Purification and biochemical characterization of dipeptidyl peptidase-II (DPP7) homologue from germinated Vigna radiata seeds

Bioorganic Chemistry 63 (2015) 132–141

Contents lists available at ScienceDirect

Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg

Purification and biochemical characterization of dipeptidyl peptidase-II (DPP7) homologue from germinated Vigna radiata seeds Tejinder Pal Khaket a,b, Suman Dhanda a, Druksakshi Jodha a, Jasbir Singh a,⇑ a b

Department of Biochemistry, Kurukshetra University, Kurukshetra, Haryana, India Department of Biotechnology, Maharishi Markandeshwar University, Ambala, Haryana, India

a r t i c l e

i n f o

Article history: Received 8 September 2015 Revised 6 October 2015 Accepted 12 October 2015 Available online 22 October 2015 Keywords: Exopeptidase Lys-Ala-4-methoxy-b-naphthylamide Vigna radiata seeds Protease DPP-II

a b s t r a c t Dipeptidyl peptidases (DPPs) are potent exopeptidases, which possess central role in proteolysis. As compared to other members of DPP family, proline containing dipeptide hydrolysing activity of DPP-II (Dipeptidyl peptidase II) is unique as it hydrolyses imino group and plays a key role in protein metabolism. In present study, DPP-II was purified from germinated moong bean seeds using acid and ammonium sulphate precipitation followed by successive chromatographies on gel filtration (pH 7.4) and cation exchanger (pH 5.9). Native PAGE and in-situ gel assay confirmed the apparent homogeneity. Purified plant DPP-II is an oligomeric enzyme with molecular weight of 97.3 kDa. Highest DPP-II activity was observed at pH 7.5 and 37 °C, with stability in the range of neutral to alkaline pH. Substrate specificity showed consequent activity for proline containing dipeptide followed by Lys-Ala and other hydrophobic dipeptides, but none of the studied endopeptidase and monopeptidase substrate was hydrolysed. Catalytic characterization with modifier studies revealed the involvement of Ser and His residues in its catalytic mechanism. Its dipeptidyl peptidase activity for proline containing dipeptide supported its role in the bioactive peptide generation and food industry. Functional studies of DPP-II revealed the significant involvement of this glycoproteinous enzyme in protein mobilization during germination. Further studies on industrial applications exploring physiological role are in progress. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Proteolysis play a significant role in various physiological processes such as removal of misfolded/abnormal proteins, maturation of the zymogen form of enzymes/peptide hormones and generation of regulatory peptides for central processes [1,2]. Among exopeptidases, DPPs are widely distributed exopeptidases that hydrolysed dipeptide moieties from the N-termini of oligopeptide chains. Among various studied DPPs, DPP-II (Dipeptidyl peptidase II) (EC: 3.4.14.2), also called quiescent cell proline dipeptidase (QPP, DPP7), is a serine protease with an aminoterminal dipeptidase activity exhibiting preference for Ala- or Pro- at the penultimate position [3–5]. Mammalian DPP-II mainly cleaves Lys-Ala, Phe-Pro and X-Pro-Pro dipeptides and its Pro hydrolysing activity may play key role in various regulatory processes by acting on a number of bioactive oligopeptides including neuropeptides, endomorphin, circulating peptide hormones, glucagon like peptides, gastric inhibitory peptide and chemokines ⇑ Corresponding author at: Department of Biochemistry, Kurukshetra University, Kurukshetra, Haryana 136119, India. E-mail address: [email protected] (J. Singh). http://dx.doi.org/10.1016/j.bioorg.2015.10.004 0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

leading to modification of their biological activities or even their inactivation [6,7]. Bista et al. [8] measured elevated mRNA of DPP-II in quiescent cells as compared to dividing cells and reduced level of DPP-II resulted in apoptosis. Thus DPP-II might be carrying some role in survival of resting cells [9]. Till now only DPP-III and IV homologues have been purified from plants [10–12] however, plant’s DPP-I and DPP-II have not been studied so far. Proteases are involved in all aspects of plant physiology however, limited studies are available about substrate specificity, physiological role or cellular location of putative proteases [13]. Moong bean (Vigna radiata) is a short duration species of Leguminosae with wide adaptability to environmental conditions. It is a major dietary staple in developing countries. Germinated moong bean seeds possess nutrient content comparable to mushroom. Higher protein content of moong bean (25–28%) further supported the study of plant proteases from moong bean. As proteolytic cleavage plays an important role in protein deposition and reactivation in seeds [14]. Therefore, in order to understand proteolysis and bioactive peptide generation during germination, it will be crucial to characterize the proteinases and peptidases involved in these processes. Though many endoprotease and few exoproteases have been studied, but DPP-II activity

T.P. Khaket et al. / Bioorganic Chemistry 63 (2015) 132–141

has not been studied in plants. To explore its importance in physiological processes and industry, purification and biochemical characterization of DPP-II activity from moong bean is of vital significance and thus this paper envisage the purification and characterization of DPP-II. 2. Materials and methods 2.1. Materials Moong bean seeds (Pusa (P) Baisakhi) were procured from Indian Agriculture Research Institute (IARI), Regional Station, Karnal (India). Lys-Ala-4-methoxy-b-naphthylamide (Lys-Ala4mbNA), synthetic substrates, CM-Sephadex, Sephadex G-100, DEAE Sephadex, Fast Garnet GBC (o-aminoazotoluene diazonium salt) were obtained from Sigma–Aldrich, USA. Tris buffer, b-mercapatoethanol, ammonium sulphate, disodium hydrogen phosphate dihydrate and sodium phosphate monobasic were obtained from Himedia India. Sodium chloride, dimethyl sulfoxide (DMSO) and HCl were obtained from Rankem and polyvinyl pyrrolidone (PVP) from SRL India. The protein samples were concentrated using Amicon stirred cell with YM 10 membrane under nitrogen pressure of 5 psi. Molecular weight marker proteins in the range of 14.3–97.4 kDa were from Bangalore Genei India. 2.2. Methods 2.2.1. Enzyme assay DPP-II activity was measured using Lys-Ala-4-methoxy-bnaphthylamide (Lys-Ala-4mbNA) as substrate. Assay buffer (875 ll) (50 mM Tris–HCl, pH 7.5) and 0.1 ml of enzyme sample was mixed and incubated at 37 °C for 10 min. The reaction was started with 25 ll of substrate (1 mg/ml DMSO) and the mixture was incubated for 20 min at 37 °C. The reaction was stopped by adding 1.0 ml of stopping reagent (1 M sodium acetate buffer, pH 4.2) and 0.5 ml coupling reagent (0.1% Fast Garnet GBC in water). The pink colour was extracted with 2.0 ml of n-butanol and estimated by recording absorbance at 520 nm. In blank, enzyme was added after the addition of coupling reagent. The enzyme activity was calculated in terms of nanomoles of -4mbNA released per min per ml enzyme. The following formula was used:9

