 74 After affinity chromatography 207 a 11.2 20

adjusted to pH 3.5 before concen-

After Sephadex G-100 chromatography 375 42.5 30 850 16.7 ~~

Homogenate supernatant from

Direct extraction with 15-ml affinity 1500 g tissue 2300 0.045 1330

resin 600 21.5 47 480 26 a

a See Results for explanation.

pH 3.5; the bulk of the enzyme elutes only after the pH change.

With partially purified thymus extracts the enzymatic activity in the second peak was typically 70- 90 % of the total applied, and the proportion of cathepsin D bound may actually be higher (see below). Even acetone-precipitated or salt-precipitated fractions from spleen which contained considerable amounts of haemoglobin separated well with 65 - 70 % of the enzyme in the second peak.

Enzyme extraction by direct addition of the resin to thymus homogenate was far faster than column chromatography, as the salt or acetone precipitation required for the latter takes at least a day. However, direct extraction with the same volume of resin isolates a lower proportion of the enzyme (Table 1). Although the use of pH-5.0 buffer to wash proteolytically inactive protein off the resin lessens the possibility of hydrolysis of the agarose-bound haemoglobin, the pH-3.5 buffer was used for this purpose without noticeable effect. The most useful preparative method proved to be direct extraction repeated four or five times on the same solution: in this way typically 85 - 90 (x (60 mg enzyme or about 3000 units) of the enzyme has been extracted with 60 ml of resin from the pH-3.5 supernatant of 1500 g thymus.

The enzyme eluted from the affinity resin is not completely pure as an almost equal amount of a single higher-molecular-weight protein is also bound and elutes with the enzyme. It is removed by gel permeation chromatography on Sephadex G-100 as demonstrated in Fig. 2. The protein in the first peak

0.6 1 t 0.5

??, 0.4

a, 0 .3

6 0.2

0.1

0

c 0

- m

i m 0

Y) D Q

Fraction number

Fig. 2. Sephadex G-100 chromatography of the enzyme from the affknity chromatographic column. The portion of the active peak which was pooled is indicated by the bar. The material in both peaks is usually light yellow. Proteolytic activity (--- .) , ’ absorbance at 280 nm (-)

has extremely little, if any, proteolytic activity against haemoglobin or bovine serum albumin at pH 3.5. The second ultraviolet-absorbing peak is asymmetric, possibly because of elution of some smaller inactive proteins.

Bearing in mind the difficulties in comparing specific activity measurements using proteins as sub- strates and varying assay methods, the specific activities of the purest samples of Sapolsky and Woessner [16] and of the present authors (up to 58 units per mg) are very similar, and both are about 30 % higher than that of Press et al. [3] and 20 ”/, higher than the purest sample of Barrett [29]. Comparison with the results



A B c 0

Fig. 3. Polyacrylamide-gel electropkoresis al p H 9.5 (gels A , B and C ) andpH4.3 (gel 0). Gel (A) shows the enzyme fraction eluted from the haemoglobin-agarose column, and gels (B) and (C) show the first and second peaks respectively from the

Sephadex (3-100 column. The same sample was used for gels (C) and (D). The uppermost of the four bands visible in gel (C) appeared infrequently and had little activity associated with it. In all gels migration was from top to bottom

Migration - Fig. 4. Polyacrylamide-gel ( p H 9.5) pattern and corresponding enzyme distribution. Of two gels run together one was stained

of others is obscured by the differences in assay methods. The activity of the enzyme (at about 0.03 pM) was, within the experimental error of 0.5 %, completely inhibited by a 20-fold molar excess of pepstatin, as expected for cathepsin D [31,32].

After Sephadex G-100 chromatography the enzyme from both tissues typically shows three principal bands on electrophoresis in polyacrylamide gels at pH 9.5 and pH 4.3, although not in the same propor- tions at the two pH values (Fig. 3). By running 30 pg of enzyme on each of two gels at pH 9.5, cutting one gel transversely, then extracting the enzyme from the segments, it was demonstrated that each of these stainable bands had activity associated with it. No

(bottom of figure) and the other sliced transversely to obtain the cathepsin D distribution (top)

significant activity was detected in any other position on the gels (Fig. 4).

Isoelectric focussing also yielded three fractions of apparent isoelectric pH values 5.6, 5.9 and 6.4 (all k 0.1) which are, within the experimental error, the same as the values of Barrett [29] for human liver cathepsin D (5.7, 6.0 and 6.5) but lower than the figures for bovine spleen recently reported by Ferguson et al. [30] (6.1, 6.3 and 6.7).

