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THE JOURNAL or BIOLOGICAL CHEMISTRY 
Vol. 251, No. 23, Issue of December 10, pp. 7593-1599, 1976 
Printed in U.S.A. 
Post-proline Cleaving Enzyme 
PURIFICATION OF THIS ENDOPEPTIDASE BY AFFINITY CHROMATOGRAPHY* 
(Received for publication, June 7, 1976) 
MASAO KOIDA AND RODERICH WALTER 
From the Department of Physiology and Biophysics, University of Illinois at the Medical Center, 
Chicago, Illinois 60612 
The endopeptidase, post-proline cleaving enzyme, has been purified 10,500-fold in an overall yield 
of 18% from lamb kidney. The enzyme possesses a specific activity of 45 ~mol/mg/min as tested with 
the substrate Z-Gly-Pro-Leu-Gly (K,,, = 6.0 x 10m5), has a molecular weight of 115,000, is comprised 
of two subunits with a molecular weight of 57,000, and exhibits maximal activity at pH 7.5 to 8.0. 
With the exception of the -Pro-Pro- linkage, the -Pro-X-peptide bond (X equals L- and n-amino acid 
residues) located internally in the peptide sequence can be hydrolyzed (cleavage occurs faster when 
X = lipophilic side chain as compared to X = acidic side chain). The appropriate -Pro-X- bonds in 
zinc-free porcine insulin, oxytocin, arginine vasopressin, angiotensin II, bradykinin-potentiating 
factor were cleaved. Human gastrin, adrenocorticotropic hormone, denatured guinea pig skin 
collagen, and ascaris cuticle collagen were not degraded. Dipeptides with the structure Z-Pro-m-X 
competitively inhibit post-proline cleaving enzyme. 
Proteolytic enzymes will attack a peptide bond, provided the 
appropriate category of amino acid side chain defined by lipo- 
philicity, steric properties etc., is present for substrate recog- 
nition (1). Although it is recognized that conformational con- 
straints within a protein substrate (2) or even a low molecular 
weight peptide substrate can modulate or even prevent the 
expected enzymatic attack (3), a direct correlation between the 
distinguishing feature of an amino acid side chain and degree 
of enzyme specificity has emerged. While chymotrypsin for 
example exhibits a limited degree of specificity, the excep- 
tional specificity of trypsin (EC 3.4.21.4) for the cleavage of the 
peptide bond at the COOH-terminal site of basic residues (4) is 
well established, and recently Armillaria melled protease (EC 
3.4 21.-) was found to catalyze hydrolysis at the NH,-terminal 
side of lysine residues (5, 6). The pyroglutamate residue is 
selectively recognized by pyrrolidone carboxylyl peptidase (7) 
and an extracellular protease from a strain of Staphylococcus 
aureus has been found to cleave the peptide bonds at the 
COOH-terminal side of both aspartate and glutamate resi- 
dues, but at certain buffer and pH conditions only at the 
glutamate residues (8). 
The proline residue with its unique aliphatic, cyclic struc- 
ture should represent a characteristic target and, indeed, exo- 
peptidases which recognize the proline residue have been de- 
scribed. These are imidopeptidase (prolidase, EC 3.4.3.7), a 
dipeptidase which can cleave the X-Pro bond (9), iminodipepti- 
dase (prolinase, EC 3.4.3.6), a dipeptidase which specifically 
attacks the Pro-X bond (lo), and proline iminopeptidase (pro- 
* This research was supported by Research Grant AM 18399 from 
the National Institute of Arthritis, Metabolism, and Digestive Dis- 
eases. 
line-peptide hydrolase, (EC 3.4.1.4)), which removes the NH2- 
terminal proline residue irrespective of the size of the sub- 
strate (11). 
A series of studies strongly suggests the existence in mam- 
malian tissue of an endopeptidase which catalyzes hydrolysis 
of the peptide bond at the carboxyl side of a proline residue 
(12-14). Since this was the first endopeptidase which exhibits 
such specificity directed at the Pro-X bond, it seemed impor- 
tant to isolate the enzyme in order to allow investigations of its 
properties. 
EXPERIMENTAL PROCEDURES 
Materials -Lamb kidneys frozen on dry ice were purchased from 
Max Insel Cohen. Newark. N. J. Human svnthetic adrenocortico- 
tropic hormone I-& (15), based on the structure of Lee et al. (16), was 
kindlv suunlied bv Dr. R. L. Colescott; human aastrin (17,18) and its 
NHz-terminal fragment, <Glu-Gly-Pro, by Dr. J. S. Morley; <Glu- 
Gly-Gly-Trp-Pro-Arg-Pro-Gly-Pro-Glu-Ile-Pro-Pro (SQ 20, 718) (19) 
bv Dr. M. A. Ondetti: zinc-free norcine insulin bv Dr. R. E. Chance; 
&tive skin collagen (M, - 3OO;OOO) of guinea pig and reduced and 
carboxvmethvlated ascaris cuticle collagen (M. - 60,000) from Dr. J. 