Activity ðnmoles=min=mlÞ ¼

3

OD520  10  2:0  10 et

 10

where e is the molar extinction coefficient of 4mbNA under assay conditions (36,600), t is reaction time in min, 2.0  103 is volume of n-butanol in litres, 10 is a multiplication factor for calculating enzyme activity per ml as 0.1 ml enzyme was used for reaction and 109 is used for converting moles into nanomoles. One unit of enzyme activity is defined as the amount of enzyme that releases one nanomole of 4-methoxy-b-naphthylamine per min from substrate(s), under the assay conditions. Specific activity is expressed as units of enzyme activity per mg of total protein. 2.2.2. Protein estimation Protein content was estimated by the method of Lowry et al. [15] using bovine serum albumin as a standard. 2.2.3. Purification of enzyme Ten percent homogenate of 48 h germinated moong bean seeds was centrifuged and pH of supernatant was lowered from 7.4 to 5.0 by dropwise addition of chilled 1 N HCl with continuous stirring. The mixture was kept overnight under refrigerated condition and thereafter, acid precipitated proteins were removed by

133

centrifugation at 4800  g for 60 min. Supernatant of acid precipitated step was subjected to ammonium sulphate fractionation (0–55%) and kept overnight in refrigerated conditions. Precipitated proteins were separated by centrifugation at 4800  g for 60 min at 4 °C. The pellet was dissolved in minimum volume of 50 mM Tris–HCl buffer pH 7.4 and loaded on Sephadex G-100 column (75  1.25 cm) that was pre-equilibrated with 50 mM Tris–HCl buffer, pH 7.4. Fractions of 3 ml each were collected at a flow rate of 0.5 ml/min. The protein content of each fraction was measured spectrophotometrically by recording absorbance at 280 nm and DPP-II activity was screened by its standard assay. Fractions having DPP-II activity were pooled, concentrated by ultra filtration and dialyzed against sodium acetate buffer (50 mM, pH 5.9). Dialyzed sample was loaded on CM-Sephadex C-50 column (30  1.25 cm) pre-equilibrated with sodium acetate buffer (50 mM, pH 5.9). The column was run at a flow rate of 1 ml/min. Fractions of 3 ml each were collected and screened for protein by observing absorbance at 280 nm. The enzyme activity was screened by standard assay of DPP-II in unbound fractions. The fractions possessing DPP-II activity were pooled, concentrated and dialyzed against Tris–HCl buffer (50 mM, pH 7.4) and stored at 4 °C. 2.2.4. Polyacrylamide gel electrophoresis (PAGE) The apparent homogeneity and purity of enzyme was checked by Davis gel electrophoresis (12%) [16]. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was run to determine the molecular weight and subunit composition of purified enzyme [17]. 2.2.5. In-situ gel assay In-situ gel assay was performed on Davis gel electrophoresis (12%). Polymerised gel was pre run first for 2 h and then purified DPP-II was loaded. After complete run, gel was cut into two halves, one was used for In-situ gel assay and other for comparative Coomassie Brilliant Blue staining. For In-situ gel assay, gel was incubated with standard assay buffer and specific substrate (Lys-Ala-4mbNA) at 37 °C and colour was developed using Fast Garnet GBC (1 mg/ml). 2.3. Peptide mass fingerprinting For peptide mass fingerprinting, purified protein was digested overnight with trypsin [matrix-assisted laser desorption ionization (MALDI) grade] at 37 °C. Then peptide mass spectra was obtained using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF) mass spectrometer (Bruker Ultraflex III TOF/TOF) and MASCOT server (www.matrixscience.com) was used to obtain the protein identity by undertaking the Peptide Mass Fingerprinting approach. 2.4. Physiochemical characterization 2.4.1. pH optima and stability Optimum pH of purified enzyme was determined by assaying it in the pH range of 4–11 using different assay buffers (50 mM) viz. sodium acetate (4.0–5.5), sodium-phosphate (6.0–6.5), Tris–HCl (7.0–8.5), glycine–NaOH (9.5–10.0) and sodium phosphate–NaOH (11.0). pH stability of purified DPP-II was also determined in the pH range of 5.5–10 with incubation at 37 °C for 10 min. Then enzyme activity was measured at optimum pH (7.4). 2.4.2. Temperature optima and stability Optimum temperature for purified enzyme was also determined in the temperature range of 0–70 °C. Temperature stability of both enzymes was determined by incubating purified enzyme

134

T.P. Khaket et al. / Bioorganic Chemistry 63 (2015) 132–141

at different temperature viz. 0°, 10°, 20°, 28°, 32°, 37°, 40°, 45°, 50°, 55°, 60°, 65° and 75 °C for 10 min then activity was measured at optimum temperature. 2.4.3. Kinetic parameters Kinetic constants viz. Km and Vmax of purified DPP-II were measured by Hanes plot [19] and Lineweaver–Burk plot [18] at 37 °C using Lys-Ala-4mbNA as substrate (conc. range 1–500 lM). 2.4.4. Substrate specificity Substrate specificity of DPP-II was studied with various chromogenic substrates [-4-methoxy-b-naphthylamide (-4mbNA) and beta-naphthylamide (-bNA)]. The enzyme was incubated at 40 °C in standard reaction mixture with different monopeptide substrates viz L-Leu-bNA, L-Val-bNA, L-Ser-bNA, H-Gly-bNA, L-Phe-bNA, L-Tyr-bNA, dipeptide substrates viz. Gly-Arg-4mbNA, Gly-Phe-bNA, Gly-Arg-bNA, Phe-Arg-bNA, Gly-Ala-bNA and various endopeptidase substrates viz. Z-Phe-Arg-bNA (Z denotes blocked N-terminal), Z-Ala-Arg-Arg-4mbNA and Z-Val-Lys-Lys-Arg4mbNA (each at final conc. of 300 lM) and relative enzyme activity was calculated with respect to Lys-Ala-4mbNA as a substrate. Blank was also prepared separately for each reaction. 2.4.5. Effect of inhibitors To predict the amino acid residues involved in enzyme catalysis, different chemical modifiers (inhibitors) were used and effect of modification was studied by enzyme assays. DPP-II was incubated with appropriate concentration of different inhibitors for 10 min. The inhibitory effect was determined by assaying the enzyme in the absence of inhibitor (control). 2.4.6. Investigation of active site by varying pH To investigate the catalytic amino acid residues present in the active site, graph was plotted between log Vmax and pH. The curve was extrapolated to find the pKa values of amino acids involved in enzyme catalysis. 2.4.7. Effect of different metal ions on DPP-II activity To study the effect of metal ions, DPP-II was incubated with different concentration of chloride salts of different metal ions viz. Ca2+, Zn2+, Co2+, Mg2+, NH+4, Ba2+, K+, Hg2+. 2.4.8. Effect of chloride on DPP-II activity To study the effect of chloride ions, purified DPP-II was made free from chloride ions by dialyzing it against 50 mM sodium phosphate pH 7.4. Dialyzed DPP-II was incubated with different concentrations of NaCl viz. 0, 10, 20, 40, 60, 80, 100, 200, 300, 400, 500, 600 and 700 mM under assay conditions, then standard DPP-II assay was run. Results were expressed as percent activity as compared to control. 2.4.9. Effect of urea Purified enzyme was pretreated with different concentration of urea viz. 0, 10, 20, 40, 60, 80, 100, 200, 300, 400, 500, 600 and 700 mM for 10 min and assay was started by adding specific substrate. Results were expressed as percent activity in comparison to control (100%). 2.4.10. Effect of organic solvents Organic solvents viz. DMSO and ethanol are commonly used solvents and also important in industrial processes, therefore, their effect was studied on DPP-II. Purified DPP-II was incubated with 0–11% (v/v) organic solvents (DMSO and ethanol) for 10 min at 37 °C, then assay was started by adding substrate. Results were expressed as percent activity with consideration of maximum activity as 100%.

