Human spleen cathepsin D: Its characterization and localization in human spleen

In?. J. &&en.

Vol. 21, No. 3, pp. 317-326, 1989

0020-7 11X/89 $3.00 + 0.00

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Copyright 0 1989 Pergamon Press plc

HUMAN SPLEEN CATHEPSIN D: ITS CHARACTERIZATION AND LOCALIZATION IN HUMAN SPLEEN KAZUMASA IKEDA,'*HARUO SUZUKI,'TADAO OKANO* and SHIGEKINAKAGAWA~~

’ apartment of Applied Chemists, School of Science and Engineering, Waseda U~v~sity, 341 Ok&o, Shinjuku-ku, Tokyo 160, Japan ‘First Department of Pathology and ‘Department of lochemistry, Nihon University Schooi of Medicine, 30-l Oyaguchikami-machi, Itabashi-ku, Tokyo 173, Japan (Received 11 July 1988) Abstract-l. The cathepsin D was purified 1830-fold under mild conditions by a rapid procedure, based on two-step affinity chromatography. 2. Its molecular weight, amino acid composition and substrate specificity were shown to display minor differences from materials of other origins. 3. Inhibition with thioi compounds was found to be a specific phenomenon of the cathepsin D from the human spleen. 4. Production of antiserum specific for purified cathepsin D was demonstrated by immunodiffusion test, an immunoadsorbent column and immunoblotting of the crude enzyme in SDS gel. 5. fn an immunocytochemical study, the antigenic sites for this enzyme were found to be localized in the reticuloendothelial system of the human spleen. 6. The role of this enzyme in human spleen cell was discussed.

anti human spleen cathepsin D serum which was identified by various techniques, the localization of cathepsin D was examined in the human spleen. In addition, based on a critical comparison of the results obtained with data reported in the literature, we discuss the animal and tissue specificity of the enzyme.

INTRODUCTION Cathepsin D (EC3.4.23.5) is a lysosomal acid proteinase (Barrett, 1971) known to be closely involved

in slow rates of tissue protein turnover (Poole, 1975). The possibility of physiologicai degradation and pathological processes has been speculated by Mego (1984), Williams ef al. (1986), Pillai er al. (1983) and Helseth and Veis (1984), but much information concerning the physiological function of cathepsin D remains to be determined. Our present knowledge of the properties of cathepsin D derives from studies on purified preparations from a wide variety of mammalian tissues including 1979), human placenta human liver (Barrett, ~Contra~tor ef ol., 1982), human gastric mucosa (Pohl et al., 198 l), porcine spleen (Huang et al., 1979) and rat spleen (Yamamoto el al., 1979). Human spleen has been also used as a source of cathepsin D for the preparation of anti cathepsin D serum and immunohistochemical research with the antiserum (Reid et al., 1986). The amount of spleen cathepsin D is the largest among all organs (Yamamoto et al., 1979). Nevertheless, the specific functions which would explain the abundance of cathepsin D in the spleen have not been identified in any previous research on spleen cathepsin D. In the present study, we investigated the properties of purified human spleen cathepsin D, and employing _.-_____ *Present address: Department

MATERIALSAND Materials

Bovine serum albumin (fraction V, 98-99% pure), bovine hemoglobin, azocasein, bovine y-globulin, human serum albumin (crystallized and lyophilized), human hemoglobin, human y-globulin, 1,2-e~xy-3-~-nitrophenoxy~propane, di~oa~tyl-DL-norleucine methylester and l-ethyl 3-(3dimethylaminopropyl~carbodiimide hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO.); Sepharose 4B and Concanavalin A-Sepharose were from Pharmacia Fine Chemicals (Piscataway, N.J.); and pepstatin A was from Peptide Institute Inc. (Osaka, Japan). All other chemicals were of analytical grade. Purification of cathepsin D in human spleen

of Applied Biological

Science, Nihon University, College of Agriculture and Veterinary Medicine, 1866 Kameino, Fujisawa-shi, Kanagawa 252, Japan. tTo whom correspondence should be addressed.