Coffey: P4C]AVP1 with a specific radioactivity’of 30 Ci/mol and a rat 
pressor activity of 416 unitslmg was available from earlier studies 
(21) as was unlabeled oxvtocin (22). The following chemicals were ~~~I 
obtained commercially as”indicated. Z-Gly-Pro-LeuIGly (Protein Re- 
search Foundation. Osaka. Janan). Z-Pro-Leu, Z-Pro-Gly, Z-Pro- 
Ala, and Ala-Pro-GIy (Fox Chemical Co.), Z-Gly-Pro-Leu (Peninsula 
Laboratories), crystalline bovine insulin, bradykinin triacetate, N- 
1 The abbreviations used follow the recommendation of the 
IUPAC-IUB Commission on Biochemical Nomenclature (20). All 
optically active amino acids are of the L configuration unless other- 
wise stated. Additional abbreviations used are: P4C]AVP, [9-glycin- 
amide-l-14C]arginine vasopressin; AVP, arginine vasopressin; Boc, 
t-butoxycarbonyl-; ONSu, succinimido-oxy-. 
7593 
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75 94 Post-proline Cleaving Enzyme 
benzoyl-Gly-Phe, Z-Pro-Leu-Gly, Leu-Gly, polylysine HBr (type VII- 
B and hydroxyapatite; type I) (Sigma Chemical Co.), human angi- 
otensin II, ammonium sulfate (enzyme grade) (Schwarz/Mann), and 
Epoxy-activated Sepharose and standard proteins for molecular 
weight determination (Pharmacia). 
Synthesis of Peptide Tested for the Inhibition of Post-proline Cleav- 
ing Enzyme-Z-n-Pro-ON&r was synthesized as reported for the L- 
isomer by Anderson et al. (23). The product was recrystallized from 
absolute ethanol, yield 55.6%; m.p. 89 to 91; laloz3 + 52” (c 2.0, 1,4- 
dioxane). 
C,, H,, N&h 
Calculated: C 59.0 H 5.24 N 8.09 
Found: C 59.0 H 5.47 N 7.87 
All the dipeptides listed in Table I, which summarizes yields, analyt- 
ical and physical properties, were prepared using the N-hydroxysuc- 
cinimide ester method (23). The Boc group of Z-Pro-Lys(Boc) was 
removed with trifluoroacetic acid (25). 
Methods -Protein determinations were performed according to 
Lowry et al. (261, using bovine serum albumin as standard. Amino 
acid analyses were carried out as described by Moore (27); peptides 
were hydrolyzed for 18 or 24 h at 110” in 6 N HCl under vacuum. 
Preparation of Z-Pro-D-Ala-Poly(Lys)-Sepharose 4B Column -The 
method employed for the activation of Sepharose 4B was based on the 
procedure described by Cuatrecasas (28). A suspension of Sepharose 
4B in water (50 ml of polymer plus 50 ml) was activated by adding 5 g 
of finely pulverized CNBr. The reaction was allowed to proceed at 20” 
for 12 min while the pH was maintained at 10.5 to 11 by addition of 4 
N NaOH. The activated Sepharose was washed with 500 ml of ice cold 
0.2 M NaHCO, buffer (pH 9.0), collected by suction and added to 50 
ml of a 1.5% solution (w/v, pH 9.0) of poly(Lys).HBr in 0.2 M 
NaHCO,. After stirring overnight at 4”, the product was collected by 
filtration, washed with 500 ml each of water, 0.1 M acetic acid 
containing 1 M NaCl, water, and 0.1 M NaHCO, (pH 8.51, and then 
suspended in 50 ml of dioxane. Z-Pro-n-Ala-ONSu (1.2 g) was dis- 
solved in 10 ml of dioxane and 5 ml of the solution was added to the 
suspension of the poly(Lys)-Sepharose with gentle stirring. After 30 
min, the rest of the solution was added. Throughout the coupling 
period the pH of the suspension was maintained at 8.5 by addition of 
0.1 N NaOH. After 30 min the product was washed with 500 ml each 
ofa 1:l mixture of dioxane and water (v/v), water, 0.1 M NaHCO,, 1 
M NaCl, and water. The washed derivative of Sepharose was kept at 
4” as a 1:l suspension (v/v) in water containing 0.02% NaN,. Follow- 
ing acid hydrolysis of the final product amino acid analysis revealed 
that 1 ml of the gel contained 23 to 30 pmol of lysine residue; and that 
80 to 88% of these residues were acylated by Z-Pro-n-Ala. 
Assay of Specific Activity of Post-proline Cleaving Enzyme- En- 
zyme activity was measured either by release of radioactively la- 
beled Arg-Gly-NH2 from [14C]AVP or by ninhydrin determination of 
Leu-Gly released from Z-Gly-Pro-Leu-Gly as described (14). 
