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. 2014 May 29;93(6):1495–1502. doi: 10.3382/ps.2013-03848

Isolation and characterization of chicken bile matrix metalloproteinase

B Packialakshmi *,†, R Liyanage ‡, K S Rasaputra †,§, Jackson O Lay Jr ‡, N C Rath §,1
PMCID: PMC4988621  PMID: 24879699

Abstract

Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies, but the significance of their expression in normal, healthy bile is not understood. We hypothesized that the MMP in bile may aid the digestion of native collagens that are resistant to conventional gastric proteases. Hence, the objective of this study was to characterize the bile MMP and check its regulation in association with dietary factors. We used substrate zymography, azocoll protease assay, and gelatin affinity chromatography to identify and purify the MMP from chicken bile. Using zymography and SDS PAGE, 5 bands at 70, 64, 58, 50, and 42 kDa were detected. The bands corresponding to 64, 50, and 42 kDa were identified as MMP2 using trypsin in-gel digestion and matrix-assisted laser desorption time-of-flight mass spectrometry and peptide mass fingerprinting. Chickens fed diets containing gelatin supplements showed higher levels of MMP expression in the bile by both azocoll assay and zymography. We conclude that the bile MMP may be associated with the digestion of collagens and other extracellular matrix proteins in avian diets.

Keywords: bile, collagen digestion, matrix metalloproteinase-2, mass spectrometry

INTRODUCTION

Bile is the hepatic fluid consisting of a complex mixture of bile acids, cholesterol derivatives, heme-derived pigments, mucins, enzymes, and protein breakdown products (Trauner and Boyer, 2003). It is also responsible for the emulsification and digestion of fat in the intestine (Tuchweber et al., 1996; Hornbuckle, 1997). Previously we showed that normal turkey bile contained substantial gelatinolytic activities that belonged to the matrix metalloproteinase (MMP) family of enzymes (Rath et al., 2001). The MMP are zinc-dependent endopeptidases that degrade extracellular matrix (ECM) and non-ECM proteins during tissue remodeling (Nagase et al., 2006; Iyer et al., 2012). The elevation of bile MMP has been generally linked to hepatic pathologies involving fibrosis, cancer, bile duct, and gall bladder diseases in humans and other mammalian models (Okada et al., 2001; Consolo et al., 2009; Kirimlioğlu et al., 2009; Hirashita et al., 2012; Syed et al., 2012). A recent study in fish showed that the elevation of their bile MMP levels was associated with aquatic pollution (Hauser-Davis et al., 2012). Nonetheless, the significance of MMP in bile fluid is not fully understood. We hypothesized that the bile MMP may serve to denature and digest native ECM proteins, such as collagens. The ECM proteins constitute major parts of animal connective tissue that are part of the natural diets of omnivorous birds (Duke, 1997; Klasing, 1998; Hauser-Davis et al., 2012). Because the native interstitial collagens can be resistant to degradation by conventional digestive proteases, the MMP in bile could likely aid their denaturation and subsequent digestion (Bornstein et al., 1966; Etherington, 1977; Chung et al., 2004). However, the exact nature of avian bile MMP is not known, because in aves, as in mammals, several classes of MMP with different substrate specificities exist (Snoek-van Beurden and Von den Hoff, 2005; Nagase and Visse, 2009; Sekhon, 2010). Therefore, the objective of the current study was to isolate and characterize the bile MMP and find whether certain feed additives can modulate their activities and digestive function.