2.4.11. Glycoprotein analysis Glycoprotein bearing hexosyl, hexuronosyl or pentosyl residues react with sulphuric acid to form furfural derivatives, which in turn react with thymol to form a chromogen that is stable for few hours at ambient temperature [20]. Carbohydrate content of the enzyme was determined by a phenol sulphuric acid method using glucose as standard [21]. 2.4.12. Storage stability of DPP-II Purified DPP-II was stored in aliquots of 100 ll in Tris–HCl (50 mM, pH 7.4 having 10% glycerol) at 20 °C. The enzyme was assayed up to 6 months at an interval of one month and residual activity was calculated. 2.5. Role of DPP-I in proteolysis 2.5.1. Effect of germination time on DPP-II activity Effect of germination time on DPP-II activity and protein content were studied in dry seeds and imbibed seeds (in deionized water for 6 h) and seeds germinated at 25 °C for 6, 8, 16, 24, 48, 72, 96, 120, 144 and 168 h. Ten percent homogenate was prepared for all dry and germinated seeds and centrifuged at 10,000  g for 10 min in a refrigerated centrifuge. DPP-II activity and protein content were determined in each supernatant. 2.5.2. Total free amino acids The content of free amino acids was estimated by ninhydrin method [22]. The concentration of free amino acids of sample was determined from the standard plot of L-glycine. The values were expressed as lg/ml. 3. Results and discussion 3.1. Purification of enzyme DPP-II was purified to apparent homogeneity using successive chromatographies up to 145.87-fold with a final yield of 19.17% and specific activity of 248.08 mol/min/mg (Table 1). Percent yield of purified plant DPP-II was higher than that of rat (kidney, brain, skin), porcine (spleen, ovary, seminal plasma, skeleton muscle), bovine (pituitary gland, dental pulp) and human (kidney, placenta), but lower than that of human seminal plasma [3,5,23–30]. Homogeneity of purified DPP-II was checked on 12% polyacrylamide gel stained with Coomassie Brilliant Blue R-250 and activity staining gel i.e. in situ gel assay (Fig. 1). Single band on gel confirmed apparent homogeneity of DPP-II. Two bands of different size were obtained for plant DPP-II on both reducing and non-reducing SDS-PAGE, thus suggesting its heterodimeric nature (Fig. 2). Same results on both reducing and non reducing conditions, thereby suggesting absence of any disulphide bond between two subunits (Fig. 2). A graph of log molecular weight versus relative mobility was plotted to calculate molecular weight of each subunit. The overall molecular weight was calculated to be 97.3 kDa with two subunits of molecular weight of 57.2 kDa and 39.8 kDa. Molecular weight of plant DPP-II is close to those of porcine spleen DPP-II [31]. However, DPP-II from other sources possessed molecular weight in the range 105–130 kDa except that of seminal plasma and rat brain DPP-II which have a molecular weight in the range of 185–220 kDa [3,5,23–30]. Plant DPP-II exists as a heterodimer with two subunits each having different mass as compared to other homodimeric forms. DPP-II from most species was reported to be homodimer with subunit’s molecular weight in the range of 50–64 kDa that varied with species [3,5,23–27, 29,30]. DPP-II from porcine seminal plasma was trimeric [28].

135

T.P. Khaket et al. / Bioorganic Chemistry 63 (2015) 132–141 Table 1 Purification of DPP-II from germinated moong beans. Steps

Total protein (mg) (mean ± S.D)

Total activity (nmol/min/ml)

Specific activity (nmol/min/mg protein)

Purification fold

% yield

Homogenate supernatant Acid-ppt supernatant 0–55% (NH4)2SO4 pellet Sephadex G-100 CM-Sephadex

2076 ± 98.4 1328 ± 16 88 ± 6.8 21 ± 3.7 2.8 ± 0.98

4144 ± 165.21 3039.41 ± 81.86 1902.07 ± 34.18 1065.41 ± 14.52 795.43 ± 9.01

1.99 2.29 21.61 50.39 284.08

1 1.15 10.85 25.32 145.87

100 73.25 45.84 25.67 19.17

Values are mean of three different experiments. Values are represented as mean ± S.D.

range. DPP-II of porcine seminal plasma was stable in the pH range of 3.5–10, ovary (pH 3–7.5), rat brain (pH 4–8) and rat kidney (pH 3.7–8.8) [25,27,35,36]. 3.3.2. Temperature optima and stability Temperature optima of purified plant DPP-II was found to be 37 °C (Fig. 5). The enzyme activity was almost lost at 65 °C. Similar results were also observed for DPP-II of other species [3,26,28,29,33]. DPP-II from Streptomyces sp. and P. gingivalis showed higher activity at 50 and 43 °C, respectively [34,35]. Activation energy of DPP-II was calculated by Arrhenius plot and was found to be 32.77 kJ/mol for Lys-Ala-4mbNA (Fig. 1 in supplementary file). These results cannot be compared because activation energy is calculated for the first time for plant DPP-II. DPP-II was found to be stable up to 50 °C in rat, human, porcine and Bos taurus [3,30,37]. DPP-II homologues were stable up to 55–60 °C depending on the source [28,32]. 3.3.3. Kinetic characterization Km and Vmax were calculated as 40 lM and 4.57 nmoles/min/ml, respectively for Lys-Ala-4mbNA by Michaelis–Menton (Fig. 2 in supplementary file), Hanes plot and Lineweaver–Burk plot (Fig. 6a and b). Same value of Km was also reported for DPP-II of human seminal plasma at pH 7.0 [33]. As kinetic constant varied with chromogen and source, varied Km values (25–555 lM) were reported for Lys-Ala-substrates in different species [25–29,33,37–39].