Throughout the purification procedure the protected tetrapeptide 
was used as the substrate with the exception of the initial extraction 
of kidney and (NH&SO, fractionation, which was followed by using 
[14C]AVP. An aliquot of 50 to 70% (NH&SO, precipitate was sub- 
jected to gel filtration on a Sephadex G-25 column (1.3 x 30 cm) to 
remove (NH&SO, and was then tested with both substrates for 
specific activity. Typical reaction mixtures consisted of 10 ~1 of a 
fraction containing less than 2 milliunits of enzyme activity (1 unit 
of enzyme activity is defined as cleavage of 1 +mol of Z-Gly-Pro-Leu- 
Gly/min at pH 7.7 and 37”) and 50 yl of 0.1 mM [‘YJIAVP or 25 ~1 of 2 
mM Z-Gly-Pro-Leu-Gly in 25 mM phosphate buffer (pH 7.7) contain- 
ing 1 m&f EDTA and 1 mM dithiothreitol. After preincubation at 37” 
for 5 min the reaction was started by addition of the substrate 
solution and after 5 or 10 min at 37”, the reaction was stopped by 
addition of 0.5 ml of a ninhydrin solution (291, or in the case of 
[‘QAVP, by acidification with 0.005 ml of 1 N acetic acid. In controls 
the substrate solution was added after the ninhydrin solution. 
For analysis of the [‘QAVP digest, lo- to 30-~1 aliquots of the 
incubation mixture was subjected to high voltage electrophoresis 
(2500 V, 1 h, pH 3.5) on Whatman No. 3MM paper and scanned for 
radioactivity. When Z-Giy-Pro-Leu-Gly was used as substrate, a 
standard curve of Leu-Gly was determined with ninhydrin in each 
assay. 
Electrophoretic Analysis of Enzyme Preparations- The purity of 
the enzyme preparation was examined by disc gel electrophoresis 
based on the method of Ornstein (30) and Davis (31). A 7% separation 
gel in 0.37 M Tris/HCl (pH 8.9) was prepared using ammonium 
persulfate as the catalyst. For pre-electrophoresis a current of 2 mA/ 
tube was applied to the gels for 1 h in 0.37 M Tris/HCl (pH 8.9) 
containing 1 mM dithiothreitol and 1 rnM EDTA. The enzyme prepa- 
ration was dissolved in 62 rnM Tris/HCl (pH 6.7) containing 1 mM 
dithiothreitol and 1 rnM EDTA and 10% sucrose (w/v). Matched pairs 
of gels were prepared. One of the gels was stained for protein in 0.1% 
Coomassie brilliant blue in 10% trichloroacetic acid, the other sliced 
into l-mm sections. The enzymatic activity of each disc, which was 
suspended in 50 ~1 of 0.2 M phosphate buffer, pH 6.8, and was then 
analyzed under standard conditions using [‘4ClAVP (diluted 1:l) as 
substrate. Incubation periods were 15, 30, or 60 min depending on 
enzyme activity and 20-~1 aliquots of the mixture were spotted on a 
Whatman No. 3MM paper and scanned for radioactivity. 
Purification Procedures of Post-proline Cleaving Enzyme- The 
method given below is designed for purification of the enzyme from 6 
to 8 lamb kidneys having a total weight of 250 to 350 g. All steps were 
carried out at 4” unless otherwise described and all centrifugations 
carried out at 24,500 x g for 15 min. During purification four kinds of 
buffers were used. Buffer A: 2 mM dithiothreitol and 2 mM EDTA in 
50 mM sodium phosphate buffer (pH 6.8); Buffer B: 1 mM dithiothrei- 
to1 and 1 rnM EDTA in 25 mM sodium phosphate (pH 6.8); Buffer C: 1 
mM dithiothreitol and 1 mM EDTA in 5 mM sodium phosphate (pH 
6.8); and Buffer D: 1 rnM dithiothreitol and 1 rnM EDTA in 50 mM 
sodium phosphate (pH 6.8). 
Extraction and Ammonium Sulfate Fractionation- Frozen lamb 
kidneys were thawed by repeated washing with distilled water, 
sliced into pieces, freed from vascular and membranous tissue, and 
weighed. Tissue was homogenized at full speed with 3 volumes (w/v) 
Dipeptide Crystallization solvent Yield 
2.Pro-Glu(OBzl) EtOAc 
% 
39 
Z-ho-Glu EtOAciEbO 
EtOAciEtO 
I-Propanol/H,O 
2.Propanolin-hexane 
Et0 
EtOAc 
EtOH/EtOAc/n-hexane 
70 134-136 C,,H,,N,O, (378.39) 57.1 5.90 1.40 57.0 5.94 7.26 
2.Pro-~.Ala 
DCHA saltb 
Z-D-Pro-~-Ala 
95 188-190 
70 159-161 
Z-Pro-Phe’ 86 124-126 C,,H,,N,O, (396.45) 66.6 6.10 
Z-Pro-Lys(Boc) 82 151-154 C,,H,,N,O, (477.56) 60.1 7.39 
z-Pro-Lys 
‘/2 H,O 
Z-Pro-o-Ala- 
ONSu.%H,O 
N-Benzvloxvcarbonvl ~1 -01 ” ” 
” Analysis performed by Rohetion Laboratories, F 
’ DCHA, dicyclohexylamine. 
r Itoh: see Ref. 24. 
TABLE I 
vldiueotides to be tested as inhibitors and lieands for affinity chromatography I 11 
Calculated 1 Found” point- 
(not car- 
reckd) 
Formula 
(molecular weight) 
Carbon Hydrogen Nitrogen Carbon 1 Hydrogen 1 Nitrogen 
133-136” C,,H,,N,O, (468.51) 64.1 
% 
6.02 
C,,H,,N,O, (501.67) 
C,,H,,N,O, (320.35) 
67.0 8.64 8.38 67.1 8.76 6.43 
60.0 6.29 8.74 60.2 6.06 8.61 
221-224 C,,H,,N,O, (387.46) 58.9 1.54 
122-125 C,,H,,N,O, (417.42) 56.3 5.67 
ham Park i.J. 
