Biol. Chem. Hoppe-Seyler Vol. 371, pp. 325-330, April 1990
Purification and Properties of Dipeptidyl Peptidase IV from Human Urine Toshiyuki CHiKUMA,Tokiko HAMA,Toshiharu NAGATSU, Masayoshi KUMEGAWA and Takeshi KATO Department of Pharmaceutical Analytical Chemistry, Showa College of Pharmaceutical Sciences; Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute ofTechnology; Department of Oral Anatomy, Josai Dental University (Received 17 November 1989)
Summary: Dipeptidyl peptidase IV (DPP-IV) (EC 3.4.14.5) has been purified from normal human urine using immunoaffinity chromatography. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular mass of urinary DPP-IVwas estimated to be 280 kDa by gel filtration. The Km value for the hydrolysis of (Gly-L-Pro)-
4-methylcoumaryl-7-amide tosylate was calculated to be 1.25 ± 0.25 x 10~4M; the pH optimum and pi were 8.7 and 6.5, respectively. These properties were the same as those of renal DPP-IV. Both renal and this urinary DPP-IV were inhibited by diisopropylfluorophosphate and activated by phospholipid. From these results we suppose that urinary DPP-IV may be derived from the kidney rather than from the blood.
Reinigung und Eigenschaften von Dipeptidyl-Peptidase IVaus menschlichem Urin Zusammenfassung: Dipeptidyl-Peptidase IV (DPPIV) (EC 3.4.14.5) wurde mit Hilfe von Immunaffinitätschromatographie aus normalem menschlichem Urin gereinigt. Das gereinigte Enzym erwies sich in der Polyacrylamid-Gelelektrophorese in Gegenwart von Natriumdodecylsulfat als einheitlich. Die Molekularmasse des DPP-IV aus Urin wurde mittels Gelfiltration zu 280 kDa bestimmt. Der #m-Wert für die
Hydrolyse von (Gly-L-Pro)-4-Methylcumaryl-7-amidtosylat war 1.25 ± 0.25 x 10~4M, das pH-Optimum lag bei 8.7 und der p/-Wert bei 6.5. Für DPP-IVaus Niere wurden die gleichen Eigenschaften gefunden. Beide Enzyme wurden durch Diisopropylfluorophosphat gehemmt und durch Phospholipid aktiviert. Aus diesen Ergebnissen schließen wir, daß die DPP-IV im Urin eher aus der Niere stammt als aus dem Blut.
Key words: Dipeptidyl peptidase IV, human urine, immunoaffinity chromatography.
Dipeptidyl peptidase IV (dipeptidyl-aminopeptidase IV, X-prolyl dipeptidyl-aminopeptidase, post proline dipeptidyl peptidase, EC 3.4.14.5) (DPP-IV) was first discovered in rat liver and kidney by Hopsu-Havu and Glenner ll] using a synthetic substrate, glycylproline /3-naphthylamide. This enzyme, purified from porcine and lamb kidney[2~4]; pig small intestinal brush
border membrane[5]; human submaxillary gland[6JJ; pig liver^; porcine pancreas^; and guinea pig intraperitoneal leukocytes^, was shown to hydrolyse yV-terminal X-Pro (X = amino acid) from pepThe physiological significance of this enzyme is not clear yet, but its activity in serum was abnormally
Enzyme: Dipeptidyl peptidase IV, dipeptidyl-peptide hydrolase (EC 3.4.14.5). Abbreviations: DPP-IV, dipeptidyl peptidase IV; Gly-Pro-MCA, (Gly-L-Pro)-4-methylcoumaryl-7-amide tosylate; AMC, 7-amino-4-methyl coumarin; DFP, diisopropylfluorophosphate; pCMB, p-chloromercuribenzoate; DTT, dithiothreitol; NEM, N-ethylmaleimide; SDS, sodium dodecyl sulfate; PBS, sodium phosphate-buffered saline; pNA, 4-nitroanilide.
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high in patients with hepatobiliary disease'11 131 and was decreased in patients with gastric cancer'11131. We also found that the enzyme activity in urine from patients with renal disease is significantly higher than its activity in normal human urine'14'. While the molecular mass of the enzyme in normal urine has been estimated to be about 400 kDa from gel filtration studies'151, there are two types of the enzyme in the urine from patients with renal disease, having molecular masses of 400 kDa and 230 kDa{14]. Therefore investigation of the excretion mechanism of the enzyme in normal urine may help to clarify the process of renal disease. In this paper, we report the purification and various properties of DPP-IVfrom normal human urine using immunoaffinity chromatography. As we have purified the enzyme from human kidney'161, we also compared the urinary enzyme with the renal one.
Materials and Methods Materials Human urine from 13 healthy male volunteers between 22 and 50 years of age was collected. Human kidney was obtained through the department of pathology of Toranomon hospital in Tokyo. (Gly-L-Pro)-4-methylcoumaryl-7-amide tosylate (Gly-Pro-MCA), 7-amino-4-methylcoumarin (AMC), and dipeptide-p-nitroanilides were obtained from the Protein Research Foundation (Osaka, Japan). Diisopropylfluorophosphate (DFP),p-chloromercuribenzoate (pCMB), dithiothreitol (DTT), W-ethylmaleimide (NFM). phosphatidylcholine, phosphatidylinositol, and a-methyl-D-mannoside were purchased from Sigma Chemical Co. (MO, U.S.A.). Sephadex G-200 and concanavalin A-Sepharose were from Pharmacia Fine Chemicals (Uppsala, Sweden). Other materials and their sources were 3,3'-diaminobentidine tetrahydrochloride (Nakarai Kagaku Co., Ltd., Tokyo, Japan), peroxydase-conjugated IgG fraction of antiserum to rabbit IgG (Seikagaku Kogyo Co., Ltd., Tokyo, Japan), complete Freunds adjuvant (Calbiochem. Berg. Corp., CA, U.S.A.), and DEAE-cellulose (Whatman, Kent, England). Enzyme and protein assay DPP-IV activity was determined at 37 °C by the fluorophotometric method of Kato et al.1'71 using Gly-Pro-MCAas substrate.The incubation mixture (100 μΐ) contained 0.5mM Gly-Pro-MCA, 0.06M Glycine/NaOH buffer, pH 8.65, water and enzyme. After the enzyme reaction was stopped by adding l m/of IM acetate buffer, pH 4.2, the fluorescence intensity was read at 460 nm with excitation at 380 nm using a Shimadzu RF-500 spectrofluorophotometer. One unit of enzyme activity was defined as the amount of enzyme catalysing the formation of 1 μητιοί of AMC/min.To study substrate specificity, 1.2mM dipeptide-4-nitroanilides, instead of 0.5mM Gly-Pro-MCA, was used as substrate; and the 4-nitroaniline liberated enzymatically from dipeptide-4-nitroanilide was assayed based on its absorbance at 380 nm. Protein was measured by the Lowry method as modified by Hartree'18' using bovine serum albumin as standard protein. Purification of DPP-IVfrom human urine Fifty liters of human urine were collected at room temperature. All of the following procedures were carried out at 0-4 °C. After the
