BIOCHEMICAL
MEDICINE
Cystine
KUNIO YOSHINOBU
20, 296-304
(1978)
Aminopeptidases Serum and HIWADA*,
from Placenta
CHIZU
INAOKAt,
Pregnancy
SAEKI-YAMAGUCHIt, AND
TATSUO
KOKUBU*
*The Second Department of Internal Medicine, Ehime University School of Medicine, Shigenobu. Onsen-gun, 791-02 Ehime, Japan, and Winical Laboratory, National Ehime Hospital, 791-02 Ehime, Japan Received
April
4, 1978
Oxytocinase activity in sera of pregnant women increases with weeks of gestation (l-9). Since Tuppy et al. (10) synthesized a substrate, L-cystine-di-fl-naphthylamide for the measurement of oxytocinase, additional synthetic substrates are available for the determination of oxytocinase activity (6,11,12). The enzyme which is measured with a synthetic substrate based on cystine is named cystine aminopeptidase (EC 3.4.11.3). In this paper we measured concurrently the enzymic activities in pregnancy sera using S-benzyl-L-cysteine-p-nitroanilide (CAP) and L-leucine-p-nitroanilide (LAP) as substrates. In addition, we separated CAP in pregnancy sera (S-CAP) from arylamidase (EC 3.4.11.2) by Sephadex G-200 gel filtration and purified CAP from normal delivery placentas (P-CAP) by ammonium sulfate precipitation, DEAE-cellulose column chromatography, and gel filtration on Sephadex G-200. Some molecular and kinetic properties of S-CAP and P-CAP were studied. MATERIALS
AND METHODS
S-Benzyl-L-cysteine-p-nitroanilide, L-leucine-p-nitroanilide, and Protein Calibration Kit, Size II were purchased from Boehringer, Mannheim, G.F.R. Ampholine, pH 3.5-5, and 6-8 were from LKB, Bromma, Sweden. Sepharose 4B, Sephadex G-200, and Blue Dextran 2000 were from Pharmacia Fine Chemicals, Dppsala, Sweden. L-Leucyl-P-naphthylamide hydrochloride, L-cystine-di+naphthylamide, and Fast Blue B salt were from Sigma Chemicals Company, St. Louis, MO. DE,, was from Whatman Biochemical Ltd., Maidstone, Kent, England. The chemicals used 296 OOO6-2944/78/0203-0296$02.00/O Copyright @ 1976 by Academic Ress. Inc. All rights of reproduction in any form reserved.
CYSTINE
AMINOPEPTIDASES
297
for polyacrylamide gel electrophoresis were from Wako Pure Chemicals Company, Osaka, Japan, p-Dimethylaminocinnamaldehyde was from Diichi Pure Chemicals Company, Tokyo, Japan. Pregnancy
and Normal
Sera
A total of 176 serum samples were collected from normal pregnant women between 2 months and term gestation, and within 1 week and 1 month after normal delivery. Eighteen normal serum samples from nonpregnant age-matched women were collected. All samples were frozen at -20°C until the time of assay. Preparation
of S-CAP
Sample
Ten serum samples from women at term gestation were mixed and each 3 ml of it was applied to a column of Sephadex G-200 (2.6 x 90 cm) at 4°C. Sodium phosphate buffer (10 mM), pH 7.3 was used for a developing buffer, and the flow rate was 20 ml/hr. Fractions (3.2 ml) were collected. The effluent volume of the enzyme peak was 166 ml. S-CAP could be separated from arylamidase (effluent volume, 206 ml). Polyacrylamide gel disc electrophoresis described below showed S-CAP sample completely free from arylamidase. Preparation
of P-CAP
Sample
All procedures were carried out at 4°C. Three placentas from normal delivery were washed with cold distilled water. The fetal membranes and the umbilical cord were dissected away from the placental mass. The homogenate in 0.1 M TrislHCl buffer, pH 7.3 was centrifuged at 1OOOgfor 20 min. Then the supernatant was adjusted to pH 5.0 with 1 M acetic acid and quickly centrifuged at 8OOOgfor 15 min. The supernatant was adjusted to pH 7.3 with 1 M NaOH and was precipitated with ammonium sulfate between 35 and 70%, DE,, column chromatography was set up to eluate P-CAP using a linear gradient elution increasing the concentration of NaCl (O-O.3 M) and the enzyme was eluted at the conductivity of 18 mu. Finally, gel filtration on a column of Sephadex G-200 (2.6 x 90 cm) was carried out. Enzyme Assay
The hydrolysis of S-benzyl-L-cysteine-p-nitroanilide and L-leucine-pnitroanilide was assayed by the method of Kuno and Haba (13). The incubation mixture contained 0.5 mM substrate 0.1 M phosphate buffer, pH 7.0 and 50 ~1 of serum or enzyme material (total volume: 1.O ml), and was incubated for 15 min. The liberatedp-nitroaniline was coupled with p-dimethylaminocinnamaldehyde and the resultant red color was read at 565 nm. The enzymic activity was expressed as nanomoles of S-benzyl-
298
HIWADA
ET AL.