METHODS

Human spleen tissues, obtained by autopsy and confirmed to be histologically normal, were stored at -20°C and used within I month of being obtained. Cathepsin D was purified from human spleen by a modification of the procedure of Afting and Becker (1981). A typical experiment with 20 g of human spleen is described. The whole procedure was carried out at 4°C. Human spleen was homogenized in 5 vol of cold pure water using a Waring blender. The homogenate was centrifuged at 10,OOOgfor 30 min. The supematant was adjusted to pH 3.5 with cont. HCI and stirred for 1 hr. The denatured protein pellet was removed by centrifugation at IOOOg for 10min. The resultant supematant was dialyzed against 0.02 M Tris-HCl buffer, pH 7.2, containing 0.5 M NaCl (Buffer A) and was

317

KAZUMASAIKEDA et al,

318

then applied to a 2.5 x 5.0 cm column of Con A-Sepharose previously equilibrated with Buffer A at a flow rate of 30 ml/hr. The column was washed with the same buffer until the A,,, of the column eluate reached a constant background level. The active protein was eluted with Buffer A containing 0.2 M a-methyl-o-glucopyranoside. The active fractions were pooled and dialyzed against 0.1 M acetate buffer, pH 4.2, containing 0.5 M NaCl (Buffer B). The dialyzed solution was applied to a 2.5 x 5.0 cm column of pepstatinSepharose equilibrated with Buffer B. The column was washed in succession with Buffer B and 0.1 M NaHCO, buffer, pH 7.1, containing 0.5 M NaCl at a flow rate of lOOml/hr. The cathepsin D activity was then eluted with 0.1 M NaHCO, buffer, pH 8.6, containing 0.5 M NaCl. The fractions which exhibited constant specific activity were collected and concentrated by ultrafiltration with a Minimodule (Asahi Chemical Industry Co. Ltd, Japan). Preparation

ofpepstatin-Sepharose

Pepstatin A was attached to aminohexyl-Sepharose synthesized according to the method of Cuatrecasas and Anfinsen (1971). 50 mg of pepstatin was dissolved in 5 ml of tetrahydrofuran/water (5 : 2, v/v). 20 g of suction-filtered Sepharose 4B previously washed in 300ml of tetrahydrofuran/water (5:2, v/v) and IOOmg of I-ethyl-3(3-dimethylaminopropyl)-carbodiimide were added to the pepstatin solution, and the pH of the solution was adjusted to 5.0 with 0.1 M HCl. The mixture was stirred gently for 6 hr at room temperature and the pH was maintained at 4.6 with 0.1 M HCl. 100 mg of l-ethyl-3 (3-dimethyl-aminopropyl)-carbodiimide was added and the coupling reaction was carried out overnight at 4°C. The gel was then washed successively with the following solutions: 500 ml of tetrahydrofuran/water (5: 2, v/v), I 1of water, 500 ml of I M NaCl and 500 ml of Buffer B. Assay of cathepsin D

Cathepsin D was measured according to the modified method of Watabe and Yago (1983) using bovine hemoglobin as the substrate. 1% substrate protein in 0.2 M citrate buffer, pH 3.5, was incubated with an appropriate amount of enzyme in a final volume of 200 ~1 for 1 hr at 37°C. The reaction was terminated by the addition of 200 ~1 of ice-cold 10% (w/v) trichloroacetic acid. The samples were centrifuged for IO min at IOOOgand the supematant was carefully removed. The amount of acid soluble peptide in the supernatant was determined by the fluorescamine method (Bohlen et al., 1973). One unit was defined as the amount of enzyme releasing 1pmol tyrosine equivalent in I hr. Electrophoretic analysis

Polyacrylamide gel electrophoresis was performed by the method of Laemmli (1970) on a 0.5 mm slab gel consisting of 12.5% gel with 3% stacking gel. The protein bands were stained with silver stain (Oakley, 1980). For molecular weight determinations, the gels were calibrated with molecular weight standards (SDS-PAGE Marker I, Seikagaku Kogyo Co. Ltd, Tokyo, Japan). lmmunoblotting procedure

Proteins on SDS-PAGE were transferred to nitrocellulose filters, and protein containing binding sites with anti human spleen cathepsin D serum were detected by the method of Towbin et al. (1979).

the protein was hydrolyzed in 6 N HCl for 24 hr at 110°C under vacuum. The analysis was carried out with a Hitachi 835 amino acid analyzer. Preparation of rabbit antiserum