Fig. 1. Davis gel electrophoresis of DPP-II. Coomassie Brilliant Blue stained (Lane 1) and activity stained enzyme (Lane 2).

3.2. Peptide mass fingerprinting and sequence comparison by MALDITOF Thirty-nine peptide fragments were obtained after trypsin digestion of DPP-II as predicted by MALDI-TOF analysis (Fig. 3). Matrix server results showed no significant similarity with any available plant protein database which also supported the novelty of DPP-II in plants.

3.3. Physicochemical characterization 3.3.1. pH optima and stability Optimum DPP-II activity was observed at pH 7.5 (Fig. 4) and only 60% activity was observed at pH 7.0 and 8.0, however it ranged between 5.0 and 6.5 for mammalian DPP-II [3,28–30,32,33] Contrarily, pH optima of DPP-II from Polyphyromonas gingivalis and Streptomyces sp. was observed in the range of 6.5–9.0 [34,35]. DPP-II was stable in the pH range of 7–8.5 with more than 90% activity (Fig. 4). DPP-II from P. gingivalis and Streptomyces sp. were also stable from neutral to the alkaline range of pH [35], whereas DPP-II from some other species was stable over acidic to alkaline

Fig. 2. SDS-PAGE (12%) of purified DPP-II. Purified DPP-II without b-ME (Lane 1), molecular weight marker (Lane 2) and purified DPP-II with b-ME (Lane 3).

136

T.P. Khaket et al. / Bioorganic Chemistry 63 (2015) 132–141

Fig. 3. Peptide mass fingerprinting of DPP-II by MALDI-TOF with spectra of trypsin digested sample.

for Lys-Ala-4mbNA substrate. These are similar to the reported values for human seminal plasma [33]. Earlier kinetic constants were measured for DPP-II using Lys-Ala and Gly-Pro with different fluorogenic and chromogenic substrates [25–27,29,33,36,38,39].

Fig. 4. pH optima and stability of DPP-II.

Fig. 5. Temperature optima and stability of DPP-II.

The reported Vmax also varied from 1.43 to 29 lmol/min/mg for different species [28,33]. Turnover rate (Kcat) and specificity constant (Kcat/Km) were calculated as 2.65 s1 and 66  103 s1 M1

3.3.4. Substrate specificity Hydrolytic activity of plant DPP-II against different synthetic substrates with different N-terminal peptide sequence was studied. Among various synthetic chromogenic substrates, plant DPP-II maximally hydrolysed Gly-Pro-bNA followed by Lys-Ala4mbNA, Ala-Ala-bNA, Asp-Ala-bNA, Gly-Ala-bNA, Leu-Ala-bNA and His-Phe-bNA (Table 2). Plant DPP-II preferentially hydrolysed Gly-Pro-bNA as compared to Lys-Ala. Similar results were also predicted previously for mammalian DPP-II [32,40]. However, preferential substrates varied from different species as observed in porcine and human which showed preferential cleavage toward Phe-Pro-bNA, Gly-Pro-MCA and Arg-Pro-pNA, respectively [26,32,33]. In addition, X-Pro cleavage, Leu-Ala-, Ala-Alahydrolysing activity was also reported for DPP-II [24,25,27,30,36]. DPP-II did not hydrolyse Gly-Arg-, Gly-Phe-, Phe-Arg-(specific substrates of DPP-I), Asp-Arg-, Ser-Met- and Arg-Arg-(specific substrates of DPP-III) [24,25,27,30,36]. Plant DPP-II did not hydrolyse any of studied aminopeptidase substrate viz. L-Trp-bNA, L-Leu-bNA, L-Val-bNA, L-Ser-bNA, H-Gly-bNA, L-Phe-bNA and L-Tyr-bNA and blocked N-terminal substrate viz. Z-Phe-Arg-bNA, Z-Ala-Arg–Arg-4mbNA and Z-Val-Lys–Lys-Arg-4mbNA. Plant DPPII did not hydrolyse the studied tripeptide (Gly-Pro-Leu-bNA). However, hydrolysis of tripeptide substrate was reported by mammalian DPP-II [26,32]. 3.3.5. Effect of inhibitors PMSF was the most potent inhibitor of plant DPP-II followed by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), Diethylpyrocarbonate (DEPC), 4-nitrophenyl iodoacetamide and puromycin. Phenylmethylsulfonyl fluoride (PMSF) inhibited up to 83% at 0.5 mM (Table 3). The degree of inhibition by PMSF is comparable to that of DPP-II from rat brain [24,36], porcine ovary [26] and human [27]. DPP-II of porcine seminal plasma [28] and rat kidney [25] was less sensitive to PMSF. AEBSF showed more than 60% inhibition at 0.05 mM concentration and up to 73% at 1 mM concentration. Our results are in agreement with previous studies

T.P. Khaket et al. / Bioorganic Chemistry 63 (2015) 132–141

137

Fig. 6. Determination of kinetic parameters for hydrolysis of Lys-Ala-4mbNA by DPP-II viz. Hanes plot (a) and Lineweaver–Burk plot (b).

on inhibitory effect of AEBSF on DPP-II from rat kidney [30] and porcine seminal plasma [28]. Both PMSF and AEBSF are specific inhibitors of serine proteases. Inhibition of plant DPP-II by serine modifying agents suggested the involvement of the serine residue in enzyme catalysis. DEPC (ethoxyformic anhydride) specifically modifies active site histidine residues (in pH range of 5.5–7.5) by substitution of one of nitrogen on the imidazole ring [41]. Inhibition of plant DPP-II by DEPC (62%) suggests that histidine might also be involved in catalysis. Pepstatin is an established modifier of aspartate residue. Plant DPP-II was slightly inhibited by pepstatin at 0.5 mM concentration. DPP-II from porcine ovary and skeleton muscle was also inhibited slightly up to 11% at 0.01 mM and 6% at 0.5 mM, by pepstatin. Puromycin showed 29% inhibition at 1 mM concentration. Comparable inhibition was also reported in rat brain DPP-II [24], but DPP-II from porcine ovary was highly sensitive toward puromycin [26]. DPP-II activity was not significantly affected by sulfhydryl reagents viz. iodoacetic acid, iodoacetamide, N-ethylmaleimide

and p-chloromercuribenzenesulfonic acid (PCMBS). DPP-II from other sources was also resistant to these agents [3,24,25,28,31,32,37,42,43]. These findings indicated that a SH-residue is neither involved in catalysis nor in regulation of DPP-II activity which was also supported by its structural analysis [44]. Inhibitory effect of PMSF, AEBSF and DEPC supported the role of serine and histidine residues in catalysis. Inhibition by pepstatin though to lower extent also hint toward involvement of aspartic acid. Structural studies also revealed that catalytic triad of Ser, Asp and His present at the active site of DPP-II [44]. 3.3.6. Amino acid residues on active site pKa values of amino acids involved in catalysis were 6.0 and 9.0 which corresponds to pKa of His and Ser, respectively (Fig. 7). Histidine is often found in the active sites of enzymes, where the imidazole ring can act as both proton acceptor and proton donor in the course of enzymatic reactions. These results are in agreement with inhibition studies.

