250
Biochbnica et Biophysica Acta, I 119 (1992) 250-255 ~:~ 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34123
Identification of immuno-reactive lipocortin l-like molecules in serum and plasma by an enzyme immunoassay for lipocortin 1 Kenji Uemura 1.2, Hiroshi Inagaki 2, Yoshiro Wada 2, Keiko Nakanishi 2, Kiyofumi Asai 2, Taiji Kato 2, Yoshihiro Ando 3 and Reiji Kannagi 3 t Department of Medicine and Pediatrics, School of Dentistr)', Aichi-Gakuin Unicersity, Nagoya (Japan), e Departments of Pediatrics attd Bioregulation Research. Nagoya City Unicersio, Medical School, Nagoya (Japan) and 3 Departments of Dermatology and Clinical Science, Faculty of Medicine, Kyoto Unirersity, Kyoto (Japan) (Received 17 August 1991) (Revised manuscript received !1 October 1991)
Key words: Annexin: Enzyme immunoassay; Lipocortin 1; Serum level; (Human)
A two-site enzyme immunoassay for lipocortin 1 (LCI) has been developed. The detection limit of LC1 was 0.2 ng/tube and the optimal assay range was 1 to 100 n g / t u b e . The assay system enabled us to identify immunoreactive lipocortin l-like molecules (IR-LCI) in human serum and plasma. Normal human serum and plasma IR-LCI concentrations were 44.6 n g / m l for males and 43.1 n g / m l for females with no significant difference between both sexes. The age-related analysis among nine age groups from newborn to 69 years old revealed that the serum or plasma level was high in infants (77.5 n g / m l for newborn and 75.6 n g / m l for 1 month-I year group), and the 40-year-old (52.2 n g / m l ) and 50-year-old (51.3 n g / m l ) groups. The major population of plasma IR-LCI was of 70 kDa in size corresponding to that of the LC1 homodimer. The present enzyme immunoassay system is sufficiently sensitive for the clinical study of LC1 in human body fluids, tissues and organs.
Introduction
Lipocortins or annexins are recognized as a family of Ca2÷-dependent phospholipid binding proteins [1,2], comprising at least six different members (LC1 to 6) [3,4]. Lipocortins are found to be a regulator of cytoskeletal organization related to the transportation of exocytic vesicles [5], a factor involved in inflammation [6], immune response [7], blood coagulation [8] or cell growth and differentiation [9,10], and a substrate for tyrosine kinase involving epidermal growth factor (EGF)-receptor [11-14]. Among the members of lipocortin family, lipocortin 1 (LC1) was initially described as an intracellular protein responsive to glucocorticoid [15-18]. After much controversy as to whether or not the synthesis of LC1 is accessible by glucocorticoid [19-21], it has recently become clear that both LCI-mRNA expression and protein synthesis are aug-
Abbreviations: LCI, lipocortin I; IR, immunoreactive. Correspondence: K. Uemura, Departments of Pediatrics and Bioregulation Research, Nagoya City University Medical School, MizuhoKu, Nagoya 467, Japan.
mented by glucocorticoid [22,23]. Concerning cellular localization of LC1, the extracellular distribution in human seminal fluid [24,25], lung lavage fluid [26] and peritoneal exudates [27] has been demonstrated and glucocorticoid also induced the secretion of LC1 from cultured squamous cells [28], whereas the biological function of extracellular LC1, apart from a variety of intracellular functions of LC1, is not yet known. None of reports, however, detects LC1 in human serum nor plasma due to lack of sensitive quantification methods. We for the first time detect immunoreactive LCl-like molecules in human serum and plasma, using a sensitive enzyme immunoassay system for LC1, enabling the measurement of a minute amount of LC1. Materials and Methods Anti human LCI antiserum 500 /~g of LC1, which were purified from human placenta according to the method previously described [29], were emulsified with 0.5 ml of complete Freund's adjuvant (Difco, U.K.) and injected subcutaneously into a New Zealand albino rabbit. Booster immunizations of 100/~g were given after 2 and 5 weeks. Anti LC1
251 antibody was raised 6 weeks after the initial immunization. The gamma-globulin fraction of anti-LC! antiserum (10 ml)was prepared by two successive steps of ammonium sulfate precipitation (411 and 33% saturation). The precipitates were collected by centrifugation (120(~)×g for 20 min), dissolvcd in 2 ml of acetate buffer, 0.1 mol/l (pH 4.5) and dialyzed against 2 changes of the same buffer (4 1 each) at 4°C for 24 h for a following pepsin digestion.
