Purification and Properties of Cathepsin D from Porcine Spleen*

Transcription

1 THE JOURNAL. OF BIOI.OGICAL CHEMIWZY Vol. 251, No. 15, Issue of Aupst 10. pp Printed m U.S.A. Purification and Properties of Cathepsin D from Porcine Spleen* (Received for publication, December 23, 1975) MADELEINE CUNNINGHAM AND JORDAN TANG From the Laboratory of Protein Studies, Oklahoma Medical Research Foundation and the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklah6ma City, Oklahoma Cathepsin D was purified from porcine spleen to near homogeneity as determined by gel electrophoresis. The isolation scheme involved an acid precipitation of tissue extract, DEAE-cellulose and Sephadex G-200 chromatography, and isoelectric focusing. The end product represented about a fold purification and about a 10% recovery. The purified enzyme was the major isoenzyme, which represented 60% of cathepsin D present in porcine spleen. Two minor isoenzymes of cathepsin D were present in small amounts. The purified enzyme resembled porcine pepsin in molecular weight (35,000), amino acid composition, and inactivation by specific pepsin inactivators. The ph activity curve of the purified enzyme showed two optima near ph 3 and 4. The relative activities at these optimal ph values were affected by salt concentration. Experimental evidence indicated that the two-optima phenomenon is a property of a single enzyme species. Although the gastric and microbial acid proteases have been extensively studied in recent years, the tissue protease cathepsin D is only understood superficially with regard to its catalytic mechanisms, its structure-function relationships, and its possible regulation in uiuo. Several reasons have contributed to our lack of knowledge of this important enzyme. First, cathepsin D is present in tissues in relatively low concentrations, and thus its purification is tedious. Although the purification of cathepsin D has been reported from a number of sources, including bovine spleen and other bovine tissues (l-7), chicken liver (8), porcine thyroid (9, lo), porcine intestinal mucosa (ll), and human red blood cells (la), the enzyme has not been obtained in large quantity. Second, conflicting reports have appeared on the molecular weight of this enzyme, ranging from 12,000 for bovine uterus enzyme (4) to 58,000 for cathepsin D from bovine spleen (1). It is not clear whether they are active fragments of cathepsin D or structurally different isoenzymes. Third, the presence of numerous isoenzyme forms adds more complications, and the number of isoenzymes appears to vary in different tissues. For example, there are three isoenzymes of cathepsin D in human and chicken liver (8) and 12 in bovine uterus (4). This confusion concerning the molecular weight and the possible existence of isoenzymes contributes to the difficulties in the structure-function studies. In view of this conflicting and confusing information, it is clear that significant progress in understanding cathepsin D structure and function can be made only if a reasonably large quantity of pure enzyme can be obtained. Additionally, a source of the cathepsin must be found from which a major isoenzyme form can be obtained for further studies. Finally, *This work was supported by Research Grants GM and AM-6487 from the National Institutes of Health the size and other characteristics of cathepsin D must be clearly documented in order to facilitate such studies. For the following reasons we have undertaken a study of cathepsin D from porcine spleen. This tissue was chosen not only because cathepsin D from porcine spleen has not been extensively studied, but also because a large supply of this organ would be available for the scale-up in the enzyme purification. Moreover, since porcine pepsin is the most extensively studied acid protease (13), a direct comparison of porcine pepsin to a cathepsin D from the same animal source would be preferable. In this paper we report the purification and properties of porcine spleen cathepsin D. The enzyme has been purified to near homogeneity. Only one major isoenzyme form was found. The size, composition, and other characteristics of porcine spleen cathepsin D are very similar to those of porcine pepsin. MATERIALS AND METHODS Purification of Cathepsin D-Fresh porcine spleen was obtained locally from Wilson Packing Co. (Oklahoma City, Okla.) and was immediately frozen and kept at 10. Three to six spleens were thawed in the refrigerator overnight for extraction the next day. After removing fat, the spleens were minced and homogenized for 2 min in a Waring Blendor in ice-cold distilled water. About 2 ml of water were mixed with each gram of minced tissue. The homogenate was centrifuged for 30 min at 16,300 x gin a GSA rotor of a Sorval RC-2B centrifuge at 4. The tissue pellet was discarded. The supernatant was decanted and dried by lyophilization. The lyophilized extract (about 30 to 40 g) was dissolved in ice-cold distilled water giving a total volume of approximately 80 to 100 ml. To this suspension, 1 N HCl was added with stirring at 4 to achieve a ph value of 3.7 to 3.8. A heavy precipitate formed in this process and the suspension was allowed to stand 1 hour at ice temoerature before centrifuging at 27,000 x g for 30 min in a Sorvall RC-2B centrifuge (SS-34 rotor). The supernatant was decanted and kept at 4. The ph of this solution was adjusted to 6.0 to 7.0 using 1 M Na,HPO,. Then