L-cysteine-p-nitroanilide or L-leucine-p-nitroanilide ute per milliliter of serum (sample). Gel Filtration
for the Determination
of Apparent
hydrolyzed Molecular
per minWeight
Sepharose 4B gel filtration was carried out on a column (2.6 x 90 cm) in 0.01 M phosphate buffer, pH 7.3, containing 0.1% sodium azide at 4”C, with an upward flow rate of 20 ml/h. The column was calibrated with the Protein Calibration Kit. Three milliliters of enzyme material were applied to a column; 3.2-ml of fractions were collected. Polyacrylamide
Gel Disc Electrophoresis
Polyacrylamide gel disc electrophoresis was carried out by the method described previously (14). Stain of enzymic activity zone in the gel was performed using L-cystine-di-pnaphthylamide or r.-leucyl-/3-naphthylamide as substrate. Isoelectric
Focusing
Isoelectric focusing was carried out according to the method of Vesterberg (15). Ten milliliters of enzyme material were mixed in the less dense solution and applied to a sucrose gradient column (LKB 110 column) with 1% Ampholine, pH 3.5-5. The starting power was 3 W and the maximum voltage was 600 V. The electrofocusing was terminated around 48 hr, 2.6-ml fractions were collected and the pH was measured at 4°C. RESULTS Serum CAP and LAP Activities in Normal and Women After Normal Delivery
Controls,
Pregnant
Women
The mean CAP and LAP activities of 18 normal controls were 12 k 1 (SE) nmol/min/ml of serum and 98 rt 4 nmol/min/ml, respectively. Both activities increased gradually with weeks of gestation as shown in Fig. 1. At term the mean CAP and LAP activities in 20 sera were 644 t 57 nmol/min/ml and 934 5 66 nmol/min/ml, respectively. Within a week after delivery both enzymic activities remained at high levels (CAP, 43 1 * 35; LAP, 601 + 43; n = 19) and a month after delivery CAP activity was still significantly higher (39 ? 4, n = 15) than normal @ < 0.001, by Student’s t test. On the other hand, the mean ratio of LAP against CAP in 18 normal controls was 8.8 + 0.5 (SE). However, the ratios decreased rapidly from 2 months (L/C = 4.2 + 0.2, n = 4) to 4 months (L/C = 2.3 -f. 0.1, n = 21) and then they decreased slowly till the term (L/C = 1.5 ? 0.03, n = 20) as shown in Fig. 2. Even 1 month after delivery, the ratio was still significantly lower (L/C = 3.1 * 0.3, n = 15) than that of controls (p < 0.001). These results strongly suggest that cystine aminopeptidase in pregnant serum hydrolyzes L-leucine-p-nitroanilide.
CYSTINE
299
AMINOPEPTIDASES
CAP 0 LAP l
a
Normal
2
3
4
5
6
7
8
Months of Gestation
9
‘p . :.
....I , ai,
10 w$/” 1M after Delivery
FIG. I. Serum CAP and LAP activities in normal pregnant women and women after normal delivery (n = 176). 0, CAP; 0, LAP.
Polyacrylamide
Disc Gel Electrophoresis
Figure 3 shows the CAP bands (CAP, and CAP,) of partially purified CAP from placenta (P-CAP). These two bands were also seen in the same positions when L-leucyl-/3-naphthylamide was used as substrate. The term pregnant serum showed these two bands in the same positions of polyacrylamide gel, but CAP, band appeared weaker than CAP, band as reported by Kleiner and Brouet-Yager (16). When L-leucyl-pnaphthylamide was used for staining enzymic activities, additional two or three bands were observed as shown by Smith (17). Apparent Molecular Weight The apparent molecular weights of S-CAP and P-CAP were estimated by gel filtration on Sepharose 4B column. The enzymic activity was monitered in the eluted fractions. The molecular weights of S-CAP and P-CAP were 340.000.
HI WADA ET AL.
I I
i
2
3 4 5 6 7 8 9 10 Within 1w ‘M Months of Gestation after Deliv.
FIG. 2. The ratio of LAP against CAP in sera from normal pregnant women and women after normal delivery (n = 176).