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enzyme was precipitated from fresh urine with (NH 4 ) 2 SO 4 at 70% saturation, the precipitate was collected by centrifugation at 15000 x g for 20 min, dissolved in 15mM phosphate buffer, pH 7.2, and dialysed extensively against 15mM phosphate buffer, pH 7.2. After centrifugation at 15000 x g for 20 min, the supernatant was applied to a DEAE-cellulose column (2.6 x 15 cm) equilibrated with 15mM phosphate buffer, pH 7.2, and eluted with 1000 ml of a linear gradient of NaCl (0 to 0.3M).The active fractions were combined. For further Chromatographie purification, the enzyme solution was dialysed against a large volume of 15mM phosphate buffer, pH 7.4, containing 0.15M NaCl. The dialysed enzyme solution was then applied on a column of concanavalin A-Sepharose (1.6 x 7.3 cm), previously equilibrated with 15mM phosphate buffer, pH 7.4, containing 0.15M NaCl. After the column was extensively washed with the same buffer, the enzyme was eluted with the same buffer containing 5% a-methyl-D-mannoside.The pooled eluate containing the enzyme was dialysed against 50mMTris/HCl buffer, pH 8.0, containing 0.15M NaCl and applied to an anti DPP-IV Sepharose 4B column (1.6 x 8.3 cm) equilibrated with the same buffer as described previously'16'. After washing the column with the equilibrating buffer, the adsorbed enzyme was eluted with 2mMTris/HCl buffer, pH 8.O.The purified sample was stored at - 80 °C until use. Determination of molecular mass The molecular mass of DPP-IV purified from human urine was determined by gel filtration on Sephadex G-200 by the method of Whitaker1191. Ferritin (480 kDa), catalase (240 kDa), bovine serum albumin (dimer 130 kDa, monomer 67 kDa), and chymotrypsinogen (25 kDa) were used as standards. Sodium dodecyl sulfate polyacrylamide gel electrophoresis Analytical gel electrophoresis was done in the presence of sodium dodecyl sulfate (SDS) as described by Laemmli120'. Protein was detected by staining with silver'21'. Isoelectric focusing The method described by Mey et al.'22' was adapted using LKB 2117 multiphor system. Fifteen μΐ of enzyme solution was applied on to a LKB ampholine PAG plate, pH 3.5-9.5.The cathodic electrode solution was IM NaOH; the anodic, IM H3PO4. After electrofocusing was carried out at 24 mA for 4 h at 4 °C, the ampholine PAG plates were cut every 5 mm. Each gel section was put into a tube containing 100 μΐ of distilled water and stored overnight. And then the activity and pH in each tube were measured to determine the isoelectric point of the enzyme. Effects of phospholipids on DPP-IV activity Phospholipids (phosphatidylcholine and phosphatidylinositol) were diluted with 0.05M sodium phosphate buffer, pH 8.0, to appropriate concentration. The mixtures containing phospholipids and enzyme were incubated at 37 °C for 10 min and sonicated for 5 min in a Branson 220 (45 KHz) to make liposomes. In this experiment, enzymes purified from both human urine and kidney were used to make them and these liposomes were assayed for enzyme activity as described under enzyme and protein assays. Effects of chemical reagents on enzymatic activity Enzyme solution was preincubated in the absence or presence of various chemicals (0.1 to ImM) in 0.06M Glycine/NaOH buffer, pH 8.65, for 10 min at 37 °C. Immediately thereafter, 0.5mM Gly-Pro-MCA (final concentration) was added to the reaction mixture and incubated at 37 ° C for 30-60 min. After stopping the reaction (1 ml of IM acetate buffer, pH 4.2), the fluorescence intensity was read as described above.
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Purification of Dipeptidyl Peptidase IVfrom Human Urine
HI 50
Fig. 1. DEAE-cellulose column chromatography. The column (2.6 x 15 cm) was equilibrated with 15mM phosphate buffer, pH 7.2. The enzyme was eluted with a linear gradient of NaCl (0 to 0.3M), and fractions of 9 ml each were collected. DPP-IV activity (· ·) and absorbance at 280 nm (O—O) were measured. Fraction II was used for further purification.
500
Immunohistochemistry of DPP-IV in human kidney Blocks of normal human kidney ( I x l x l cm) were frozen at - 80 °C until use. Antiserum was prepared as described previously1 161. Sections were cut 20 π\μ thick with a cryostat and processed for immunocytochemical staining using the indirect peroxidase method.The sections were washed in O.OlM sodium phosphate-buffered saline (PBS) and incubated for 10 min in PBS containing either a 50-fold dilution in PBS of anti-human kidney DPP-IV serum or a 10-fold diluted preimmune serum from rabbits. After the first incubation, the sections were washed with PBS and then incubated for 10 min at room temperature with goat anti-rabbit IgG serum conjugated to horseradish peroxidase. Finally sections were washed in PBS, stained in Karnovsky solution'231, mounted in glycerol glass slides and viewed by a photomicroscope.
Results Purification of DPP-IV from human urine DPP-I Vwas successively purified from normal human urine. The enzyme was precipitated from urine at 70% (NH4)2SO4 saturation with a recovery of 80%. The precipitate was suspended in 15mM phosphate buffer, pH 72 and applied to a DEAE-cellulose column using a linear gradient of NaCl (0-0.3M) for elu-
tion (Fig. 1). Enzyme activity was eluted at 0.04M (fraction I) and O.lM NaCl (fraction II). Fraction II was dialysed and thereafter applied on a concanavalin A-Sepharose column to further purify the enzyme, because Fukasawa et al. previously reported that DPP-IV from pig kidney contained 18.3% of carbohydrate consisting of mannose, galactose etc.[3]. By this chromatography the enzyme activity was purified about 3-fold. After dialysing with a large amount of 50mM Tris/HCl buffer, pH 8.0, this enzyme was further purified by anti DPP-IVSepharose 4B column chromatography. Unadsorbed fractions showed no activity and human urinary DPP-IV was eluted with high yield by reducing the ionic strength with 2mM Tris/HCl buffer, pH 8.O. Total purification was achieved about 1500-fold with a recovery of 2.9% from the ammonium sulfate fractionation step (Table 1). Specific activity of the purified enzyme was 48.5 μιηο1/(ππη χ mg protein) using Gly-Pro-MCA at pH 8.65. This final purified sample showed to be homogeneous by polyacrylamide gel electrophoresis using silver stain.