Preparation o f enzyme immunoassay system A sandwich enzyme immunoassay system for LCI was composed of the antibody (Fab'),-immobilized on a solid-phase and the antibody Fab' fragments labeled with fl-D-galacto.~,idase from l:3cheriehia coli as previously reported [3~)]. One half of the antibody (Fab'L fragments, which had been isolated from the pepsin digests of anti LCI antibody with a Sephadex GI50 column (2.6 × 40 cm) and successively with a LC2-affinity column comprising of LC2 conjugated on CNBractivated Sepharose 4B (Pharmacia, Sweden) to remove cross-reacting antibody to LC2, were immobilized non-covalently on polystyrene beads (3.18 mm diameter, Precision Plastic Ball, Chicago, IL, U.S.A.). LC2-affinity column was prepared by coupling I00 ~g of LC2, which was copurified with LCI from human placenta, to 0.5 g of CNBr-activated Sepharose 4B (Pharmacia). The other half of the (Fab') 2 fraction, after dialysis against 0.1 mol/I acetate buffer (pH 5.0) for 16 h, was reduced to Fab' fragments by 15 mmol/I 2-mercaptoethylamine (Sigma) at 37°C for 3(1 min. The Fab' fragments were then separated from the reducing reagent by Bio-Gel P-10 (Bio-Rad) column (2 × 30 cm) equilibrated with acetate buffer (pH 5.0). The exposed sulfhydryi groups (1.5 mol/Fab' fragment) of the Fab' fragment were blocked by adding 10-times the excess amount of a bifunctional cross-linker, N,N'-o-phenylenedimaleimide (Aldrich Chemical, Milwaukee, Wl, U.S.A.) at 37°C for 20 min. The maleimidated-Fab' fragments were separated from the excess dimaleimide on Bio-Gel P-10 column (2 × 30 cm), equilibrated with 0.1 mol/I sodium phosphate buffer (pH 6.3) and conjugated with 2.5 mg of fl-o-galactosidase (BoehringerMannheim, Germany) at 40°C for 16 h. The .8-0galactosidase-labeled Fab' fragment was separated on a Bio-Gel A-5m (Bio-Rad) column (2 × 40 cm) equilibrated with sodium phosphate buffer, 0.01 mol/1 (pH 7.0) containing 0.1 mol/I NaCI, 1 mmol/l MgCI 2, ! g/! bovine serum albumin (BSA) and 1 g/i sodium azide (buffer A), as a major peak of galactosidase activity. Immunoassay A polystyrene bead coated with (Fab') z was incubated in duplicate with various amounts of standard LCI (0-1 / ~ g / t u b e ) o r test sample in 0.4 mi of sodium
phosphate buffer, 0.01 mol/I (pH 7.0) containing 0.1 molfl NaCI, I mmolfl MgCI 2, 5 g f l gelatin (Difeo), 1 g / I bovine serum albumin, and i g f l sodium azide (buffer G) in a test tube with continuous shaking for 4 h at 30°C. After washing three times with 1.0 ml of cold buffer A, the bead was transferred to a second test tube containing 2 mU (I U = I #mol produet/min) of the Fab'-fl-o-galactosidase complex in 0.2 ml of buffer A and incubated at 4°C for 16 h. After washing three times with 1.0 ml of cold buffer A, the bead was transferred to the third test tube containing 0.1 ml of buffer A. The enzyme reaction at 30°C was started by adding 5(1 p~! of 0.3 mmolfl 4-methylumbelliferyl-fl-ogalaetoside (Koch-Light, Suffolk, U.K.) as substrate, and stopped after 1 h by adding 2.5 ml of 0.1 mol/I glyeine-NaOH buffer (pH 10.3). The fluoreseenee intensity of 4-mcthylumbelliferone in the reaetion mixture was measured with a fluorescenee speetrophotometer (Hitachi, Model No. 650-40) at 360 nm exeitation and 450 nm emission, against 100 nmol/! of 4-methylumbelliferone (Wako Pure Chemieal Industries, Osaka, Japan) as standard, fl-D-galaetosidase bound to the bead was expressed as the fluoreseenee intensity of 4-methylumbelliferone produeed in a l-h reaetion from 0.1 mmol/I 4-methylumbelliferyl-~-D-galaetoside. A fluoreseenec intensity of 100 was equivalent to I • 10-7 mol/I 4-mcthylumbelliferone.
