January 2003
Volume 44, Issue 1
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Biochemistry and Molecular Biology  |   January 2003
Localization and Activity of Membrane Dipeptidase in Bovine Ciliary Epithelium
Author Affiliations
  • Hiromi Ikeda
    From the Department of Ophthalmology and Visual Science, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
  • Masamichi Ueda
    Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan; and the
  • Hiroshi Kobayashi
    Department of Ophthalmology, Saga Medical University, Saga, Japan.
  • Yoshihito Honda
    From the Department of Ophthalmology and Visual Science, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
Investigative Ophthalmology & Visual Science January 2003, Vol.44, 37-43. doi:10.1167/iovs.02-0216
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      Hiromi Ikeda, Masamichi Ueda, Hiroshi Kobayashi, Yoshihito Honda; Localization and Activity of Membrane Dipeptidase in Bovine Ciliary Epithelium. Invest. Ophthalmol. Vis. Sci. 2003;44(1):37-43. doi: 10.1167/iovs.02-0216.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate the localization and the activity of membrane dipeptidase (MDP) in the bovine eye.

methods. A monoclonal antibody (mAb), 49C mAb, raised against bovine ciliary process was used to examine the localization of MDP. Conversion of leukotriene (LT)D4 to LTE4 was evaluated by enzyme-linked immunosorbent assay for LTE4. Hydrolytic activity (β-lactamase activity) was evaluated with a fluorometric assay. To clarify the contribution of MDP to conversion of LTD4 and β-lactamase activity, we separated MDP from other enzymes by 49C mAb-conjugated gel.

results. The antigenic molecule of 49C mAb was shown to be MDP by amino acid sequencing. MDP was immunohistochemically detected in the ciliary pigmented and nonpigmented epithelial cells. Conversion of LTD4 to LTE4 in the ciliary process was much greater than that of the neural retina (NR). β-Lactamase activity in the ciliary process was apparent, but that in the NR or the retinal pigment epithelium was negligible. Approximately 100% of β-lactamase activity in the ciliary process was catalyzed by the 49C mAb-bound fraction. Conversion of LTD4 was catalyzed by the 49C mAb-bound fraction (55% of total activity) and by the unbound fraction (45% of total activity).

conclusions. This study produced the first evidence of the presence of MDP in ciliary epithelial cells. The ciliary epithelium converts LTD4 to LTE4 and shows β-lactamase activity. Conversion of LTD4 is catalyzed by at least two enzymes, and a major part of the conversion is induced by MDP.