Antibodies to the purified human spleen cathepsin D were raised in a rabbit. Purified cathepsin D in saline was emulsified with an equal volume of complete Freund’s adjuvant to give a concentration of 100 pg/ml. One ml of the emulsion was injected intradermally each week for 4 consecutive weeks. Serum was prepared from samples bled through the ear vein after another 6days. the presence of cathepsin D antibodies in the rabbit serum was evaluated by the Ouchterlony double diffusion test. Preparation Sepharose

of anti human spleen cathepsin D serum-

Anti cathepsin D serum was directly coupled to BrCNactivated Sepharose 4B at 4°C. 300 ~1 of the antiserum was mixed with 5 ml of 0. I M NaHCO,, pH 9.5, and 5 g of BrCN-activated Sepharose 4B which was synthesized and equilibrated with 0.1 M NaHCO,, pH 9.5, according to the method of Cuatrecasas and Anfinsen (1971). Coupling was carried out for 20 hr under gentle stirring. The gel was filtered and treated with 20 ml of 1 M Tris-HCl buffer. DH 8, overnight. The anti cathepsin D serum-Sepharose’was then sequentially washed with 200 ml of 1 M NaCl, 200 ml of 0.1 M acetate buffer. DH 5. and 500 ml of 0.9% NaCl in 10 mM phosphate buffer, pH 7.4 (Buffer C). Immunoafinity column chromatography

Two milliliters of the enzyme solution purified partially up to the Con A step described above, was dialyzed against Buffer C at 4°C and mixed with 5 ml of wet anti cathepsin D serum-Sepharose gel which had been washed with 200 ml of cold Buffer C. Gentle stirring was carried out for 40 hr at 4°C and the mixture was then packed into a column (1 x 6 cm). After washing the column with 20 ml of Buffer C, cathepsin D was eluted with 0.1 M citrate buffer, pH 2.5. Immunocyrochemistry

A small tissue sample of macroscopically normal spleen obtained at autopsy within 3 hr after the death of the patient, was quenched rapidly in dry ice-acetone. Sections of 5 pm were cut in a cryostat at - 20°C and dried overnight at 37°C. Immunocytochemical staining was carried out with the anti human spleen cathepsin D serum according to the indirect conjugate procedure (Nakane and Pierce, 1966). The sections washed for IOmin by immersion in cold phosphate buffer saline were incubated at room temperature for I hr with anti human spleen cathepsin D serum or normal rabbit serum as a negative control, which was diluted 1: 10 with phosphate buffer saline, respectively. After washing in phosphate buffer saline, the sections were incubated with peroxidase-labeled goat anti rabbit IgG serum diluted 1: 100 with phosphate buffer saline for 30 min at room temperature and washed in cold phosphate buffer saline. Staining of the peroxidase complex adsorbed on the sections was accomplished by incubation for 15 min in 3,3’-diaminobenzidine and 0.005% H,O,. After this reaction had been stopped by washing in cold phosphate buffer saline, staining of the nuclei with hematoxylin was performed for 5 sec. RESULTS

Protein delermination

Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. Amino acid analysis

Purified human spleen cathepsin D was dialyzed against distilled water and lyophilized. For the amino acid analysis,

PuriJication

of human

spleen cathepsin

D

Results for a typical purification of human spleen cathepsin D are summarized in Table 1. The enzyme

was purified 1830-fold as compared to the specific activity in the initial homogenate with an overall yield of 30.7%. Affinity chromatography on pepstatin-

319

Human spleen cathepsin D Table I. Purification of cathcpsin D from human spleen

Total Purification step Initial homogenate Acidification Con A-Sepharose Pepstatin-Sepharose

protein (ma)

14,400 6600 10.1 2.38

Sepharose allowed the removal of many proteins from the column by washing with 0.1 M NaHCO, buffer, pH 7.1, containing 0.5 M NaCl (Fig. 1). On SDS-PAGE, the purified enzyme showed two protein components which appeared as double bands with mobilities corresponding to approx. mol. wt 32,000 and 15,000 (Fig. 2). The major component reacted to the anti human spleen cathepsin D serum on immunoblotting, but the minor faint component did not. The reactivity by immunoblotting with the crude enzyme was similar, but there was an additional, minor reaction with 2 bands of approx. 53,000 and 47,OOOmol. wt (Fig. 3). The amino acid composition of human spleen cathepsin D is shown in Table 2. For comparison, the amino acid compositions of human liver and porcine spleen cathepsin D are also presented. Human and porcine spleen, and human liver cathepsin D clearly have quite similar compositions. Major differences are observed only in the amounts of methionine. Substrate speciJcity