2 Cathepsin D from Porcine Spleen 4529 the preparation was dialyzed for 36 to 48 hours against several changes of 6 liters of sodium phosphate buffer, ph 7. The crude enzyme solution was then applied to a DEAE-cellulose column (2 x 50 cm), which was equilibrated and eluted with M sodium phosphate buffer at ph 7. Fractions of 3.6 to 3.8 ml were collected. The active fractions (see Results ) were pooled, dialyzed against water, and lyophilized. The pool from the DEAE-cellulose column was then applied to a Sephadex G-200 column (2.6 x 41.5 cm) equilibrated with 0.1 M sodium phosphate buffer, ph 6.8. Fractions of 1.4 ml were collected. The pooled active fractions from the Sephadex chromatography occasionally were applied to a CM-cellulose column which was equilibrated with M sodium phosphate buffer, ph 7. The enzyme was eluted from the CM-cellulose column with a linear gradient of 0 to 0.15 M NaCl in the same buffer. Proteolytically active fractions were pooled, dialyzed against water, and lyophilized. Isoelectric focusing was performed in an LKB 8100 column (LKB Instruments, Rockville, Md.). Ampholines with a ph range of 6 to 8 (LKB Produkter AB Bromma, Sweden) were used in a sucrose gradient, One-milliliter fractions were collected at the end of the isoelectric focusing, and the ph of each fraction was determined. Proteolytically active fractions were pooled, dialyzed 3 days against water at 4, and lyophilized. Assay of Cathepsin D Actiuity-The hemoglobin assay described determines the acid proteolytic activity in preparations from homogenized spleen. Before purification the assay probably measured cathepsin E as well as D. The assay was initiated by the addition of a small aliquot of enzyme to a mixture of 0.2 ml of 0.5 M sodium formate buffer, ph 3.2, and 0.2 ml of 5% bovine hemoglobin which was preincubated at 37. The reaction was stopped after 30 min of incubation at this temperature with the addition of 2.5 ml of 3% trichloroacetic acid. The precipitated mixture was allowed to stand for 10 min at room temperature and then was filtered through Whatman No. 50 filter paper. The absorbance of the filtrate was measured at 280 nm on a Zeiss model PMQ3 spectrophotometer. The enzyme unit was defined as the increase in optical density at 280 nm. Polyacrylamide Gel Electrophoresis-Proteins were separated by polyacrylamide gel disc electrophoresis according to the method of Davis (14). Approximately 50 pg of protein were applied in sample gels. Electrophoresis was performed in a Canalco model 1200 apparatus (Canalco, Rockville, Md.). All reagents for the electrophoresis were obtained from Canalco, and stock solutions were mixed to obtain 7% acrylamide containing 2.63% N,N-methylenebisacrylamide. Gels were fixed in 20% sulfosalicylic acid for at least 18 hours and were stained in 0.25% Coomassie blue in water. Destaining was accomplished in 7% acetic acid. Some of the unstained gels were sectioned, and the sections were eluted in M sodium phosphate buffer and assayed for enzyme activity. Molecular Weight-Molecular weights of purified preparations were determined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate according to the method of Weber and Osborn (15). Proteins with known molecular weights were used as standards. They were 23,800 for trypsin (16), 34,600 for pepsin (17), and 68,000 for serum albumin (18). The molecular weight of cathepsin D was determined from the linear semilogarithmic plot. Molecular weight was also determined from the elution volume of proteins from Sephadex gel filtration experiments. Proteins of known molecular weight were chromatographed to obtain a standard curve from which the molecular weight of cathepsin D was estimated according to the procedure described by Andrews (19). Fluorescamine Determination-When the acetylated CNBr fragment of human hemoglobin 01 chain (peptide D) was digested with cathepsin D, the newly exposed NH, termini were measured by the fluorescamine reaction. To 2 ml of 0.1 M borate buffer, ph 8.0, 0.1 ml of digest was added. Fluorescamine (Roche Diagnostics; Division of Hoffman-LaRoche Inc.; Nutley, N. J.) was