Isoelectric
Point
Both S-CAP and P-CAP revealed a shoulder on the basic side of the main peak at pH 4.48. The pH in the fraction of shoulder was 4.62. This represents two conformational forms of cystine aminopeptidase suggested by Sjiiholm and Yman (18). Heat Denaturation The enzyme solutions of S-CAP and P-CAP were incubated at 60°C in 0.1 M phosphate buffer, pH 7.0, and the samples were removed after the stated times. The remaining enzymic activities of both CAPS were measured using S-benzyl+cysteine-p-nitroanilide and L-leucine-p-nitroanilide as substrate. Both CAPS were inactivated similarly regardless of substrates (Fig. 4). Kinetic Studies K, values for S-CAP and P-CAP were determined from LineweaverBurk plots in 9.1 M phosphate buffer, pH 7.0. Both K, values of S-CAP and P-CAP for the hydrolysis of S-benzyl-L-cysteine-p-nitroanilide were 1.4 x 1O-4 M and those for the hydrolysis of L-leucine-p-nitroanilide were 2.3 x 1O-4 M as shown in Fig. 5. The ratios of hydrolysis of L-leucine-p-nitroanilide against S-benzyl-Lcysteine-p-nitroanilide in 0.1 v phosphate buffer, pH 7.0, by S-CAP and P-CAP were identical and 1.4. Both S-CAP and P-CAP were inhibited 80% of enzymic activities by 5
CYSTINE
AMINOPEPTIDASES
301
FIG. 3. Polyacrylamide gel (6.5%) electrophoresis of P-CAP. Electrophoresis was carried out in 0.01 M Tris/glycine buffer, pH 8.3. for 2 hr at 2 mA per gel. Enzyme activity was stained with L-cystine-di-/3-naphthylamide and Fast Blue B salt.
mM EDTA with both substrates, S-benzyl-L-cysteine-p-nitroanilide and L-leucine-p-nitroanilide. Mg2+ and Co2+ (1 mM) did not affect enzymic activities of S-CAP and P-CAP with both substrates, but 1 mM Zn*+ inhibited 36% and 1 mM Mn*+ inhibited 40% of enzymic activities of S-CAP and P-CAP with both substrates. DISCUSSION
Our results of molecular and kinetic properties of S-CAP and P-CAP indicate that S-CAP is identical with P-CAP. CAP was a metalloenzyme. Molecular weights of human cystine aminopeptidases from placenta (19) and retroplacental serum (20) were determined to be 320,000 and 325,000 by gel filtration, respectively. Molecular weights of S-CAP and P-CAP determined by us with Sepharose 4B gel filtration were 340,000 and the value was fairly consistent with those by others. The molecular weight of
302
HIWADA
ET AL.
s &lOO.c.-> G cl .-u E
i? 50: .P .-c “E i!
I
I I I I I 10 20 30 Incubation time(min)
FIG. 4. Effect of heat on enzymic activities of S-CAP and P-CAP. Each sample was incubated at 60°C in 0.1 M phosphate buffer, pH 7.0. Samples were removed at various times and the enzymic activities were assayed with S-benzyl+-cysteine-p-nitroanilide or L-leucine-p-nitroanilide. Each dot shows the mean of three determinations. 0, S-CAP assayed with S-benzyl-L-cysteine-p-nitroanilide; 0, S-CAP assayed with L-leucine-pnitroanilide; A, P-CAP assayed with S-benzyl-r-cysteine-p-nitroanilide; A, P-CAP assayed with L-leucine-p-nitroanilide.
2
4
6
8
10
12
FIG. 5. Lineweaver-Burk plots for the hydrolysis of S-benzyl-L-cysteine-p-nitroanilide and L-leucine-p-nitroanilide by S-CAP and P-CAP in 0.1 M phosphate buffer, pH 7.0, at 37°C. The reaction time was 15 min. Each dot shows the mean of duplicate determinations. O-O, S-CAP assayed with S-benzyl-L-cysteine-p-nitroanihde; A-A. P-CAP assayed with S-benzyl-L-cysteine-p-nitroanilide: O-0, S-CAP assayed with L-leucine-pnitroanilide; A-A, P-CAP assayed with r-leucine-p-nitroanilide.