MATERIALS AND METHODS

Chemicals and Reagents

Azocoll (mesh >50, EMD Millipore, Billerica, MA), BCA protein assay reagent (Pierce, Rockford, IL), calf skin soluble type I collagen (Worthington Biochemical Corporation, Lakewood, NJ), dithiothreitol (DTT; Omnipur, EMD Millipore), GM 6001, E-64 {N-[N-(L-3-trans-carboxyirane-2-carbonyl)-L-leucyl]-agmatine, a cysteine protease inhibitor; EMD Millipore}, iodoacetamide (ICN Biomedicals Inc., Aurora, OH), Omix Tips C18 (Varian, Palo Alto, CA), page ruler protein molecular weight markers (ThermoScientific, Rockford, IL), Spectra/Por molecular weight cut-off (MWCO) 3500 dialysis membrane (Spectrum Medical Industries Inc., Fort Mill, SC), gelatin sepharose 4B (GE Healthcare, Buckinghamshire, United Kingdom), SilverQuest silver staining kit (Invitrogen, Carlsbad, CA), trypsin gold, mass spectrometry grade (Promega, Madison, WI), chromatography mini column (VWR, Radnor, PA), and Ultrafree-MC 10000 NMWL filter unit (EMD Millipore) were purchased from their respective vendors. The poultry diets were made according to NRC specifications (NRC, 1994). The feed supplements were obtained from local suppliers, except for the beef gelatin, which was purchased from Lucky Vitamin (Conshohocken, PA; luckyvitamin.com). All other chemicals and reagents, including porcine skin gelatin type A, 4-aminophenylmercuric acetate (APMA), phenylmethyl sulfonyl fluoride (PMSF), leupeptin, and α-cyano-4-hydroxycinnamic acid were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO).

Bile Collection

The animal protocols were approved by the Institutional Animal Usage and Care Committee of the University of Arkansas. Bile was obtained from euthanized chickens using sterile syringes, centrifuged at 21,000 × g for 20 min at 4°C, and the supernatant was stored at −20°C until further analyses. Protein content was determined by the micro-BCA method and absorbance at 280 nm (A280) as necessary.

Gelatin and Collagen Zymography

Zymography was performed using 10% PAGE containing 0.1% porcine gelatin or 0.08% type I skin collagen, as described previously (Rath et al., 2001). Intact bile or the gelatin-sepharose affinity purified bile proteins were mixed with nonreducing Laemmli sample buffer (Sigma-Aldrich) and electrophoresed at constant voltage of 100 V in a Novex gel apparatus (Invitrogen). The gels were washed twice with 2.5% triton X-100 (Sigma-Aldrich) for 15 min each, and incubated in a buffer consisting of 50 mM Tris, 200 mM NaCl, 10 mM CaCl2, and 0.05% Brij-35, pH 7.6 (incubation buffer; IB) for 5 to 8 h at 37°C. The gels were stained with Coomassie brilliant blue R250 (Sigma-Aldrich) to visualize gelatinolytic and collagenlytic bands. The SDS-PAGE gels were stained with SilverQuest silver staining kit and documented using Gel Logic GL2200 (Carestream Health Inc., Rochester, NY). The approximate molecular weight (MW) of the bands, and their intensities were determined using the Gel Logic system. The MMP activities of samples were expressed as sum of their band intensities per microgram of protein. To test for the activation of enzyme, the bile samples were incubated with 1 mM APMA for 30 to 60 min at 37°C.

Azocoll Assay

The MMP activity was determined using the azocoll method of Jiang et al., (2007) with modifications. Ten microliters of bile sample containing 5 to 10 µg of equivalent protein were added to 190 µL of azocoll suspension (3 mg/mL of IB) and incubated with or without any inhibitor at 37°C for 15 h. The blanks consisted of azocoll reaction mixture to which bile was added after incubation. The tubes were centrifuged for 5 min at 2,000 × g at room temperature, transferred to 96-well microtiter plates, and the color was read at 520 nm. Each sample was assayed in triplicate. Different protease inhibitors, 20 mM EDTA (divalent ion chelator and MMP inhibitor), 1 mM PMSF (serine protease inhibitor), 20 μM E64 (cysteine protease inhibitor), 10 µM leupeptin (cysteine, serine, threonine protease inhibitor), and 20 µM GM 6001 (MMP inhibitor), were used to identify the nature of the bile proteases. The percentages of enzyme activities were calculated with respect to controls.