5.98 63.8 
7.07 
8.80 
10.8 
9.85 
66.4 6.39 6.84 
59.6 7.48 8.39 
58.8 
56.3 
% 
6.06 
7.31 
5.79 
5.90 
10.55 
9.91 
0.R 
[al.” 
- 103 
Cc 1.9, MeOH) 
-46 
(c 1.4, MeOH) 
-96 
(c 2.1, MeOH) 
+141 
Cc 2.1, MeOH) 
-16 
(c 1, EtOH) 
-26 
(c 1, EtOH) 
-64 
(c 1, IN AcOH) 
f21 
(c 2, MeOH) 
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Post-proline Cleaving Enzyme 7595 
of Buffer A in a Vir-Tis model 45 homogenizer for four 30-s periods, 
stirred at 22” for 1 h, and centrifuged. The pellet was washed with 
300 ml of Buffer A and to the combined supernatant fractions, solid 
ammonium sulfate was slowly added with constant stirring until 
50% saturation (313 a/liter at room temperature) was reached. After 
stirring at 22” for 2 hy the precipitate which formed was separated by 
centrifugation, washed with 200 ml of Buffer A saturated to 50% with 
ammonium sulfate and the ammonium sulfate concentration in the 
combined supernatant fraction was raised to 65% by gradual addi- 
tion of solid ammonium sulfate (101 g/ml) while being stirred at 22” 
for 1 h. The suspension was kept overnight and precipitate formed 
was collected bv centrifueation. 
First DEAK-Sephader A-50 Column Chromatography- The 
(NH&SO, precipitate was dissolved in Buffer B (4 g/50 ml). After 
removal of insoluble material by centrifugation, the supernatant 
was desalted on a Sephadex G-25 column (2.5 x 90 cm) equilibrated 
with Buffer B. The active fractions were combined and applied to a 
column (2.5 x 35 cm) of DEAE-Sephadex A-50 equilibrated with 
Buffer B. The column was eluted with a linear gradient established 
using 500 ml of Buffer B and 500 ml of Buffer B containing 0.25 M 
sodium chloride. Fractions of 11 ml were collected using a flow rate of 
45 ml/h. Fractions containing enzyme activity were combined and 
the enzyme protein was either precipitated by dialyzing against 9 
volumes of 80% saturated ammonium sulfate solution or concen- 
trated by ultrafiltration using an Amicon cell with a PM-30 mem- 
brane. 
Hydroxyapatite Column Chromatography -A column of hydroxy- 
apatite cellulose (2.5 x 35 cm) was prepared according to Massey (32) 
from 15 ml of hvdroxvapatite and 24 g of Whatman CF-11 cellulose. 
The crude enzyme preparation obtained after the first ion exchange 
chromatography was dissolved in 10 ml of Buffer C and desalted on a 
Sephadex-G-25 column(2.5 x 35 cm). The active fraction was then 
applied to the hydroxyapatite cellulose column and after washing 
the column with 1 bed volume of Buffer C, the column was eluted 
using a linear gradient established with 406 ml of Buffer C and 400 
ml of 0.15 M sodium phosphate buffer (pH 6.8) containing 1 mM 
dithiothreitol and 1 rnG EDTA. The flow rate was 40 ml/h and 11-ml 
fractions were collected. The fractions containing the enzyme activ- 
itv were combined and the enzvme nrotein Dreciuitated as described. 
“Affinity Chromatography Foilowed by Ion Exchange Chromatogra- 
ph.y- The enzyme precipitate was dissolved in 3 ml of Buffer B and 
.after removal of insoluble material by centrifugation, the clear 
supernatant was desalted on a Sephadex G-25 column (1.3 x 30 cm) 
eauilibrated with Buffer D. The active fractions were combined and 
applied to a column of Z-Pro-n-Ala-poly(Lys)-Sepharose (0.9 x 15 cm) 
equilibrated with Buffer D. After washing with 20 ml of Buffer D, 
the column was eluted with 25 ml of Buffer D containing the inhibi- 
tor Z-Pro-Phe (2 mg/ml). The flow rate was 25 ml/h. The fractions 
containing the enzyme were combined and applied to a DEAE- 
Sephadex A-50 column (0.9 x 5 cm) equilibrated with Buffer D. The 
column was eluted with a linear gradient established with 50 ml of 
Buffer D and 50 ml of Buffer D containing 0.3 M NaCl. The flow rate 
was 9.5 ml/h and 2.5ml fractions were collected. The fractions 
containing the enzyme were combined and the enzyme protein was 
recovered as a precipitate as described above. The precipitate was 
dissolved in 1 ml of water containina 1 rnM dithiothreitol. and 1 mM 
sodium EDTA (pH 7.0) and centrifuged. The clear supernatant was 
desalted on a Sephadex G-25 column (0.9 x 15 cm) which had been 
equilibrated with water containing dithiothreitol and EDTA. The 
eluate was collected in 0.5-ml fractions and the enzymatically active 
fractions were kept frozen at -70”. 