Membrane dipeptidase (MDP; referred to as dehydropeptidase-I, renal dipeptidase, or microsomal dipeptidase; EC 3.4.13.19) is a glycosyl phosphatidylinositol (GPI)-anchored ectoenzyme 1 that is involved in the metabolism of glutathione, leukotriene (LT)D4, and certain β-lactam antibiotics. The cysteinyl leukotrienes (LTC4, LTD4, and LTE4), which are potent mediators of inflammatory responses, 2 are sequentially degraded by two enzymes: γ-glutamyl transpeptidase (GGTP), which catalyzes the conversion of LTC4 to LTD4, and MDP, which catalyzes the hydrolysis of LTD4 to LTE4. 3 4 5 With regard to glutathione (γ-glutamyl cysteinyl glycine), the degradation is initiated by GGTP to form cysteinyl-bis-glycine (cys-bis-gly) and cysteinyl-glycine (cys-gly) 3 and MDP was suggested to metabolize cys-bis-gly into its constituent amino acids. 5 6 MDP is the only known mammalian enzyme to exhibit β-lactamase activity. 7 8 MDP catalyzes the hydrolysis of some β-lactam antibiotics, carbapenems such as imipenem, that have potent activity against a wide spectrum of bacteria. 7 8 9 MDP appears to be relatively unique among the mammalian peptidases in hydrolyzing peptides with either a dehydro bond (e.g., glycyl-dehydro-phenylalanine) or D-amino acid in the C-terminal position (e.g., Gly-D-Phe). 10 11  
The activity of MDP is widely distributed to various organs and has been detected in the lung, 12 kidney, 13 14 pancreas, 15 and small intestine. 16 With regard to the metabolisms of glutathione and cysteinyl leukotrienes which are mediated through the successive actions of GGTP and MDP, the expressions of these two enzymes are not concordant among various tissues. In the experiment of the MDP knock-out mouse, Habib et al. 16 demonstrated that the conversion of LTD4 to LTE4 and the degradation of cys-bis-gly generated from glutathione are catalyzed by at least two alternative pathways (one of which is MDP), and the degree of the contribution of MDP to LTD4 conversion is different in each organ. 
Although the metabolisms of cysteinyl leukotrienes and glutathione have been reported in the eye, the physiological roles remain unclarified. The localization and activity of GGTP were investigated in the ocular tissues, 17 18 19 20 but the presence of MDP in the eye has not been studied to date. As the result of screening of antibodies raised against bovine ciliary epithelium proteins, we established the monoclonal antibody (mAb) for MDP. In this study, we demonstrated for the first time the presence of MDP in bovine eye and the potential activities of MDP in bovine ciliary cells. 
Materials and Methods
Tissues
Bovine eyes were obtained from a local slaughterhouse. A razor blade was used to cut around the entire globe just posterior to the ora serrata. The lens, lens capsule, and zonulae were removed exposing the ciliary process. Bovine kidney and pancreas were obtained from the same slaughterhouse. 
Production of Monoclonal Antibody
The ciliary processes were dissected from the excised eyeball and minced with scissors. The ciliary processes included mainly the epithelium and partially the ciliary stroma. They were incubated in RPMI 1640 medium (Nissui, Tokyo, Japan) containing 0.2% collagenase (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and 0.01% DNase I (Sigma, St. Louis, MO), at 37°C for 30 minutes. After the undissociated materials were discarded, the cell suspension was collected and centrifuged for 5 minutes at 160g. Some of the cells were washed twice and suspended in phosphate-buffered saline and immediately used for immunization, and the rest were stored in liquid nitrogen for further immunization. Eight-week-old BALB/c mice were injected intraperitoneally with 2 × 106 cells every 3 weeks for 9 weeks and then killed with chloroform, and the spleen was obtained. Animals were managed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The spleen cells of an immunized mouse were fused with X63Ag8.653 mouse myeloma cells, using 50% polyethylene glycol 1500 (BDH Chemicals, Poole, UK) on the third day after the last immunization. 21 The fused cells were cultured in RPMI medium supplemented with 15% fetal calf serum, growth factor-containing medium (10% BM-Condimed H1; Roche Molecular Biochemicals, Mannheim, Germany), and hypoxanthine aminopterin thymidine (Flow Co., Ayrshire, Scotland, UK) on 96-well plates. An indirect immunofluorescence method with frozen sections of bovine tissue, as described later, was used for screening the supernatants of growing hybridomas. The positive hybridomas were cloned twice and injected intraperitoneally into mice previously treated with 2,6,10,14-tetramethylpentadecane (Pristane; Tokyo Kasei Co., Tokyo, Japan). IgG was purified from ascitic fluid with immobilized protein A (Affi-Gel Protein A; Bio-Rad, Hercules, CA). Ig isotype was determined using an isotype-specific antibody for mouse mAbs (Serotec, Ltd., Oxford, UK). 
Indirect Immunofluorescence Staining
The tissue was embedded in OCT compound (Tissue-Tek; Miles Scientific, Naperville, IL) and snap frozen in liquid nitrogen. Frozen tissues were sliced to a thickness of 8 μm with a cryostat microtome (Microm HM 500M; Carl Zeiss, Oberkochen, Germany), immediately air dried on coated (Neoprene; Nisshin EM, Tokyo, Japan) glass slides, and fixed in acetone at −20°C for 5 minutes. The slides were incubated with 49C mAb (5 μg/mL, diluted in culture medium) or anti-trinitrophenyl (TNP) mAb (IgG1, 5 μg/mL, for a negative control) 22 for 30 minutes at room temperature. After a wash in phosphate-buffered saline, the slides were incubated with fluorescein isothiocyanate-conjugated second antibody (diluted 1:50; Dako Japan, Kyoto, Japan) for 40 minutes at room temperature in the dark. The slides were then washed, mounted with aqueous mounting medium (Perma Fluor; Immunon, Pittsburgh, PA) and examined under a fluorescence microscope (Nikon, Tokyo, Japan). 
Purification of 49C Antigen
The ciliary processes (0.5 g) were homogenized (Polytron homogenizer; Kinematica AG, Luzern, Switzerland) in 5 mL of ice-cold 40 mM phosphate buffer (pH 7.3) containing 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40 (Iwai Chemicals, Tokyo, Japan), and protease inhibitors, including 1 mM amidinophenyl methanesulfonyl fluoride hydrochloride and 10 μg/mL each of leupeptin and pepstatin (Peptide Institute, Osaka, Japan). After centrifugation (10,000g, 30 minutes, 4°C), the concentration of Nonidet P-40 in the lysate was reduced by dilution to 0.3%. The supernatant was passed through a separation column of activated gel (Affi-Gel 10; Bio-Rad) that was conjugated with anti-TNP mouse IgG1 mAb (2 mg IgG/mL gel). The effluent was then incubated with the activated gel (0.2 mL Affi-Gel 10; Bio-Rad) that was conjugated with 49C mAb (2 mg IgG/mL gel) at 4°C for 3 hours. After sufficient washing with the buffer, the antigen was eluted with 0.5 M NH4OH containing 0.1% Nonidet P-40. The eluate was evaporated under reduced pressure at room temperature. The samples were dissolved in lysis buffer with 0.1 M dithiothreitol (Wako Pure Chemical Industries, Ltd.) and separated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in the gel were visualized with a silver stain kit (Wako Pure Chemical Industries, Ltd.). 
Partial Amino Acid Sequencing of the Protein Purified from Bovine Ciliary Process
Bovine ciliary processes (3 g) were homogenized (Polytron; Kinematica AG) in 30 mL of ice-cold 40 mM phosphate buffer containing 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, and 1 mM amidinophenyl methanesulfonyl fluoride hydrochloride, as described in the prior section. After absorbing nonspecific binding with anti-TNP mAb-conjugated gel, the lysate was incubated with 1 mL activated gel (Affi-Gel 10; Bio-Rad) conjugated with 49C mAb at 4°C for 3 hours. After the gel was washed, the antigenic molecules were eluted as just described. The purified antigen was dissolved in lysis buffer for SDS-PAGE. After electrophoresis, the proteins in the polyacrylamide gel were transblotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore Corp., Bedford, MA) in Tris-boric acid buffer including 0.03% SDS and 10% methanol. The protein on the PVDF membrane was stained with Coomassie blue (Nacalai Tesque, Kyoto, Japan), the 48.0- and 43.7-kDa protein bands were obtained, and then the N-terminal amino acid sequences were analyzed. In some experiments, the proteins in the polyacrylamide gel were stained with Coomassie blue and the 48.0-kDa band was obtained. After treatment with lysylendopeptidase, the fragments released from the 48.0-kDa protein were collected and separated by reversed-phase high-performance liquid chromatography for amino acid sequencing. The N-terminal amino acid sequence of each polypeptide was analyzed (sequencing was performed by the APRO Life Science Institute, Tokushima, Japan). The Swiss Prot databases were used in the analysis of amino acid sequence homology (http://www.expasy.org provided in the public domain by the Swiss Institute of Bioinformatics, Geneva, Switzerland). 
β-Lactamase Assay
Samples.