The purified enzyme showed a pH optimum of 3.5 for hydrolysis of all tested substrates: human hemoglobin, human serum albumin and human y-immunoglobulin (Fig. 4). Table 3 summarizes the hydrolysis of six blood proteins by human spleen cathepsin D. The highest activity was found with hemoglobin, whereas bovine serum albumin and human or bovine y-immunoglobulin gave a lower activity at the optimum pH. pH stability of human spleen cathepsin D

The purified human spleen cathepsin D was most stable at pH 3.5 among the three tested pH levels.

1

I

Total activitvtU1

Sp. act. W/ma)

Purification t-fold)

I820 932 868 560

0.129 0.141 85.9 235

I

RecOVCrY wn)

loo

1.10 668 1830

51.1 41.1 30.7

PH Fig. 4. pH activity profile of human spleen cathepsin D using substrates of human origin. The concentrations of substrates were 1%. Assays for cathepsin D activity were performed as described under Materials and Methods. Values are presented as percentages of the activity at the optimum pH for each of the substrates. Human hemoglobin (0). human serum albumin (0) and human y-globulin (A). Table 2. Amino acid composition of human spleen cathepsin D Content (mol/100mol Amino acid ASX Thr Ser Glx Pro Cly Ala Vdl CYS Met Ile Leu Tyr Phe LYS His Arg Trp

Human spleen cathepsin D

of glycine)

Human liver cathepsin Da

Porcine spleen cathepsin Db

7s

80 52 64 89 58 IO0 49 78 I9 3s 55 88 52 38 63 13 25 IO

83 58 63 78 54 100 47 73 I8 3 54 83 38 38 55 I8 24 ND’

‘.bValues obtained from Barrett (1979) and (1984).respectively;‘not determined.

48 61 70 so 100 43 70 18 20 58 70 31 31 52 IS 20 IO Shewale and

Table 3. Substrate specificity of purified human

Tang

spleen

cathepsin D Substrate

Fmction number

Fig. I. Chromatography of human spleen cathepsin D on a pepstatin-Sepharose column. The elution conditions were as described under Materials and Methods. Fractions of 5 ml were collected. Absorbance at 280nm (0) and cathepsin D activity (A).

Bovine hemoglobin Bovine serum albumin Bovine 7 -globulin Human hemoglobin Human serum albumin Human y-globulin Aaocasein

Activity (%) 100 I6 2.5 130 71 2.9 I2

Concentration of substrates were I %. All activities are relative to that obtained with human hemoglobin which is set at 100. The activity was measured as described under Materials and Methods.

320

KAZUMASA IKEDA et

al.

1.0

O’

E i B

z .Z .t

z 0.05 1

Q 0.5

0

0

10

20

30

The

40

5

10

15

20

Fraction number

50

(min)

Fig. 5. Effect of pH on human spleen cathepsin D stability. The purified enzyme solutions dialyzed against 50mM citrate buffer, pH 3.5 (O), 50 mM citrate buffer, pH 5.0 (a), or 50 mM phosphate buffer, pH 7.4 (A), were incubated at 37°C for different periods of time as indicated. Cathepsin D activity was measured as described under Materials and Methods.

Fig. 7. Immunoaffinity chromatography of human spleen cathepsin D. The elution conditions were as described under Materials and Methods. Fractions of 1 ml were collected at a flow rate of 1ml/hr. Absorbance at 280 nm (0) and cathepsin D activity (A).

3.5 under the given conditions after a 45 min incubation period (Fig. 5). At neutral pH, the cathepsin D activity was unstable as compared to those at acidic pHs at 37°C.

column did not adhere to anti cathepsin D serum-Sepharose but about 80% of the applied cathepsin D activity did adhere. The cathepsin D activity emerged as a sharp peak which contained 70% of the applied activity. It should be noted also that cathepsin D did not bind to a control (normal rabbit serum reacted) Sepharose 4B column under any conditions tested.