prepared in spectral grade acetone in a concentration of 0.1 mg/ml. One milliliter of fluorescamine solution was added to the borate buffer solution and mixed well. Samples were read immediately in an Aminco Bowman fluorometer model with an excitation wavelength of 390 nm and an emission wavelength of 475 nm. Slits used in the fluorometer were Nos. 4, 3, 3,4, and 4, and the instrument was set at 80% sensitivity. A standard plot of the relative intensities of 200 pm01 to 50 nmol of Glu-Glu was used to determine the nanomoles of new NH, terminus produced. Isoelectric Focusing in Polyacrylamide Gels--Isoelectric focusing was performed in polyacrylamide gels according to the method of Wrigley (20). Samples were focused for 3 hours, and gels were removed from the tubes and either stained or sectioned. Sections (2 mm) were eluted in water and the ph and cathepsin D activity were measured. The gels were stained in aqueous 0.15% bromphenol blue after being fixed for 30 min in 10% trichloroacetic acid. Amino Acid Analysis-Amino acids were determined with a Spinco model 120B amino acid analyzer. A modified range card in the recorder permitted quantitative analysis in the range from to 0.10 rmol of amino acid. Analysis was performed according to the accelerated procedure of Spackman (21). The samples were hydrolyzed in 5.7 N HCl in sealed evacuated tubes for 24 hours at 110 * 2. After removal of the HCl under reduced pressure, the residues were dissolved in 0.2 M sodium citrate buffer, ph 2.2. Aliquots of the samples were analyzed. Tryptophan was determined in the amino acid analyzer after hydrolysis with methanesulfonic acid according to the method of Liu and Chang (22) Isolation of Cathepsin D Substrate from CNBr-fragments of Human Hemoglobin-A peptide containing the NH,-terminal 32 residues of the cy chain of human hemoglobin was isolated. The detailed procedure is described in the supplement to this paper. The structure of this peptide is Val-Leu-Ser-Pro-Ala-Asp-Lys-Thr-Asn-Val-Lys-Ala-Ala- Trp-Gly-Lys-Val-Gly-Ala-His-Ala-Gly-Glu-Tyr-Gly-Ala-Glu-Ala-Leu- Glu-Arg-Hse. Digestion of Oxidized Insulin A and B Chains by Cathepsin D-The digestion mixture contained 20 mg of A or B chain and 700 and 500 pg, respectively, of cathepsin D in 2 ml of 0.4 M ammonium acetate buffer, ph 3.2. The sample was incubated at 37 for 43 hours, frozen at dry ice temperature, and lyophilized. RESULTS Purification of Cathepsin D A summary of a representative purification is shown in Table I. In all, six steps were involved with a final purification of about 1000-fold and with at least 10% recovery. The chromatographic pattern of the acid supernatant on the DEAE-cellulose column is shown in Fig. 1A. The cathepsin D activity was eluted in M sodium phosphate buffer. A minor activity peak was eluted in the same buffer containing 0.1 M NaCl. This peak was assumed to be cathepsin E, which is known to absorb on a DEAE-cellulose column and to elute only at higher concentrations of salt (23). The first active fraction was subjected to further purification on Sephadex G-200 (Fig. 1B) and by isoelectric focusing (Fig. 1C). The last step yielded a major protein and activity peak at ph An apparent broad shoulder of this activity peak can be seen on the low ph side, and this was reproducible in all other isoelectric focusing experiments. This shoulder probably represents the presence of some isoenzyme forms which will be discussed further in the isoenzyme section ( Results ). Since the total units of activity in this shoulder region were minimal, the material from this region was not further investigated. The homogeneity of the final preparation was tested by polyacrylamide gel electrophoresis. Only one major band was observed (Fig. 2). A few light bands of contaminants were frequently seen, but judging from the intensities of these bands, they were present in very low quantities. PolyacryIamide gels were sectioned, eluted and tested for proteolytic activity. As can be seen in Fig. 2, the activity was associated with a protein band. Sectioned gels from a preparation after Some of the data are presented as a miniprint supplement immediately following this paper. (Figs. Sl through S6 and Tables SI through SV will be found on pp ) For the convenience of those who prefer to obtain the supplementary material in the form of 17 pages of full size photocopies, they are available as JBC Document Number 76M-296. Orders should specify the title, authors, and reference to this paper, the JCB Document Number, and the number of copies desired. Orders should be addressed to The Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Maryland 20014, and must be accompanied by a remittance to the order of the Journal in the amount of $2.50 per set of photocopies.