The characteristic gel pattern of three principal bands has been obtained repeatedly for both the spleen and thymus enzymes, whether the protein was isolated from fresh tissue within 4 h (and run on gels within 13.5 h), or more slowly from tissues that had been



Fig. 5. Dodecylsulphate ( B ) and alkaline ( A ) gels of cathepsin D isolated by ajftnity Chromatography from thymus extracts that had stood jor a day at 4 "C after adjusting to p H 3.5. The arrows indicate the bands of molecular weight 42000 (top) and 27000 on the gel at the right

stored at -30 "C for several months. Addition of inhibitors (10 mM iodoacetate and 1 pM p-chloro- mercuribenzoate) of the other well-characterized proteinases and peptidases present in these tissues (cathepsins A, B, and C), throughout the preparation was without effect. The appearance of multiple forms of cathepsin D is therefore not the result of degradation in vitro by any of the other three cathepsins. The gel pattern was however markedly altered if the enzyme was not extracted from the solution immediately after adjustment to pH 3.5; the appearance of addi- tional bands can be seen by comparing Fig. 3 and 5.

Dodecylsulphate gel electrophoresis of the enzyme (Fig. 6) revealed a single polypeptide of apparent molecular weight 42000 f 4000 as the dominant chain : there is also a trace of a polypeptide of 27 000 and an even smaller amount of a polypeptide at 14000.

The constancy of this gel pattern after various methods of sample preparation for electrophoresis strongly suggests that the reaction between dodecylsulphate and the protein had reached equilibrium, and therefore that the enzyme had been fully dissociated into its subunits. For comparison with this pattern, Fig. 7 shows the corresponding pattern for a sample

Fig. 6. Dodecylsulphate - polyacrylarnide-gel electrophoresis of the jirst (gel A ) and second (gel B ) peaks from the Sephadex G-100 column. The first peak often gave a single band of apparent molecular weight 120000. The polypeptide of molecular weight 14000 which was seen in extremely small amounts in most preparations is not visible in the photograph (gel B). The standard proteins, which are marked arrows in gel (C), have from top to bottom molecular weights of 69000, 43000,25700, and 14300

initially purified by conventional techniques [15] and then subjected to affinity and Sephadex G-100 chromatography before electrophoresis. Many smaller peptides are evident, with one of molecular weight near 27 000 being dominant. When the tissue extracts were allowed to stand for 1-2 days at 4 "C after adjustment to pH 3.5, the enzyme finally isolated by affinity chromatography also contained appreciable amounts of the smaller polypeptides (Fig. 5). The small amount of polypeptide of molecular weight greater than 42 000 on the dodecylsulphate gels in Fig. 5 and 7 is attributable to incomplete resolution of the two peaks on Sephadex G-100 chromatography (cf. Fig. 2) in these instances.

The protein from the first Sephadex G-100 gel permeation chromatographic peak often barely enter-



Fig. 7. Dodecylsulphate-polyacrylamide gel electrophoresis of bovine spleen cathepsin D ( A ) initially purijkd by conventional techniques (see Results) and then by afiinity and Sephadex

G-100 chromatography. The standard proteins (B) are as for Fig. 6

ed the 10% polyacrylamide gels used for dodecylsulphate electrophoresis and necessitated the use of gels containing 5 7; acrylamide. Extrapolation of the standard line, which should introduce little error (e.g. [33]), yielded a molecular weight of 120000 f 12000 for the polypeptide of this protein, but in many preparations this protein dissociated into approximately equal amounts of polypeptides with molecular weights near 60000 and 27000.

DISCUSSION

Affinity chromatography on haemoglobin-agarose clearly affords a rapid and simple purification of cathepsin D. Up to 20mg of the enzyme has been isolated in a single step with a purification of up to 500-fold. In the presence of the moderate amounts of haemoglobin present in spleen extracts even after partial purification by precipitation a reasonable separation is still possible, despite the competition between free and agarose-bound haemoglobin for the enzyme. In many of the experiments several grams of protein were passed down the column which contained less than 300 mg of immobilized haemoglobin: asso- ciation ofsome of the enzyme with the first protein peak is therefore expected. Indeed, the recovery of haemoglobin-digesting activity is necessarily less than com- plete as it can be shown by the addition of pepstatin (0.3 - 1 pM) to the homogenates (containing about 0.02 pM enzyme) that only about 90 % of this activity

is inhibited, the remainder presumably being due to enzymes other than cathepsin D which are not bound to the affinity resin under the conditions used here. The highest overall yields were obtained by repeated extraction of thymus supernatants, where the principal losses were caused by coprecipitation of the enzyme with other proteins upon the addition of the pH-3.5 buffer.