Inhibition of human spleen cathepsin D

Immunocytochemistry

The effects of various compounds on the purified enzyme are summarized in Table 4. The enzyme was potently inhibited by 10 pg/ml pepstatin and 10 pg/ml diazoacetyl-DL-norleucine methylester. The SH compounds, cysteine, mercaptoethylamine and glutathione, also inhibited about 50% of the control activity at 5 mM.

In the human spleen, antigenic sites for cathepsin D were demonstrated sporadically as immunodeposits of 3,3’-diaminobenzidine in large single cells around follicular areas, but not in the masses of small and middle lymphocytes in the white pulp (Fig. 8). The cells seemed to belong to the reticuloendothelial system based on their location.

About 40% of the cathepsin D activity was lost at pH

Detection of anti human spleen cathepsin D serum

Both the crude and purified human spleen cathepsin D were tested against a control serum and an antiserum by the Ouchterlony immunodiffusion technique (Fig. 6). Two sharp precipitin lines, indicating reactions of identity, were observed only with the antiserum. Potency of the antibodies to human spleen cathepsin D was confirmed using antiserum affinity chromatography (Fig. 7). Under the stated conditions, most of the proteins in the pool from the Con A Table

4. Inhibition

of human

spleen

cathepsin

D activity Activitv

Concentration

Inhibitor

(%)

10mM

100

Cystcine

5lllM

43

Mcrcaptoethylamine

5mM

57

GSH

5mM

48

lodoacctic

acid

10 leg/ml

Pcpstatin

2

Diazoacetyl-oL-norleucine methylester

10 fig/ml

II

IO uniml

100

l,2-Epoxy-3-(p-nitrophenoxy) orooane Cathepsin under tration

D activities Materials

and Methods,

of inhibitor.

percentage value

were measured

relative

represents

The

containing

cathepsin

to the activity

the average

in the assay mixture

D activity without

described

the indicated any inhibitors.

of 3 experiments.

concen-

is expressed

as a Each

DISCUSSION

Human spleen has been used as an enzyme source for cathepsin D (Reid et al., 1986) but the purification procedures and enzyme characteristics have not been described in detail. Our purification procedure for human spleen cathepsin D involved a combination of two affinity chromatographies, using Con ASepharose and pepstatin-Sepharose, by a modification of the method of Afting and Becker (1981). Pepstatin-Sepharose has been synthesized by several workers (Huang et al., 1979; Murakami and Inagami, 1975; Whitaker and Seyer, 1979; Linde and Perslinden, 1978; Kazakova and Orekhovich, 1976). Only a small amount of pepstatin could be dissolved in the reaction solvents they mentioned and so that the adsorption potential of the given pepstatinSepharose appeared to be low. We employed a solvent in which more than 10 mM of pepstatin and a water soluble carbodiimide could be dissolved, and succeeded in synthesizing an affinity matrix binding a high level of pepstatin. Afting and Becker (1981) successfully performed pepstatin-Sepharose affinity column chromatography using 6 M urea solution as the washing buffer of the column. However, a high concentration of urea might uncouple the hydrogen

42 .9 K

,28 .6K

-14 .3K

Fig. 2. SDS-PAGE analysis of purified human spleen cathepsin D. (A) Silver stained gel of the Con A-Sepharose fraction of human spleen cathepsin D (lane 1), the finally purified human spleen cathepsin D (lane 2) and molecular weight standard (lane 3). (B) Immunoblotting of the parallel gel of the gel used in part (A) above. Con A-Sepharose fraction (lane 4) and tinally purified enzyme (lane 5).

321

‘,&@_:

L

g”;.

57.2 K

.a&-42.9K

-

28.6 K

-

14.3K

Fig. 3. Antigenic components against anti human spleen cathepsin D serum in human spleen extract. (A) Silver stained gel of human spleen extract. The soluble fraction of human spleen homogenized in water with Politron was prepared as the human spleen extract. (B) Immunoblotting of the parallel gel.

322

Fig. 6. Immunodiffusion analysis of human spleen cathepsin D. The Ouchterlony double diffusion test was carried out at 4°C for 20 hr. Well 1, rabbit anti human spleen cathepsin D serum; well 2, purified enzyme; well 3, acid extract of human spleen; and well 4, a control serum. Each well contained lO/rl of sample.