CYSTINE
AMINOPEFTIDASES
303
cystine aminopeptidase from monkey placenta was determined to be 87,000 by gel filtration in the presence of 5 mM mercaptoethanol and 83,000 by SDS polyacrylamide gel electrophoresis (21). Since human cystine aminopeptidase is a glycoprotein (22,23), its molecular weight obtained from gel filtration might be an overestimate as suggested by Ward and Arnott (24). Alternatively, human cystine aminopeptidase might tend to make a polymeric form. CAP samples prepared from pregnancy serum (S-CAP) and placenta (P-CAP) hydrolyzed L-leucine-p-nitroanilide. There is a possibility that CAP samples from pregnancy serum and placenta are contaminated with an enzyme which hydrolyzes L-leucine-p-nitroanilide. However, it is not probable, because the enzyme materials showed the same behaviors with regard to heat denaturation, inhibition by EDTA and effects of divalent cations whichever S-benzyl-L-cysteine-p-nitroanilide or L-leucine-pnitroanilide was used as substrate. Furthermore, two electrophoretic CAP bands on polyacrylamide gel were also stained using L-leucyl-Pnaphthylamide as substrate. A purified cystine aminopeptidase from monkey placenta hydrolyzed L-leucyl-/3-naphthylamide more rapidly than L-cystine-di-/3-naphthylamide (21). Thus, an increase of LAP activity in pregnancy serum is mainly caused by an increase in cystine aminopeptidase. It is clear from our results and others (25,26) that cystine aminopeptidase in pregnancy serum derives from placenta. Oya et al. (27) have reported that cystine aminopeptidase is a lysosomal enzyme in placenta. But there is still a possibility that lysosomal fraction prepared by them was contaminated with secreting granules containing cystine aminopeptidase. Serum cystine aminopeptidase in pregnant women has been shown to reflect the functional status of the placenta (2,3,9,28). This suggests that normally functioning placenta secretes cystine aminopeptidase into the mother’s blood. If this is the case, the physiological significance ofcystine aminopeptidase in pregnant women remains to be studied again. SUMMARY
We simultaneously measured the enzymic activities in 142 pregnancy sera, 34 sera from women after normal delivery, and 18 sera from normal nonpregnant women using S-benzyl-L-cysteine-p-nitroanilide (CAP) and L-leucine-p-nitroanilide (LAP). We confirmed that both enzymic activities increased with weeks of gestation. The mean ratio of LAP against CAP in normal controls was 8.8 f 0.5 (SE) (n = 18). However, the ratio decreased with weeks of gestation and at term the mean ratio was 1.5 ? 0.03 (n = 20). Partially purified cystine aminopeptidase from pregnancy serum was identical with that purified partially from placenta with respect to elec-
304
HIWADA
ET AL.
trophoretic mobility, isoelectric point, apparent molecular weight, heat denaturation, K, value, and effects of EDTA and divalent cations. It was concluded that cystine aminopeptidase hydrolyzed L-leucine-pnitroanilide. An increase of LAP activity in pregnant serum was mainly attributed to an increase in cystine aminopeptidase. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Melander, S. E. J., Nature (London) 191, 176 (1961). Babuna, C., and Yenen, E., Amer. J. Obstet. Gynecol. 95, 925 (1966). Hensleigh, P. A.. and Krantz, K. E., Amer. J. Obsret. Gynecol. 107, 1233 (1970). Chapman, L., Silk, E., Skupny, A., Tooth, E. A.. and Barnes. A., J. Obstet. Gyned. Brit. Commonwealth 78, 435 (1971). Curzen, P., and Varme, R., Amer. .I. Obstet. Gynecol. 115, 929 (1973). Usategui-Gomez, M., Tarbutton, P.. Yeager, F., and Fernandez-de Castro, A., Clin. Chim. Acta 47, 409 (1973). Kleiner, H., and Brouet-Yager, M., Clin. Chim. Acra 48, 299 (1973). Christensen, A., Acta Endocrinol. ?S, 189 (1974). Spellacy, W. N., Usategui-Gomez, M.. and Fernandez-de Castro, A.. Amer. J. Obstet. Gynecol., 127, 10 (1977). Tuppy, H., and Nesvadba, H., Monatsh. Chem. 88, 977 (1957). Wintersberger, E., Milller-Hartburg, W., and Tuppy. H., C/in. Chim. Acta 14, 786 (1966). van Oudhensden, A. P. M., Z. Klin. Chem. K/in. Biochem. 10, 345 (1972). Konno. M., and Haba, S., Jap. J. Clin. Chem. 1, 83 (1977) [in Japanese]. Hiwada, K., Yamaguchi, C.. Inaoka, Y., and Kokubu, T.. C/in. Chim. Acta 75, 31 (1977). Vesterberg, O., in “Methods in Enzymology” (W. B. Jakoby, Ed.), Vol. 22, p. 389. Academic Press, New York/London, 1971. Kleiner, H., and Brouet-Yager, M., Clin. Chim. Acta 40, 177 (1966). Smith, E. E., Biochem. Med. 7, 253 (1973). Sjoholm, I., and Yman, L., Acta Pharmacol. Suecica 3, 377 (1966). Oya, M., Yoshino. M.. and Mizutani, S., Experienria 31, 1019 (1975). Yman, L., and Sjoholm, I., Acta Pharmacol. Suecica 4, 13 (1967). Hayashi, M.. and Oshima, K., J. Biochem. 80, 389 (1976). Tuppy, M., Wiesbauer, U.. and Wintersberger, E., Monatsh. Chem. 94, 321 (1963). Sjiiholm, I., and Yman, L., Acra Pharmacol. Suecica 3, 389 (1%6). Ward, D. N.. and Arnott, M. S., Anal. Biochem. 12, 296 (1%5). Page, E. W., Titus, M. A., Mohun, G.. and Glendening. M. B., Amer. J. Obsfet. Gynecol. 82, 1090 (1961). Oya, M., Yoshino, M., Mizutani, S., and Wakabayashi, T.. Gynecol. Invest. 5, 276 (1974). Oya, M., Wakabayashi, T.. Yoshino, M., and Mizutani. S., Physiol. Chem. Physics. 8, 327 (1976). Josephides, E. C. H., and Turkington, V. E., J. Obsfet. Gynecol. Brit. Commonwealth 74, 251 (1967).