Human senml and plasma specbnens Normal human serum and plasma (from EDTA blood) were obtained from the outpatients free of any infectious disease for adults and children (older than l month), from full-term newborn babies and umbilical cords. For a partial purification of LCl-like molecules from blood collected in the presence of 0.1% sodium citrate, 201) ml of plasma were applied on a column of DEAE Sephacel (Pharmacia) equilibrated with 20 mmol/! Tris-HCi buffer (pH 7.5) containing l mmol/I EGTA, l m m o l / l EDTA and 50 mmol/l NaCI. The flow-through fraction of DEAE Sephacel column, wherein CaCI 2 was added to make its final concentration above 5 mmoi/I after dialysis vs. 20 mmol/I imidazole-HC! buffer (pH 7.5) containing 1 mmol/I EGTA, l mmol/! EDTA, 50 mmol/! NaCI and ! mmol/l 2-mercaptoethanol, was loaded on a phospholipid-CPG column (l × 6 cm) comprising phosphatidylcholine (PC) and phosphatidylserine (PS) (PC:PS = 5:1), which was prepared according to the previous method [29] and equilibrated with 20 mmol imidazoleHCi buffer (pH 7.5) containing 100 mmol/! NaCl, l mmol/I CaC! 2 and l mmol/l 2-mercaptoethanol. After thoroughly washing the column, the adsorbed proteins on the column were eluted with 20 mmol/I imidazole-HCl buffer (pH 7.5) containing 100 mmoi/I NaCl, 2 mmol/I EGTA and 1 mmol/l 2-mercaptoethanol. The fractions with the corresponding reten-
252 tion time to that for placental LCI were pooled and used for Western blot analyses.
Western blot analysis SDS-polyacrylamide gel electrophoresis (SDSPAGE) was performed by the use of 12% gels accordingto Laemmli:s method [31]. Samples on SDS-PAGE were transferred to polyvinylidene difluoride (PVDF) membrane (immobilon-P, Millipore, U.K.) by the standard methods, which were processed under the manufactural protocols of Blotting detection kit (Amersham) for rabbit antibodies, employing anti LC1 antibody. Cross-linked LCI by human erythrocyte transglutaminase was prepared as described previously [32] and used for Western blot analysis as the standard LCI comprising monomer (37 kDa), dimer (70 kDa) and tetramer (140 kDa) of LCI.
TABLE I LereL$ o f IR-LCI in serunt or plasma from t'arious age groups
All data represent mean_+ S.D. Diiferences from the 20-29-year-old group * P < I].115 and * * P < 0.01 (Student's t-test). Age
Number
IR-LCI concentration (ng/ml)
total (male, male female) Umbilical cord 1-311 days 1-12 months I - 9years 10-19years 211-29years 3()-39 years 411-49 years 511-59 years 60-69 years Total
Ii 8 19(13,6) 18(111,8) 24(15,9) 18 (%91 28(17,11) 29(15,14) 30116,14) 24 (13,11) 2119
female
total
106,5 _+70.2 * * 77,5 _+50.2 * * 63.4_+31.1 i112.[)_+74.3 75,6+52.2 ** 41.3_+32.2 34.2_-/-211.1 38,1+27.7 411.9_+27.5 28.1_+ 14.6 36,1-+24.3 28.9_+ 12.4 27.4_+ 8.6 28.1_+ 10.7 34.5_+21.8 35.1_+12.2 34.7_+19.5 48.1_+35.9 56.6_+46.11 52.2-+41.3" 58.5_+22.8 43.1-+ 17.6 51,3+21.6 ** 31.1 -+ 14.8 33.3_+24.1 34,3_+ 19.6 44.6 + 28.4
43.1 _+36.9
48.5 _+39.2
Results
Sensitit'ity and precision of the assay for LCI The immunochemical reactivity between purified LCI and LCl-specific antibody isolated by lipocortin 2 (LC2)-affinity column was examined by a double immunodiffusion technique and Western blot analysis. There was no detectable cross-reactivity with LC2 on both two methods (data not shown). Fig. 1 shows the standard curve of LCI and the cross-reactivity with LC2 on the assay system. The absence of cross-reactiv,ity with LC2 (0.1 ng to 1.0 /.~g/tube) was further iconfirmed in the assay system. The limit of detection, corresponding to 2 S.D. higher than the mean fluores-
50(3
0/0/O
/;/ //
20C
o/
c_ ¢,
10C
u~
~ o
5o
20
I
10 .7
I
I
10 .'6
10-5
I
10-4
I
10.3
(mg I tube} Fig. I. Standard curves of LCi and LC2. Standard curve of purified LCI ( o o ) and LC2 (zx zx) on the assay system. To estimate the recovery of added standard LCI in the serum, the indicated amounts of LCI (e e) in the presence of serum (50 / d / t u b e , comprising 19.3 ng IR-LCI/ml separately estimated by the assay) were subjected to the assay.
