Rapid purification of dipeptidyl peptidase IV from rat liver plasma membrane

Biochimica et Biophysica Acta 924 (1987) 543-547 Elsevier

543

BBA 22752

Rapid purification of dipeptidyl peptidase IV from rat liver plasma membrane *

Sabine Hartel a, Christoph Hanski Christiane Hoffmann a, Jost Mauck

a, a

Wolfgang Kreisel b, and Werner Reutter a

a Institut fftr Molekularbiologie und Biochemie der Freien Unioersitiit, Berlin (Germany) and b Medizinische Klinik, Klinikum der Albert-Ludwigs- Universitiit, Freiburg (F.R.G.)

(Received 16 December 1986)

Key words: Dipeptidyl peptidase IV; Proteinase purification; (Rat liver plasma membrane)

Dipeptidyi peptidase IV (EC 3.4.14.5) was solubilized from rat liver plasma membranes with sulphobetaine 14 and purified by successive affinity chromatography on Con A-Sepharose, wheat germ ieetin-Sepharose and arglnine-Sepharose columns. The specific activity of the final preparation was 49. 4 /tmol Gly-Pro p-nitroanilide/min per mg protein, representing a 1098-fold purification of the homogenate. SDS-POlyacrylamide gel eleetrophoresis of the arglnine-Sepharose eluate showed a single protein band with a molecular weight of 105 000. The isoelectric point was determined to be 3.9 under non-denaturing conditions with sulphobetaine 14. The preparation was free of post-proline cleaving enzyme. The content of aminopeptidase M was 0.2% of the total protein.

Introduction Dipeptidyl peptidase IV (EC 3.4.14.5) is a widely distributed exopeptidase which was originally isolated from rat liver and kidney [1,2] and, more recently, from bovine liver [3]. Due to its specificity for Gly-Pro dipeptides, its possible involvement in collagen metabolism was suggested [4,5]. Recently, it was shown that dipeptidyl peptidase IV is involved in cell-matrix interaction in culture [6]. Moreover, dipeptidyl peptidase IV levels are greatly decreased during hepatoma growth [7,8]. These findings prompted experi* Dedicated to Professor Fleckenstein on the occasion of his 70th birthday. Abbreviations: Con A, Concanavalin A; EDTA, ethylenediaminetetraacetate, sodium salt; IgG, immunoglobulinG; PMSF, phenylmethylsulphonyl fluoride. Correspondence: S. Hartel, Institut ffir Molekularbiologie und Biochemie der Freien Universitiit, Arnimallee 22, D-1000 Berlin 33, Germany.

ments to biochemically characterize the enzyme further. Our main interest is primarily focussed on its oligosaccharide structure, which might be altered during malignancy. The analysis of the oligosaccharide structure requires several milligrams of pure enzyme. Therefore, an efficient purification method was developed. Materials and Methods Animals and isolation of the liver plasma membrane Male Wistar rats (Ivanovas, Kisslegg, F.R.G.) weighing about 160-180 g each were used. Liver plasma membranes were isolated by zonal centrifugation as described by Pfleger et al. [9] and modified by Biichsel et al. [10]. The purity of the membrane preparation was routinely checked by assay of marker enzymes as reported earlier [11]. Determination of the enzyme activities The activity of dipeptidyl peptidase IV was determined with tosyl-Gly-Pro p-nitroanilide

0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

544

(Bachem, Bubendorf, Switzerland) as the substrate [12]; the specific activity was calculated from a standard curve of p-nitroaniline (Serva, Heidelberg, F.R.G.) solution in 0.1 M Tris-HC1 (pH 8.0). Aminopeptidase M activity was measured with alanine p-nitroanilide (Serva, Heidelberg, F.R.G.) as the substrate [13]. The determination of the activity of post-proline cleaving enzyme was carfled out with carboxybenzoxy-Gly-Pro p-nitroanilide (Bachem, Bubendorf, Switzerland) as the substrate [14].