Table 1. Purification of DPP-IVfrom human urine. Stage
Protein [mg]
Total activity [U]
Specific activity [U/mg]
Yield [%]
Purification (-fold)
(NH4)2SO4(0-70%) DEAE-cellulose Concanavalin A-Sepharose Anti-DPP-IVSepharose
2046 286.8 41.3 0.04
67.7 15.3 6.16 1.94
0.033 0.053 0.150 48.5
100 23 9.1 2.9
1 1.6 4.5 1465
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Effect ofpH on the activity of DPP-IVpurified from human urine When Gly-Pro-MCA was used as substrate, the optimum pH was about at 8.7 (Fig. 2), as described previously|16]. When Glycine/NaOH buffer was used, the enzyme activity was higher than when Tris/maleate buffer was employed. These results were similar to those obtained from DPP-IV purified from human kidney(16]. Effects of phospholipids on D P P-IV activity We examined the effects of phospholipids on DPP-IV activity at several concentrations (final concentrations ranging from 0.001% to 0.5%) (Table 2). Control tubes contained only 0.05M sodium phosphate buffer instead of phospholipid solution. As shown in Table 2, the activity of DPP-IV purified from both human urine and kidney associated with phospholipids showed an about 50% increase compared with the control. Phosphatidylcholine at a concentration of 0.01% showed the highest increase of DPP-IV activity in both human urine and kidney. On the other hand, phosphatidylinositol at concentrations ranging from 0.001% to 0.1% showed the same activity in human urine and tends to increase the activity in human kidney, respectively. When we examined the effect of Ca2® in these studies, no change was noted (data not shown). 0.06M Glycine/NaOH buffer O.OSMTris/Maleate buffer Ο—Ο
0.8
0.6
Hh
8
pH
10
Fig. 2. The effect of pH on DPP-IVactivity. Tris/maleate buffer, pH 7.0-9.0 (O—O) and Glycine/NaOH buffer, pH 8.0-10.0 (·—·) were used.
Table 2. Effects of phospholipids on activity of DPP-IV purified from human urine and kidney. Phospholipids
3
Final concentration
DPP-IVactivity [% of control]
[%]
Human urine3
Human kidneyb
Phosphatidylcholine
0.5 0.1 0.05 0.01 0.005 0.001
108 ± 14 138 ± 22 148 ± 25 175 ± 19 150 ± 18 159 ± 25
104 ± 10 138 ± 15 145 ± 17 150 ± 17 124 ± 13 125 ± 9
Phosphatidylinositol
0.1 0.05 0.01 0.005 0.001
146 ±22 144 ±24 141 ± 14 144 ±25 140 ± 17
161 ± 19 166 ± 20 148 ± 16 142 ± 16 137 ± 14
Control (100%) = 48.5 ,umol/(min χ mg protein). Control (100%) = 54.8/imol/(min x mg protein).
Effects of chemical reagents on DPP-IVactivity We examined the effects of various reagents on DPPIV activity. DPP-IV activity was not affected by several cations (Ca2®, Mg2®, Mn2®, and K®), EDTA, and thiol reagents (pCMB, DTT, and NEM). Furthermore, DPP-IVactivity was not inhibited by puromycin or bacitracin. But DFP, a serine protease inhibitor, inhibited the enzyme activity completely at O.lmM, indicating that this enzyme is one of the serine enzymes. Properties of D P P-IV purified from human urine Some enzymatic and physicochemical properties of the enzyme were studied. The isoelectric point of the enzyme was 6.5, and the Km value using Gly-Pro-MCA as substrate was 1.25 ± 0.25 x 10~4M. The molecular mass of the enzyme was estimated to be about 280000 Da by Sephadex G-200 gel filtration using the method of Whitaker[19]. On the other hand, this enzyme fraction gave a single band on polyacrylamide gel electrophoresis in the presence of SDS, and a molecular mass of about 110-130 kDa was estimated. These results indicate that DPP-IV in human urine is composed of a single identical subunit corresponding to the materials of 110-130 kDa. Therefore, DPP-IV in human urine is dimer of an identical subunit. Substrate specificity of urinary DPP-IV Seven kinds of dipeptide-4-nitroanilide (pNA) (Gly-Pro-, Lys-Pro-, Arg-Pro-, Ala-Ala-, Ala-Gly-, GlyBrought to you by | Purdue University Libraries Authenticated Download Date | 6/10/15 5:04 PM
Vol. 371 (1990)
Purification of Dipeptidyl-Peptidase IVfrom Human Urine
Leu-, and Gly-Hyp-pNA) were examined as substrates. The urinary DPP-IV was specific for derivatives with a proline residue as the second amino acid from the N-terminus, as was found with DPP-IV of other tissues13'5'7'10·16'241. The hydrolysis of Lys-Pro-, and Arg-Pro-pNA by the enzyme were 136% and 95% of that of Gly-Pro-pNA, respectively. On the other hand, other substrates Ala-GIy-, Gly-Leu-, and Gly-Hyp-pNA showed no detectable activity. The hydrolysis of Ala-Ala-pNA was 8% of that of Gly-Pro-pNA. Immunohisto chemistry of DPP-IV in human kidney Localization of DPP-IV in human kidney was investigated by using an indirect peroxidase staining method using anti-human renal DPP-I Vantibodies coupled to peroxidase. The region of DPP-IV localization was stained dark brown, using preimmune rabbit serum as a control. No specific staining was seen. On the other hand, when anti-DPP-IV was used, renal tubules in the human kidney slices were stained specifically indicating that DPP-IV exists mostly in renal tubules, and the glomerular region was also slightly stained suggesting that DPP-IV may also exist in renal glomeruli (data not shown).
Discussion Although the physiological role of DPP-I Vis not yet clear, one function of the enzyme may be the cleavage of N-terminal glycylproline from the peptides derived from collagen. Glycylproline is hydrolysed to glycine and proline by imidopeptidase^. In the present study, DPP-IV from human urine was homogeneously purified for the first time and its properties were investigated. Immunoaffinity chromatography of anti DPP-IVSepharose 4B was very effective for purifying the enzyme. By DEAE-cellulose chromatography, two active fractions were obtained (Fig. 1). In the preliminary experiments, fraction II was adsorbed to the anti DPP-IV Sepharose 4B column. Therefore we used fraction II for further purification. As there is a report that pig kidney DPP-IV exists in renal brush border membranes[2], we assume that the enzyme activity is affected by phospholipids. As shown in Table 2, DPP-IV activity from both human urine and kidney sources was enhanced by phospholipids. Although the mechanisms of enhancement are not clear, DPP-IV might be immobilized on the phospholipid membranes similar to those of renal brush border.