β-Lactamase assay was evaluated from the fluorometric detection of D-phenylalanine (D-Phe) released from the substrate Gly-D-Phe according to the method of Heywood and Hooper, 11 with minor modifications. Bovine ciliary process, neural retina (NR), or retinal pigment epithelium (RPE) partially containing choroid (0.5 g each) was homogenized at 4°C in 0.5 mL of 0.1 M Tris-HCl (pH 8.0). The resultant supernatant was diluted with buffer to 35 mg/mL of the protein concentration. 
β-Lactamase Activity in the Ciliary Process, the NR, or the RPE.
The resultant supernatant (5 μL) was diluted to 90 μL with 0.1 M Tris-HCl buffer and preincubated at 37°C for 3 minutes. The reaction was started by adding 10 μL Gly-D-Phe (10 mM; Sigma) in buffer to a final concentration of 1 mM. After incubation at 37°C, the reaction was terminated by heating at 100°C for 4 minutes. After a brief centrifugation, the supernatant was used for the fluorometric assay. All samples were assayed in duplicate from three experiments. 
Fluorometric Assay.
The fluorometric assay mix contained 24 μL flavin adenine dinucleotide (FAD, 10 mg/mL; Sigma), 40 μL peroxidase (type VI, 2500 U/mL 0.1 M Tris-HCl [pH 8.0]; Sigma), 1.2 mL D-amino acid oxidase (type I, 2.6 U/mL; Biozyme Laboratories, Gwent, UK) and 2.5 mL p-hydroxyphenylacetic acid (5 mg/mL; Sigma) made up to 6.0 mL with 0.1 M Tris-HCl (pH 8.0). D-Phe (Nacalai Tesque) was used as a standard. To each sample to be quantified, an equal amount of the fluorometric assay mix was added, and they were incubated in the dark at 37°C. After 60 minutes, the fluorescence of each sample was measured in a fluorescence spectrophotometer (model F-2000; Hitachi, Tokyo, Japan) at an excitation of 317 nm and an emission of 414 nm. Enzyme activity was expressed as moles of D-Phe released from the substrate Gly-D-Phe. Protein concentrations were measured using a detergent-compatible protein assay kit (Bio-Rad). 
Inhibitory Effect of 49C mAb on β-Lactamase Activity.
To ascertain whether 49C mAb inhibits MDP activity, 49C mAb dialyzed against 0.1 M Tris-HCl buffer was added to the resultant supernatant (5 μL) of ciliary process and diluted to 90 μL with 0.1 M Tris-HCl buffer. After 60 minutes, the a 90-μL sample was incubated with 10 μL Gly-D-Phe (10 mM) for 20 minutes at 37°C. Preincubation, addition of Gly-D-Phe, and the termination were performed as described earlier. The final concentration of 49C mAb was 30 or 100 μg/mL in a final reaction volume of 0.1 mL. After brief centrifugation, the supernatant was used for the fluorometric assay. All samples were assayed in duplicate from three experiments. 
Contribution of MDP to β-Lactamase Activity in the Ciliary Process.
To certify the contribution of MDP to β-lactamase activity in the ciliary process, we separated MDP from other enzymes by activated gel (Affi-Gel 10; Bio-Rad) conjugated with 49C mAb. The resultant supernatant of the ciliary process obtained, as described earlier, was mixed with octyl-β-d-glucopyranoside (Polyscience, Inc., Warrington, PA) at 120 mM for 1 hour at 4°C to solubilize MDP from the membrane. 3 After centrifugation at 15,000g, the supernatant (2 μL, protein concentration; 35 mg/mL) was mixed with 30 μL of the affinity medium conjugated with 49C mAb or with anti-TNP mAb at 4°C overnight. After the addition of 90 μL buffer and centrifugation at 10,000g, 90 μL of the supernatant was used to react with Gly-D-Phe for the β-lactamase assay. The gel was washed with buffer to remove unbound enzymes, and buffer was added to a final reaction volume of 90 μL for the β-lactamase assay. A 90-μL sample was incubated with 10 μL Gly-D-Phe (10 mM) for 20 minutes at 37°C. Preincubation, the addition of Gly-D-Phe, and termination were performed as just described. After brief centrifugation, the supernatant was used for the fluorometric assay. All samples were assayed in duplicate from three experiments. 
Bioconversion of LTD4 to LTE4 and Evaluation of LTE4
Samples.
Bovine ciliary process or NR was homogenized at 4°C in 0.5 mL of 20 mM phosphate buffer consisting of 150 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2 and then centrifuged at 10,000g for 10 minutes at 4°C. The resultant supernatant was diluted with buffer to 35 mg/mL of concentrated protein. 
Bioconversion of LTD4 to LTE4 in the Ciliary Process or the NR.
The reaction mixture containing 10 μL of the resultant supernatant and 80 μL of the phosphate buffer was preincubated at 37°C for 3 minutes. The reaction was initiated by the addition of 10 μL LTD4 (10,000 pg/mL; Cayman Chemical, Ann Arbor, MI) and after incubation at 37°C (for 0, 2, 5, or 10 minutes) was terminated by cooling at 0°C on ice. All samples were assayed by enzyme-linked immunosorbent assay (ELISA) in duplicate from three experiments. 
Contribution of MDP to Bioconversion of LTD4 to LTE4 in the Ciliary Process.
The resultant supernatant of the ciliary process obtained as described earlier was mixed with octyl-β-d-glucopyranoside at 120 mM for 1 hour at 4°C. After centrifugation at 15,000g, 2 μL of the supernatant (protein concentration; 35 mg/mL) was mixed with 30 μL of the activated gel (Affi-Gel 10) conjugated with 49C mAb or with anti-TNP mAb at 4°C overnight. After the addition of 90 μL of buffer and centrifugation at 10,000g, 90 μL of the supernatant was used for the bioconversion assay of LTD4, as just described. The gel was washed with buffer to remove unbound enzymes, and buffer was added to a final reaction volume of 90 μL. The 90-μL sample was incubated with 10 μL LTD4 (10,000 pg/mL) for 10 minutes at 37°C. Preincubation, the addition of LTD4, and the termination were as described earlier. All samples were assayed by ELISA in triplicate from two experiments. 
Measurement of LTE4 by Leukotriene E4 ELISA.
Ten microliters of the resin suspension of cysteinyl-leukotriene affinity sorbent (Cayman Chemical) was added to each 100-μL sample and stirred for 60 minutes. After a brief centrifugation, the supernatant was removed. The pelleted resin was washed with 1 mL buffer by centrifugation and resuspended in 0.5 mL of cold methanol for 5 minutes with vigorous mixing. The methanol extraction procedure was performed twice. The methanol in the supernatant was collected and evaporated under reduced pressure. 
LTE4 was quantitated using a leukotriene E4 ELISA kit (Cayman Chemical). The ELISA procedure was conducted as instructed by the manufacturer. The evaporated residue was resuspended in 50 μL of the provided enzyme immunoassay buffer, and 50 μL of the provided leukotriene E4-acetylcholinesterase conjugate (LTE4 tracer) and 50 μL of leukotriene E4 antiserum were added. These were incubated for 18 hours at room temperature in the enzyme immunoassay plate. The plate was washed with buffer, and Ellman reagent, which contains the substrate to acetylcholinesterase, was added to each well. The reagent was developed for 90 minutes. Optical densities (ODs) were determined for each well by using a spectrophotometer set to 412 nm. With this assay kit, LTE4 could be measured in quantities ranging from 10 to 1000 pg/mL. The bioconvertibility of LTD4 to LTE4 was expressed as picograms per milliliter. Protein concentrations were measured with detergent-compatible protein assay kit. 
Results
Expression Profiles of 49C Antigen in Bovine Eye and Other Organs
One hybridoma, 49C, which belongs to the IgG1 isotype, was selected by immunohistochemistry. In the bovine eye, 49C antigen was detected in the cytoplasm of the ciliary pigmented epithelial (PE) cells and nonpigmented epithelial (NPE) cells (Fig. 1) . The antigen was highly expressed in the pars plicata and moderately in the pars plana. At the ora serrata, the expression of antigen in the PE and the NPE cells became weaker and was nonexistent in the retina. The weak expression of 49C antigen in the PE and NPE cells of the iris extended approximately 1 mm from the root of the iris. Beyond this point, iridial cells showed no expression. No other intraocular structures demonstrated the expression. The 49C antigen was highly expressed in the renal proximal tubular cells of the kidney and in the acinar cells of the pancreas (data not shown). 
49C Antigen Purified from Bovine Ciliary Process
49C antigen was purified from bovine ciliary process by immunoaffinity chromatography. The SDS-PAGE profile of the affinity-purified antigen showed specific protein bands at 48.0 and 43.7 kDa in reducing conditions (Fig. 2) . The purification was independently performed four times with high reproducibility. 
Partial Amino Acid Sequencing of the Protein Purified from Bovine Ciliary Process
The 48.0- and 43.7-kDa proteins were affinity purified from bovine ciliary process and transblotted onto a PVDF membrane. Partial amino acid sequencing of the 48.0- and 43.7-kDa proteins showed that each sequence of 10 amino acids from the N terminus was identical. The 48.0-kDa protein in the polyacrylamide gel was treated with lysylendopeptidase, and we obtained four polypeptides. A homology search showed that five peptide sequences were highly homologous to sheep MDP 23 (Fig. 3)
β-Lactamase Assay to Measure the Hydrolysis of Gly-D-Phe
β-Lactamase Activity in the Ciliary Process, NR, or RPE.