Affinity chromatography Plasma membranes (111.2 mg obtained from 100 g liver) were solubilized for 1 h at 25°C in 0.01 M Tris-HC1 (pH 7.2) containing 0.15 M NaC1, 2 mM MgC12, 1 mM CaC12, 0.1% (w/v) (2.7 mM) sulphobetaine 14 (Calbiochem, Frankfurt, F.R.G.) and 15 mM NaN 3, followed by centrifugation for 1 h at 100000 × g at 4°C. The supernatant was applied to a Con A-Sepharose column. The column was washed with solubilization buffer and eluted in 5 ml fractions with the same buffer supplemented with 0.2 M methyl aglucopyranoside. Fractions containing the enzyme activity were pooled and rechromatographed on a wheat germ lectin-Sepharose column. The elution was carried out with 0.2 M N-acetylglucosamine in solubilization buffer. Fractions containing the enzyme activity were pooled again and applied to an arginine-Sepharose column (5 × 1 cm). The sample was eluted by a linear salt gradient from 0 to 0.15 M NaC1 in 0.01 M Tris-HC1 (pH 7.2) containing 0.1% sulphobetaine 14. The derivatized Sepharoses were obtained from Pharmacia (Heidelberg, F.R.G.). Electrophoretic determination of the purity of the enzyme Samples were boiled in sample buffer (3% SDS, 5% mercaptoethanol, 10% glycerol, 0.01% bromphenol blue in 0.062 M Tris-HCl (pH 6.8)) for 5 min and analysed in SDS-polyacrylamide gels according to Laemmli [15]. Gels were stained in 0.1% Coomassie G 250 and destained as described [11]. Destained rod gels were scanned in a computing spectrophotometer (DU 8 Beckman, Fullerton, CA U.S.A.).

Isoelectric focussing Isoelectric focussing was carried out in a 3% rod polyacrylamide gel containing Servalyte 2-11 (Serva, Heidelberg, F.R.G.) and 0.1% sulphobetaine 14. After 900 volt. h the gel was sliced, and in each homogenized slice the pH and the enzyme activity were determined. Production of antiserum The anti-rat-liver dipeptidyl peptidase IV antiserum was raised against the purified enzyme in a rabbit. 10 /~g protein were dispersed in complete Freund's adjuvant (Gibco, Grand Island, NY U.S.A.) and injected intradermally. The boosting followed 21, 28 and 38 days thereafter. 1 week after the last injection, serum was collected. The immunoglobulin fraction was isolated from 5 ml of the antiserum on a protein A-Sepharose affinity column (Pharmacia, Heidelberg, F.R.G.), lyophilized, and reconstituted with 0.01 M Tris-HC1 (pH 7.4). Blotting and immunolocafization of the antigen The samples (plasma membranes or arginineSepharose eluate) were separated on SDS gel elec~ophoresis and transferred to nitrocellulose (Schleicher and Schi~ll, Dassel, F.R.G.) as described by Towbin et al. [17]. After transfer of the protein, the nitrocellulose paper was incubated with phosphate-buffered saline containing 3% bovine serum albumin for 12 h. The immunolocalization of the antigen was carried out by modifying the method of Pauli et al. [18]. The nitrocellulose paper was cut into strips and incubated overnight at 4°C with the purified rabbit antibody. The strips were then washed with phosphate-buffered saline containing 0.1% Tween 20. The antibody was detected with a fl-galactosidase-linked donkey anti-rabbit IgG (Amersham, Braunschweig, F.R.G.), and with 5-bromo-4chloro-3-indolyl fl-D-galactoside (Biomol, Ilvesheim, F.R.G.) as the substrate. Results and Discussion

Isolation of the dipeptidyl peptidase IV from rat liver The isolation of dipeptidyl peptidase IV from rat liver was achieved by solubilization of the

545 TABLE I PURIFICATION OF DIPEPTIDYL PEPTIDASE IV FROM RAT LIVER One unit of dipeptidyl peptidase IV was defined as the amount of the enzyme hydrolysing 1 #mole Gly-Pro p-nitroanilide per min at 37°C. Isolation step

Total protein (mg)

Total activity (units)

Specific activity (units/mg)

Recovery (%)

Purification (fold)

Homogenate Plasma membranes Plasma membranes, solubilized Con A-Sepharose eluate Wheat germ lectin-Sepharose eluate Arginine-Sepharose eluate

13 970 111.2 110.6 3.0 1.8 0.26

628.6 65.7 68.1 57.9 51.1 12.9

0.045 0.591 0.616 19.5 28.6 49.4

100 10.5 11 9.2 8.1 2.1

1 13.1 14 443.2 650.0 1097.8

plasma membrane preparation with sulphobetaine 14 and subsequent affinity chromatography steps on Con A-Sepharose, wheat germ lectin-Sepharose and arginine-Sepharose columns as summarized in Table I. The specific activity of the enzyme in crude homogenate was 44.8 U / g protein. About 70% was sedimentable and represents the membranebound fraction. By use of zonal gradient centrifugation about 11% of the total amount of plasma membranes were obtained [19]. The preparation contained 10.5% of the total dipeptidyl peptidase activity, indicating that most of the enzyme was bound to the cell membranes and only small amounts were in the Golgi or in vesicles [20]. Dipeptidyl peptidase IV was found to constitute 0.09% of the total liver protein and 1.2% of the membrane preparation, which corroborated the results obtained by quantitative immunoprecipitation [21]. The enzyme was solubilized totally from the membrane fraction with 0.1% sulphobetaine 14, a mild zwitterionic detergent, which did not interfere with any of the chromatographic steps. In contrast to most non-ionic detergents, it was a great advantage that sulphobetaine 14 could also be easily removed by dialysis. Since the intact structure of the protein was essential for production of antibodies, prior autolysis was avoided. This procedure produces an enzyme in its active form, but devoid of the hydrophobic fragment by which it is anchored in the membrane [22]. Rat liver dipeptidyl peptidase IV has 8-15 sugar chains of complex-type and probably a small number of high-mannose-type chains [23]. Based