Affinity Purification of Bile MMP

Pooled samples of chicken bile were dialyzed against IB using 3500 MWCO Spectra/Por membrane with 3 successive changes. Affinity purification was performed on a gelatin-sepharose column following the procedure of Zhang and Gottschall (1997). Briefly, 1 mL of gelatin sepharose beads was loaded in a minicolumn, equilibrated with IB twice with the dialyzed bile added to cover the bed volume, and incubated for 4 h at 4°C. Unbound materials were eliminated with 3 successive washings with IB. The bound materials were eluted with 1mL of IB containing 10% dimethyl sulfoxide. The eluants were concentrated using 10,000 MWCO Ultrafree-MC Millipore filter. The protein content of the retentate was determined by A280 and subjected to PAGE and gelatin zymography. The zymogram gels were incubated for 3 h.

In-Gel Digestion

The silver-stained protein bands were excised using a spot picker (The Gel Company, San Francisco, CA), transferred to sterile tubes, destained, and washed with water twice for 15 min. The gel pieces were then washed for 1 h sequentially with 25 mM ammonium bicarbonate (NH4HCO3) and 50% acetonitrile (ACN) in 50 mM and vacuum dried. The dried gel pieces were subjected to reduction with 10 mM DTT at 56°C for 1 h followed by alkylation with 55 mM iodoacetamide for 1 h in the dark at room temperature. Trypsin (20 ng/µL) was added in volume enough to hydrate the gel pieces in ice for 20 min followed by the addition of 50 µL of 25 mM NH4HCO3 before overnight incubation at 37°C. Gel pieces without any protein were treated identically as control to subtract artifact-associated peaks. The digested peptides were eluted from gels with 5% formic acid in 50% ACN for 15 min twice in an ultrasonic water batch (Branson 3200, Branson Ultrasonics, Danbury, CT). The extracted peptides were concentrated using a CentriVap centrifugal vacuum concentrator (Labconco, Kansas City, MO) and desalted using Omix C18 tips. Aliquots of desalted solutions were mixed with equal volumes of saturated α-cyano-4-hydroxycinnamic acid prepared in 1:1 ratio of water and ACN containing 0.1% formic acid then spotted on a Bruker MTP384 target plate (Bruker Daltonics, Billerica, MA) to identify the peptides using matrix-assisted laser desorption (MALDI) time-of-flight (TOF) mass spectrometry and peptide mass fingerprinting (Rosenfeld et al., 1992; Hellman et al., 1995).

Mass Spectrometry

Mass spectra were obtained in reflector positive ion mode using a Bruker Daltonics Ultraflex II MALDI-TOF/TOF mass spectrometer. The background peaks present in trypsin-treated control gel pieces were removed and the MALDI peptide mass fingerprint (PMF) was subjected to tandem MS/MS using MALDI LIFT-TOF/TOF (Bruker Daltonics). Bruker Biotools 3.1 was used to combine PMF and LIFT-MS/MS data and searched against National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) nonredundant Gallus gallus database using the Mascot 2.2 search engine to identify the protein(s). Single missed cleavage, fixed carbamidomethylation of cysteine, variable methionine oxidation, 100 ppm error at the MS level, and 0.5 Da error at the MS/MS level were used during the database search.

Effect of Dietary Additives on Bile MMP

Fifty male broiler chickens from a local hatchery were randomly assigned to 5 groups and received feed according to NRC specifications (NRC, 1994), with or without specified supplements, and ad libitum water. The control birds received a normal diet whereas the rest of the groups received supplements consisting 4% of either beef gelatin, skim milk powder, rice powder, or lard from d 1 through 43. Feed was withdrawn overnight before euthanasia to retain bile in the gall bladder. The birds were weighed before killing by carbon dioxide asphyxiation, and the bile was collected from the gall bladder as described previously. The protein content of bile was determined by micro-BCA method. The MMP activity was estimated by azocoll assay and zymogram densitometry. The azocoll assay was performed in triplicate using 5 bile samples per group and incubated for 10 h. The groups were compared using the optical density of the dye released per microgram of protein. In densitometry, the activities were calculated as gelatinolytic intensities per microgram of protein.