Activity was evaluated from the fluorometric detection of D-Phe released from the substrate Gly-D-Phe. The amount of D-Phe in the supernatant of the homogenate of the ciliary process increased during the incubation period, and the release rate of D-Phe between 5 and 30 minutes was 3.1 μM/min (Fig. 4) . In contrast, MDP activity in the homogenate of the NR or the RPE was very low, and the release rate of D-Phe was 0.1 in the NR and 0.1 μM/min in the RPE. 
Effect of 49C mAb on β-Lactamase Activity.
The hydrolysis of Gly-D-Phe in the supernatant of the homogenate of ciliary process was slightly inhibited by 49C mAb. Figure 5 shows that concentration of mAb in 100 μg/mL exhibited the maximum inhibitory activity. 
Contribution of MDP to β-Lactamase Activity in the Ciliary Process.
The amount of D-Phe released by MDP bound to 49C mAb-conjugated gel was 31.4 ± 7.9 μM, and that by the fraction unbound to 49C mAb-conjugated gel was −0.1 ± 1.2 μM (Fig. 6) . D-Phe released by the fraction bound to the control mAb (anti-TNP mAb)-conjugated gel was 2.8 ± 1.7 μM, and that by the unbound fraction was 47.0 ± 6.2 μM. Approximately 100% of β-lactamase activity in the supernatant of the homogenate of the ciliary process was catalyzed by the fraction bound to 49C mAb-conjugated gel and little activity by the unbound fraction. 
Bioconversion of LTD4 to LTE4 and Evaluation of LTE4
Bioconversion of LTD4 to LTE4 in the Ciliary Process or the NR.
LTE4 converted from LTD4 in the supernatant of the homogenate of the ciliary process or the NR was quantitated with a leukotriene E4 ELISA kit. The amount of LTE4 in the homogenate of the ciliary process increased proportionally to the incubation time, between 2 and 10 minutes. The increase of LTE4 in the NR was markedly less than that of the ciliary process (Fig. 7) . The conversion rate of LTD4 to LTE4 between 2 and 10 minutes was 38.2 pg/mL per minute in the ciliary process and 8.6 pg/mL per minute in the NR. The conversion rate in the ciliary process was approximately 4.4-fold higher than that in the NR. 
Contribution of MDP to Bioconversion of LTD4 to LTE4 in the Ciliary Process.
LTE4 converted from LTD4 by MDP bound to 49C mAb-conjugated gel was 715 ± 131 pg/mL, and that converted by the fraction unbound to 49C mAb-conjugated gel was 596 ± 248 pg/mL (Fig. 8) . LTE4 converted from LTD4 by the fraction bound to anti-TNP-conjugated gel was 167 ± 76 pg/mL, and that converted by the fraction unbound to anti-TNP conjugated gel was 1229 ± 251 pg/mL. The amount of LTE4 converted by MDP bound to 49C mAb-conjugated gel was 55% of the total converted by the fraction bound and unbound to 49C mAb-conjugated gel, whereas that converted by the unbound fraction was 45%. 
Discussion
In this study, we raised an mAb, 49C, which recognizes an antigen localized in the cytoplasm of bovine ciliary PE and NPE cells. The antigenic molecules purified from bovine ciliary process by immunoaffinity chromatography consisted of two proteins of 43.7 and 48.0 kDa. The sequences of 10 amino acids from the 17th to the 26th residues of the N-terminal region of the purified proteins of 48.0 and 43.7 kDa were identical, and the amino acid sequences of four polypeptides from the 48.0-kDa protein were highly homologous to sheep MDP 23 (Fig. 3) , which suggests that both purified proteins are MDP with the same epitope against 49C mAb. It is unclear how MDP is metabolized in the ciliary epithelium, but it is possible that the 43.7-kDa protein may be the degradation product of the 48.0-kDa protein. Thus, it is suggested that the 49C antigen specifically expressed in the ciliary PE and the NPE cells was MDP. This is the first study to demonstrate the presence of MDP in the ciliary epithelium among mammalian species. 
MDP is the only known mammalian peptidase capable of hydrolyzing peptides containing D-amino acid, 10 and it is believed to metabolize LTD4 to LTE4. 3 We evaluated β-lactamase activity and conversion of LTD4 to LTE4 in the ciliary process. β-Lactamase-induced release of D-Phe from Gly-D-Phe was found to be high in the ciliary process and negligible in the NR or the RPE. The enzyme-mediated conversion of LTD4 to LTE4 was significantly higher in the ciliary process than in the NR. Habib et al. 16 demonstrated that the conversion of LTD4 to LTE4 and the degradation of cys-bis-gly generated from glutathione were catalyzed by at least two alternative pathways (one of which is MDP) in experiments using the MDP-knockout mouse. To clarify the contribution of MDP in the ciliary process, we separated MDP from other enzymes using 49C mAb-conjugated gel. We demonstrated that approximately 100% of β-lactamase activity in the ciliary supernatant was catalyzed by the fraction bound to 49C mAb-conjugated gel (Fig. 6) . Conversely, LTD4 conversion was catalyzed by MDP (55%) and other enzymes (45%; Fig. 8 ). Because MDP binds specifically to 49C mAb, these results suggest that β-lactamase assay is specific to MDP and that the contribution of MDP to LTD4 conversion was about half of the total activity of the LTD4 conversion in the ciliary process. In the present study, we examined whether 49C mAb inhibits β-lactamase activity in the ciliary process (Fig. 5) . The maximum inhibitory activity of 49C mAb (100 μg/mL) was 15% of β-lactamase activity. Because the β-lactamase assay is specific for MDP activity, LTD4 conversion by MDP was expected to be inhibited by 49C mAb as much as β-lactamase activity was. However, LTD4 conversion by other enzymes was not inhibited by 49C mAb. Taking these findings into consideration, the maximum inhibition of LTD4 conversion by 49C mAb may have been less than 8% in the present study, and LTD4 conversion by MDP was approximately 60% of the total activity. Regarding the minor activity by the anti-TNP-bound fraction shown in Figures 6 and 8 , it is suggested that the enzyme activity was catalyzed by enzymes (MDP or others) that were aggregated during incubation with gel and stuck in the spaces among particles of gels. 
In the MDP-knockout mouse study, 16 the mutant mice retained partial ability to convert LTD4 to LTE4, ranging from 80% to 90% of the wild-type levels in the small intestine and liver to 16% in kidney and 40% in lung, heart, and pancreas. In the present study, we showed that LTD4 conversion remained in the supernatant of the homogenate of the ciliary process after removing MDP with 49C mAb-conjugated gel. To clarify the function of MDP in the ocular tissue, determination of the inflammatory response or biochemical analysis of MDP-knockout mouse eyes is needed. 
GGTP and MDP act in concert to metabolize cysteinyl leukotrienes and glutathione in various tissues. 5 6 Although GGTP has been extensively studied in the eye, 17 18 19 MDP has not been studied so far. The immunohistochemical localization and the activity of GGTP was demonstrated in the ciliary epithelial cells. 20 Shichi et al. 20 showed that the NPE cells in porcine ciliary processes were labeled uniformly and intensely by fluorescent anti-rat GGTP antibodies, whereas only the plasma membrane (on the stroma side) was labeled in the PE cells. Furthermore, they reported that the GGTP activity of microsomes from the NPE cells was 23 times higher than that of microsomes from the PE cells. The present findings and those of their studies suggest the existence and activity of GGTP and MDP in the ciliary epithelial cells. It is likely that these two enzymes are involved in the conversion of LTC4, through LTD4, to LTE4 in the eye. The significance of this conversion is that LTE4 is 10 to 100 times less active than LTD4 in inducing inflammation. 24 25 Several experiments have been conducted to investigate whether cysteinyl leukotrienes induce inflammatory reactions in the eye. LTC4 and LTD4, when injected intracamerally, showed no influence on intraocular pressure, aqueous humor protein, or leukocyte concentration. 26 Topical application and intravitreous injection of LTD4 did not promote albumin leakage into the aqueous humor. 27 LTC4 was not detected in eyes graded as having clinically moderate or severe experimental autoimmune uveitis, and GGTP was significantly elevated in serum samples. 28 These studies did not find inflammatory reactions induced by LTC4 or LTD4. It is suggested that they may have measured LTC4 or LTD4 after GGTP and MDP were already converting LTC4, through LTD4, to LTE4 in the ciliary epithelium and that MDP may inactivate LTD4 to prevent aqueous humor inflammation. 
MDP is a widely distributed enzyme and MDP activity has been reported in various tissues. 16 The present immunohistochemical studies showed that 49C antigen was detected in the proximal tubular cells of the kidney and the acinar cells of the pancreas, where the distribution of MDP has been reported. 29 30 The ciliary epithelium in the eye may have the function of metabolizing glutathione, similar to the proximal tubular cells in the kidney. 29 In addition to the activity as a dipeptidase, recent studies have demonstrated nonenzymatic functions of MDP as a lipoprotein-binding protein in the lung 31 and a membrane-stabilizing protein in the zymogen granule membrane of pancreatic acinar cells. 30 It is not known whether any of these reactions is particularly important in the function of ciliary cells. Further investigations of MDP activity are warranted to help in understanding the physiological role of the ciliary epithelial cells in the eye. 
 