cence intensity in 10 zero standard samples, was 0.2 ng/tube. The optimal detection range was 1-100 ng/tube, it is known that macromolecular proteins in serum or plasma occasionally interfere with solid phase enzyme immunoassay systems, so buffer G containing gelatin (5 g/I) was used instead of buffer A to reduce a nonspecific interference as described by Kato et al. [30]. The standard curve in the presence of either Ca -'+ (1 mmol/l) or EDTA (1 mmol/I) was essentially identical to that in the absence, whereas the apparent /3-galactosidase activity was inhibited to 77 + 13% of control by Ca -'+ addition as described by Rickenberg [33], indicating no antigenic alteration of LCI due to calcium binding as observed in S100 protein [34]. The recovery of added standard LC1 (1-100 ng/tube) to a constant amount of human serum (50 # l / t u b e ) was 100.7 + 10.8% (n = 10) and the standard curve in the presence of serum was parallel to the original one without serum. A sample volume of 50 txl/tube for serum or plasma was used for the following assay of LCI. The precision was tested by assaying 3 samples 10 times in one assay Cintra-assay) and 3 samples in 10 consecutive assays (inter-assay). The coefficients of variation in all instances were less than 14% (data not shown).
lmmuno-reactit'e LCl-like molecules in huntan serum attd plasma proteins The serum or plasma concentration of immunoreactive LCl-like molecules (IR-LC1) in the control subjects was determined among nine age groups from newborn to 69 years old and cord blood (Table I). Plasma IR-LCI level was high in the cord blood which might be derived from placenta rich in LCI contents. No statistically significant difference between both sexes was detected among all age groups (44.6 ng/ml
253 b
c [kDa)
r
#
,---,-
- - 9 7 . 4
~.~
-
6t5.2
-- 45
........... 1 4 . 4
Fig. 2. Western blot analysis. The LCl-like molecules in plasma were partially purified as described in 'Materials and Methods" and subjecled to Western blot analysis after the development on SDS-PAGE. Lane a, partially purified LCl-like substances (equivalent to ~ ng of LCI on the assay system), lane b. standard LCI (511 ng of LCI) and lane c, cross-linked LCI by transglutaminase (20 ng of LCI ). Phosphorylase t97.4 kDaL bovine serum albumin (66.2 kDa), ovalbumin 145.11 kDaL carbonic anhydrase 131.11 kDal, soybean trypsin inhibitor (21.5 kDa) and lysozyme (14.4 kDa) were used as molecular weight standards.
for male and 43.1 ng/ml for female) nor even within each age group. The age groups younger than 1 year and middle ages from 40-50-years-old showed significantly high contents as compared with the 20-year-old age group. There was no difference in IR-LCI levels between normal serum (14.8 + 6.8 ng/ml, n = 10) and plasma (16.7 _+_4.2 ng/ml, n = 10) from the identical
donor, nor alteration of serum IR-LCI level during the incubation periods at 4°C from 0 to 48 h before blood cell separation by centrifugation (Table Ill. As shown in Fig. 2, Western blot analyses of the partially purified I R-LCI from plasma predominantly visualized the band of dimeric form of IR-LCI with a molecular weight of 70 kDa, whereas the preadsorbed antibodies by LCI could visualized the band of neither LCI including its polymers nor IR-LC1 components in plasma (data not shown). The electrophoretic pattern of serum on Western blot analysis was identical to that of plasma (data not shown). There were detectable minor bands possibly corresponding to a monomeric LCI (37 kDa) and processed fragments of LC1 by some proteinases. Although the blood incubation at 24°C for 48 h 2-fold elevated the apparent serum IR-LCI level, the dimeric form as well as the monomeric form entirely disappeared and shifted in to the fragments with smaller molecular sizes on SDS-PAGE. Discussion
There are numerous reports on the intracellular functions of lipocortins including the cytoskeletal reorganization for endocytic or exocytic movements of vesicles [5] and the suppression of eicosanoid production resulting from its inhibitory action on phospholipase A2 [35]. In contrast, Pepinsky et al. first demonstrated that LC1 was also extracellularly secreted into peritoneal exudate and proposed the possibility of extracellular functions of LCI [28]. in support of this idea, LC1 was detcctcd in human lung lavage fluid [26], seminal fluid [24,25] and the conditioned media of squamous cell carcinoma S q C C / Y I differentiated by glucocorticoid [28], whereas the extracellular functions were not