Cathepsin Activity of Bile

To demonstrate that the gelatinolytic activities were not related to cathepsin, the bile samples were electrophoresed in duplicate using a gelatin-containing gel and divided into 2 halves to develop zymogram. One half of the gel was incubated in MMP IB and the other half in a cathepsin incubation buffer (100 mM Na phosphate, 1 mM EDTA, and 2 mM DTT, pH 5.5). Gels were equilibrated with cathepsin incubation buffer for 30 min, replaced with fresh buffer, and incubated for 5 h at 37°C (Wilder et al., 2011). The cathepsin and MMP zymogram were visually compared.

Statistics

The results from quantitative assays, such as the effect of various inhibitors on azocoll protease activity and the densitometry, were presented as mean ± SEM. Software from SAS (SAS Institute Inc., Cary, NC) was used to perform a 1-way ANOVA and Duncan’s t-test. A P-value of ≤0.05 was considered to be significant.

RESULTS

Zymography

Gelatin zymography showed 5 gelatinolytic bands corresponding to approximate MW of 70, 64, 58, 50, and 42 kDa respectively (Figure 1a), whereas the collagen zymography showed only 4 bands. Due to differential mobility of MW standards in collagen zymography, an approximate alignment with gelatin showed only 4 bands corresponding to 70, 64, 58, and 42 kDa, respectively (Figure 1b). Incubation with APMA for 30 or 60 min resulted in similar profiles, showing 2 major bands corresponding to 64 and 42 kDa (Figure 2).

Figure 1.

Figure 1.

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Substrate zymography of chicken bile using (a) gelatin and (b) collagen. MW = molecular weight.

Figure 2.

Figure 2.

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Gelatinolytic profiles of bile matrix metalloproteinases with or without aminophenylmercuric acetate (APMA) activation. Lane 1 is bile incubated without APMA for 60 min; lanes 2, 3, and 4 are bile incubated for 0, 30, and 60 min, respectively, with APMA. MW = molecular weight.

Effect of Inhibitors on Azocoll Protease Activity

Azocoll proteolysis was inhibited by both MMP inhibitors, EDTA and GM 6001, but not by serine or cysteine protease inhibitors, such as E64, leupeptin, and PMSF (Figure 3). Cathepsin activity was not evident in the samples analyzed (data not shown).

Figure 3.

Figure 3.

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Effects of different protease inhibitors on azocoll protease activity. An asterisk (*) indicates P ≤ 0.05 (n = 3). E-64 = N-[N-(l-3-trans-carboxyirane-2-carbonyl)-l-leucyl]-agmatine, a cysteine protease inhibitor; GM 6001, a collagenase inhibitor (EMD Millipore, Billerica, MA); PMSF = phenylmethyl sulfonyl fluoride.

Affinity Purification and Molecular Characterization of MMP

The bands corresponding to 64, 50, and 42 kDa (Figure 4) were all identified as type IV collagenase preproprotein (NCBI reference sequence NP_989751.1) based on their PMF as listed in Table 1. The band at 64 kDa was identified using 20 peaks in the PMF (Figure 5) and LIFT-TOF/TOF (MS/MS) data corresponding to 5 peptide peaks at m/z 1,071, 1,563, 1,579, 1,670, and 2,107, respectively. Mascot search of the combined PMF (MS) and LIFT-TOF/TOF (MS/MS) data showed a significant protein hit (P ≥ 0.05, Mascot score 253; Matrixscience, 2013), with a sequence coverage of 29% and matching peptides distributed from 1 to 646 of the amino acid sequence. The peak at m/z 1,670 (Figure 6) had the highest Mascot ion score, 82, compared with the peaks at 2,107, 1,580, 1,564, and 1,071 with ion scores of 57, 28, 26, and 10 respectively. The band at 50 kDa was identified using 17 peaks (Table 1), including the same 5 peptides with LIFT-TOF/TOF (MS/MS) data. This identification was based on a lower Mascot score (250), with a 25% sequence coverage and peptide hits covering protein sequence from positions 1 to 590 (Table 1). Similarly the band at 42 kDa was identified based on a lower Mascot score of 220, sequence coverage of 26%, and peptide hits distributed from 1 to 492 of the amino acid sequence.