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DPP-IV has been reported to be a serine enzyme but to be neither a metal nor a sulfhydryl enzyme^. DPPIV purified from human urine was also completely inhibited by DFP but was affected by neither any sulfhydryl reagents nor cations. These results indicate that DPP-IV from human urine is also a serine enzyme but not a sulfhydryl enzyme. DPP-IVpurified from the human urine rapidly hydrolysed 4-nitroanilides of Lys-Pro, Gly-Pro and Arg-Pro, but also very slowly hydrolysed the 4-nitroanilide of Ala-Ala. These results confirmed our previous findings on the substrate specificity of DPP-IV, namely that the enzyme catalyses the hydrolysis of amide bonds of peptides with a proline and alanine residues in the penultimate position17·16·24'251. To clarify the origin of urinary DPP-IV, we compared the properties with those of DPP-IV purified from human kidney. Except for the high molecular mass found in native urine, both enzymes have the same properties; they are inhibited by DFP, enhanced by phospholipids, and have the same p/ and optimum pH of 6.5 or 8.7, respectively. Substrate specificity is also similar to both enzymes. These properties of urinary DPP-IV were also nearly identical with those of DPP-IV purified from other human tissues, namely submaxillary gland[?1, placenta[26], and lymphocytes^. From the immunohistochemial study, this enzyme is mostly found in renal tubule cells, but it is probable that this enzyme also exists, to a lesser degree, in renal glomeruli. As mentioned above, urinary DPP-I Vis very similar to renal DPP-IV, and the former enzyme may come from both renal tubuli and glomeruli. By Sephadex G-200 gel filtration, urinary DPP-IV was eluted at the position corresponding to material with an approximate molecular mass of 280 kDa, a value considerably lower than that of 400 kDa, previously reported for the normal human urine enzyme[15]. We suppose that this discrepancy may be derived from the following different conditions. In the previous study, urine was directly subjected to column chromatography on Sephadex G-200 for determination of the molecular mass, but in this study we used any ionic treatment such as (NH4)2SO4 precipitation and DEAE-cellulose column chromatography for purification of the enzyme. Therefore there is a possibility that DPP-IV in urine is present as a polymer or is conjugated with some other macromolecules and becomes disassociated during the purification procedures. Further investigations on the molecular mass of urinary DPPIVis needed. Brought to you by | Purdue University Libraries Authenticated Download Date | 6/10/15 5:04 PM
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In the present experiments, we found another type of Gly-Pro-MCA-hydrolysing enzyme on DEAE-cellulose column chromatography (fraction I).The isolation of fraction I enzyme is currently under investigation.
References 1
Hopsu-Havu, V. K. & Glenner, G. G. (1966) Histochemie 7, 197-201. Kenny, A.J., Booth, A.G., George, S.G., Ingram, J.,Kershaw, D., Wood, E.J. & Young, A.R. (1976) Biochem. J. 155, 169-182. 3 Fukasawa, K.M., Fukasawa, K. & Harada, M. (1978) Biochim. Biophys. Acta 535, 161-166. 4 Yoshimoto,T. &Walter, R. (1977) Biochim. Biophys. Acta 485, 391-401. 5 Svensson, B., Danielsen, M., Staun, M., Jeppesen, L., Noren, O. & Sjöström, H. (1978) Eur. J. Biochem. 90, 489-498. 6 Oya, H., Nagatsu, I. & Nagatsu, T. (1972) Biochim. Biophys. Acta 258, 591-599. 7 Kojima, K., Hama,T., Kato,T. & Nagatsu, T. (1980) /. Chromatogr. 189, 233-240. 8 Fukasawa, K.M., Fukasawa, K., Hiraoka, B.Y. & Harada, M. (1981) Biochim. Biophys. Acta 657,179-189. 9 Yoshimoto,T., Kita,T., Ichinose, M. &Tsuru, D. (1982) J. Biochem. 92, 275-282. 10 Kudo, M., Nakamura,T. & Koyama, J. (1985)7. Biochem. 97,1211-1218. 2
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11 Nagatsu, L, Nagatsu,T. &Yamamoto,T. (1968) Experientia 24, 347-348. 12 Hino, M., Fuyamada, H., Nagatsu,T., Kurokawa, S. & Okuyama, S. (1976) Clin. Chim. Acta 67,103-105. 13 Hino, M., Fuyamada, H., Hayakawa, T., Nagatsu, T., Oya, H., Nakagawa, Y., Takemoto, T. & Sakakibara, S. (1976) Clin. Chem. 22, 1256-1261. 14 Hama, T., Nagatsu, T., Kobayashi, S., Azuma, S., MiyachuT., Kumagai, Y. & Kato,T. (1981) Clin. Chim. Acta 113, 217-221. 15 Kato,T.. Hama,T., Kojima, K., Nagatsu,T. & Sakakibara, S. (1978) Clin. Chem. 24, 1163-1166. 16 Hama,T., Okada, M., Kojima, K., Kato,T., Matsuyama, M. & Nagatsu,T. (1982) Mol. Cell. Biochem. 43, 35-42. 17 Kato,T., Nagatsu,T., Kimura,T. & Sakakibara, S. (1978) Biochem. Med. 19,351-359. 18 Hartree, E.F. (1972) Anal. Biochem. 48, 422-427. 19 Whitaker, J.R. (1963) Anal. Chem. 35,1950-1953. 20 Laemmli, U.K. (1970) Nature (London) 227, 680-685. 21 Oakley, B.R., Kirsch, D.R. & Morris, N.R. (1980) Anal. Biochem. 105,361-363. 22 Mey, J.D., Vandesande, F. & Dierickx, K. (1974) Cell Tiss. Res. 153,531-543. 23 Graham, R.C. & Karnovsky, M.J. (1966) J. Histochem. Cytochem. 14, 291-302. 24 Nagatsu, T., Hino, M., Fuyamada, H., Hayakawa, T., Sakakibara, S., Nakagawa,Y. &Takemoto,T. (1976)Anal. Biochem. 74, 466-476. 25 Kato,T., Nagatsu,T., Kimura,T. & Sakakibara, S. (1978) Experientia 34, 319-320. 26 Püschel, G., Mentlein, R. & Heymann, E. (1982) Eur. J. Biochem. 126, 359-365. 27 Mentlein, R., Rix, H., Feller, A.C. & Heymann, E. (1986) Biomed. Biochim. Acta 45, 567-574.
Toshiyuki Chikuma, Ph.D.*, Department of Pharmacheutical Analytical Chemistry, Showa College of Pharmaceutical Sciences, 5-l-8Tsurumaki, Setagaya-Ku,Tokyo 154; Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama 227; Department of Oral Anatomy, Josai Dental University, Sakado 350-02, Japan. * To whom correspondence should be sent.