MEDICINE
Cystine
KUNIO YOSHINOBU
20, 296-304
(1978)
Aminopeptidases Serum and HIWADA*,
from Placenta
CHIZU
INAOKAt,
Pregnancy
SAEKI-YAMAGUCHIt, AND
TATSUO
KOKUBU*
*The Second Department of Internal Medicine, Ehime University School of Medicine, Shigenobu. Onsen-gun, 791-02 Ehime, Japan, and Winical Laboratory, National Ehime Hospital, 791-02 Ehime, Japan Received
April
4, 1978
Oxytocinase activity in sera of pregnant women increases with weeks of gestation (l-9). Since Tuppy et al. (10) synthesized a substrate, L-cystine-di-fl-naphthylamide for the measurement of oxytocinase, additional synthetic substrates are available for the determination of oxytocinase activity (6,11,12). The enzyme which is measured with a synthetic substrate based on cystine is named cystine aminopeptidase (EC 3.4.11.3). In this paper we measured concurrently the enzymic activities in pregnancy sera using S-benzyl-L-cysteine-p-nitroanilide (CAP) and L-leucine-p-nitroanilide (LAP) as substrates. In addition, we separated CAP in pregnancy sera (S-CAP) from arylamidase (EC 3.4.11.2) by Sephadex G-200 gel filtration and purified CAP from normal delivery placentas (P-CAP) by ammonium sulfate precipitation, DEAE-cellulose column chromatography, and gel filtration on Sephadex G-200. Some molecular and kinetic properties of S-CAP and P-CAP were studied. MATERIALS
AND METHODS
S-Benzyl-L-cysteine-p-nitroanilide, L-leucine-p-nitroanilide, and Protein Calibration Kit, Size II were purchased from Boehringer, Mannheim, G.F.R. Ampholine, pH 3.5-5, and 6-8 were from LKB, Bromma, Sweden. Sepharose 4B, Sephadex G-200, and Blue Dextran 2000 were from Pharmacia Fine Chemicals, Dppsala, Sweden. L-Leucyl-P-naphthylamide hydrochloride, L-cystine-di+naphthylamide, and Fast Blue B salt were from Sigma Chemicals Company, St. Louis, MO. DE,, was from Whatman Biochemical Ltd., Maidstone, Kent, England. The chemicals used 296 OOO6-2944/78/0203-0296$02.00/O Copyright @ 1976 by Academic Ress. Inc. All rights of reproduction in any form reserved.
CYSTINE
AMINOPEPTIDASES
297
for polyacrylamide gel electrophoresis were from Wako Pure Chemicals Company, Osaka, Japan, p-Dimethylaminocinnamaldehyde was from Diichi Pure Chemicals Company, Tokyo, Japan. Pregnancy
and Normal
Sera
A total of 176 serum samples were collected from normal pregnant women between 2 months and term gestation, and within 1 week and 1 month after normal delivery. Eighteen normal serum samples from nonpregnant age-matched women were collected. All samples were frozen at -20°C until the time of assay. Preparation
of S-CAP
Sample
Ten serum samples from women at term gestation were mixed and each 3 ml of it was applied to a column of Sephadex G-200 (2.6 x 90 cm) at 4°C. Sodium phosphate buffer (10 mM), pH 7.3 was used for a developing buffer, and the flow rate was 20 ml/hr. Fractions (3.2 ml) were collected. The effluent volume of the enzyme peak was 166 ml. S-CAP could be separated from arylamidase (effluent volume, 206 ml). Polyacrylamide gel disc electrophoresis described below showed S-CAP sample completely free from arylamidase. Preparation
of P-CAP
Sample
All procedures were carried out at 4°C. Three placentas from normal delivery were washed with cold distilled water. The fetal membranes and the umbilical cord were dissected away from the placental mass. The homogenate in 0.1 M TrislHCl buffer, pH 7.3 was centrifuged at 1OOOgfor 20 min. Then the supernatant was adjusted to pH 5.0 with 1 M acetic acid and quickly centrifuged at 8OOOgfor 15 min. The supernatant was adjusted to pH 7.3 with 1 M NaOH and was precipitated with ammonium sulfate between 35 and 70%, DE,, column chromatography was set up to eluate P-CAP using a linear gradient elution increasing the concentration of NaCl (O-O.3 M) and the enzyme was eluted at the conductivity of 18 mu. Finally, gel filtration on a column of Sephadex G-200 (2.6 x 90 cm) was carried out. Enzyme Assay
The hydrolysis of S-benzyl-L-cysteine-p-nitroanilide and L-leucine-pnitroanilide was assayed by the method of Kuno and Haba (13). The incubation mixture contained 0.5 mM substrate 0.1 M phosphate buffer, pH 7.0 and 50 ~1 of serum or enzyme material (total volume: 1.O ml), and was incubated for 15 min. The liberatedp-nitroaniline was coupled with p-dimethylaminocinnamaldehyde and the resultant red color was read at 565 nm. The enzymic activity was expressed as nanomoles of S-benzyl-
298
HIWADA
ET AL.