Determination of Molecular Weight of Purified Enzyme - The mo- 
lecular weight of the purified enzyme was estimated by gel filtration 
on a Sephadex G-150 column (35 x 2.5 cm) according to Andrews (33) 
and by sodium dodecyl sulfate gel electrophoresis as described by 
Weber and Osborn (34). A total volume of 75 yl containing 20 pg of 
protein was applied. A current of 2.5 mA per gel (5.5 x 0.5 mm) was 
used. Rabbit muscle aldolase, bovine hemoglobin, ovalbumin, chy- 
motrypsinogen, and ribonuclease were used-as standards. 
Effect of pH on Enzyme Activity- For pH studies, the following 
buffers were used: 0.1 i sodium citrate in the pH range of 3.5 to 5.5; 
0.1 M sodium phosphate from pH 6.0 to 8.0; and 0.1 M sodium borate 
from pH 8.25 to 9.5. The reaction mixture consisted of 50 ~1 of the 
desired buffer containing 1 mM dithiothreitol and 1 rnM EDTA, 10 ~1 
of a 5 mM solution of Z-Gly-Pro-Leu-Gly and 2 milliunits of enzyme 
activity. The reaction was allowed to proceed for 5 min at 37” and was 
stopped by the addition of 1.0 ml of ninhydrin reagent. The stock 
solution of Z-Gly-Pro-Leu-Gly (25 mM) was prepared by the addition 
of 0.1 N NaOH to a suspension of peptide in water until a final pH of 
7.0 was reached. 
Kinetic Studies of Oligopeptidyl Substrates and Inhibitors- For 
kinetic studies the react& mixture (200 ~1) contained 1 to 4 milli- 
units of enzyme activity and 6.4 to 10 x lo2 nmol of peptide (the 
substrate alone or substrate plus inhibitor) in 25 rnM sodium phos- 
phate buffer (pH 7.7) containing 1 mM dithiothreitol and 1 mM 
EDTA. The reaction was carried out for 2 min at 37” and terminated 
by addition of 0.5 ml of ninhydrin reagent. 
Identification of Cleavage Products of Naturally Occurring Pep- 
tides by Post-proline Clea&g Enzyme - Peptide (0.5 to 1.0 pmol) was 
dissolved in 200 ~1 of 0.1 M ammonium bicarbonate (pH 7.8) contain- 
ing 1 mM dithiothreitol and 1 mM EDTA and incubated with 1.0 unit 
of the enzyme (20 ~1) at 22” for 24 h. In the case of native guinea pig 
skin collagen. 10 mg of protein was suspended in 2.5 ml of 0.5 M 
acetic acidand denatured-by incubating the suspension at 45” for 2 h. 
The protein was then dialyzed overnight against 1000 ml of 0.1 M 
phosphate buffer, pH 7.7. The suspended materials were removed by 
centrifuging at 12,000 x g for 10 min. The protein concentration of 
the clear sunernatant was 3.2 me/ml. To follow the course of dises- 
tion, 5-~1 aliquots were taken from the reaction mixture at 1, 2, 4, 8, 
and 24 h. mixed with 15 ~1 of 1 M NH,OH and the eluate lyophilized. 
The amino acid composition of the eluted peptide was analyzed. In 
case of gastrin, adrenocorticotropic hormone, denatured guinea pig 
collagen, and ascaris cuticle collagen, which were not cleaved by the 
enzyme after 24 h of digestion, 1.0 unit of fresh enzyme was added to 
the reaction mixture and the reaction continued for another 24 h. 
Aliquots (1 ~1) of the reaction mixture were taken at 1, 2, 4, 8, 24, 
and 48 h and spotted on a Silica Gel G plate for thin layer chromatog- 
raphy. After chromatographic development with the-solvent system 
n-butyl alcohol/acetic acid/water (4/1/l, v/v/v), the plate was devel- 
oped with chloride/tolidine reagent (35). 
RESULTS 
As can be seen in Fig. 1, gradient elution from DEAE- 
Sephadex A-50 (36) resulted in two peaks capable of inactivat- 
ing [‘YJAVP. While the first peak released labeled Arg-Gly- 
NH, and Gly-NH2 from [14ClAVP, the second produced only 
Arg-Gly-NH,. Moreover, only fractions of the latter peak re- 
leased Leu-Gly from Z-Gly-Pro-Leu-Gly. Therefore, the second 
peak contained the post-proline cleaving enzyme. The yield of 
the enzyme at this step was consistently greater than 100% 
(e.g. in one experiment 160% and 140% in terms of cleavage of 
Z-Gly-Pro-Leu-Gly and [14C]AVP, respectively), possibly re- 
vealing the presence of enzyme inhibitor or activation of a 
precursor of the enzyme. 