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Purification and Properties of Dipeptidyl Peptidase IV from Human Urine Toshiyuki CHiKUMA,Tokiko HAMA,Toshiharu NAGATSU, Masayoshi KUMEGAWA and Takeshi KATO Department of Pharmaceutical Analytical Chemistry, Showa College of Pharmaceutical Sciences; Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute ofTechnology; Department of Oral Anatomy, Josai Dental University (Received 17 November 1989)
Summary: Dipeptidyl peptidase IV (DPP-IV) (EC 3.4.14.5) has been purified from normal human urine using immunoaffinity chromatography. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular mass of urinary DPP-IVwas estimated to be 280 kDa by gel filtration. The Km value for the hydrolysis of (Gly-L-Pro)-
4-methylcoumaryl-7-amide tosylate was calculated to be 1.25 ± 0.25 x 10~4M; the pH optimum and pi were 8.7 and 6.5, respectively. These properties were the same as those of renal DPP-IV. Both renal and this urinary DPP-IV were inhibited by diisopropylfluorophosphate and activated by phospholipid. From these results we suppose that urinary DPP-IV may be derived from the kidney rather than from the blood.
Reinigung und Eigenschaften von Dipeptidyl-Peptidase IVaus menschlichem Urin Zusammenfassung: Dipeptidyl-Peptidase IV (DPPIV) (EC 3.4.14.5) wurde mit Hilfe von Immunaffinitätschromatographie aus normalem menschlichem Urin gereinigt. Das gereinigte Enzym erwies sich in der Polyacrylamid-Gelelektrophorese in Gegenwart von Natriumdodecylsulfat als einheitlich. Die Molekularmasse des DPP-IV aus Urin wurde mittels Gelfiltration zu 280 kDa bestimmt. Der #m-Wert für die
Hydrolyse von (Gly-L-Pro)-4-Methylcumaryl-7-amidtosylat war 1.25 ± 0.25 x 10~4M, das pH-Optimum lag bei 8.7 und der p/-Wert bei 6.5. Für DPP-IVaus Niere wurden die gleichen Eigenschaften gefunden. Beide Enzyme wurden durch Diisopropylfluorophosphat gehemmt und durch Phospholipid aktiviert. Aus diesen Ergebnissen schließen wir, daß die DPP-IV im Urin eher aus der Niere stammt als aus dem Blut.
Key words: Dipeptidyl peptidase IV, human urine, immunoaffinity chromatography.
Dipeptidyl peptidase IV (dipeptidyl-aminopeptidase IV, X-prolyl dipeptidyl-aminopeptidase, post proline dipeptidyl peptidase, EC 3.4.14.5) (DPP-IV) was first discovered in rat liver and kidney by Hopsu-Havu and Glenner ll] using a synthetic substrate, glycylproline /3-naphthylamide. This enzyme, purified from porcine and lamb kidney[2~4]; pig small intestinal brush
border membrane[5]; human submaxillary gland[6JJ; pig liver^; porcine pancreas^; and guinea pig intraperitoneal leukocytes^, was shown to hydrolyse yV-terminal X-Pro (X = amino acid) from pepThe physiological significance of this enzyme is not clear yet, but its activity in serum was abnormally
Enzyme: Dipeptidyl peptidase IV, dipeptidyl-peptide hydrolase (EC 3.4.14.5). Abbreviations: DPP-IV, dipeptidyl peptidase IV; Gly-Pro-MCA, (Gly-L-Pro)-4-methylcoumaryl-7-amide tosylate; AMC, 7-amino-4-methyl coumarin; DFP, diisopropylfluorophosphate; pCMB, p-chloromercuribenzoate; DTT, dithiothreitol; NEM, N-ethylmaleimide; SDS, sodium dodecyl sulfate; PBS, sodium phosphate-buffered saline; pNA, 4-nitroanilide.
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high in patients with hepatobiliary disease'11 131 and was decreased in patients with gastric cancer'11131. We also found that the enzyme activity in urine from patients with renal disease is significantly higher than its activity in normal human urine'14'. While the molecular mass of the enzyme in normal urine has been estimated to be about 400 kDa from gel filtration studies'151, there are two types of the enzyme in the urine from patients with renal disease, having molecular masses of 400 kDa and 230 kDa{14]. Therefore investigation of the excretion mechanism of the enzyme in normal urine may help to clarify the process of renal disease. In this paper, we report the purification and various properties of DPP-IVfrom normal human urine using immunoaffinity chromatography. As we have purified the enzyme from human kidney'161, we also compared the urinary enzyme with the renal one.
Materials and Methods Materials Human urine from 13 healthy male volunteers between 22 and 50 years of age was collected. Human kidney was obtained through the department of pathology of Toranomon hospital in Tokyo. (Gly-L-Pro)-4-methylcoumaryl-7-amide tosylate (Gly-Pro-MCA), 7-amino-4-methylcoumarin (AMC), and dipeptide-p-nitroanilides were obtained from the Protein Research Foundation (Osaka, Japan). Diisopropylfluorophosphate (DFP),p-chloromercuribenzoate (pCMB), dithiothreitol (DTT), W-ethylmaleimide (NFM). phosphatidylcholine, phosphatidylinositol, and a-methyl-D-mannoside were purchased from Sigma Chemical Co. (MO, U.S.A.). Sephadex G-200 and concanavalin A-Sepharose were from Pharmacia Fine Chemicals (Uppsala, Sweden). Other materials and their sources were 3,3'-diaminobentidine tetrahydrochloride (Nakarai Kagaku Co., Ltd., Tokyo, Japan), peroxydase-conjugated IgG fraction of antiserum to rabbit IgG (Seikagaku Kogyo Co., Ltd., Tokyo, Japan), complete Freunds adjuvant (Calbiochem. Berg. Corp., CA, U.S.A.), and DEAE-cellulose (Whatman, Kent, England). Enzyme and protein assay DPP-IV activity was determined at 37 °C by the fluorophotometric method of Kato et al.1'71 using Gly-Pro-MCAas substrate.The incubation mixture (100 μΐ) contained 0.5mM Gly-Pro-MCA, 0.06M Glycine/NaOH buffer, pH 8.65, water and enzyme. After the enzyme reaction was stopped by adding l m/of IM acetate buffer, pH 4.2, the fluorescence intensity was read at 460 nm with excitation at 380 nm using a Shimadzu RF-500 spectrofluorophotometer. One unit of enzyme activity was defined as the amount of enzyme catalysing the formation of 1 μητιοί of AMC/min.To study substrate specificity, 1.2mM dipeptide-4-nitroanilides, instead of 0.5mM Gly-Pro-MCA, was used as substrate; and the 4-nitroaniline liberated enzymatically from dipeptide-4-nitroanilide was assayed based on its absorbance at 380 nm. Protein was measured by the Lowry method as modified by Hartree'18' using bovine serum albumin as standard protein. Purification of DPP-IVfrom human urine Fifty liters of human urine were collected at room temperature. All of the following procedures were carried out at 0-4 °C. After the