on this glycoprotein structure, chromatography on Con A-Sepharose and wheat germ lectin-Sepharose was chosen [24.25]. Under optimal conditions, dipeptidyl peptidase IV totally bound to concanavalin A or wheat germ lectin, the recovery in the eluates being 84% and 88%, respectively. The binding of dipeptidyl peptidase IV to arginine-Sepharose and its elution by a salt gradient from 0 to 0.15 M NaCI is less well understood. At pH 7, arginine-Sepharose is a positively charged matrix, and could bind the negatively charged dipeptidyl peptidase IV molecules as a result of ionic interactions. However, other serine proteinases also bind to arginine-Sepharose [26,27]. Therefore it is possible that the negatively charged serine in the active centre is also involved in the binding to arginine. This notion is supported by reports on the binding of dipeptidyl peptidase IV to Gly-Pro-Sepharose, which probably involved the active centre and was desorbed by 0.05 M NaC1 [28]. The purification of dipeptidyl peptidase IV was monitored by determination of the enzymatic activity (Table I) and by means of SDS-polyacrylamide gel electrophoresis (Fig. 1). The increase of the specific activity in the preparation was concomitant with the emergence of the prominent 105-110-kDa protein band in the gel. The molecular mass of the enzyme in the native state was found to be 220 kDa [12]. The two subunits, which are not bound by disulfide bridges [29,30], dissociate under denaturing conditions and appear as a single protein band in SDS-gel electrophoresis.

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Fig. 1. SDS-polyacrylamide gel electrophoresis of each purification step. A, solubilized plasma membranes (50 /~g); B, Con A-Sepharose eluate (50 #g); C, wheat germ lectin-Sepharose eluate (25 #g); D, arginine-Sepharose eluate (8 /Lg). Samples were separated on rod gel, stained and destained. The protein profile was recorded with a densitometer.

Criteria of purity The purified enzyme had a specific activity of 49.4 U/rag, which was considerably higher than the value obtained previously by immunoprecipitation (10 U/mg) [31]. During the purification procedure dipeptidyl peptidase IV was separated from aminopeptidase M after chromatography on arginine-Sepharose, and from post-proline cleaving enzyme after isolation of the plasma membranes. The specific activity of aminopeptidase M, determined with 0.2 mM alanine p-nitroanilide, was 0.086 U / m g protein, and post-proline cleaving enzyme, measured with 1 mM carboxybenzoxy-Gly-Pro p-nitroanilide was not detectable after incubation for 4 h. The purified enzyme was

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remarkably stable in 8 M urea. After an overnight incubation in the presence of 8 M urea, a residual enzyme activity of 58% was obtained, confirming the absence of post-proline cleaving enzyme, which is urea-sensitive [14]. Isoelectric focussing of dipeptidyl peptidase IV in the presence of 0.1% lllOl~lr,

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Fig. 3. Detection of dipeptidyl peptidase IV on a nitrocellulose blot of plasma membranes with: 1, solubilized plasma membranes (50 /~g); 2, arginine-Sepharose eluate (8 /Lg) amido black-stained; 3, solubilized plasma membranes (50/zg); and 4, arginine-Sepharose eluate (8 pg) immunostained with polyclonal antiserum produced against the purified enzyme.

547 sulphobetaine 14 showed a peak of activity at a p I of 3.9 (Fig. 2). The polyclonal antiserum p r o d u c e d in rabbit against the purified enzyme precipitated 96% of the enzymatic activity and recognized a single protein b a n d on nitrocellulose blots with plasma membranes as antigen (Fig. 3), thus confirming the purity of the preparation used for immunization. The present procedure permits a rapid preparation of 240 # g of dipeptidyl peptidase IV with high purity per 100 mg plasma membranes. The m e t h o d can be applied to isolation of dipeptidyl peptidase IV from rat liver and tissues where the enzyme has similar structural properties.

Acknowledgements

8 9 10 11 12 13 14 15 16 17 18

T h e authors thank Professor Dr. C. Bauer and Dr. Dj. Josic for valuable discussions. This work was supported b y the Deutsche Forschungsgemeinschaft B o n n - B a d Godesberg (Re 523/2-1) and the F o n d s der Chemischen Industrie Frankfurt/Main.

References

19 20 21 22 23 24 25

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