3 l 4530 Cathepsin D from Porcine Spleen TABLE I Purification of porcine spleen cathepsin D step Fraction Total units Total protein Specific activity RWOWry Purification rm w Pd%? w -fold 1 Extract , Lyophilizate , Acid supematant , DEAE-cellulose Sephadex G Isoelectric focusing A M NaCl a b C d FRACTION NUMBER FIG. 1. A, DEAE-cellulose column chromatography of spleen acid supernatant. Elution was with sodium phosphate buffer, ph 7; B, Sephadex G-260 column chromatography of DEAE-cellulose-purified cathepsin D in 0.1 M sodium phosphate buffer, ph 6.8; C, preparative isoelectric focusing of the Sephadex fraction of cathepsin D in an ampholyte ph gradient from ph 6 to 8. -, optical density at 280 nm; --- cathepsin D activity;., ph. The horizontal bar underneath the peak represents the pooled fractions. Sephadex G-200 chromatography and after isoelectric focusing showed that the activity migrated to the same position in both cases. Characterization Studies ph Optimum-The optimum ph for cathepsin D activity was tested using purified enzyme and hemoglobin substrate in the ph range from 2 to 5. Two optima at ph 3.4 and 3.8 were i\. I / : /*,I \ A,* _-.--._ :p: I I I I I CM FROM ORIGIN FIG. 2. Polyacrylamide gel patterns of cathepsin D fractions. fop to bottom: a, acid supernatant; b, DEAE-cellulose fraction; c, Sephadex G-200 fraction; d, isoelectrically focused fraction. Arrow indicates the position of cathepsin D. Graph demonstrates the activity of 2-mm sections of polyacrylamide gels of cathepsin D fractions; O-0, isoelectrically focused fraction and l , Sephadex G-200 fraction. seen with either 0.5 M sodium acetate or formate buffers (Fig. 3). When the buffer concentration was changed to 0.1 M, a shift in the ph profile was observed and the ph optimum was approximately 2.9. The optimum at ph 4 was still present, but greatly reduced in size (Fig. 3). This shift in the ph dependence curve was apparently an effect of salt concentration. Addition of 0.4 M NaCl to 0.1 M formate or acetate buffer produced a ph profile similar to that of 0.5 M formate or acetate buffer. If the cation of the buffer was varied to K+ or NH,+, no difference was seen. In order to determine whether the salt had an effect on substrate or enzyme, another smaller substrate, acetylated hemoglobin peptide D (see Materials and Methods ), was used in the presence of two different salt concentrations at P *\.