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enzyme was precipitated from fresh urine with (NH 4 ) 2 SO 4 at 70% saturation, the precipitate was collected by centrifugation at 15000 x g for 20 min, dissolved in 15mM phosphate buffer, pH 7.2, and dialysed extensively against 15mM phosphate buffer, pH 7.2. After centrifugation at 15000 x g for 20 min, the supernatant was applied to a DEAE-cellulose column (2.6 x 15 cm) equilibrated with 15mM phosphate buffer, pH 7.2, and eluted with 1000 ml of a linear gradient of NaCl (0 to 0.3M).The active fractions were combined. For further Chromatographie purification, the enzyme solution was dialysed against a large volume of 15mM phosphate buffer, pH 7.4, containing 0.15M NaCl. The dialysed enzyme solution was then applied on a column of concanavalin A-Sepharose (1.6 x 7.3 cm), previously equilibrated with 15mM phosphate buffer, pH 7.4, containing 0.15M NaCl. After the column was extensively washed with the same buffer, the enzyme was eluted with the same buffer containing 5% a-methyl-D-mannoside.The pooled eluate containing the enzyme was dialysed against 50mMTris/HCl buffer, pH 8.0, containing 0.15M NaCl and applied to an anti DPP-IV Sepharose 4B column (1.6 x 8.3 cm) equilibrated with the same buffer as described previously'16'. After washing the column with the equilibrating buffer, the adsorbed enzyme was eluted with 2mMTris/HCl buffer, pH 8.O.The purified sample was stored at - 80 °C until use. Determination of molecular mass The molecular mass of DPP-IV purified from human urine was determined by gel filtration on Sephadex G-200 by the method of Whitaker1191. Ferritin (480 kDa), catalase (240 kDa), bovine serum albumin (dimer 130 kDa, monomer 67 kDa), and chymotrypsinogen (25 kDa) were used as standards. Sodium dodecyl sulfate polyacrylamide gel electrophoresis Analytical gel electrophoresis was done in the presence of sodium dodecyl sulfate (SDS) as described by Laemmli120'. Protein was detected by staining with silver'21'. Isoelectric focusing The method described by Mey et al.'22' was adapted using LKB 2117 multiphor system. Fifteen μΐ of enzyme solution was applied on to a LKB ampholine PAG plate, pH 3.5-9.5.The cathodic electrode solution was IM NaOH; the anodic, IM H3PO4. After electrofocusing was carried out at 24 mA for 4 h at 4 °C, the ampholine PAG plates were cut every 5 mm. Each gel section was put into a tube containing 100 μΐ of distilled water and stored overnight. And then the activity and pH in each tube were measured to determine the isoelectric point of the enzyme. Effects of phospholipids on DPP-IV activity Phospholipids (phosphatidylcholine and phosphatidylinositol) were diluted with 0.05M sodium phosphate buffer, pH 8.0, to appropriate concentration. The mixtures containing phospholipids and enzyme were incubated at 37 °C for 10 min and sonicated for 5 min in a Branson 220 (45 KHz) to make liposomes. In this experiment, enzymes purified from both human urine and kidney were used to make them and these liposomes were assayed for enzyme activity as described under enzyme and protein assays. Effects of chemical reagents on enzymatic activity Enzyme solution was preincubated in the absence or presence of various chemicals (0.1 to ImM) in 0.06M Glycine/NaOH buffer, pH 8.65, for 10 min at 37 °C. Immediately thereafter, 0.5mM Gly-Pro-MCA (final concentration) was added to the reaction mixture and incubated at 37 ° C for 30-60 min. After stopping the reaction (1 ml of IM acetate buffer, pH 4.2), the fluorescence intensity was read as described above.
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Purification of Dipeptidyl Peptidase IVfrom Human Urine
HI 50
Fig. 1. DEAE-cellulose column chromatography. The column (2.6 x 15 cm) was equilibrated with 15mM phosphate buffer, pH 7.2. The enzyme was eluted with a linear gradient of NaCl (0 to 0.3M), and fractions of 9 ml each were collected. DPP-IV activity (· ·) and absorbance at 280 nm (O—O) were measured. Fraction II was used for further purification.
500
Immunohistochemistry of DPP-IV in human kidney Blocks of normal human kidney ( I x l x l cm) were frozen at - 80 °C until use. Antiserum was prepared as described previously1 161. Sections were cut 20 π\μ thick with a cryostat and processed for immunocytochemical staining using the indirect peroxidase method.The sections were washed in O.OlM sodium phosphate-buffered saline (PBS) and incubated for 10 min in PBS containing either a 50-fold dilution in PBS of anti-human kidney DPP-IV serum or a 10-fold diluted preimmune serum from rabbits. After the first incubation, the sections were washed with PBS and then incubated for 10 min at room temperature with goat anti-rabbit IgG serum conjugated to horseradish peroxidase. Finally sections were washed in PBS, stained in Karnovsky solution'231, mounted in glycerol glass slides and viewed by a photomicroscope.
Results Purification of DPP-IV from human urine DPP-I Vwas successively purified from normal human urine. The enzyme was precipitated from urine at 70% (NH4)2SO4 saturation with a recovery of 80%. The precipitate was suspended in 15mM phosphate buffer, pH 72 and applied to a DEAE-cellulose column using a linear gradient of NaCl (0-0.3M) for elu-
tion (Fig. 1). Enzyme activity was eluted at 0.04M (fraction I) and O.lM NaCl (fraction II). Fraction II was dialysed and thereafter applied on a concanavalin A-Sepharose column to further purify the enzyme, because Fukasawa et al. previously reported that DPP-IV from pig kidney contained 18.3% of carbohydrate consisting of mannose, galactose etc.[3]. By this chromatography the enzyme activity was purified about 3-fold. After dialysing with a large amount of 50mM Tris/HCl buffer, pH 8.0, this enzyme was further purified by anti DPP-IVSepharose 4B column chromatography. Unadsorbed fractions showed no activity and human urinary DPP-IV was eluted with high yield by reducing the ionic strength with 2mM Tris/HCl buffer, pH 8.O. Total purification was achieved about 1500-fold with a recovery of 2.9% from the ammonium sulfate fractionation step (Table 1). Specific activity of the purified enzyme was 48.5 μιηο1/(ππη χ mg protein) using Gly-Pro-MCA at pH 8.65. This final purified sample showed to be homogeneous by polyacrylamide gel electrophoresis using silver stain.