138

T.P. Khaket et al. / Bioorganic Chemistry 63 (2015) 132–141

Table 2 Substrate specificity of purified DPP-II.

Table 4 Effect of metal ions on DPP-II activity.

Substrates

% activity

Metal ions

Concentration (mM)

% activity

Gly-Pro-bNA Lys-Ala-4mbNA Ala-Ala-bNA Asp-Ala-bNA Gly-Ala-bNA Leu-Ala-bNA His-Phe-bNA Asp-Arg-NA Leu-Ser-4mbNA Gly-Arg-4mbNA Gly-Phe-bNA Gly-Arg-bNA Phe-Arg-bNA Ser-Met-bNA Ser-Tyr-bNA Arg-Arg-4mbNA Gly-Pro-Leu-bNA Z-Phe-Arg-bNA Z-Ala-Arg-Arg-4mbNA Z-Val-Lys-Lys-Arg-4mbNA L-Trp-bNA L-Leu-bNA L-Val-bNA L-Ser-bNA H-Gly-bNA L-Phe-bNA L-Tyr-bNA

128 ± 2.5 100 33.86 ± 1.14 27 ± 0.98 26 ± 0.62 18 ± 0.87 16 ± 1.12 NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL

Control FeCl3

0 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2

100 93.74 ± 0.45 93.9 ± 0.12 97.74 ± 0.7 156 ± 1.21 99 ± 0.3 93 ± 0.88 89 ± 0.98 77 ± 2.09 69 ± 0.48 121.87 ± 1.42 83 ± 0.69 75 ± 1.18 131 ± 2.01 69.2 ± 0.78 62.5 ± 0.76 121.87 ± 1.08 93.9 ± 0.98 76 ± 0.54 150 ± 1.12 93 ± 0.34 82.96 ± 0.12 109.37 ± 0.16 80.17 ± 0.48 78.41 ± 0.92 134.37 ± 1.08 98.84 ± 0.12 79.55 ± 0.87 107.81 ± 0.45 109.27 ± 0.86 82.96 ± 0.72 121.87 ± 0.99 93.35 ± 0.18 65.91 ± 0.3 121.87 ± 0.88 87 ± 0.46 76.14 ± 0.66 107.81 ± 0.87 82 ± 1.46 75 ± 0.86 125 ± 1.14 89 ± 0.12 81.82 ± 0.45

Values are mean of three different experiments. Values are represented as mean ± S.D.

CuCl2

HgCl2

FeSO4

ZnCl2

MgCl2

KCl

NH4Cl

BaCl2

CaCl2

MnCl2

3.3.7. Effect of metal ions Effect of metal ions on DPP-II is shown in Table 4. DPP-II activity increased in the presence of all studied cations at 0.05 mM concentration except Fe3+ and Hg2+. Cu2+ resulted in a maximum increase (56%) followed by K+, Ni+, Ba2+ Zn2+, Co2+, Cd2+, Fe2+, Mg2+, Mn2+, , Ni2+, Ca2+ and Li+ as compared to control. Fe3+ and Hg2+ decreased DPP-II activity by 6% and 11%, respectively, even at 0.05 mM concentration. More than 0.2 mM concentration of metal ions showed an inhibitory effect on plant DPP-II which was also reported earlier for DPP-II from other sources [24–26,28,30–32,37,42,45,46]. In rat

NiCl2

LiCl2

CdCl2

Values are mean of three different experiments. Values are represented as mean ± S.D.

Table 3 Effect of different inhibitors on activity of purified DPP-II. Inhibitors

Concentration

% inhibition

Control PMSF

0 0.5 mM 1 mM 0.05 mM 0.1 mM 0.5 mM 1 mM 1% 5% 0.5 mM 1 mM 0.5 mM 1 mM 0.5 mM 1 mM 0.05 mM 0.1 mM 0.5 mM 0.1 mM 0.5 mM 1 mM 0.5 mM 1 mM

0 83 ± 1.2 87 ± 0.82 62 ± 0.88 68 ± 0.52 69 ± 0.5 73 ± 0.23 62 ± 0.87 63 ± 0.65 23 ± 0.98 31 ± 0.72 14 ± 1.4 29 ± 0.39 3 ± 0.36 6 ± 0.88 0 7.78 ± 0.2 3 ± 0.13 0 7 ± 0.65 9.5 ± 0.98 0 2 ± 0.18

AEBSF

DEPC 4-Nitrophenyl iodoacetamide Puromycin DTNB Leupeptin Pepstatin 4-Choloromercuribenzoic acid Iodoacetate NEM

Values are mean of three different experiments. Values are represented as mean ± S.D.

Fig. 7. Investigation of catalytic residues of DPP-II.

mast cells, Zn2+ inhibited approximately 85% activity at 10 mM [47] but in rabbit lung Zn2+ inhibited approximately 90% of DPP-II activity at 1 mM [48].