Figure 1.
 
The expression of 49C antigen in bovine ciliary cells detected by the indirect immunofluorescence method. The 49C mAb was raised against the ciliary process, and immunohistochemistry was performed. Serial sections of (AC) the anterior pars plicata of the ciliary process, (DF) the posterior pars plicata, and (GI) the ora serrata. Staining was with (A, D, G) hematoxylin and eosin, (B, E, H) 49C mAb, (C, F, I), and anti-TNP mAb (negative control). Fluorescence in the PE and NPE cells was intense in the pars plicata, moderate in the pars plana, and negative in the retina. Separated NR from the posterior part of the retina is visible in the lower left corner of (G). Scale bar, 100 μm.
Figure 1.
 
The expression of 49C antigen in bovine ciliary cells detected by the indirect immunofluorescence method. The 49C mAb was raised against the ciliary process, and immunohistochemistry was performed. Serial sections of (AC) the anterior pars plicata of the ciliary process, (DF) the posterior pars plicata, and (GI) the ora serrata. Staining was with (A, D, G) hematoxylin and eosin, (B, E, H) 49C mAb, (C, F, I), and anti-TNP mAb (negative control). Fluorescence in the PE and NPE cells was intense in the pars plicata, moderate in the pars plana, and negative in the retina. Separated NR from the posterior part of the retina is visible in the lower left corner of (G). Scale bar, 100 μm.
Figure 2.
 