TABLE I1 Stability of IR-LCI in serum fiYml preincubated blood under tile conditions with i'aried temperatures and periods All serum samples were prepared by centrifugation (8011x g, 15 min, 4°C) subsequent to the blood prcincubation under the conditions with the indicated varied temperatures and periods. * Serum was obtained from incubated bhmd at 24°C for 311 min. (Sample ! )
(Sample 2)
Biochbnica et Biophysica Acta, I 119 (1992) 250-255 ~:~ 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34123
Identification of immuno-reactive lipocortin l-like molecules in serum and plasma by an enzyme immunoassay for lipocortin 1 Kenji Uemura 1.2, Hiroshi Inagaki 2, Yoshiro Wada 2, Keiko Nakanishi 2, Kiyofumi Asai 2, Taiji Kato 2, Yoshihiro Ando 3 and Reiji Kannagi 3 t Department of Medicine and Pediatrics, School of Dentistr)', Aichi-Gakuin Unicersity, Nagoya (Japan), e Departments of Pediatrics attd Bioregulation Research. Nagoya City Unicersio, Medical School, Nagoya (Japan) and 3 Departments of Dermatology and Clinical Science, Faculty of Medicine, Kyoto Unirersity, Kyoto (Japan) (Received 17 August 1991) (Revised manuscript received !1 October 1991)
Key words: Annexin: Enzyme immunoassay; Lipocortin 1; Serum level; (Human)
A two-site enzyme immunoassay for lipocortin 1 (LCI) has been developed. The detection limit of LC1 was 0.2 ng/tube and the optimal assay range was 1 to 100 n g / t u b e . The assay system enabled us to identify immunoreactive lipocortin l-like molecules (IR-LCI) in human serum and plasma. Normal human serum and plasma IR-LCI concentrations were 44.6 n g / m l for males and 43.1 n g / m l for females with no significant difference between both sexes. The age-related analysis among nine age groups from newborn to 69 years old revealed that the serum or plasma level was high in infants (77.5 n g / m l for newborn and 75.6 n g / m l for 1 month-I year group), and the 40-year-old (52.2 n g / m l ) and 50-year-old (51.3 n g / m l ) groups. The major population of plasma IR-LCI was of 70 kDa in size corresponding to that of the LC1 homodimer. The present enzyme immunoassay system is sufficiently sensitive for the clinical study of LC1 in human body fluids, tissues and organs.
Introduction
Lipocortins or annexins are recognized as a family of Ca2÷-dependent phospholipid binding proteins [1,2], comprising at least six different members (LC1 to 6) [3,4]. Lipocortins are found to be a regulator of cytoskeletal organization related to the transportation of exocytic vesicles [5], a factor involved in inflammation [6], immune response [7], blood coagulation [8] or cell growth and differentiation [9,10], and a substrate for tyrosine kinase involving epidermal growth factor (EGF)-receptor [11-14]. Among the members of lipocortin family, lipocortin 1 (LC1) was initially described as an intracellular protein responsive to glucocorticoid [15-18]. After much controversy as to whether or not the synthesis of LC1 is accessible by glucocorticoid [19-21], it has recently become clear that both LCI-mRNA expression and protein synthesis are aug-
Abbreviations: LCI, lipocortin I; IR, immunoreactive. Correspondence: K. Uemura, Departments of Pediatrics and Bioregulation Research, Nagoya City University Medical School, MizuhoKu, Nagoya 467, Japan.
mented by glucocorticoid [22,23]. Concerning cellular localization of LC1, the extracellular distribution in human seminal fluid [24,25], lung lavage fluid [26] and peritoneal exudates [27] has been demonstrated and glucocorticoid also induced the secretion of LC1 from cultured squamous cells [28], whereas the biological function of extracellular LC1, apart from a variety of intracellular functions of LC1, is not yet known. None of reports, however, detects LC1 in human serum nor plasma due to lack of sensitive quantification methods. We for the first time detect immunoreactive LCl-like molecules in human serum and plasma, using a sensitive enzyme immunoassay system for LC1, enabling the measurement of a minute amount of LC1. Materials and Methods Anti human LCI antiserum 500 /~g of LC1, which were purified from human placenta according to the method previously described [29], were emulsified with 0.5 ml of complete Freund's adjuvant (Difco, U.K.) and injected subcutaneously into a New Zealand albino rabbit. Booster immunizations of 100/~g were given after 2 and 5 weeks. Anti LC1
251 antibody was raised 6 weeks after the initial immunization. The gamma-globulin fraction of anti-LC! antiserum (10 ml)was prepared by two successive steps of ammonium sulfate precipitation (411 and 33% saturation). The precipitates were collected by centrifugation (120(~)×g for 20 min), dissolvcd in 2 ml of acetate buffer, 0.1 mol/l (pH 4.5) and dialyzed against 2 changes of the same buffer (4 1 each) at 4°C for 24 h for a following pepsin digestion.