4 Cathepsin D from Porcine Spleen 4531 TIME OF DIGESTION imini FIG. 3 (left). ph optima of purified cathepsin D. Sodium formate or acetate buffer, 0.1 M (O--3); 0.5 M (a- - -0); 0.1 M sodium formate or acetate +0.4 M sodium chloride (04). FIG. 4 (right). Digestion of acetylated peptide D by cathepsin D with fluorescamine determination of hydrolysis. Hydrolysis at ph 2.8 and 4.0 in 0.1 M and 0.5 M buffers; 04, 0.1 M, ph 2.8; l M, ph 2.8; O-O, 0.1 M, ph 4.0; O...O, 0.1 M, ph 4.0. varying ph values. The results in Fig. 4 showed no significant difference of the initial hydrolysis rate in the two salt concentrations, 0.1 M and 0.5 M. The salt effect observed with the hemoglobin substrate, at both ph 2.8 and 4.0, must be due to conformational changes in the protein substrate. When the ph optimum was measured using peptide D, a bimodal curve similar to that using hemoglobin substrate was again seen (not shown). Therefore, further experiments were performed to determine whether two species of enzyme might be present. The enzyme was treated with low concentrations of an irreversible inhibitor, diazoacetyl-dl-norleucine methyl ester to inactivate approximately one-half of the enzyme activity. The ph activity curve was again produced using hemoglobin substrate with 0.5 M buffer. The ratio of the proteolytic activity at the peaks near ph 3.0 and ph 4.0, however, remained the same as controls untreated with inactivator. Partial inactivation of the enzyme by acid or alkali at ph 2.0 and ph 10.0 likewise left the ratio of the two peaks unaltered. These results suggest that the bimodal curve seen in the ph optimum experiments was probably the activity of a single cathepsin D and was not due to the presence of a second enzyme species. Molecular Weight-The molecular weight of isoelectrically focused cathepsin D was determined by different methods. The result of electrophoresis in sodium dodecyl sulfate-polyacrylamide gels is shown in Fig. 5. The molecular weight of the enzyme was determined to be 34,000 to 35,000. Since previous studies (10) have suggested that porcine thyroid cathepsin D was a dimer near ph 7 to 7.5 and a monomer near ph 3 to 4, the enzyme was chromatographed on Sephadex G-200 in buffers of various ph values. The elution position of the enzyme corresponded to a molecular weight of 35,000 as compared to proteins of known molecular weight (Fig. 6). When a crude enzyme preparation was chromatographed separately at ph 4 or 7, the enzyme activity was observed in the same elution volume (as the position shown in Fig. lb), indicating that no change in molecular weight had taken place. Occasionally, very minor activity peaks could be seen at positions corresponding to molecular weights of approximately 21,000 and 43,000. The presence of these minor peaks had no apparent relationship with the ph of the elution buffer. Furthermore, when electrofocused cathepsin D was chromatographed again on Sephadex G-200, the activity was found at the identical elution position as that observed for less purified preparations. g RELATIVE MOBILITY FIG. 5. Molecular weight determination of cathepsin D as a function of mobility in sodium dodecyl sulfate-polyacrylamide gels z E 260 I 240 ui P F IJj 140 a, 120 > \ LYSOZYME (14.400) CATHEPSIN D pti7 ( CATHEPSIN D ph4 (34.500) 4LBUMIN ). \ \ PEPSIN ) MOLECULAR WEIGHT FIG. 6. Molecular weight determination of cathepsin D by elution from a Sephadex G-200 column (2.6 x 41.5 cm) in 0.1 M sodium phosphate or acetate buffer, ph 6.8 or 4.0, respectively. Amino Acid Compositon-The amino acid composition of cathepsin D from porcine spleen is shown in Table II. Zsoenzymes-Reports of isoenzyme forms of cathepsin D have been numerous (1, 4, 8). In the purification described here, we observed one major form of cathepsin D containing approximately 50 to 60% of the total activity. However, several minor isoenzyme forms were also observed. First, as described in the molecular weight determinations, two minor activity peaks were found in the chromatographic pattern of cathepsin D on Sephadex G-200. Second, when the partially purified enzyme from the DEAE-cellulose fraction (step 4, Table I) was separated