T.P. Khaket et al. / Bioorganic Chemistry 63 (2015) 132–141

139

However, Co2+ and Cd2+, strongly inhibited DPP-II from human kidney and bovine anterior pituitary (3) at 1 mM [45]. The inhibitory effect of Hg2+ on plant DPP-II was comparable to the data obtained for DPP-II from other sources [28,30,31,45]. On the other hand, DPP-II from rat kidney [25] and porcine ovary [26] were completely inhibited at 1 mM Hg2+. Ethylenediaminetetraacetic acid (EDTA) resulted in a 39% decrease in DPP-II activity even at 10 mM concentration (Fig. 3 in supplementary file). Only 10% activity was lost at 1 mM EDTA. Slight decrease in DPP-II activity in rat was also observed while porcine skeleton muscle, ovary, bovine pituitary and rat kidney DPP-II were not inhibited even up to 5 mM [3,24,26,28–32]. 3.3.8. Effect of chloride ions DPP-II activity slightly decreased in the presence of NaCl and more than 10% decrease was observed at 10 mM NaCl (Fig. 4 in supplementary file). NaCl also showed varied inhibitory effect (11–73%) up to 100 mM NaCl on DPP-II isolated from human, rat, porcine and bovine [3,24,26,28,32,33,49]. Plant DPP-II retained approximately 66% activity up to 1 M NaCl. In porcine seminal plasma and ovary, NaCl reduced only 11% and 14% activity, even at 100 mM and 400 mM concentration [26,28]. In rat brain, NaCl reduced approximately 26% DPP-II activity at 50 mM concentration [24] but DPP-II from enamel was more sensitive for NaCl and 73% inhibition was observed at 100 mM NaCl [49]. 3.3.9. Effect of urea on DPP-II activity Enzyme activity initially increased and reached maximum at 0.2 M urea concentration and then gradually declined (Fig. 5 in supplementary file). Initial increase in DPP-II activity by urea may be because of alteration of the active site by denaturant, due to which enzyme’s active site appears more open and flexible [50]. Increase in enzyme activity in the presence of urea is also reported for DPP-III [12]. The activity of adenylate kinase and carbamyl phosphate synthase increased many folds up to 1 M urea due to conformational changes that led to their activation [50]. But at 2 M urea, only 33.54% activity remained. At this concentration, urea might have severely altered amino acids of active site which caused complete denaturation and as a result enzyme rigorously lost its activity. 3.3.10. Effect of organic solvents on DPP-II activity DPP-II activity increased with increase in organic solvents concentration and was maximum at 7% organic solvents (DMSO and ethanol) but decreased thereafter with further increase in the concentration of organic solvents (Fig. 6 in supplementary file). DPP-II retained 55% activity, even at 14% DMSO. There are no such reports to compare the effect of organic solvents on DPP-II activity. Initial increase in DPP-II activity with increase in organic solvent concentration may be due to the increased substrate solubility and altered hydrophobic sites on an enzyme that induces at more favourable conformation at the enzyme’s active site [51,52]. But higher organic solvent concentration may severely disturb conformation of hydrophobic residues which subsequently result in decreased enzyme activity [51].

Fig. 8. Glycoprotein staining for DPP-II.

was comparable to that of DPP-II from rat (14%) [29]. Native DPP-II was reported to be a glycoprotein with mannose and glucosamine moieties [26,29–31,36,45]. Chiravuri et al. [53] suggested that N-glycosylation was necessary for its activity. Six glycosylation sites were predicted in amino acid sequence of rat DPP-II [30]. Further studies are needed for elucidation of nature of linkage between the carbohydrate(s) and protein to explain the function of glycosylation. 3.3.12. Storage stability Approximately 93% and 50% activity was left after one and six months of storage, respectively. Our results are also supported by previous studies on storage stability of DPP-II from other sources. Maes et al. [33] reported DPP-II stability for at least 4 months in the presence of 1 mg/ml BSA at 4 °C, 20 °C or 80 °C and in the presence of 0.1% tween 20 at 80 °C (95–105% residual activity). The rat kidney enzyme (1 mg/ml in 10 mM sodium phosphate buffer, pH 7.6, containing 150 mM NaCl and 2 mM mercaptoethanol) was stable at 20 °C for 3 months without any loss of activity, but after one year the activity dropped to 70% of original activity [28]. Rat DPP-II was stable for several months at 20 °C to 30 °C [28,30,36]. 3.4. Studies on physiological role of plant DPP-II

3.3.11. Glycoprotein analysis Carbohydrate content of purified enzyme was studied by phenol–sulphuric method and carbohydrate content was measured from standard graph of known glucose concentration. The estimated carbohydrate content was 12% for DPP-II. Dark brown spots of DPP-II on native gel confirmed the glycosylation of plant DPP-II (Fig. 8). Both spectrophotometric and gel analysis confirmed that plant DPP-II is a glycoprotein. The carbohydrate content of plant DPP-II

3.4.1. Germination and proteolysis Attempts were made to study physiologic role of plant DPP-II, thereby effect of germination time on protein content, amino acid content and activity of DPP-II were studied and correlated. Protein content decreased significantly with germination and had negative correlation with germination time, which clearly supported proteolysis on germination (Fig. 7 in supplementary file). Storage protein mobilization is widely reported process in germination as it

140

T.P. Khaket et al. / Bioorganic Chemistry 63 (2015) 132–141

is supposed to provide raw material for growth of embryonic plant [53,54]. In contrast to protein content, free amino acid content increased up to 168 h, thus positively correlated with germination time (Fig. 8 in supplementary file). Decrease in total protein content and increase in free amino acid content are attributed to proteolysis in which precursor polypeptides are processed by limited proteolysis to mature subunits of reserve proteins in storage tissue cells of the developing seeds. This limited proteolysis induced conformational changes of storage proteins, thus open them to attack by additional indoor and exopeptidases which degrade the protein reserves completely. Proteases that catalyse limited cleavage or complete degradation are synthesized as precursors which also undergo stepwise limited proteolysis when they are formed in cotyledons of developing or germinating seeds [54]. In general, this processing transforms enzymatically inactive proenzymes into active proteases. While proteases are involved in most cellular processes, proteolysis still is not a well understood process in plant biology. Very little is known about substrate specificity, physiological role or cellular location of many putative proteases known or the processes they are involved in [55,56]. The initial germination period (7 days from imbibition) was suggested to be the rapid stage of storage protein hydrolysis [57]. DPP-II activity initially increased up to 72 h, but decreased slightly thereafter (Fig. 9). To study the relation of DPP-II activity and proteolysis (decrease in protein content and increase in free amino acid content), their correlation analysis was performed. Correlation analysis of DPP-II with time showed a significant correlation (r > 0.6) with germination time. Significant negative correlation (r > 0.7) exists between protein content and enzyme activity of DPP-II. Statistical analysis revealed a significant correlation between proteolysis and DPP-II activity. During germination, DPP-I activity was negatively correlated with protein content (r > 0.7) and positively correlated with amino acid content (r > 0.5). This significant correlation between the activity of DPP-II, protein content and free amino acid content suggests direct involvement of DPP-II in protein mobilization during germination. Results revealed involvement of DPP-II in initial seed storage protein mobilization either its direct involvement in final stage of hydrolysis or in activation of other proteases which play significant role in cell signaling and seed differentiation. Both maintenance of cellular homeostasis and regulation of multiple cellular functions require a continuous turnover of intracellular proteins. It is, therefore, expected that regulated protein degradation plays a crucial

Fig. 9. Effect of time of germination on DPP-II activity.