Table 1. Purification of DPP-IVfrom human urine. Stage
Protein [mg]
Total activity [U]
Specific activity [U/mg]
Yield [%]
Purification (-fold)
(NH4)2SO4(0-70%) DEAE-cellulose Concanavalin A-Sepharose Anti-DPP-IVSepharose
2046 286.8 41.3 0.04
67.7 15.3 6.16 1.94
0.033 0.053 0.150 48.5
100 23 9.1 2.9
1 1.6 4.5 1465
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T. Chikuma,T. Hama,T. Nagatsu, M. Kumegawa andT. Kato
Effect ofpH on the activity of DPP-IVpurified from human urine When Gly-Pro-MCA was used as substrate, the optimum pH was about at 8.7 (Fig. 2), as described previously|16]. When Glycine/NaOH buffer was used, the enzyme activity was higher than when Tris/maleate buffer was employed. These results were similar to those obtained from DPP-IV purified from human kidney(16]. Effects of phospholipids on D P P-IV activity We examined the effects of phospholipids on DPP-IV activity at several concentrations (final concentrations ranging from 0.001% to 0.5%) (Table 2). Control tubes contained only 0.05M sodium phosphate buffer instead of phospholipid solution. As shown in Table 2, the activity of DPP-IV purified from both human urine and kidney associated with phospholipids showed an about 50% increase compared with the control. Phosphatidylcholine at a concentration of 0.01% showed the highest increase of DPP-IV activity in both human urine and kidney. On the other hand, phosphatidylinositol at concentrations ranging from 0.001% to 0.1% showed the same activity in human urine and tends to increase the activity in human kidney, respectively. When we examined the effect of Ca2® in these studies, no change was noted (data not shown). 0.06M Glycine/NaOH buffer O.OSMTris/Maleate buffer Ο—Ο
0.8
0.6
Hh
8
pH
10
Fig. 2. The effect of pH on DPP-IVactivity. Tris/maleate buffer, pH 7.0-9.0 (O—O) and Glycine/NaOH buffer, pH 8.0-10.0 (·—·) were used.
Table 2. Effects of phospholipids on activity of DPP-IV purified from human urine and kidney. Phospholipids
3
Final concentration
DPP-IVactivity [% of control]
[%]
Human urine3
Human kidneyb
Phosphatidylcholine
0.5 0.1 0.05 0.01 0.005 0.001
108 ± 14 138 ± 22 148 ± 25 175 ± 19 150 ± 18 159 ± 25
104 ± 10 138 ± 15 145 ± 17 150 ± 17 124 ± 13 125 ± 9
Phosphatidylinositol
0.1 0.05 0.01 0.005 0.001
146 ±22 144 ±24 141 ± 14 144 ±25 140 ± 17
161 ± 19 166 ± 20 148 ± 16 142 ± 16 137 ± 14
Control (100%) = 48.5 ,umol/(min χ mg protein). Control (100%) = 54.8/imol/(min x mg protein).
Effects of chemical reagents on DPP-IVactivity We examined the effects of various reagents on DPPIV activity. DPP-IV activity was not affected by several cations (Ca2®, Mg2®, Mn2®, and K®), EDTA, and thiol reagents (pCMB, DTT, and NEM). Furthermore, DPP-IVactivity was not inhibited by puromycin or bacitracin. But DFP, a serine protease inhibitor, inhibited the enzyme activity completely at O.lmM, indicating that this enzyme is one of the serine enzymes. Properties of D P P-IV purified from human urine Some enzymatic and physicochemical properties of the enzyme were studied. The isoelectric point of the enzyme was 6.5, and the Km value using Gly-Pro-MCA as substrate was 1.25 ± 0.25 x 10~4M. The molecular mass of the enzyme was estimated to be about 280000 Da by Sephadex G-200 gel filtration using the method of Whitaker[19]. On the other hand, this enzyme fraction gave a single band on polyacrylamide gel electrophoresis in the presence of SDS, and a molecular mass of about 110-130 kDa was estimated. These results indicate that DPP-IV in human urine is composed of a single identical subunit corresponding to the materials of 110-130 kDa. Therefore, DPP-IV in human urine is dimer of an identical subunit. Substrate specificity of urinary DPP-IV Seven kinds of dipeptide-4-nitroanilide (pNA) (Gly-Pro-, Lys-Pro-, Arg-Pro-, Ala-Ala-, Ala-Gly-, GlyBrought to you by | Purdue University Libraries Authenticated Download Date | 6/10/15 5:04 PM
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Purification of Dipeptidyl-Peptidase IVfrom Human Urine
Leu-, and Gly-Hyp-pNA) were examined as substrates. The urinary DPP-IV was specific for derivatives with a proline residue as the second amino acid from the N-terminus, as was found with DPP-IV of other tissues13'5'7'10·16'241. The hydrolysis of Lys-Pro-, and Arg-Pro-pNA by the enzyme were 136% and 95% of that of Gly-Pro-pNA, respectively. On the other hand, other substrates Ala-GIy-, Gly-Leu-, and Gly-Hyp-pNA showed no detectable activity. The hydrolysis of Ala-Ala-pNA was 8% of that of Gly-Pro-pNA. Immunohisto chemistry of DPP-IV in human kidney Localization of DPP-IV in human kidney was investigated by using an indirect peroxidase staining method using anti-human renal DPP-I Vantibodies coupled to peroxidase. The region of DPP-IV localization was stained dark brown, using preimmune rabbit serum as a control. No specific staining was seen. On the other hand, when anti-DPP-IV was used, renal tubules in the human kidney slices were stained specifically indicating that DPP-IV exists mostly in renal tubules, and the glomerular region was also slightly stained suggesting that DPP-IV may also exist in renal glomeruli (data not shown).
Discussion Although the physiological role of DPP-I Vis not yet clear, one function of the enzyme may be the cleavage of N-terminal glycylproline from the peptides derived from collagen. Glycylproline is hydrolysed to glycine and proline by imidopeptidase^. In the present study, DPP-IV from human urine was homogeneously purified for the first time and its properties were investigated. Immunoaffinity chromatography of anti DPP-IVSepharose 4B was very effective for purifying the enzyme. By DEAE-cellulose chromatography, two active fractions were obtained (Fig. 1). In the preliminary experiments, fraction II was adsorbed to the anti DPP-IV Sepharose 4B column. Therefore we used fraction II for further purification. As there is a report that pig kidney DPP-IV exists in renal brush border membranes[2], we assume that the enzyme activity is affected by phospholipids. As shown in Table 2, DPP-IV activity from both human urine and kidney sources was enhanced by phospholipids. Although the mechanisms of enhancement are not clear, DPP-IV might be immobilized on the phospholipid membranes similar to those of renal brush border.