in isoelectric focusing in a ph 3.5 to 10.0 gradient, three proteolytically active peaks were observed (Fig. 7). The major peak, which had an isoelectric point of 7.68, was essentially the same as that for the final preparation of cathepsin D. Two minor peaks appeared at isoelectric points of 7.35 and Some of these minor isoenzyme forms were probably removed with Sephadex G-200. Therefore, they appeared only as a shoulder in the final purification step (Fig. 1C). In order to find out whether these isoenzyme forms could be artifacts produced in the acid precipitation step of the purification, the original extract and the acid supernatant (steps 1 and 2 in Table I) were subjected to isoelectric focusing in polyacrylamide gels. The patterns of the proteolytic activity were identical to that shown in Fig. 7, indicating that these isoenzyme forms were not artifacts produced during the acid precipitation step (step 3, Table I). Enzyme Stability and Inhibition Studies-Purified cathepsin D was stable upon freezing and at temperatures up to 50, which was found to be the optimum temperature for enzyme activity. The enzyme was most stable at ph values near neutral, and various divalent and monovalent cations had no significant effect on enzyme activity (see Fig. S6 and Table 105

5 4532 Cathepsin D from Porcine Spleen Amino acid 24.Hr hydrolysis Lysine Histidine Arginine Tryptoph& Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Total TABLE Amino acid composition of porcine spleen cathepsin D II 4%Hr hydrolysis 72.Hr hydrolysis Final Nearest integer Porcine pepsin Bovine chymosin residues/molecule *Taken from Foltmann s chymosin sequence (B. Foltmann, personal communication). c Assuming a molecular weight of 35,000 for cathepsm D, as found in this work. The final values are the averages with standard deviations FRACTION NUMBER FIG. 7. Isoelectrically focused DEAE-cellulose fraction of cathepsin D in a gradient from ph 3.5 to 10.0; -, optical density at 280 nm; ---, cathepsin D activity;., ph. SIV ). Specific pepsin inactivator, diazoacetylnorleucine methyl ester, inactivated the enzyme rapidly. Several epoxide pepsin inactivators, including 1,2-epoxy-3-(p-nitrophenoxy) propane which is a specific inactivator of pepsin (24-26), inactivated cathepsin D. In order to achieve sufficient inactivator concentration, 5% ethanol was added to the solutions. These results are documented in the miniprint (Fig. S5 and Table SV). Specificity-The sites of cleavage by cathepsin D were studied on three oligopeptides, oxidized A and B chains of insulin and peptide D, a cyanogen bromide fragment from the LY chain of human hemoglobin. The findings about the hydrolytic sites are summarized in Fig. 8 while the evidence is documented in the miniprint which follows the main text. DISCUSSION Porcine spleen cathepsin D is apparently very similar to porcine pepsin in several aspects. Firstly, the molecular weight of 35,000 is nearly the same as 34,600 found from the pepsin * * * i * * i A i * zt i l ~ The final values were obtained from extrapolation to zero time of hydrolysis. Half-cysteine was determined as cysteic acid after oxidations with performic acid. The fmal values were the average values of the 48.hour and 72- hour hydrolysis samples. sequence (17). Secondly, the amino acid compositions of the two enzymes show general similarities. Among the similar residue numbers, half-cystine and tryptophan are known to be conservative in evolution. Bovine chymosin, which is structurally homologous to pepsin, also shows a strong similarity to porcine cathepsin D in amino acid composition (Table II). Thirdly, cathepsin D is inhibited by specific pepsin inactivators. Both diazoacetylnorleucine methyl ester and 1,2-epoxy& (p-nitrophenoxy)propane are active center-directed reagents (24-26) which are known to esterify, respectively, active site aspartyl residues 32 and 215 in the pepsin molecule (17). These results indicate that the catalytic mechanisms of the two enzymes are similar. Finally, the specificities of the two enzymes are generally similar in the cleavage of peptide bonds adjacent to the hydrophobic amino acid residues (Fig. 8). Considerable