Preparation o f enzyme immunoassay system A sandwich enzyme immunoassay system for LCI was composed of the antibody (Fab'),-immobilized on a solid-phase and the antibody Fab' fragments labeled with fl-D-galacto.~,idase from l:3cheriehia coli as previously reported [3~)]. One half of the antibody (Fab'L fragments, which had been isolated from the pepsin digests of anti LCI antibody with a Sephadex GI50 column (2.6 × 40 cm) and successively with a LC2-affinity column comprising of LC2 conjugated on CNBractivated Sepharose 4B (Pharmacia, Sweden) to remove cross-reacting antibody to LC2, were immobilized non-covalently on polystyrene beads (3.18 mm diameter, Precision Plastic Ball, Chicago, IL, U.S.A.). LC2-affinity column was prepared by coupling I00 ~g of LC2, which was copurified with LCI from human placenta, to 0.5 g of CNBr-activated Sepharose 4B (Pharmacia). The other half of the (Fab') 2 fraction, after dialysis against 0.1 mol/I acetate buffer (pH 5.0) for 16 h, was reduced to Fab' fragments by 15 mmol/I 2-mercaptoethylamine (Sigma) at 37°C for 3(1 min. The Fab' fragments were then separated from the reducing reagent by Bio-Gel P-10 (Bio-Rad) column (2 × 30 cm) equilibrated with acetate buffer (pH 5.0). The exposed sulfhydryi groups (1.5 mol/Fab' fragment) of the Fab' fragment were blocked by adding 10-times the excess amount of a bifunctional cross-linker, N,N'-o-phenylenedimaleimide (Aldrich Chemical, Milwaukee, Wl, U.S.A.) at 37°C for 20 min. The maleimidated-Fab' fragments were separated from the excess dimaleimide on Bio-Gel P-10 column (2 × 30 cm), equilibrated with 0.1 mol/I sodium phosphate buffer (pH 6.3) and conjugated with 2.5 mg of fl-o-galactosidase (BoehringerMannheim, Germany) at 40°C for 16 h. The .8-0galactosidase-labeled Fab' fragment was separated on a Bio-Gel A-5m (Bio-Rad) column (2 × 40 cm) equilibrated with sodium phosphate buffer, 0.01 mol/1 (pH 7.0) containing 0.1 mol/I NaCI, 1 mmol/l MgCI 2, ! g/! bovine serum albumin (BSA) and 1 g/i sodium azide (buffer A), as a major peak of galactosidase activity. Immunoassay A polystyrene bead coated with (Fab') z was incubated in duplicate with various amounts of standard LCI (0-1 / ~ g / t u b e ) o r test sample in 0.4 mi of sodium
phosphate buffer, 0.01 mol/I (pH 7.0) containing 0.1 molfl NaCI, I mmolfl MgCI 2, 5 g f l gelatin (Difeo), 1 g / I bovine serum albumin, and i g f l sodium azide (buffer G) in a test tube with continuous shaking for 4 h at 30°C. After washing three times with 1.0 ml of cold buffer A, the bead was transferred to a second test tube containing 2 mU (I U = I #mol produet/min) of the Fab'-fl-o-galactosidase complex in 0.2 ml of buffer A and incubated at 4°C for 16 h. After washing three times with 1.0 ml of cold buffer A, the bead was transferred to the third test tube containing 0.1 ml of buffer A. The enzyme reaction at 30°C was started by adding 5(1 p~! of 0.3 mmolfl 4-methylumbelliferyl-fl-ogalaetoside (Koch-Light, Suffolk, U.K.) as substrate, and stopped after 1 h by adding 2.5 ml of 0.1 mol/I glyeine-NaOH buffer (pH 10.3). The fluoreseenee intensity of 4-mcthylumbelliferone in the reaetion mixture was measured with a fluorescenee speetrophotometer (Hitachi, Model No. 650-40) at 360 nm exeitation and 450 nm emission, against 100 nmol/! of 4-methylumbelliferone (Wako Pure Chemieal Industries, Osaka, Japan) as standard, fl-D-galaetosidase bound to the bead was expressed as the fluoreseenee intensity of 4-methylumbelliferone produeed in a l-h reaetion from 0.1 mmol/I 4-methylumbelliferyl-~-D-galaetoside. A fluoreseenec intensity of 100 was equivalent to I • 10-7 mol/I 4-mcthylumbelliferone.