Figure 4.

Figure 4.

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Gelatin zymogram (a) and silver-stained protein bands (b) of gelatin-sepharose affinity purified bile.

Table 1.

List of peptides observed in 64, 50, and 42 kDa bands and expected m/z of peptides from matrix metllaproteinase (MMP2)

Peptide observed
Position Sequence Expected (m/z) (MH+) 64 kDa band (m/z) (MH+)1 50 kDa band (m/z) (MH+) 42 kDa band (m/z) (MH+)
1–12 MKTHSVFGFFFK 1,475.7 1,475.7 1,475.7 1,475.7
99–112 CGNPDVANYNFFPR 1,670.7 1,670.7 1,670.8 1,670.7
125–143 IIGYTPDLDPETVDDAFAR 2,108.0 2,108.0 2,108.1 2,108.0
144–155 AFKVWSDVTPLR — 1,418.7 — 1,418.7
147–155 VWSDVTPLR 1,072.5 1,072.5 1,072.4 1,072.5
159–172 INDGEADIMINFGR 1,564.7 1,564.7 1,564.7 1,564.7
159–172 INDGEADIMINFGR oxidation(M) 1,580.7 1,580.7 1,580.7 1,580.7
173–184 WEHGDGYPFDGK 1,407.6 1,407.6 1,407.6 1,407.6
250–260 NDGFLWCSTTK 1,328.6 1,328.6 — 1,328.6
261–289 DFDADGKYGFCPHESLFTMGGNGDGQPCK oxidation (M) — — 3,223.6 3,223.6
294–307 FQGQSYDQCTTEGR 1,676.7 1,676.7 1,676.7 1,676.6
313–322 WCGTTEDYDR 1,302.6 — 1,302.5 1,302.5
370–382 LWCASTSSYDDDR 1,575.6 1,575.7 — 1,575.6
370–383 LWCASTSSYDDDRK 1,703.7 1,703.7 1,703.7 1,703.7
474–485 HDIVFDGVAQIR 1,369.6 1,369.8 — 1,369.6
474–492 HDIVFDGVAQIRGEIFFFK — — 2,238.2 2,238.2
486–494 GEIFFFKDR 1,158.6 1,158.6 — 1,158.5
523–534 IDAVYESPQDEK 1,393.6 1,393.7 — 1,393.6
558–570 KLTSLGLPPDVQR 1,423.8 — — 1,423.7
559–570 LTSLGLPPDVQR 1,295.7 1,295.6 — 1,295.7
583–590 TYIFSGDR 958.4 958.5 — 958.4
637–646 DQYYLQMEDK 1,332.5 — — 1,332.5
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1Protonated molecule.

Figure 5.

Figure 5.

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Peptide mass fingerprint (PMF) of the 64-kDa protein band. An asterisk (*) indicates the peptide peaks were subjected to MS/MS fragmentation. Values in parentheses correspond to the amino acid sequences of chicken MMP2. Color version available in the online PDF.

Figure 6.

Figure 6.

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Tandem mass spectrometry of m/z 1,670.66 peptides showing b and y fragmentation ions derived from N and C terminus of the peptide, respectively, with corresponding sequence information, using a 1-letter amino acid code. Color version available in the online PDF.

Effect of Feed Supplements on Bile MMP Activity

Chickens fed diets containing 4% gelatin showed a statistically significant increase in MMP activities based on azocoll assay (Figure 7). Both gelatin and milk powder supplements increased gelationolytic activities determined by zymogram densitometry (Figure 8).