SDS-PAGE analysis of the antigenic molecules purified with 49C mAb-conjugated activated gel. Purification of 49C antigen was performed by immunoaffinity chromatography. The purified 49C antigen was visualized by silver staining after 12% SDS-PAGE in reducing conditions. Lane 1: molecular size markers (97.4, 66.2, 45, and 31 kDa from top to bottom); lane 2: proteins purified with 49C mAb from bovine ciliary process; lane 3: proteins purified with anti-TNP mAb from bovine ciliary process; lane 4: the eluate from the 49C-conjugated activated gel without incubation in the tissue lysate; lane 5: the eluate from the anti-TNP-conjugated activated gel without incubation in the tissue lysate. In lane 2, specific protein bands of 43.7 (open arrowhead) and 48.0 kDa (filled arrowhead) were observed.
Figure 2.
 
SDS-PAGE analysis of the antigenic molecules purified with 49C mAb-conjugated activated gel. Purification of 49C antigen was performed by immunoaffinity chromatography. The purified 49C antigen was visualized by silver staining after 12% SDS-PAGE in reducing conditions. Lane 1: molecular size markers (97.4, 66.2, 45, and 31 kDa from top to bottom); lane 2: proteins purified with 49C mAb from bovine ciliary process; lane 3: proteins purified with anti-TNP mAb from bovine ciliary process; lane 4: the eluate from the 49C-conjugated activated gel without incubation in the tissue lysate; lane 5: the eluate from the anti-TNP-conjugated activated gel without incubation in the tissue lysate. In lane 2, specific protein bands of 43.7 (open arrowhead) and 48.0 kDa (filled arrowhead) were observed.
Figure 3.
 
Partial amino acid sequencing analysis of the proteins purified from bovine ciliary process. The N-terminal amino acid sequences of the 48.0- and the 43.7-kDa protein and internal amino acid sequences of four polypeptides from the 48.0-kDa protein were compared with the homologous sequences deduced from the cDNA of sheep MDP. 23 The amino acid sequences from the N terminus of the 48.0- and 43.7-kDa proteins were identical. Five amino acid sequences determined from the 48.0-kDa protein were highly homologous to sheep MDP. Identical amino acids are indicated by asterisks, similarities by dots. The numbers beside the amino acid sequences of sheep MDP represent the sequence position of the amino acids from the N terminus. The percentage of identical amino acid residues is indicated.
Figure 3.
 
Partial amino acid sequencing analysis of the proteins purified from bovine ciliary process. The N-terminal amino acid sequences of the 48.0- and the 43.7-kDa protein and internal amino acid sequences of four polypeptides from the 48.0-kDa protein were compared with the homologous sequences deduced from the cDNA of sheep MDP. 23 The amino acid sequences from the N terminus of the 48.0- and 43.7-kDa proteins were identical. Five amino acid sequences determined from the 48.0-kDa protein were highly homologous to sheep MDP. Identical amino acids are indicated by asterisks, similarities by dots. The numbers beside the amino acid sequences of sheep MDP represent the sequence position of the amino acids from the N terminus. The percentage of identical amino acid residues is indicated.
Figure 4.
 
β-Lactamase activity in the homogenate of the ciliary process, the NR, or the RPE. The time course of D-Phe release from Gly-D-Phe (final reaction concentration; 1 mM) was measured with the supernatant (protein concentration; 35 mg/mL) from the homogenate of the ciliary process (▪), the NR (•), or the RPE (▴). The rate of release of D-Phe in the supernatant (5 μL) from the homogenate of the ciliary process was constant between 5 and 30 minutes. The amount of D-Phe after 10 minutes’ incubation increased proportionally to the amount of supernatant of the homogenate of the ciliary process added (0, 0.5, 1.5, 5, and 15 μL; data not shown). The released D-Phe also increased proportionally to the concentration of Gly-D-Phe added (0.2, 0.5, 1, 2, and 5 mM, data not shown). No significant enzyme activity was detected in either the supernatant of the NR or the RPE.
Figure 4.
 
β-Lactamase activity in the homogenate of the ciliary process, the NR, or the RPE. The time course of D-Phe release from Gly-D-Phe (final reaction concentration; 1 mM) was measured with the supernatant (protein concentration; 35 mg/mL) from the homogenate of the ciliary process (▪), the NR (•), or the RPE (▴). The rate of release of D-Phe in the supernatant (5 μL) from the homogenate of the ciliary process was constant between 5 and 30 minutes. The amount of D-Phe after 10 minutes’ incubation increased proportionally to the amount of supernatant of the homogenate of the ciliary process added (0, 0.5, 1.5, 5, and 15 μL; data not shown). The released D-Phe also increased proportionally to the concentration of Gly-D-Phe added (0.2, 0.5, 1, 2, and 5 mM, data not shown). No significant enzyme activity was detected in either the supernatant of the NR or the RPE.
Figure 5.
 
The inhibitory effect of 49C mAb on the activity of β-lactamase in the ciliary process. The hydrolysis of Gly-D-Phe in the supernatant of the homogenate of ciliary processes incubated with 30 or 100 μg/mL 49C mAb retained 86% or 85% of the amount of D-Phe release without 49C, respectively.
Figure 5.
 
The inhibitory effect of 49C mAb on the activity of β-lactamase in the ciliary process. The hydrolysis of Gly-D-Phe in the supernatant of the homogenate of ciliary processes incubated with 30 or 100 μg/mL 49C mAb retained 86% or 85% of the amount of D-Phe release without 49C, respectively.
Figure 6.
 
Contribution of MDP to β-lactamase activity in the ciliary process. The supernatant (protein concentration; 35 mg/mL) after solubilizing MDP by octyl-β-d-glucopyranoside was mixed with 49C mAb-conjugated gel to separate MDP from other enzymes. Most of the β-lactamase activity was catalyzed by the fraction bound to 49C mAb-conjugated gel, but there was almost no activity by the control fraction (anti-TNP) mAb-conjugated gel.
Figure 6.
 
Contribution of MDP to β-lactamase activity in the ciliary process. The supernatant (protein concentration; 35 mg/mL) after solubilizing MDP by octyl-β-d-glucopyranoside was mixed with 49C mAb-conjugated gel to separate MDP from other enzymes. Most of the β-lactamase activity was catalyzed by the fraction bound to 49C mAb-conjugated gel, but there was almost no activity by the control fraction (anti-TNP) mAb-conjugated gel.
Figure 7.
 