Further Purification of Post-proline Cleaving Enzyme by 
Affinity Chromatography - Affinity chromatography using 
Sepharose as the carrier for the column, poly(Lys) as the 
bridge, and the inhibitor Z-Pro-n-Ala as the ligand, was most 
successful among a number of combinations of spacer groups 
and ligands tested. A major increase in the specific activity of 
the post-proline cleaving enzyme preparation was obtained 
(Fig. 2). Z-Pro-Phe or Z-Pro-Glu (5 mM) were found to be most 
successful in eluting the enzyme. The final step, a rechroma- 
tography on the DEAE-Sephadex A-50 column, removed the 
dipeptide used for enzyme elution and to a certain extent other 
proteins absorbed on the affinity column. 
Criteria for the Purity of Post-proline Cleaving Enzyme- 
Progress in the purification of the enzyme was followed by 
analytical disc gel electrophoresis (Fig. 3). The post-proline 
cleaving enzyme preparation after the affinity chromatogra- 
phy step had an average specific activity of 45 units/mg. The 
preparations revealed two sharp minor bands with slightly 
lower migration rates than a third band (Fig. 3). The latter is 
the major band and a scan of the gel is shown in Fig. 3. 
Analysis of l-mm gel slices using YCIAVP as substrate 
showed that enzymatic activity was associated only with the 
major band. 
Molecular Weight Estimate - The molecular weight of the 
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7596 Post-proline Cleaving Enzyme 
IO0 
7.5 
? 
FIG. 1. First DEAE-Sephadex A-50 
.! 
column chromatography of post-proline 
z 5.c b 
E 
I 
2! 5. 
J. 
!- 
26 b 6 
FRKTION N”Mt3Ei’ 
80 
AMINOPEPTIDASE 
CLEAVING ENZYME 
‘= 
O< 
Z 
MIGRATION 
3 
G 
FIG. 3. Scan of polyacrylamide gel electrophoretic pattern of post- 
proline cleaving enzyme at the final stage of purification. The inset 
4 
shows the progress of purification and migration pattern ofpost- 
proline cleaving enzyme determined by analytical gel electrophore- 
I 
sis. A, sample after first DEAE-Sephadex A-50 chromatography; B, 
after hydroxyapatite step; C, after DEAE-Sephadex A-50 chromatog- 
raphy following affinity chromatography. Enzyme activity is only 
3 associated with the major band in C. The scan corresponds to Sample 
c. 
FRACllON NUMBER 
FIG. 2. Affinity column chromatography of post-proline cleaving 
enzyme by using Z-Pro-n-Ala-poly(Lys)-Sepharose 4B. The arrow 
indicates start of elution of the enzyme with 5 mM Z-Pro-Phe. 
enzyme at the final stage of purification was estimated to be 
115,000 by gel filtration on a Sephadex G-150 column. Gel 
electrophoresis in the presence of sodium dodecyl sulfate indi- 
cates an average molecular weight of 57,000 for post-proline 
cleaving enzyme monomer. 
Stability and Storage of Enzyme - The purified enzyme dis- 
solved in 1 mM EDTA and dithiothreitol at pH 7.0 may be 
stored frozen at -20 to -70” for several months without any 
detectable loss of activity. Lyophilized material had lost about 
10% of its activity after 1 day and 50% after 1 week when kept 
atroomtemperature. 
Profile of Enzyme Activity as Function of pH- The activity 
of the enzyme was examined in citrate, phosphate, and borate 
buffers covering a pH range of 3.5 to 9.5. Maximal activity was 
observed in the range of pH 7.5 to 8.0 with Z-Gly-Pro-Leu-Gly 
as substrate, identical to earlier observations (14). Thus, all 
experiments described below were carried out at pH 7.7 to 7.8. 
Interaction of Oligopeptides with Post-proline Cleaving En- 
zyme - All N-blocked dipeptides used with the general formula 
Z-Pro-X were not cleaved by post-proline cleaving enzyme, i.e. 
Z-Pro-m-Ala, Glu, Leu, Lys, Phe. Even when the incubation 
time was extended from 4 to 50 min no detectable cleavage 
occurred. However, these Z-Pro-X peptides were found to com- 
petitively inhibit the cleavage of Z-Gly-Pro-Leu-Gly (Table II). 
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Post-proline Cleaving Enzyme 7597 
Z-Pro-Glu was the strongest inhibitor with a Ki value of 1.3 x 
10m5. Z-D-Pro-D-Ala and Bz-Gly-Phe were found to be neither 
substrates nor inhibitors. Other amino acids and peptides 
which were neither cleaved nor inhibited the enzyme included 
proline, Z-Pro, Ala-Pro-Gly, Pro-Leu-Gly, Gly-Pro-Hyp, Leu- 
Gly-NH2, formyl-Pro-Leu-Gly-NH,, and Pro-Leu-Gly-NH,. 
Moreover, while Z-Pro-Leu-Gly was found to be an inhibitor, 
Z-Gly-Pro-Leu was a good substrate. Z-Gly-Pro-Leu-Gly-Pro 
was an even better substrate and the best was Z-Gly-Pro-Leu- 
Gly with a K, of 6.0 x 10-5. 
TABLE II 
Znhibition of post-proline cleaving enzyme by N-blocked dipeptides 
Per cent inhibition0 KC 
Z-Pro-Leu 4.0 nd.” 
Z-Pro-D-Leu 7.8 n.d. 