role during growth and development in all organisms [58]. Mammalian homologues of DPP-II were also suggested to be involved in lysosomal proteolysis [59]. When germinating moong bean seeds were subjected to salinity stress, significant increase in DPP-II activity was observed (unpublished work). It suggests the potential role of DPP-II in stress management. Further studies are in progress in our lab. 4. Conclusion DPP-II is purified to apparent homogeneity from germinated moong bean seeds. It is an oligomer with molecular mass of 97.3 kDa and works optimally at pH 7.5 for hydrolysis of Lys-Ala-4mbNA. It is a serine protease with cleaving ability toward hydrophobic amino acid residues containing dipeptides. Its dipeptidyl aminopeptidase activity supported its role in the activation of other proteases and bioactive peptide generation and consequently in industrial food processing in presence of other proteases. Further studies are needed to confirm its physiological role. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bioorg.2015.10. 004. References [1] N. Gonzalez-Rabade, J.A. Badillo-Corona, J.S. Aranda-Barradas, M.C. OliverSalvador, Production of plant proteases in vivo and in vitro-a review, Biotechnol. Adv. 29 (2011) 983–996. [2] J.M. Palma, L.M. Sandalio, F.J. Corpas, M.C. Romero-Puertas, I. McCarthy, L.A. Del Rio, Plant proteases, protein degradation, and oxidative stress: role of peroxisomes, Plant Physiol. Biochem. 40 (2002) 521–530. [3] J.K. McDonald, T.J. Reilly, B.B. Zeitman, S. Ellis, Dipeptidyl arylamidase II of the pituitary. Properties of lysylalanyl-beta-naphthylamide hydrolysis: inhibition by cations, distribution in tissues, and subcellular localization, J. Biol. Chem. 243 (1968) 2028–2037. [4] R. Underwood, M. Chiravuri, H. Lee, T. Schmitz, A.K. Kabcenell, K. Yardley, B.T. Huber, Sequence, puri-fication, and cloning of an intracellular serine protease, quiescent cell proline dipeptidase, J. Biol. Chem. 274 (1999) 34053–34058. [5] M.B. Maes, S. Scharpe, I. De Meester, Dipeptidyl pepti-dase II (DPP-II), a review, Clin. Chim. Acta 380 (2007) 31–49. [6] K. Augustyns, P. Van der Veken, K. Senten, A. Haemers, The therapeutic potential of inhibitors of dipeptidyl peptidase IV (DPP IV) and related prolinespecific dipeptidyl aminopeptidases, Curr. Med. Chem. 12 (2005) 971–998. [7] O.V. Danilova, A.K. Tai, D.A. Mele, M. Beinborn, A.B. Leiter, A.S. Greenberg, J.W. Perfield, J. Defuria, P.S. Singru, R.M. Lechan, B.T. Huber, Neurogenin 3-specific dipeptidyl peptidase-2 deficiency causes impaired glucose tolerance, insulin resistance, and visceral obesity, Endocrinology 150 (2009) 5240–5248. [8] P. Bista, D.A. Mele, D.V. Baez, B.T. Huber, Lymphocyte quiescence factor Dpp2 is transcriptionally activated by KLF2 and TOB1, Mol. Immunol. 45 (2008) 3618– 3623. [9] M. Chiravuri, T. Schmitz, K. Yardley, R. Underwood, Y. Dayal, B.T. Huber, A novel apoptotic pathway in quiescent lymphocytes identified by inhibition of a post-proline cleaving aminodipeptidase: a candidate target protease, quiescent cell proline dipeptidase, J. Immunol. 163 (1999) 3092–3099. [10] A. Davy, K.K. Thomsen, M.A. Juliano, L.C. Alves, I. Svendsen, D. Simpson, Purification and characterization of barely dipeptidyl peptidase IV, Plant Physiol. 122 (2000) 425–431. [11] D. Jodha, P. Attri, T.P. Khaket, J. Singh, First report of DPP-III in plants: its subcellular localization in germinating mung bean (Vigna radiata) seeds and distribution in plant parts, Appl. Biol. Res. 14 (2012) 100–103. [12] D. Jodha, P. Attri, T.P. Khaket, J. Singh, Isolation, purification and biochemical characterization of dipeptidyl peptidase-III from germinated Vigna radiata seeds, Process Biochem. 48 (2013) 730–737. [13] R.A.L. Van der Hoorn, Plant proteases: from phenotypes to molecular mechanisms, Annu. Rev. Plant Biol. 59 (2008) 191–223. [14] B.B. Singh, G.P. Dixit, P.K. Katiyar. Vigna research in India (25 years of research achievements). All India Coordinated Research Project on MULLaRP, IIPR, Kanpur-India, 2010. [15] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [16] B.J. Davis, Disc electrophoresis II, methods and applications to human serum proteins, Ann. Acad. Sci. NY 121 (1964) 404–427. [17] U.K. Laemmli, Cleavage of structural proteins during the assembly of bacteriophage T4, Nature 227 (1970) 680–685.

T.P. Khaket et al. / Bioorganic Chemistry 63 (2015) 132–141 [18] C.S. Hanes, Studies on plant amylase: the effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley, Biochem. J. 26 (1932) 1406–1421. [19] H. Lineweaver, D. Burk, The determination of enzyme dissociation constants, J. Am. Chem. Soc. 56 (1934) 658–666. [20] J.E. Gander, Gel protein stains: glycoproteins, Methods Enzymol. 104 (1984) 447–449. [21] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric Method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. [22] W. Troll, R.K. Cannan, A modified photometric ninhydrin method for the analysis of amino and imino acids, J. Biol. Chem. 200 (1953) 803–811. [23] J.K. McDonald, F.H. Leibach, R.E. Grindeland, S. Ellis, Purification of dipeptidyl aminopeptidase II (dipeptidyl arylami-dase II) of the anterior pituitary gland. Peptidase and dipeptide esterase activities, J. Biol. Chem. 243 (1968) 4143– 4150. [24] K. Imai, T. Hama, T. Kato, Purification and properties of rat brain dipeptidyl aminopeptidase, J. Biochem. (Tokyo) 93 (1983) 431–437. [25] K. Fukasawa, K.M. Fukasawa, B.Y. Hiraoka, M. Harada, Purification and properties of dipeptidyl peptidase II from rat kidney, Biochim. Biophys. Acta 745 (1983) 6–11. [26] D.A. Eisenhauer, J.K. McDonald, A novel dipeptidyl peptidase II from the porcine ovary. Purification and characterization of a lysosomal serine protease showing enhanced specificity for prolyl bonds, J. Biol. Chem. 261 (1986) 8859– 8865. [27] T. Sakai, K. Kojima, T. Nagatsu, Rapid chromatographic purification of dipeptidyl-aminopeptidase II from human kidney, J. Chromatogr. 416 (1987) 131–137. [28] K. Huang, M. Takagaki, K. Kani, I. Ohkubo, Dipeptidyl peptidase II from porcine seminal plasma: purification, characterization, and its homology to granzymes, cytotoxic cell proteinases (CCP1-4), Biochim. Biophys. Acta 1290 (1996) 149–156. [29] K.M. Fukasawa, K. Fukasawa, K. Higaki, N. Shiina, M. Ohno, S. Ito, J. Otogoto, N. Ota, Cloning and functional expression of rat kidney dipeptidyl peptidase II, Biochem. J. 353 (2001) 283–290. [30] H. Araki, Y. Li, Y. Yamamoto, M. Haneda, K. Nishi, R. Kikkawa, I. Ohkubo, Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase, J. Biochem. (Tokyo) 129 (2001) 279–288. [31] K.R. Lynn, The isolation and some properties of dipeptidyl peptidases II and III from porcine spleen, Int. J. Biochem. 23 (1991) 47–50. [32] M.A. Sentandreu, F. Toldra, Partial purification and characterisation of dipeptidyl peptidase II from porcine skeletal muscle, Meat Sci. 57 (2001) 93–103. [33] M.B. Maes, A.M. Lambeir, K. Gilany, K. Senten, P. Van der Veken, B. Leiting, K. Augustyns, S. Scharpe, I. De Meester, Kinetic investigation of human dipeptidyl peptidase II (DPP-II)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell proline dipeptidase (QPP)/dipeptidyl pep-tidase 7 (DPP7), Biochem. J. 386 (2005) 315–324. [34] S. Murao, T. Kitade, H. Oyama, T. Shin, Isolation and characterization of a dipeptidylamino peptidase from Streptomyces sp. WM-23, Biosci. Biotechnol. Biochem. 58 (1994) 1545–1546. [35] A. Banbula, P. Mak, M. Smoluch, J. Travis, J. Potempa, Arginine-specific cysteine proteinase from Porphyromonas gingivalis as a convenient tool in protein chemistry, Biol. Chem. 382 (2001) 1399–1404. [36] R. Mentlein, G. Struckhoff, Purification of two dipeptidyl aminopeptidases II from rat brain and their action on proline-containing neuropeptides, J. Neurochem. 52 (1989) 1284–1293. [37] V.K. Hopsu-Havu, C.T. Jansen, M. Jarvinen, Partial purification and characterization of an acid dipeptide naphthylamidase (carboxytripeptidase) of the rat skin, Arch. Klin. Exp. Dermatol. 236 (1970) 282–296.