323

Fig. 8. Immunocytochemical hum an spleen were stained

demonstration of cathepsin D in human spleen. 5 pm Sections with the anti human spleen cathepsin D serum as described under and Methods (A x 40, B x 200).

324

of . frozen M Iaterials

325

Human spleen cathepsin D bonds between the light chain and heavy chain in the cathepsin D molecule during the chromatography (Huang et al., 1979). We therefore excluded all proteins except for cathepsin D from the pepstatin column with pH change of the washing buffer (Fig. 1). The purified enzyme revealed two double bands on SDS-PAGE. This was similar to the protein pattern reported for human liver cathepsin D purified without a pepstatin affinity column by others (Barrett, 1979). The production of anti human spleen cathepsin D serum raised in a rabbit was demonstrated by both the immunodiffusion method and immunoadsorbent column chromatography. The antiserum did not inhibit the cathepsin D activity but cathepsin D was specifically adsorpted onto the immuno affinity column. The crude enzyme purified by the previous step of pepstatin-Sepharose chromatography showed a single immunoreactive band on immunoblotting (Fig. 2). On SDS-PAGE of the final purified enzyme, apparent double protein bands reacted with the antiserum, suggesting that the smaller molecular weight band represents decomposed protein of native cathepsin D formed in the course of purification with pepstatin-Sepharose. Three immunoreactive compounds corresponding to mol. wt 53,000, 47,000 and 32,000, were obtained in the SDS gel from acid extract of human spleen (Fig. 3). The two minor bands among them were eliminated by Con A-Sepharose column chromatography, suggesting that these two proteins were not so highly glycosylated and apparently corresponded to a precursor and an intermediate type of cathepsin D which have been reported in cultured human fibroblasts (Gieselmann et al., 1985). Cathepsin D activity was rather unstable at neutral or alkaline pH compared to acidic pH (Fig. 5). Denaturation of the enzyme eluted from pepstatinSepharose may occur in alkaline buffer solution for the elution. On the other hand, Barth and Afting (1984) have reported that end-p-N-acetylglucosaminidase H could cleave the oligosaccharide moiety in pig myometrium cathepsin D and after such treatment, the enzyme showed a higher electrophoretic mobility on SDS-PAGE. Over 50% of the /I-N-acetylglucosaminidase in initial homogenate of human spleen remained in the crude enzyme purified partially to the Con A-Sepharose step (data not shown) and this activity might remove the oligosaccharide moiety from cathepsin D during dialysis in pH 4.2 buffer since cathepsin D might behave as a preferential substrate at that pH. The amino acid composition of purified human spleen cathepsin D is similar to that reported for human liver (Barrett, 1979) or porcine spleen (Shewale and Tang, 1984), with a major difference being observed only in the amounts of methionine. As is the case with cathepsin D of other origins, human spleen cathepsin D was inhibited with pepstatin which is a potent inhibitor of aspartyl proteases (Umezawa, 1976). The amino acid sequence of cathepsin D from porcine spleen is closely homologous with that of pepsin (Shewale and Tang, 1984). Diazoacetyl-DL-norleucine methylester and 1,2epoxy-3Qnitrophenoxy) propane inhibit pepsin by reacting with different aspartyl residues of the