Figure 7.

Figure 7.

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Effect of different feed supplements on bile protease activity indicated by optical density (OD) of azocoll hydrolysis associated dye release (n = 5). Dissimilar letters (a,b) indicate significant statistical differences at P ≤ 0.05.

Figure 8.

Figure 8.

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Effect of different feed supplements on bile gelatinolytic activity analyzed by densitometry. Dissimilar letters (a–c) over bars indicate significant differences (P ≤ 0.05).

DISCUSSION

Bile plays an important role in digestive physiology, especially in the emulsification and digestion of fat (Tuchweber et al., 1996). We hypothesized that biliary MMP perhaps aids in the digestion of native collagenous proteins, which constitute a significant fraction of body proteins of both vertebrates and invertebrates (Duke, 1997; Engel, 1997; Klasing, 1998). Although collagens are assumed to be digested by gastric enzymes at low pH, the fibrous interstitial collagens are generally resistant to proteases, such as pepsin, chymotrypsin, and trypsin, unless denatured (Bornstein et al., 1966; Harkness et al., 1978). Cysteine cathepsins, which can digest collagens under low pH, are not known to occur in normal gastric secretions other than in some pathological conditions (Nagy et al., 1997; Reiser et al., 2010). The mammalian MMP, such as MMP1 and 3, but not MMP2, are capable of denaturing and cleaving interstitial, fibrillar collagens under neutral pH conditions (Fields, 2013). Our results showed that MMP protein bands in avian bile belonged to 72 kDa type IV collagenase, also known as MMP2, or gelatinase A (Aimes and Quigley, 1995; Hahn-Dantona et al., 2000; Murphy and Nagase, 2008). The lower MW bands (50 and 42 kDa) appear to be the truncated version of the same MMP2 because they lacked sequence coverage in the C-terminal domain compared with 64-kDa band (Table 1). In MMP2, the signal, propeptide and catalytic domains are located near the N terminus and the hemopexin domain at the C terminus that binds substrates and tissue inhibitors of MMP (Patterson et al., 2001; Morgunova et al., 2002; Piccard et al., 2007). Chicken MMP2 has collagenase activity because it can denature and cleave triple helical collagens to produce characteristic three-fourths and one-fourth fragments (Aimes and Quigley, 1995; Hahn-Dantona, Aimes and Quigley, 2000; Patterson, Atkinson, Knauper and Murphy, 2001); hence, it is capable of denaturing and digesting fibrillar collagens.

Next we asked if MMP was critical to the digestive process of the birds, then posited that it may be modulated by their dietary factors resulting in an increase of their digestive activities. Diet-induced adaptive modulation of digestive enzymes has been observed in birds (Karasov and Hume, 1997; Karasov et al., 2011; Brzęk et al., 2013). Although direct correlation of dietary protein content and digestive proteases were demonstrated in mammals (Hara et al., 2000), such a correlation has not been established in birds. Our results with azocoll protease activity assay and densitometry of gelatinolytic bands showed that the gelatin-supplemented diets increased bile MMP levels, although a modest increase by milk was observed. Nonetheless, the gelatin-induced elevation of bile MMP suggests that food types may regulate adaptive modulation of their function.

Apart from collagen digestion, we cannot exclude the possibility of other gastrointestinal functions of MMP, such as their ability to activate of growth factors, antimicrobial proteins, and other proteases, including MMP and several receptor proteins, which need to be explored in the context of digestive physiology (Amadesi and Bunnett, 2004; Sengupta and MacDonald, 2007; Nagase and Visse, 2009). In conclusion, our results show that chicken bile constitutively secretes type IV collagenase (MMP2), which can then be modulated by their dietary constituents, and these enzymes possibly help in the digestion of ECM proteins in their diets.