L-cysteine-p-nitroanilide or L-leucine-p-nitroanilide ute per milliliter of serum (sample). Gel Filtration
for the Determination
of Apparent
hydrolyzed Molecular
per minWeight
Sepharose 4B gel filtration was carried out on a column (2.6 x 90 cm) in 0.01 M phosphate buffer, pH 7.3, containing 0.1% sodium azide at 4”C, with an upward flow rate of 20 ml/h. The column was calibrated with the Protein Calibration Kit. Three milliliters of enzyme material were applied to a column; 3.2-ml of fractions were collected. Polyacrylamide
Gel Disc Electrophoresis
Polyacrylamide gel disc electrophoresis was carried out by the method described previously (14). Stain of enzymic activity zone in the gel was performed using L-cystine-di-pnaphthylamide or r.-leucyl-/3-naphthylamide as substrate. Isoelectric
Focusing
Isoelectric focusing was carried out according to the method of Vesterberg (15). Ten milliliters of enzyme material were mixed in the less dense solution and applied to a sucrose gradient column (LKB 110 column) with 1% Ampholine, pH 3.5-5. The starting power was 3 W and the maximum voltage was 600 V. The electrofocusing was terminated around 48 hr, 2.6-ml fractions were collected and the pH was measured at 4°C. RESULTS Serum CAP and LAP Activities in Normal and Women After Normal Delivery
Controls,
Pregnant
Women
The mean CAP and LAP activities of 18 normal controls were 12 k 1 (SE) nmol/min/ml of serum and 98 rt 4 nmol/min/ml, respectively. Both activities increased gradually with weeks of gestation as shown in Fig. 1. At term the mean CAP and LAP activities in 20 sera were 644 t 57 nmol/min/ml and 934 5 66 nmol/min/ml, respectively. Within a week after delivery both enzymic activities remained at high levels (CAP, 43 1 * 35; LAP, 601 + 43; n = 19) and a month after delivery CAP activity was still significantly higher (39 ? 4, n = 15) than normal @ < 0.001, by Student’s t test. On the other hand, the mean ratio of LAP against CAP in 18 normal controls was 8.8 + 0.5 (SE). However, the ratios decreased rapidly from 2 months (L/C = 4.2 + 0.2, n = 4) to 4 months (L/C = 2.3 -f. 0.1, n = 21) and then they decreased slowly till the term (L/C = 1.5 ? 0.03, n = 20) as shown in Fig. 2. Even 1 month after delivery, the ratio was still significantly lower (L/C = 3.1 * 0.3, n = 15) than that of controls (p < 0.001). These results strongly suggest that cystine aminopeptidase in pregnant serum hydrolyzes L-leucine-p-nitroanilide.
CYSTINE
299
AMINOPEPTIDASES
CAP 0 LAP l
a
Normal
2
3
4
5
6
7
8
Months of Gestation
9
‘p . :.
....I , ai,
10 w$/” 1M after Delivery
FIG. I. Serum CAP and LAP activities in normal pregnant women and women after normal delivery (n = 176). 0, CAP; 0, LAP.
Polyacrylamide
Disc Gel Electrophoresis
Figure 3 shows the CAP bands (CAP, and CAP,) of partially purified CAP from placenta (P-CAP). These two bands were also seen in the same positions when L-leucyl-/3-naphthylamide was used as substrate. The term pregnant serum showed these two bands in the same positions of polyacrylamide gel, but CAP, band appeared weaker than CAP, band as reported by Kleiner and Brouet-Yager (16). When L-leucyl-pnaphthylamide was used for staining enzymic activities, additional two or three bands were observed as shown by Smith (17). Apparent Molecular Weight The apparent molecular weights of S-CAP and P-CAP were estimated by gel filtration on Sepharose 4B column. The enzymic activity was monitered in the eluted fractions. The molecular weights of S-CAP and P-CAP were 340.000.
HI WADA ET AL.
I I
i
2
3 4 5 6 7 8 9 10 Within 1w ‘M Months of Gestation after Deliv.
FIG. 2. The ratio of LAP against CAP in sera from normal pregnant women and women after normal delivery (n = 176).