Bioconversion of LTD4 to LTE4 in the ciliary process and the NR. The time course of LTE4 converted from LTD4 in the supernatant of the homogenate of the ciliary process (▪) or the NR (•) was measured with a leukotriene E4 ELISA kit. After the addition of LTD4 (final reaction concentration; 1000 pg/mL), the rate of LTE4 increase in the ciliary process was constant between 2 and 10 minutes. The conversion rate in the ciliary process was markedly higher than that in the NR. Data at time zero represent the amount of LTE4 in samples chilled in an ice-water bath.
Figure 7.
 
Bioconversion of LTD4 to LTE4 in the ciliary process and the NR. The time course of LTE4 converted from LTD4 in the supernatant of the homogenate of the ciliary process (▪) or the NR (•) was measured with a leukotriene E4 ELISA kit. After the addition of LTD4 (final reaction concentration; 1000 pg/mL), the rate of LTE4 increase in the ciliary process was constant between 2 and 10 minutes. The conversion rate in the ciliary process was markedly higher than that in the NR. Data at time zero represent the amount of LTE4 in samples chilled in an ice-water bath.
Figure 8.
 
Contribution of MDP to the bioconversion of LTD4 to LTE4 in the homogenate of the ciliary process. The supernatant (protein concentration; 35 mg/mL) after solubilizing MDP by octyl-β-d-glucopyranoside was mixed with 49C mAb-conjugated gel to separate MDP from other enzymes. The amount of LTE4 converted by the fraction bound to 49C mAb-conjugated gel was larger than that converted by the fraction unbound to 49C mAb-conjugated gel. In the control experiment with anti-TNP mAb, most of the activity was catalyzed by the fraction unbound to anti-TNP mAb-conjugated gel.
Figure 8.
 
Contribution of MDP to the bioconversion of LTD4 to LTE4 in the homogenate of the ciliary process. The supernatant (protein concentration; 35 mg/mL) after solubilizing MDP by octyl-β-d-glucopyranoside was mixed with 49C mAb-conjugated gel to separate MDP from other enzymes. The amount of LTE4 converted by the fraction bound to 49C mAb-conjugated gel was larger than that converted by the fraction unbound to 49C mAb-conjugated gel. In the control experiment with anti-TNP mAb, most of the activity was catalyzed by the fraction unbound to anti-TNP mAb-conjugated gel.
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Figure 1.
 
The expression of 49C antigen in bovine ciliary cells detected by the indirect immunofluorescence method. The 49C mAb was raised against the ciliary process, and immunohistochemistry was performed. Serial sections of (AC) the anterior pars plicata of the ciliary process, (DF) the posterior pars plicata, and (GI) the ora serrata. Staining was with (A, D, G) hematoxylin and eosin, (B, E, H) 49C mAb, (C, F, I), and anti-TNP mAb (negative control). Fluorescence in the PE and NPE cells was intense in the pars plicata, moderate in the pars plana, and negative in the retina. Separated NR from the posterior part of the retina is visible in the lower left corner of (G). Scale bar, 100 μm.
Figure 1.
 
The expression of 49C antigen in bovine ciliary cells detected by the indirect immunofluorescence method. The 49C mAb was raised against the ciliary process, and immunohistochemistry was performed. Serial sections of (AC) the anterior pars plicata of the ciliary process, (DF) the posterior pars plicata, and (GI) the ora serrata. Staining was with (A, D, G) hematoxylin and eosin, (B, E, H) 49C mAb, (C, F, I), and anti-TNP mAb (negative control). Fluorescence in the PE and NPE cells was intense in the pars plicata, moderate in the pars plana, and negative in the retina. Separated NR from the posterior part of the retina is visible in the lower left corner of (G). Scale bar, 100 μm.
Figure 2.
 
SDS-PAGE analysis of the antigenic molecules purified with 49C mAb-conjugated activated gel. Purification of 49C antigen was performed by immunoaffinity chromatography. The purified 49C antigen was visualized by silver staining after 12% SDS-PAGE in reducing conditions. Lane 1: molecular size markers (97.4, 66.2, 45, and 31 kDa from top to bottom); lane 2: proteins purified with 49C mAb from bovine ciliary process; lane 3: proteins purified with anti-TNP mAb from bovine ciliary process; lane 4: the eluate from the 49C-conjugated activated gel without incubation in the tissue lysate; lane 5: the eluate from the anti-TNP-conjugated activated gel without incubation in the tissue lysate. In lane 2, specific protein bands of 43.7 (open arrowhead) and 48.0 kDa (filled arrowhead) were observed.
Figure 2.
 
SDS-PAGE analysis of the antigenic molecules purified with 49C mAb-conjugated activated gel. Purification of 49C antigen was performed by immunoaffinity chromatography. The purified 49C antigen was visualized by silver staining after 12% SDS-PAGE in reducing conditions. Lane 1: molecular size markers (97.4, 66.2, 45, and 31 kDa from top to bottom); lane 2: proteins purified with 49C mAb from bovine ciliary process; lane 3: proteins purified with anti-TNP mAb from bovine ciliary process; lane 4: the eluate from the 49C-conjugated activated gel without incubation in the tissue lysate; lane 5: the eluate from the anti-TNP-conjugated activated gel without incubation in the tissue lysate. In lane 2, specific protein bands of 43.7 (open arrowhead) and 48.0 kDa (filled arrowhead) were observed.
Figure 3.
 
Partial amino acid sequencing analysis of the proteins purified from bovine ciliary process. The N-terminal amino acid sequences of the 48.0- and the 43.7-kDa protein and internal amino acid sequences of four polypeptides from the 48.0-kDa protein were compared with the homologous sequences deduced from the cDNA of sheep MDP. 23 The amino acid sequences from the N terminus of the 48.0- and 43.7-kDa proteins were identical. Five amino acid sequences determined from the 48.0-kDa protein were highly homologous to sheep MDP. Identical amino acids are indicated by asterisks, similarities by dots. The numbers beside the amino acid sequences of sheep MDP represent the sequence position of the amino acids from the N terminus. The percentage of identical amino acid residues is indicated.
Figure 3.
 