variation exists among the values reported for the molecular weight of cathepsin D. Keilovh reported a molecular weight of 58,000 (27). This value was confirmed by the results of Press et al. (l), and Andrews (28) obtained a value of 45,000. Smaller molecular weights were reported by Smith et al. (10) (-21,000) and by Sapolsky and Woessner (4). In the latter case, a group of isoenzymes demonstrated about 12 different molecular weights ranging from 13,000 to 42,000. Facing these conflicting reports, possible sources of error should be carefully examined. (a) The dimerization of cathepsin D, such as is the case for the enzyme from thyroid (lo), would not be a factor here. We obtained virtually the same molecular weight for the enzyme in the presence and absence of sodium dodecyl sulfate. (b) The possibility that smaller molecular weight fragments had resulted from autodigestion of the enzyme was carefully examined. We have shown that the elution position of cathepsin D in gel filtration is the same for the enzyme in the crude extract and in the final purified product. Therefore, no change of molecular weight due to the

6 Cathepsin D from Porcine Spleen 4533 Insulin A-Chain so3 so3 10 so, 15 so, H-Gly-lle-Val-Glu-GIn-C:s-C~~-Alo-Ser-V~l-C~~-S~~-L~~-ly~-G~ -L~~-Gl~-A~ -Ty~-C~~-A~~-OH 4t It h.ulin B-Chain 1 5 so so, H-Ph~-V~l-A~~-Gl~-Hi~-L~~-C~~-Gly-S~~-Hi~-L~~-V~l-Gl~-Al~-L~~-Ty~-L~~-V~l-C~~-Gly-Gl~-A~~-Gly-Ph~-Phc-Tyr-Thr-Pro~Ly~~A~a~OH t 4 f +4 44f Peptide D H-Val-Leu-Ser-Pro-Ala-Asp-L~-Thr-Arn-Val-Lys-Ala-Ala-Trp-Gly-Ly~-Val-Gly-Al~-Hi~-Al~-Gly-Gl~-Ty~~Gly-Al~-G~~~A~~~L~~~Gl~~A~~~ Native peptide Acetylated peptide. r FIG. 8. Specificity of porcine spleen cathepsin D. The specificity of the enzyme is shown in the sites of cleavage in four peptides; oxidized A chain of bovine insulin, oxidized B chain of bovine insulin, peptide D, and acetylated peptide D (note that in the last case the NH, terminus as well as the c-nh, groups of lysine residues are autodigestion had taken place during the purification. In addition, purified cathepsin D was incubated in acid before gel filtration. The same elution position was found with some loss of total activity. No evidence of cathepsin D of smaller size was found. (c) The 35,000 molecular weight which we obtained represents the size of the major isoenzyme form. As described under Results, only very minor activities were found to be associated with molecular weights of 43,000 and 21,000. The question of isoenzymes of cathepsin D also warrants some consideration. In addition to the two minor isoenzymes with different molecular weights, at least two minor activity peaks with apparently lower isoelectric points were observed in the isoelectric focusing. These isoenzyme forms also appear to be genuine in that their presence was not altered at the different stages of purification. The nature of these isoenzyme forms is not clear. However, our results show clearly that in the extract of porcine spleen, only one predominant form of cathepsin D was found which had a molecular weight of 35,000 and an isoelectric point at ph 7.6. In this study highly purified cathepsin D was obtained from porcine spleen in a relatively simple six-step purification. Although alternatives were studied, isoelectric focusing as a final step always produced the most desirable material with at least 10% recovery and over IOOO-fold purification. The relative purity of the preparation depended on whether or not the focused material had been pooled conservatively or liberaily. Although trace amounts of impurities were usually present in the final preparation (Fig. 4), this procedure has the simplicity suitable for a scaled-up purification required for structurefunction studies of cathepsin D. The bimodal ph dependence curve of porcine spleen cathepsin D raised the interesting point of whether the preparation contained two enzymes with different ph optima. This question <s compn cated by the different responses of enzyme activity near ph 3 and 4 to two salt concentrations (Fig. 3). (Slight shifts of optima in the higher salt concentrations were not considered to be significant and are excluded from the current discussion.) However, when an oligopeptide (peptide D) was used as substrate instead of hemoglobin, the salt effect diminished (Fig. 4), suggesting that this effect was on the hemoglobin substrate. Three lines of evidence suggest that the bimodal ph curve was a property of a single cathepsin D. (a) Hse-OH acetylated). The vertical arrr~u~s below the sequences indicate the relative preference of the hydrolyzed site. They are assigned either from the peptide yields or from the carboxypeptidase A digestions (see miniprint for detail ). 