Human senml and plasma specbnens Normal human serum and plasma (from EDTA blood) were obtained from the outpatients free of any infectious disease for adults and children (older than l month), from full-term newborn babies and umbilical cords. For a partial purification of LCl-like molecules from blood collected in the presence of 0.1% sodium citrate, 201) ml of plasma were applied on a column of DEAE Sephacel (Pharmacia) equilibrated with 20 mmol/! Tris-HCi buffer (pH 7.5) containing l mmol/I EGTA, l m m o l / l EDTA and 50 mmol/l NaCI. The flow-through fraction of DEAE Sephacel column, wherein CaCI 2 was added to make its final concentration above 5 mmoi/I after dialysis vs. 20 mmol/I imidazole-HC! buffer (pH 7.5) containing 1 mmol/I EGTA, l mmol/! EDTA, 50 mmol/! NaCI and ! mmol/l 2-mercaptoethanol, was loaded on a phospholipid-CPG column (l × 6 cm) comprising phosphatidylcholine (PC) and phosphatidylserine (PS) (PC:PS = 5:1), which was prepared according to the previous method [29] and equilibrated with 20 mmol imidazoleHCi buffer (pH 7.5) containing 100 mmol/! NaCl, l mmol/I CaC! 2 and l mmol/l 2-mercaptoethanol. After thoroughly washing the column, the adsorbed proteins on the column were eluted with 20 mmol/I imidazole-HCl buffer (pH 7.5) containing 100 mmoi/I NaCl, 2 mmol/I EGTA and 1 mmol/l 2-mercaptoethanol. The fractions with the corresponding reten-
252 tion time to that for placental LCI were pooled and used for Western blot analyses.
Western blot analysis SDS-polyacrylamide gel electrophoresis (SDSPAGE) was performed by the use of 12% gels accordingto Laemmli:s method [31]. Samples on SDS-PAGE were transferred to polyvinylidene difluoride (PVDF) membrane (immobilon-P, Millipore, U.K.) by the standard methods, which were processed under the manufactural protocols of Blotting detection kit (Amersham) for rabbit antibodies, employing anti LC1 antibody. Cross-linked LCI by human erythrocyte transglutaminase was prepared as described previously [32] and used for Western blot analysis as the standard LCI comprising monomer (37 kDa), dimer (70 kDa) and tetramer (140 kDa) of LCI.
TABLE I LereL$ o f IR-LCI in serunt or plasma from t'arious age groups
All data represent mean_+ S.D. Diiferences from the 20-29-year-old group * P < I].115 and * * P < 0.01 (Student's t-test). Age
Number
IR-LCI concentration (ng/ml)
total (male, male female) Umbilical cord 1-311 days 1-12 months I - 9years 10-19years 211-29years 3()-39 years 411-49 years 511-59 years 60-69 years Total
Ii 8 19(13,6) 18(111,8) 24(15,9) 18 (%91 28(17,11) 29(15,14) 30116,14) 24 (13,11) 2119
female
total
106,5 _+70.2 * * 77,5 _+50.2 * * 63.4_+31.1 i112.[)_+74.3 75,6+52.2 ** 41.3_+32.2 34.2_-/-211.1 38,1+27.7 411.9_+27.5 28.1_+ 14.6 36,1-+24.3 28.9_+ 12.4 27.4_+ 8.6 28.1_+ 10.7 34.5_+21.8 35.1_+12.2 34.7_+19.5 48.1_+35.9 56.6_+46.11 52.2-+41.3" 58.5_+22.8 43.1-+ 17.6 51,3+21.6 ** 31.1 -+ 14.8 33.3_+24.1 34,3_+ 19.6 44.6 + 28.4
43.1 _+36.9
48.5 _+39.2
Results
Sensitit'ity and precision of the assay for LCI The immunochemical reactivity between purified LCI and LCl-specific antibody isolated by lipocortin 2 (LC2)-affinity column was examined by a double immunodiffusion technique and Western blot analysis. There was no detectable cross-reactivity with LC2 on both two methods (data not shown). Fig. 1 shows the standard curve of LCI and the cross-reactivity with LC2 on the assay system. The absence of cross-reactiv,ity with LC2 (0.1 ng to 1.0 /.~g/tube) was further iconfirmed in the assay system. The limit of detection, corresponding to 2 S.D. higher than the mean fluores-
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20
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I
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10-5
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10-4
I
10.3
(mg I tube} Fig. I. Standard curves of LCi and LC2. Standard curve of purified LCI ( o o ) and LC2 (zx zx) on the assay system. To estimate the recovery of added standard LCI in the serum, the indicated amounts of LCI (e e) in the presence of serum (50 / d / t u b e , comprising 19.3 ng IR-LCI/ml separately estimated by the assay) were subjected to the assay.