Z-Pro-Ala 21 3.0 x 10-a 
Z-Pro-D-Ala 20 3.1 x 10-d 
Z-Pro-Phe 33 2.9 x 10-S 
Z-Pro-Glu 48 1.3 x 10-S 
Z-Pro-Glu(OBz1) 22 n.d. 
Z-Pro-Gly 11 n.d. 
Z-Pro-Lys(Boc) 22 nd. 
Z-D-Pro-D-Ala 0 
Bz-Gly-Phe 0 
n The incubation mixture (100 ~1, final volume) contained 6.4 
milliunits of enzyme activity, 100 nmol of Z-Gly-Pro-Leu-Pro in the 
presence or absence of 120 nmol of N-blocked dipeptide. Incubation 
was at 37” for 5 min. The reaction was stopped by addition of 0.5 ml of 
ninhydrin reagent. 
b n.d., not determined. 
Digestion of Naturally Occurring Peptides by Post-proline 
Cleaving Enzyme - The following peptides were tested: zinc- 
free porcine insulin, oxytocin, vasopressin, angiotensin II, 
bradykinin, bradykinin-potentiating factor, human adreno- 
corticotropic hormone, human gastrin, denatured collagens. 
Products formed as a result of peptide digestion by purified 
post-proline cleaving enzyme and criteria used for identifica- 
tion of catabolites are listed in Table III. 
DISCUSSION 
The purification scheme found to give the best results to 
date yields a post-proline cleaving enzyme of a 10,500-fold 
purification on an average (Table IV). The affinity chromatog- 
raphy step proved to be the most important in increasing the 
state of purity of the post-proline cleaving enzyme prepara- 
tion. 
A number of oligopeptides and naturally occurring peptides 
were tested with post-proline cleaving enzyme in order to re- 
evaluate earlier substrate-specificity studies with partially 
purified enzyme. It was confirmed that peptides without pro- 
line residues were not degraded, while all types of -Pro-X- 
bonds, with the exception of a -Pro-Pro- bond, can be hydro- 
lyzed although there are considerable differences in rates. The 
rate of cleavage is fastest when X is a lipophilic residue, slower 
in case of a basic residue and slowest when an acidic group is 
present in the vicinal residue. In order to obtain detectable 
cleavage the proline residue cannot occupy the NH,-terminal 
position of an unprotected peptide. 
The inability of the post-proline enzyme preparation to 
cleave Bz-Gly-Phe, a standard substrate for carboxypeptidase 
TABLE III 
Sites of hydrolysis of naturally occurring peptides by post-proline cleaving enzyme 
Substrate and site of cleavage 
Insulin (Zn*+-free) 
1 
Oxytocin, Cys-Tyr-Ile-Gln-Asn-ys-Pro-Leu-Gly-NH, 
1 
Angiotensin II, Asp-Arg-Val-Tyr-Be-His-Pro-Phe 
1 1 
Bradykinin, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 
Identified product(s) and amino 
acid composition 
Lys-Ala 
.95 1.0 
Leu-Gly-NH, 
1.0 .96 
NH,-terminal heptapeptide 
Phe 
Asp-Arg-Val-Tyr-Ile-His-Pro 
1.0 1.0 .89 .80 .74 .83 1.0 
Arg-Pro-Pro 
.98 2.0 
Electrophoretic 
mobility” 
CR,) 
0.61b 
0.550 
0.19b 
0.13” 
0.33 
0.52 
1 1 1 
Bradykinin-<Glu-Gly-Gly-Trp-Pro-Arg-Pro-Gly-Pro-Glu-Ile-Pro-Pro 
potentiating factor. 
Gly-Phe-Ser-Pro 0.26 
1.0 1.0 .94 1.0 
Phe-Arg 0.50 
1.0 1.0 
pGlu-Gly-Gly-Tip-Pro 
1.0 2.0 1.0 
-0.02 
Arg-Pro 
1.0 1.0 
Gly-Pro (trace) 
Glu-Be-Pro-Pro (trace) 
Gly-Pro-Glu-Ile-Pro-Pro 
1.1 3.0 1.0 .97 
n R, is expressed in relation to the rate of migration of Gly-NH,. For experimental detail, see text. 
* The electrophoretic mobility of this peptide was also compared with authentic material. 
0.56 
0.28 
0.20 
0.24 
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7598 Post-proline Cleaving Enzyme 
TABLE IV 
Summary of purification of post-proline cleaving enzyme 
Enzyme activity was measured in 25 rnM sodium phosphate buffer, pH 7.7, containing 1 rnM dithiothreitol and EDTA using the substrate 
Z-Gly-Pro-Leu-Gly in a final concentration of 1.6 mM. 
Purification steu Total or&in Sue&c activitv Total activitv Yield Purification 
Tissue extract (23,500 x g; super) 
(NH&SO, fractionation (50-70%) 
DEAE-Sephadex A-50 
Hydroxyapatite 
Affinity and DEAE-Sephadex A-50 
chromatography 
g 
38 
4.0 
0.25 
2.5 x 10-a 
6.4 x lo-” 
A (3’7), and Leu-Gly-NH, demonstrates the absence of carboxy- 
peptidase, aminopeptidase, and aminodipeptidase activity. 