4, high yield sites; f, medium yield sites; and f, low yield sites. No evidence of further separation of two enzyme forms in the final preparation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing in columns or polyacrylamide, and ion exchange chromatography on CM-cellulose columns was found. (b) The ph dependence curve at each stage of the six purification steps was virtually unchanged. (c) Enzyme partially inactivated withdiazoacetylnorleucine methyl ester also produced a typical bimodal ph dependence curve with the same activity ratios at the two optimal ph values. Admittedly, none of this evidence represents a definitive proof. Due to the lack of evidence to support the two enzyme hypothesis, we suggest that this is the property of a single enzyme. It is interesting to note that cathepsin D purified from human and chick liver by Barrett (29) had a similar ph-dependent activity curve. Acknowledgments-We thank Jean A. Hartsuck for stimulating discussions and for constructive criticism and Fred Lee for assisting in peptide D purification. The excellent technical assistance of Judy Cline, Re& Richmond, Ken Jackson, and Paul Lanier is deeply appreciated. Appreciation is also expressed to Jeanie Schneider for skillful preparation of the manuscript. REFERENCES 1. Press, E. M., Porter, R. R., and Cebra, J. (1960) &&em. J. 74, Anson, M. L. (1939) J. Gen. Physiol. 22, KeilovQ, H., Markovic, O., and Keil, B. (1969) Coil. Czech. Chem. Comm. 34, Sapolsky, A. I., and Woessner, J. F., Jr. (1972) J. Biol. Chem. 247, Smith, R., and Turk, V. (1974) Eur. J. Biochem. 48, Turk, V., Kregar, I., Gubensec, F., and Lebez, D. (1969) Enzymologia 36, Ferguson, J. B., Andrews, J. R., Voynick, I. M., and Fruton, J. S. (1%3( d. f%l: cr?iem. 248, b7ui-67@3 8. Barrett, A. J. (1970) Biochem. J. 117, Kress, L. F., Peanasky, R. J., and Klitgaard, H. M. (1966) Biochim. Biophys. Acta 113, Smith, G. D., Murray, M. A., Nichol, L. W., and Trikojus, V. M. (1969) Biochim. Biophys. Acta 171, Kregar, I., Turk, V., and Lebez, D. (1967) Enzymologia 33, Reichelt, D., Jocobsohn, E., and Haschen, R. J. (1974) Biochim. Biophys. Acta 341, Fruton, J. S. (1971) in The Enzymes (Bayer, P. D., ed) Vol. 3, p. 152, Academic Press, New York

7 4534 Cathepsin D from Porcine Spleen 14. Davis, B. J. (1964) Ann. N. Y. Acad. Sci Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, Kay, C. M., Smillie, L. B.. and Hilderman. F. A. (1961) J. Biol. &m. 236, Tang, J., Sepulveda, P., Marciniszyn, J., Jr., Chen, K. C. S., Huang, W-Y., Tao, N., Liu, D., and Lanier, J. P. (1973) Proc. 25. N&l. Acad. Sci. U. S. A. 70, Tanford, C. K., Karvahara, K., and Lapanje, S. (1967) J. Am. 27. Chem. Sot. 89, Andrews. P. (1964) Biochem. J Wrigley, C. W. (1968) J. Chromat&. 36, Spackman, D. H. (1963) Fed. Proc. 22, Liu, T. Y., and Chang, Y. H. (1971) J. Biol. Chem. 246, Lapresle, C. (1971) in Tissue Proteinases (Barrett, A. J., and Dingle, J. T., eds) pp , North-Holland Publishing Co., Amsterdam Rajagopalan, T. G., Stein, W. H., and Moore, S. (1966) J. Biol. Chem. 241, Tang, J. (1971) J. Biol. Chem. 246, Chen, K. C. S.. and Tanp.., J. (1972) J. Biol. Chem Keilovi, H. (1971) in Tissue Proteincues (Barrett, A. J., and Dingle, J. T., eds) North-Holland Publishing Co.. Amsterdam Andre&s, P. (1965) Biochem. J. 96, Barrett, A. J. (1971) in Tissue Proteinases (Barrett, A. J., and Dingle, J. T., eds) North-Holland Publishing Co., Amsterdam

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10 Purification and properties of cathepsin D from porcine spleen. M Cunningham and J Tang J. Biol. Chem. 1976, 251: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at