It is more usual to use an immobilized inhibitor for the affinity chromatography of enzymes ; indeed the use of pepstatin-agarose for the purification of rennin from partially purified fractions has been reported [34,35]. Preliminary experiments (R. Smith and F. GubenSek, unpublished) using pepstatin-Se- pharose to isolate cathepsin D indicate that the product is identical to that obtained using haemoglobin- agarose and that the proportion of enzyme bound under equivalent conditions is similar. Recently a-chymotrypsin, containing only small amounts of impuri- ties, has been purified using resins containing this enzyme’s substrate, L-tryptophan methyl ester, bound covalently through aminocaproate to agarose resin [36]. After the first passage of a-chymotrypsin through the affinity column all of the substrate was degraded; however the resin, then containing bound product, still retarded enzyme subsequently passed through it. In the present experiments samples of haemoglobin- agarose resin have been used repeatedly with little decline in their ability to bind cathepsin D, and it may be that this enzyme is also bound to both intact and partially hydrolyzed substrate : the complex



nature of the substrate precludes a simple demon- stration of binding to a previously cleaved portion of the molecule. The bound haemoglobin should however be hydrolyzed very slowly at the temperature used for chromatography.

The nature of the larger protein that co-elutes with cathepsin D from the affinity resin is at present unknown. It appears not to have activity like cathepsin D or cathepsin E [37] as it has negligible activity at acid pH against both haemoglobin (Fig. 2) and bovine serum albumin: the activity it shows could be accounted for by contamination with as little as 0.05 % of cathepsin D.

For different tissues widely varying numbers of forms of cathepsin D have been reported (see e.g. [6]). Several authors (e.g. [3,16,29]) have tried to ensure that the number of forms isolated is indeed the number existing in vivo and in each case have established that the major forms at least were present in their homogenates. This has led to the supposition that degradation of the enzyme in vitro is insignificant. However, comparison of the subunit structures of the enzymes isolated by these authors with that obtained by affinity chromatography shows that the observation of the same number of forms in the homogenate supernatant as are finally isolated is insufficient evidence for the absence of scission in vitro of some peptide bonds in the molecule.

Data on the subunit structure of the enzyme come only from a few recent studies using dodecylsulphate gel electrophoresis. The bovine uterus enzyme studied by Sapolsky and Woessner [16] contained a considerable amount (> 30%) of polypeptide of molecular weight (M,) 27000 in addition to a 42000-M, chain and smaller amounts of a 13 000-M, chain. The enzyme isolated by Barrett (private communication) from various tissues contains only the two smaller chains and bovine spleen cathepsin D isolated by Ferguson et al. [30] showed only the 28000-M, polypeptide. Similar conventional preparations from bovine spleen and thymus in this laboratory [15] have yielded an enzyme similar to that from uterus, the 27000-M, polypeptide being a major component. The above observations are in sharp contrast to the composition of the enzyme rapidly isolated by affinity chromatography which shows only extremely small amounts of polypeptides other than the 42000-M, chain (Fig. 6) under diverse isolation conditions.

It could be argued that the data of Barrett and of Sapolsky and Woessner pertain to enzymes of different tissues which need not have completely the same properties as bovine spleen or thymus enzyme. How- ever, the same criticism can not hold for the other two studies [15,30]. The possibility that the smaller chains are simply not bound as tightly to the affinity resin is

obviated by the finding that after several conventional purification steps [15] the spleen cathepsin finally purified by affinity chromatography, followed by Sephadex G-100 chromatography or by preparative electrophoresis [15], contains considerable amounts of 27000-M, and smaller-molecular-weight polypeptides in addition to the 42000-M, chain'. These smaller peptides, as noted above, are also obtained by affinity chromatography if the tissue extracts are allowed to stand at pH 3.5 for a day.

Further, the electrophoretic pattern of the enzyme on acid, alkaline, and dodecylsulphate-polyacrylamide gels was unaffected by the inclusion or omission of the preliminary acetone or salt precipitations used in many of the affinity chromatographic preparations and one can therefore infer that no loss of particular enzyme forms occurs during these steps. The conclu- sion that degradation of the enzyme in vitro is significant seems therefore inescapable : the occurrence of minor degradation in vivo is not excluded.

The generous gift of pepstatin from Prof. H. Umezawa is gratefully acknowledged. This work was supported by funds from the Boris KidriE Foundation and the National Science Foundation (U.S.A.), research grant no. GF-31389.

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The chain of 58000-60000 M , which also appeared in the material isolated by preparative electrophoresis [I51 has not been obtained in any of the pure enzyme preparations using affinity chromatography and Sephadex G-100 chromatography. However, an inactive chain of this molecular weight is often removed in the latter step (Fig. 6).

____


254 R. Smith and V. Turk: Affinity Chromatography of Cathepsin D

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R. Smith’s present address : Department of Physical Chemistry, University of Sydney, Sydney, New South Wales, Australia 2006 V. Turk, Oddelek za Biokemijo, lnstitut “Joief Stefan”, Univerza v Ljubljani, PoStni Predal 199, YU-61001 Ljubljana, Yugoslavia


Cathepsin D: Rapid Isolation by Affinity Chromatography on Haemoglobin-Agarose Resin

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