Therefore, it is concluded that the release of the COOH- 
terminal phenylalanine moiety from angiotensin II, which 
was also observed with the partially purified preparation (141, 
was caused by the post-proline cleaving enzyme and not by a 
contaminating activity. Endopeptidases are known to be capa- 
ble of releasing terminal amino acid residues, although gener- 
ally the rate of cleavage is considerably reduced. 
The collagenase substrate, Z-Gly-Pro-Leu-Gly-Pro, is de- 
graded by post-proline cleaving enzyme; however, the products 
formed are Z-Gly-Pro and Leu-Gly-Pro, which are different 
from those produced by collagenase (38). Z-Gly-Pro-Leu-Gly, 
which has been used as a substrate throughout the purifica- 
tion, has an even higher affinity than the collagenase sub- 
strate for post-proline cleaving enzyme, with an apparentK,,, 
of 6.0 x 10-j. An interesting observation is that Z-Gly-Pro-Leu 
acts as a good substrate while Z-Pro-Leu-Gly is found to be an 
exti amely poor substrate and inhibits the enzymatic cleavage 
of Z-Gly-Pro-Leu-Gly. This finding suggests that the presence 
of the urethane-type protecting group at the NH,-terminal 
side of the proline residue is an important structural require- 
ment for a peptide to be an inhibitor. An additional require- 
ment for inhibition seems to be the L configuration of the 
proline since Z-n-Pro-n-Ala failed to inhibit the enzyme. On 
the other hand, the configuration of the X residue does not 
affect the inhibitory property (nor the substrate property) (14) 
of the peptides. On the basis of the information gathered 
(Table II) it appears possible to develop potent inhibitors based 
on the general structure Z-Pro-X, with X being an acidic 
residue. 
Finally, a series of naturally occurring peptides were stud- 
ied as possible substrates of post-proline cleaving enzyme (Ta- 
ble III). Whenever cleavage did occur it was without exception 
by hydrolysis of the peptide bond at the carboxyl side of proline 
residues. Thus, in case of zinc-free porcine insulin only the 
Pro-Lys bond was cleaved. Earlier results with oxytocin (13, 
14), arginine vasopressin (391, and angiotensin II (39) were 
confirmed and extended. The results with bradykinin reveal 
that the Pro-Phe peptide bond is more rapidly cleaved than the 
Pro-Gly bond. Judging from the rate of appearance of products 
in the case of bradykinin-potentiating factor, the Pro-Arg and 
Pro-Gly bonds are cleaved fairly rapidly and at about the same 
rate, while scission of the Pro-Glu peptide linkage proceeds at 
an exceedingly slow rate. The Pro-Pro bonds in the two pep- 
tides are not attacked. The enzyme seems to degrade low 
molecular weight peptides faster than the large molecules 
where Pro-X bonds located toward the end of the COOH- 
terminal of the sequence are preferred for cleavage. This is for 
example the case with insulin and it may also explain the 
unitslmg units 96 -fold 
4.3 x 10-a 163 100 1 
1.3 x 10-Z 52 32 3 
0.33 82 50 76 
1.9 47 29 4.4 x 102 
45 29 18 1.05 x 104 
resistance of the Pro-X bonds in gastrin, adrenocorticotropic 
hormone, and denatured collagens to enzymatic attack. 
The data presented, along with those obtained previously, 
reveal that the post-proline cleaving enzyme is an endopepti- 
dase which exhibits a strict substrate specificity for the -Pro- 
X-peptide bond while appearing to be sensitive to the tertiary 
structure of the substrate. The species, organ and cellular 
distribution, and possible physiological role of this enzyme is 
currently under investigation. 
Acknowledgments -The preparation and generous supply of 
many of the oligopeptides by Dr. Ronald Orlowski is very 
much appreciated. The authors are thankful to Ms. M. Fischl 
for excellent and thoughtful technical assistance and to Mr. 
Chris Botos for performing amino acid analyses. Discussions 
with Dr. T. Audhya were most helpful. We are indebted to 
Dr. R. L. Colescott, Armour Pharmaceutical Company, Kan- 
kakee, Ill.; Dr. J. S. Morley, Imperial Chemical Industries, 
Ltd., Mereside Alderley Park, England; Dr. M. A. Ondetti, 
E. R. Squibb and Sons, Inc., Princeton, N. J.; Dr. R. E. 
Chance, Eli-Lilly Research Laboratories., Indianapolis, Ind., 
and Dr. J. Coffey, Hoffmann-La Roche, Nutley, N. J. for their 
most helpful gifts of peptides. 
1. 
2. 
3. 
4. 
5. 
6. 
7. 
8. 
9. 
10. 
11. 
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Bergmann, M. (1942) Adu. Enzymol. 2, 49-68 
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Doonan, S., and Fahmy, H. M. A. (1975) Eur. J. Biochem. 56, 
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Doolittle, R. F., and Armentrout, R. W. (1968) Biochemistry ‘7, 
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Houmard, J., and Drapeau, G. R. (1972) Proc. Natl. Acad. Sci. 
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M Koida and R Walter
chromatography.
Post-proline cleaving enzyme. Purification of this endopeptidase by affinity
1976, 251:7593-7599.J. Biol. Chem. 
 
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