Isoelectric
Point
Both S-CAP and P-CAP revealed a shoulder on the basic side of the main peak at pH 4.48. The pH in the fraction of shoulder was 4.62. This represents two conformational forms of cystine aminopeptidase suggested by Sjiiholm and Yman (18). Heat Denaturation The enzyme solutions of S-CAP and P-CAP were incubated at 60°C in 0.1 M phosphate buffer, pH 7.0, and the samples were removed after the stated times. The remaining enzymic activities of both CAPS were measured using S-benzyl+cysteine-p-nitroanilide and L-leucine-p-nitroanilide as substrate. Both CAPS were inactivated similarly regardless of substrates (Fig. 4). Kinetic Studies K, values for S-CAP and P-CAP were determined from LineweaverBurk plots in 9.1 M phosphate buffer, pH 7.0. Both K, values of S-CAP and P-CAP for the hydrolysis of S-benzyl-L-cysteine-p-nitroanilide were 1.4 x 1O-4 M and those for the hydrolysis of L-leucine-p-nitroanilide were 2.3 x 1O-4 M as shown in Fig. 5. The ratios of hydrolysis of L-leucine-p-nitroanilide against S-benzyl-Lcysteine-p-nitroanilide in 0.1 v phosphate buffer, pH 7.0, by S-CAP and P-CAP were identical and 1.4. Both S-CAP and P-CAP were inhibited 80% of enzymic activities by 5
CYSTINE
AMINOPEPTIDASES
301
FIG. 3. Polyacrylamide gel (6.5%) electrophoresis of P-CAP. Electrophoresis was carried out in 0.01 M Tris/glycine buffer, pH 8.3. for 2 hr at 2 mA per gel. Enzyme activity was stained with L-cystine-di-/3-naphthylamide and Fast Blue B salt.
mM EDTA with both substrates, S-benzyl-L-cysteine-p-nitroanilide and L-leucine-p-nitroanilide. Mg2+ and Co2+ (1 mM) did not affect enzymic activities of S-CAP and P-CAP with both substrates, but 1 mM Zn*+ inhibited 36% and 1 mM Mn*+ inhibited 40% of enzymic activities of S-CAP and P-CAP with both substrates. DISCUSSION
Our results of molecular and kinetic properties of S-CAP and P-CAP indicate that S-CAP is identical with P-CAP. CAP was a metalloenzyme. Molecular weights of human cystine aminopeptidases from placenta (19) and retroplacental serum (20) were determined to be 320,000 and 325,000 by gel filtration, respectively. Molecular weights of S-CAP and P-CAP determined by us with Sepharose 4B gel filtration were 340,000 and the value was fairly consistent with those by others. The molecular weight of
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s &lOO.c.-> G cl .-u E
i? 50: .P .-c “E i!
I
I I I I I 10 20 30 Incubation time(min)
FIG. 4. Effect of heat on enzymic activities of S-CAP and P-CAP. Each sample was incubated at 60°C in 0.1 M phosphate buffer, pH 7.0. Samples were removed at various times and the enzymic activities were assayed with S-benzyl+-cysteine-p-nitroanilide or L-leucine-p-nitroanilide. Each dot shows the mean of three determinations. 0, S-CAP assayed with S-benzyl-L-cysteine-p-nitroanilide; 0, S-CAP assayed with L-leucine-pnitroanilide; A, P-CAP assayed with S-benzyl-r-cysteine-p-nitroanilide; A, P-CAP assayed with L-leucine-p-nitroanilide.
2
4
6
8
10
12
FIG. 5. Lineweaver-Burk plots for the hydrolysis of S-benzyl-L-cysteine-p-nitroanilide and L-leucine-p-nitroanilide by S-CAP and P-CAP in 0.1 M phosphate buffer, pH 7.0, at 37°C. The reaction time was 15 min. Each dot shows the mean of duplicate determinations. O-O, S-CAP assayed with S-benzyl-L-cysteine-p-nitroanihde; A-A. P-CAP assayed with S-benzyl-L-cysteine-p-nitroanilide: O-0, S-CAP assayed with L-leucine-pnitroanilide; A-A, P-CAP assayed with r-leucine-p-nitroanilide.