cence intensity in 10 zero standard samples, was 0.2 ng/tube. The optimal detection range was 1-100 ng/tube, it is known that macromolecular proteins in serum or plasma occasionally interfere with solid phase enzyme immunoassay systems, so buffer G containing gelatin (5 g/I) was used instead of buffer A to reduce a nonspecific interference as described by Kato et al. [30]. The standard curve in the presence of either Ca -'+ (1 mmol/l) or EDTA (1 mmol/I) was essentially identical to that in the absence, whereas the apparent /3-galactosidase activity was inhibited to 77 + 13% of control by Ca -'+ addition as described by Rickenberg [33], indicating no antigenic alteration of LCI due to calcium binding as observed in S100 protein [34]. The recovery of added standard LC1 (1-100 ng/tube) to a constant amount of human serum (50 # l / t u b e ) was 100.7 + 10.8% (n = 10) and the standard curve in the presence of serum was parallel to the original one without serum. A sample volume of 50 txl/tube for serum or plasma was used for the following assay of LCI. The precision was tested by assaying 3 samples 10 times in one assay Cintra-assay) and 3 samples in 10 consecutive assays (inter-assay). The coefficients of variation in all instances were less than 14% (data not shown).
lmmuno-reactit'e LCl-like molecules in huntan serum attd plasma proteins The serum or plasma concentration of immunoreactive LCl-like molecules (IR-LC1) in the control subjects was determined among nine age groups from newborn to 69 years old and cord blood (Table I). Plasma IR-LCI level was high in the cord blood which might be derived from placenta rich in LCI contents. No statistically significant difference between both sexes was detected among all age groups (44.6 ng/ml
253 b
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Fig. 2. Western blot analysis. The LCl-like molecules in plasma were partially purified as described in 'Materials and Methods" and subjecled to Western blot analysis after the development on SDS-PAGE. Lane a, partially purified LCl-like substances (equivalent to ~ ng of LCI on the assay system), lane b. standard LCI (511 ng of LCI) and lane c, cross-linked LCI by transglutaminase (20 ng of LCI ). Phosphorylase t97.4 kDaL bovine serum albumin (66.2 kDa), ovalbumin 145.11 kDaL carbonic anhydrase 131.11 kDal, soybean trypsin inhibitor (21.5 kDa) and lysozyme (14.4 kDa) were used as molecular weight standards.
for male and 43.1 ng/ml for female) nor even within each age group. The age groups younger than 1 year and middle ages from 40-50-years-old showed significantly high contents as compared with the 20-year-old age group. There was no difference in IR-LCI levels between normal serum (14.8 + 6.8 ng/ml, n = 10) and plasma (16.7 _+_4.2 ng/ml, n = 10) from the identical
donor, nor alteration of serum IR-LCI level during the incubation periods at 4°C from 0 to 48 h before blood cell separation by centrifugation (Table Ill. As shown in Fig. 2, Western blot analyses of the partially purified I R-LCI from plasma predominantly visualized the band of dimeric form of IR-LCI with a molecular weight of 70 kDa, whereas the preadsorbed antibodies by LCI could visualized the band of neither LCI including its polymers nor IR-LC1 components in plasma (data not shown). The electrophoretic pattern of serum on Western blot analysis was identical to that of plasma (data not shown). There were detectable minor bands possibly corresponding to a monomeric LCI (37 kDa) and processed fragments of LC1 by some proteinases. Although the blood incubation at 24°C for 48 h 2-fold elevated the apparent serum IR-LCI level, the dimeric form as well as the monomeric form entirely disappeared and shifted in to the fragments with smaller molecular sizes on SDS-PAGE. Discussion
There are numerous reports on the intracellular functions of lipocortins including the cytoskeletal reorganization for endocytic or exocytic movements of vesicles [5] and the suppression of eicosanoid production resulting from its inhibitory action on phospholipase A2 [35]. In contrast, Pepinsky et al. first demonstrated that LC1 was also extracellularly secreted into peritoneal exudate and proposed the possibility of extracellular functions of LCI [28]. in support of this idea, LC1 was detcctcd in human lung lavage fluid [26], seminal fluid [24,25] and the conditioned media of squamous cell carcinoma S q C C / Y I differentiated by glucocorticoid [28], whereas the extracellular functions were not
TABLE I1 Stability of IR-LCI in serum fiYml preincubated blood under tile conditions with i'aried temperatures and periods All serum samples were prepared by centrifugation (8011x g, 15 min, 4°C) subsequent to the blood prcincubation under the conditions with the indicated varied temperatures and periods. * Serum was obtained from incubated bhmd at 24°C for 311 min. (Sample ! )
(Sample 2)