329
DPP-IV has been reported to be a serine enzyme but to be neither a metal nor a sulfhydryl enzyme^. DPPIV purified from human urine was also completely inhibited by DFP but was affected by neither any sulfhydryl reagents nor cations. These results indicate that DPP-IV from human urine is also a serine enzyme but not a sulfhydryl enzyme. DPP-IVpurified from the human urine rapidly hydrolysed 4-nitroanilides of Lys-Pro, Gly-Pro and Arg-Pro, but also very slowly hydrolysed the 4-nitroanilide of Ala-Ala. These results confirmed our previous findings on the substrate specificity of DPP-IV, namely that the enzyme catalyses the hydrolysis of amide bonds of peptides with a proline and alanine residues in the penultimate position17·16·24'251. To clarify the origin of urinary DPP-IV, we compared the properties with those of DPP-IV purified from human kidney. Except for the high molecular mass found in native urine, both enzymes have the same properties; they are inhibited by DFP, enhanced by phospholipids, and have the same p/ and optimum pH of 6.5 or 8.7, respectively. Substrate specificity is also similar to both enzymes. These properties of urinary DPP-IV were also nearly identical with those of DPP-IV purified from other human tissues, namely submaxillary gland[?1, placenta[26], and lymphocytes^. From the immunohistochemial study, this enzyme is mostly found in renal tubule cells, but it is probable that this enzyme also exists, to a lesser degree, in renal glomeruli. As mentioned above, urinary DPP-I Vis very similar to renal DPP-IV, and the former enzyme may come from both renal tubuli and glomeruli. By Sephadex G-200 gel filtration, urinary DPP-IV was eluted at the position corresponding to material with an approximate molecular mass of 280 kDa, a value considerably lower than that of 400 kDa, previously reported for the normal human urine enzyme[15]. We suppose that this discrepancy may be derived from the following different conditions. In the previous study, urine was directly subjected to column chromatography on Sephadex G-200 for determination of the molecular mass, but in this study we used any ionic treatment such as (NH4)2SO4 precipitation and DEAE-cellulose column chromatography for purification of the enzyme. Therefore there is a possibility that DPP-IV in urine is present as a polymer or is conjugated with some other macromolecules and becomes disassociated during the purification procedures. Further investigations on the molecular mass of urinary DPPIVis needed. Brought to you by | Purdue University Libraries Authenticated Download Date | 6/10/15 5:04 PM
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In the present experiments, we found another type of Gly-Pro-MCA-hydrolysing enzyme on DEAE-cellulose column chromatography (fraction I).The isolation of fraction I enzyme is currently under investigation.
References 1
Hopsu-Havu, V. K. & Glenner, G. G. (1966) Histochemie 7, 197-201. Kenny, A.J., Booth, A.G., George, S.G., Ingram, J.,Kershaw, D., Wood, E.J. & Young, A.R. (1976) Biochem. J. 155, 169-182. 3 Fukasawa, K.M., Fukasawa, K. & Harada, M. (1978) Biochim. Biophys. Acta 535, 161-166. 4 Yoshimoto,T. &Walter, R. (1977) Biochim. Biophys. Acta 485, 391-401. 5 Svensson, B., Danielsen, M., Staun, M., Jeppesen, L., Noren, O. & Sjöström, H. (1978) Eur. J. Biochem. 90, 489-498. 6 Oya, H., Nagatsu, I. & Nagatsu, T. (1972) Biochim. Biophys. Acta 258, 591-599. 7 Kojima, K., Hama,T., Kato,T. & Nagatsu, T. (1980) /. Chromatogr. 189, 233-240. 8 Fukasawa, K.M., Fukasawa, K., Hiraoka, B.Y. & Harada, M. (1981) Biochim. Biophys. Acta 657,179-189. 9 Yoshimoto,T., Kita,T., Ichinose, M. &Tsuru, D. (1982) J. Biochem. 92, 275-282. 10 Kudo, M., Nakamura,T. & Koyama, J. (1985)7. Biochem. 97,1211-1218. 2
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11 Nagatsu, L, Nagatsu,T. &Yamamoto,T. (1968) Experientia 24, 347-348. 12 Hino, M., Fuyamada, H., Nagatsu,T., Kurokawa, S. & Okuyama, S. (1976) Clin. Chim. Acta 67,103-105. 13 Hino, M., Fuyamada, H., Hayakawa, T., Nagatsu, T., Oya, H., Nakagawa, Y., Takemoto, T. & Sakakibara, S. (1976) Clin. Chem. 22, 1256-1261. 14 Hama, T., Nagatsu, T., Kobayashi, S., Azuma, S., MiyachuT., Kumagai, Y. & Kato,T. (1981) Clin. Chim. Acta 113, 217-221. 15 Kato,T.. Hama,T., Kojima, K., Nagatsu,T. & Sakakibara, S. (1978) Clin. Chem. 24, 1163-1166. 16 Hama,T., Okada, M., Kojima, K., Kato,T., Matsuyama, M. & Nagatsu,T. (1982) Mol. Cell. Biochem. 43, 35-42. 17 Kato,T., Nagatsu,T., Kimura,T. & Sakakibara, S. (1978) Biochem. Med. 19,351-359. 18 Hartree, E.F. (1972) Anal. Biochem. 48, 422-427. 19 Whitaker, J.R. (1963) Anal. Chem. 35,1950-1953. 20 Laemmli, U.K. (1970) Nature (London) 227, 680-685. 21 Oakley, B.R., Kirsch, D.R. & Morris, N.R. (1980) Anal. Biochem. 105,361-363. 22 Mey, J.D., Vandesande, F. & Dierickx, K. (1974) Cell Tiss. Res. 153,531-543. 23 Graham, R.C. & Karnovsky, M.J. (1966) J. Histochem. Cytochem. 14, 291-302. 24 Nagatsu, T., Hino, M., Fuyamada, H., Hayakawa, T., Sakakibara, S., Nakagawa,Y. &Takemoto,T. (1976)Anal. Biochem. 74, 466-476. 25 Kato,T., Nagatsu,T., Kimura,T. & Sakakibara, S. (1978) Experientia 34, 319-320. 26 Püschel, G., Mentlein, R. & Heymann, E. (1982) Eur. J. Biochem. 126, 359-365. 27 Mentlein, R., Rix, H., Feller, A.C. & Heymann, E. (1986) Biomed. Biochim. Acta 45, 567-574.
Toshiyuki Chikuma, Ph.D.*, Department of Pharmacheutical Analytical Chemistry, Showa College of Pharmaceutical Sciences, 5-l-8Tsurumaki, Setagaya-Ku,Tokyo 154; Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama 227; Department of Oral Anatomy, Josai Dental University, Sakado 350-02, Japan. * To whom correspondence should be sent.
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