enzyme, respectively (Bayliss et al., 1969, Chen and Tang, 1972). These two aspartyl residues are identical between pepsin and cathepsin D from porcine spleen (Shewale and Tang, 1984). 1,2-Epoxy-3-(p-nitrophenoxy) propane reacts with the aspartyl residue at position 32 in the pepsin molecule (Chen and Tang, 1972), which corresponds to that at position 33 in the light chain of porcine cathepsin D. Human spleen cathepsin D was strongly inhibited with diazoacetylDr.-norleucine methylester but not with 1,2-epoxy-3(p-nitrophenoxy) propane, suggesting that the amino acid residue located at this position of the light chain of human spleen cathepsin D is not related to its proteolytic activity. Kooistra et al. (1982) have reported that thiol compounds do not inhibit cathepsin D. However, our studies indicate that cysteine, mercaptoethylamine and glutathione do inhibit human spleen cathepsin D, especially glutathione which inhibited it at the level of a physiological concentration. Mego (1984) reported a stimulatory effect of thiols on the degradation of bovine serum albumin with mouse cathepsin D due to disulfide-bond reaction in the substrate. However, our results indicate that thiols inhibit the degradation of bovine serum albumin with human spleen cathepsin D (data not shown). These inhibitions suggest that thiol is more effective on the disulfide bonds of human spleen cathepsin D than on those of serum albumin, and cathepsin D may be regulated with thiols in human spleen lysosomes. The antigenic sites for cathepsin D were not in lymphocytes of the human spleen (Fig. 8). The finding that y-globulin was not degraded by human spleen cathepsin D supports the hypothesis that cathepsin D may not fulfill so much of its proteolytic function in the white pulp. Stained cells in the white pulp as found by Reid et al. (1986) were not observed in our stained sections. Cathepsin D contained in macrophages contributes to the digestion of erythrocyte proteins endocytosed by macrophages (Dingle et al., 1973). In our stained sections of human spleen, all cells which had presented in sinusoids disappeared during the immunostaining procedure. Free macrophages were not found in the sections and the presence of antigenic sites for cathepsin D was not confirmed in free macrophages. Cells containing abundant cathepsin D in the splenic cords appeared to be fixed macrophages or reticular cells which constituted the so-called reticuloendothelial system. The activity of cathepsin D in alveolar macrophages is almost 10 times higher than that in resident peritoneal macrophages (Orlowski et al., 1984), which allows us to conjecture on the specific amount of cathepsin D activity in a splenic macrophage. To determine the origin of the abundant cathepsin D contained in the spleen, we are now undertaking immunocytochemical localization studies at the electron microscopic level. REFERENCES

Afting E. G. and Becker M. L. (1981) Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield. Biochem. J. 197, 519-522. Barrett A. J. (1971)Purification and properties of cathepsin

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KAZUMASAI t(EDA

D from liver of chicken, rabbit and man. In Tissue Proteinaees (Edited by Barrett A. J. and Dingle J. T.), pp. 209-248. North Holland, Amsterdam. Barrett A. J. (1979) Human cathepsin D. Adu. exp. Med. Biol. 95, 291-300. Barth R. and Afting E. G. (1984) Cathepsin D from pig myometrium. Biochem. J, 219, 899-904. Bayliss R. S., Knowles J. R. and Wybrandt G. B. (1969) An aspartic acid residue at the active site of pepsin. Biochem. J. 113, 377-386. Bohlen P., Stein S., Dairman W. and Udenfriend S. (1973) Fluorometric assay of proteins in the nanogram range. Archs Biochem. Biophys. 155, 213-220. Cfien K. C. S. and Tang J. (1972) Amino acid sequence around the epoxy-reactive residues in pepsin. J. biol. Chem. 247, 25662574. Contractor S. F., Manson R. M. and Oakey M. (1982) Purification and properties of human placental cathepsin D. Piaeenta 3, 45-56. Cuatrecasas D. and Anfinsen C. B. (1971) Affinity chromatography. Meth. Enzym. 22, 345-378. Dingie J. T., Poole A. R., Lazarus G. S. and Barrett A. J. (1973) Immunoinhibition of intra~iiuiar urotein digestion in macrophages, J. exp. Med. 137, 1i24-1141. Gieselmann V., Hasilik A. and Figura K. (1985) Processing of human cathensin D in lysosomes in vitro. J. Mol. Chem. 260, 3215-3220: _ Helseth D. J. Jr and Veis A. (1984) Cathepsin D-mediated processing of procollagen: lysosomal enzyme involvement in secretory processing of procollagen. Proc. natn. Acad. Sci. U.S.A. 81, 3302-3306. Huang J. S., Huang S. S. and Tang J. (1979) Cathepsin D isozymes from porcine spleens. J. biof. Chem. 254, 11405-i 1417. Kaxakova 0. V. and Orekhovich V. N. (1976) Crystallization of cathepsin D. Biochem. biouhvs. - f Res Commun. 72. 747-752. Kooistra T., Millard C. and Lloyd B. (1982) Role of thiols in degradation of proteins by cathepsins. Biochem. J. 204, 471-477. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature 227, 680-685. Linde A. and Persliden B. (1978) Purification of cathepsin D by AH-Sepharose affinity chromatography. Prep. B&hem. 8, 23 i-240. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin reagent. J. biol. Chem. 193, 265-275. Mego 1. L. (1984) Role of thiols, pH and cathepsin D in the

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