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cystine aminopeptidase from monkey placenta was determined to be 87,000 by gel filtration in the presence of 5 mM mercaptoethanol and 83,000 by SDS polyacrylamide gel electrophoresis (21). Since human cystine aminopeptidase is a glycoprotein (22,23), its molecular weight obtained from gel filtration might be an overestimate as suggested by Ward and Arnott (24). Alternatively, human cystine aminopeptidase might tend to make a polymeric form. CAP samples prepared from pregnancy serum (S-CAP) and placenta (P-CAP) hydrolyzed L-leucine-p-nitroanilide. There is a possibility that CAP samples from pregnancy serum and placenta are contaminated with an enzyme which hydrolyzes L-leucine-p-nitroanilide. However, it is not probable, because the enzyme materials showed the same behaviors with regard to heat denaturation, inhibition by EDTA and effects of divalent cations whichever S-benzyl-L-cysteine-p-nitroanilide or L-leucine-pnitroanilide was used as substrate. Furthermore, two electrophoretic CAP bands on polyacrylamide gel were also stained using L-leucyl-Pnaphthylamide as substrate. A purified cystine aminopeptidase from monkey placenta hydrolyzed L-leucyl-/3-naphthylamide more rapidly than L-cystine-di-/3-naphthylamide (21). Thus, an increase of LAP activity in pregnancy serum is mainly caused by an increase in cystine aminopeptidase. It is clear from our results and others (25,26) that cystine aminopeptidase in pregnancy serum derives from placenta. Oya et al. (27) have reported that cystine aminopeptidase is a lysosomal enzyme in placenta. But there is still a possibility that lysosomal fraction prepared by them was contaminated with secreting granules containing cystine aminopeptidase. Serum cystine aminopeptidase in pregnant women has been shown to reflect the functional status of the placenta (2,3,9,28). This suggests that normally functioning placenta secretes cystine aminopeptidase into the mother’s blood. If this is the case, the physiological significance ofcystine aminopeptidase in pregnant women remains to be studied again. SUMMARY
We simultaneously measured the enzymic activities in 142 pregnancy sera, 34 sera from women after normal delivery, and 18 sera from normal nonpregnant women using S-benzyl-L-cysteine-p-nitroanilide (CAP) and L-leucine-p-nitroanilide (LAP). We confirmed that both enzymic activities increased with weeks of gestation. The mean ratio of LAP against CAP in normal controls was 8.8 f 0.5 (SE) (n = 18). However, the ratio decreased with weeks of gestation and at term the mean ratio was 1.5 ? 0.03 (n = 20). Partially purified cystine aminopeptidase from pregnancy serum was identical with that purified partially from placenta with respect to elec-
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trophoretic mobility, isoelectric point, apparent molecular weight, heat denaturation, K, value, and effects of EDTA and divalent cations. It was concluded that cystine aminopeptidase hydrolyzed L-leucine-pnitroanilide. An increase of LAP activity in pregnant serum was mainly attributed to an increase in cystine aminopeptidase. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Melander, S. E. J., Nature (London) 191, 176 (1961). Babuna, C., and Yenen, E., Amer. J. Obstet. Gynecol. 95, 925 (1966). Hensleigh, P. A.. and Krantz, K. E., Amer. J. Obsret. Gynecol. 107, 1233 (1970). Chapman, L., Silk, E., Skupny, A., Tooth, E. A.. and Barnes. A., J. Obstet. Gyned. Brit. Commonwealth 78, 435 (1971). Curzen, P., and Varme, R., Amer. .I. Obstet. Gynecol. 115, 929 (1973). Usategui-Gomez, M., Tarbutton, P.. Yeager, F., and Fernandez-de Castro, A., Clin. Chim. Acta 47, 409 (1973). Kleiner, H., and Brouet-Yager, M., Clin. Chim. Acra 48, 299 (1973). Christensen, A., Acta Endocrinol. ?S, 189 (1974). Spellacy, W. N., Usategui-Gomez, M.. and Fernandez-de Castro, A.. Amer. J. Obstet. Gynecol., 127, 10 (1977). Tuppy, H., and Nesvadba, H., Monatsh. Chem. 88, 977 (1957). Wintersberger, E., Milller-Hartburg, W., and Tuppy. H., C/in. Chim. Acta 14, 786 (1966). van Oudhensden, A. P. M., Z. Klin. Chem. K/in. Biochem. 10, 345 (1972). Konno. M., and Haba, S., Jap. J. Clin. Chem. 1, 83 (1977) [in Japanese]. Hiwada, K., Yamaguchi, C.. Inaoka, Y., and Kokubu, T.. C/in. Chim. Acta 75, 31 (1977). Vesterberg, O., in “Methods in Enzymology” (W. B. Jakoby, Ed.), Vol. 22, p. 389. Academic Press, New York/London, 1971. Kleiner, H., and Brouet-Yager, M., Clin. Chim. Acta 40, 177 (1966). Smith, E. E., Biochem. Med. 7, 253 (1973). Sjoholm, I., and Yman, L., Acta Pharmacol. Suecica 3, 377 (1966). Oya, M., Yoshino. M.. and Mizutani, S., Experienria 31, 1019 (1975). Yman, L., and Sjoholm, I., Acta Pharmacol. Suecica 4, 13 (1967). Hayashi, M.. and Oshima, K., J. Biochem. 80, 389 (1976). Tuppy, M., Wiesbauer, U.. and Wintersberger, E., Monatsh. Chem. 94, 321 (1963). Sjiiholm, I., and Yman, L., Acra Pharmacol. Suecica 3, 389 (1%6). Ward, D. N.. and Arnott, M. S., Anal. Biochem. 12, 296 (1%5). Page, E. W., Titus, M. A., Mohun, G.. and Glendening. M. B., Amer. J. Obsfet. Gynecol. 82, 1090 (1961). Oya, M., Yoshino, M., Mizutani, S., and Wakabayashi, T.. Gynecol. Invest. 5, 276 (1974). Oya, M., Wakabayashi, T.. Yoshino, M., and Mizutani. S., Physiol. Chem. Physics. 8, 327 (1976). Josephides, E. C. H., and Turkington, V. E., J. Obsfet. Gynecol. Brit. Commonwealth 74, 251 (1967).