141

[38] A. Stockel-Maschek, C. Mrestani-Klaus, B. Stiebitz, H. Demuth, K. Neubert, Thioxo amino acid pyrrolidides and thiazolidides: new inhibitors of proline specific peptidases, Biochim. Biophys. Acta 1479 (2000) 15–31. [39] S.G. Sharoyan, A.A. Antonyan1, S.S. Mardanyan, G. Lupidi, M.M. Cuccioloni, M. Angeletti, G. Cristalli, Complex of dipeptidyl peptidase II with adenosine deaminase, Biochemistry (Moscow) 73 (2008) 943–949. [40] B. Leiting, K.D. Pryor, J.K. Wu, F. Marsilio, R.A. Patel, C.S. Craik, J.A. Ellman, R.T. Cummings, N.A. Thornberry, Catalytic properties and inhibition of prolinespecific dipeptidyl peptidases II, IV and VII, Biochem. J. 371 (2003) 525–532. [41] E.W. Miles, Modification of histidyl residues in proteins by diethypyrocarbonate, Methods Enzymol. 47 (1977) 431. [42] J.K. McDonald, C. Schwabe, Dipeptidyl peptidase II of bovine dental pulp. Initial demonstration and characterization as a fibroblastic, lysosomal peptidase of the serine class active on collagen-related peptides, Biochim. Biophys. Acta 616 (1980) 68–81. [43] S. Lampelo, K. Lalu, T. Vanha-Perttula, Bio-chemical studies on dipeptidyl peptidases I to IV of the human placenta, Placenta 8 (1987) 389–398. [44] G.A. Bezerra, E. Dobrovetsky, A. Dong, A. Seitova, L. Crombett, I.M. Shewchuk, A.M. Hassell, S.M. Sweitzer, T.D. Sweitzer, P.J. McDevitt, K.O. Johanson, K.M. Kennedy-Wilson, D. Cossar, A. Bochkarev, K. Gruber, S. DhePaganon, Structures of human DPP7 reveal the molecular basis of specific inhibition and the architectural diversity of proline-specific peptidases, PLoS One 7 (2012) e43019. [45] D. Mantle, Characterization of dipeptidyl and tripeptidyl aminopeptidases in human kidney soluble fraction, Clin. Chim. Acta 196 (1991) 135–142. [46] P. Cummins, P. Mulcahy, B. O’Connor, Identification of a dipeptidyl aminopeptidase type-II in the cytosolic fraction of bovine brain, Biochem. Soc. Trans. 20 (1991) 56S. [47] G. Struckhoff, E. Heymann, Rat peritoneal mast cells release dipeptidyl peptidase II, Biochem. J. 236 (1986) 215–219. [48] A. Paszkowski, H. Singh, G. Kalnitsky, Properties of dipeptidyl peptidase I from rabbit (Oryctolagus cuniculus) lungs, Acta Biochim. Pol. 30 (1983) 363– 380. [49] J.R. Smid, P.A. Monsour, E.M. Rousseau, W.G. Young, Cytochemical localization of dipeptidyl peptidase II activity in rat incisor tooth ameloblasts, Anat. Rec. 233 (1992) 493–503. [50] Z. Hongjie, P. Xianrning, Z. Junmei, H. Kihara, Activation and conformational changes of adenylate kinase in urea solution, Sci. China C Life Sci. 41 (1998) 245–250. [51] S. Barberis, E. Quiroga, S. Morcelle, N. Priolo, J.M. Luco, J. Mol. Catal. B: Enzymes 38 (2006) 95–103. [52] S. Dhanda, H. Singh, J. Singh, Hydrolysis of various bioactive peptides by goat brain dipeptidylpeptidase-III, Cell Biochem. Funct. 23 (2008) 339–345. [53] M. Chiravuri, F. Agarraberes, S.L. Mathieu, H. Lee, B.T. Huber, Vesicular localization and characterization of a novel post-proline-cleaving aminodipeptidase, quiescent cell proline dipeptidase, J. Immunol. 165 (2000) 5695–5702. [54] A. Helenius, M. Aebi, Roles of N-linked glycans in the endoplasmic reticulum, Annu. Rev. Biochem. 73 (2004) 1019–1049. [55] K. Muntz, Proteases and proteolytic cleavage of storage proteins in developing and germinating dicotyledonous seeds, J. Exp. Bot. 47 (1996) 605–622. [56] A.L. Tan-Wilson, K.A. Wilson, Mobilization of seed protein reserves, Physiol. Plant 145 (2012) 140–153. [57] M.J. Chrispeels, D. Boutler, Control of storage protein metabolism in the cotyledons of germinating mung beans: role of endopeptidase, Plant Physiol. 55 (1981) 1031–1037. [58] Y. Zhang, Z. Zhao, Y. Xue, Roles of proteolysis in plant self-incompatibility, Annu. Rev. Plant Biol. 60 (2009) 21–42. [59] H. Hellmann, M. Estelle, Plant development: regulation by protein degradation, Science 297 (2002) 793–797.