Partial amino acid sequencing analysis of the proteins purified from bovine ciliary process. The N-terminal amino acid sequences of the 48.0- and the 43.7-kDa protein and internal amino acid sequences of four polypeptides from the 48.0-kDa protein were compared with the homologous sequences deduced from the cDNA of sheep MDP. 23 The amino acid sequences from the N terminus of the 48.0- and 43.7-kDa proteins were identical. Five amino acid sequences determined from the 48.0-kDa protein were highly homologous to sheep MDP. Identical amino acids are indicated by asterisks, similarities by dots. The numbers beside the amino acid sequences of sheep MDP represent the sequence position of the amino acids from the N terminus. The percentage of identical amino acid residues is indicated.
Figure 4.
 
β-Lactamase activity in the homogenate of the ciliary process, the NR, or the RPE. The time course of D-Phe release from Gly-D-Phe (final reaction concentration; 1 mM) was measured with the supernatant (protein concentration; 35 mg/mL) from the homogenate of the ciliary process (▪), the NR (•), or the RPE (▴). The rate of release of D-Phe in the supernatant (5 μL) from the homogenate of the ciliary process was constant between 5 and 30 minutes. The amount of D-Phe after 10 minutes’ incubation increased proportionally to the amount of supernatant of the homogenate of the ciliary process added (0, 0.5, 1.5, 5, and 15 μL; data not shown). The released D-Phe also increased proportionally to the concentration of Gly-D-Phe added (0.2, 0.5, 1, 2, and 5 mM, data not shown). No significant enzyme activity was detected in either the supernatant of the NR or the RPE.
Figure 4.
 
β-Lactamase activity in the homogenate of the ciliary process, the NR, or the RPE. The time course of D-Phe release from Gly-D-Phe (final reaction concentration; 1 mM) was measured with the supernatant (protein concentration; 35 mg/mL) from the homogenate of the ciliary process (▪), the NR (•), or the RPE (▴). The rate of release of D-Phe in the supernatant (5 μL) from the homogenate of the ciliary process was constant between 5 and 30 minutes. The amount of D-Phe after 10 minutes’ incubation increased proportionally to the amount of supernatant of the homogenate of the ciliary process added (0, 0.5, 1.5, 5, and 15 μL; data not shown). The released D-Phe also increased proportionally to the concentration of Gly-D-Phe added (0.2, 0.5, 1, 2, and 5 mM, data not shown). No significant enzyme activity was detected in either the supernatant of the NR or the RPE.
Figure 5.
 
The inhibitory effect of 49C mAb on the activity of β-lactamase in the ciliary process. The hydrolysis of Gly-D-Phe in the supernatant of the homogenate of ciliary processes incubated with 30 or 100 μg/mL 49C mAb retained 86% or 85% of the amount of D-Phe release without 49C, respectively.
Figure 5.
 
The inhibitory effect of 49C mAb on the activity of β-lactamase in the ciliary process. The hydrolysis of Gly-D-Phe in the supernatant of the homogenate of ciliary processes incubated with 30 or 100 μg/mL 49C mAb retained 86% or 85% of the amount of D-Phe release without 49C, respectively.
Figure 6.
 
Contribution of MDP to β-lactamase activity in the ciliary process. The supernatant (protein concentration; 35 mg/mL) after solubilizing MDP by octyl-β-d-glucopyranoside was mixed with 49C mAb-conjugated gel to separate MDP from other enzymes. Most of the β-lactamase activity was catalyzed by the fraction bound to 49C mAb-conjugated gel, but there was almost no activity by the control fraction (anti-TNP) mAb-conjugated gel.
Figure 6.
 
Contribution of MDP to β-lactamase activity in the ciliary process. The supernatant (protein concentration; 35 mg/mL) after solubilizing MDP by octyl-β-d-glucopyranoside was mixed with 49C mAb-conjugated gel to separate MDP from other enzymes. Most of the β-lactamase activity was catalyzed by the fraction bound to 49C mAb-conjugated gel, but there was almost no activity by the control fraction (anti-TNP) mAb-conjugated gel.
Figure 7.
 
Bioconversion of LTD4 to LTE4 in the ciliary process and the NR. The time course of LTE4 converted from LTD4 in the supernatant of the homogenate of the ciliary process (▪) or the NR (•) was measured with a leukotriene E4 ELISA kit. After the addition of LTD4 (final reaction concentration; 1000 pg/mL), the rate of LTE4 increase in the ciliary process was constant between 2 and 10 minutes. The conversion rate in the ciliary process was markedly higher than that in the NR. Data at time zero represent the amount of LTE4 in samples chilled in an ice-water bath.
Figure 7.
 
Bioconversion of LTD4 to LTE4 in the ciliary process and the NR. The time course of LTE4 converted from LTD4 in the supernatant of the homogenate of the ciliary process (▪) or the NR (•) was measured with a leukotriene E4 ELISA kit. After the addition of LTD4 (final reaction concentration; 1000 pg/mL), the rate of LTE4 increase in the ciliary process was constant between 2 and 10 minutes. The conversion rate in the ciliary process was markedly higher than that in the NR. Data at time zero represent the amount of LTE4 in samples chilled in an ice-water bath.
Figure 8.
 
Contribution of MDP to the bioconversion of LTD4 to LTE4 in the homogenate of the ciliary process. The supernatant (protein concentration; 35 mg/mL) after solubilizing MDP by octyl-β-d-glucopyranoside was mixed with 49C mAb-conjugated gel to separate MDP from other enzymes. The amount of LTE4 converted by the fraction bound to 49C mAb-conjugated gel was larger than that converted by the fraction unbound to 49C mAb-conjugated gel. In the control experiment with anti-TNP mAb, most of the activity was catalyzed by the fraction unbound to anti-TNP mAb-conjugated gel.
Figure 8.
 
Contribution of MDP to the bioconversion of LTD4 to LTE4 in the homogenate of the ciliary process. The supernatant (protein concentration; 35 mg/mL) after solubilizing MDP by octyl-β-d-glucopyranoside was mixed with 49C mAb-conjugated gel to separate MDP from other enzymes. The amount of LTE4 converted by the fraction bound to 49C mAb-conjugated gel was larger than that converted by the fraction unbound to 49C mAb-conjugated gel. In the control experiment with anti-TNP mAb, most of the activity was catalyzed by the fraction unbound to anti-TNP mAb-conjugated gel.
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