Acknowledgments

We thank Scott Zornes, Sonia Tsai, and Wally McDonner (USDA-Agricultural Research Service, Poultry Production and Product Safety Research Unit) for assistance. Part of the study was carried out in the Statewide Mass Spectrometry Facility supported by NIH grant P30 GM103450 to the University of Arkansas.

REFERENCES

  1. Aimes R. T., Quigley J. P. Matrix metalloproteinase-2 is an interstitial collagenase. Inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments. J. Biol. Chem. 1995;270:5872–5876. doi: 10.1074/jbc.270.11.5872. [DOI] [PubMed] [Google Scholar]
  2. Amadesi S., Bunnett N. Protease-activated receptors: Protease signaling in the gastrointestinal tract. Curr. Opin. Pharmacol. 2004;4:551–556. doi: 10.1016/j.coph.2004.08.004. [DOI] [PubMed] [Google Scholar]
  3. Bornstein P., Kang A. H., Piez K. A. The limited cleavage of native collagen with chymotrypsin, trypsin, and cyanogen bromide. Biochemistry. 1966;5:3803–3812. [Google Scholar]
  4. Brzęk P., Ciminari M. E., Kohl K. D., Lessner K., Karasov W. H., Caviedes-Vidal E. Effect of age and diet composition on activity of pancreatic enzymes in birds. J. Comp. Physiol. B. 2013;183:685–697. doi: 10.1007/s00360-012-0731-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chung L., Dinakarpandian D., Yoshida N., Lauer-Fields J. L., Fields G. B., Visse R., Nagase H. Collagenase unwinds triple-helical collagen prior to peptide bond hydrolysis. EMBO J. 2004;23:3020–3030. doi: 10.1038/sj.emboj.7600318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Consolo M., Amoroso A., Spandidos D. A., Mazzarino M. C. Matrix metalloproteinases and their inhibitors as markers of inflammation and fibrosis in chronic liver disease. Int. J. Mol. Med. 2009;24:143–152. doi: 10.3892/ijmm_00000217. [DOI] [PubMed] [Google Scholar]
  7. Duke G. E. Gastrointestinal physiology and nutrition in wild birds. Proc. Nutr. Soc. 1997;56:1049–1056. doi: 10.1079/pns19970109. [DOI] [PubMed] [Google Scholar]
  8. Engel J. Versatile collagens in invertebrates. Science. 1997;277:1785–1786. doi: 10.1126/science.277.5333.1785. [DOI] [PubMed] [Google Scholar]
  9. Etherington D. J. Collagen degradation. Ann. Rheum. Dis. 1977;36:14–17. [Google Scholar]
  10. Fields G. B. Interstitial collagen catabolism. J. Biol. Chem. 2013;288:8785–8793. doi: 10.1074/jbc.R113.451211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hahn-Dantona E. A., Aimes R. T., Quigley J. P. The isolation, characterization, and molecular cloning of a 75-kDa gelatinase B-like enzyme, a member of the matrix metalloproteinase (MMP) family. An avian enzyme that is MMP-9-like in its cell expression pattern but diverges from mammalian gelatinase B in sequence and biochemical properties. J. Biol. Chem. 2000;275:40827–40838. doi: 10.1074/jbc.M006234200. [DOI] [PubMed] [Google Scholar]
  12. Hara H., Ohyama S., Hira T. Luminal dietary protein, not amino acids, induces pancreatic protease via CCK in pancreaticobiliary-diverted rats. Am. J. Physiol. Gastrointest. Liver Physiol. 2000;278:G937–G945. doi: 10.1152/ajpgi.2000.278.6.G937. [DOI] [PubMed] [Google Scholar]
  13. Harkness M. L., Harkness R. D., Venn M. F. Digestion of native collagen in the gut. Gut. 1978;19:240–243. doi: 10.1136/gut.19.3.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hauser-Davis R. A., Lima A. A., Ziolli R. L., Campos R. C. First-time report of metalloproteinases in fish bile and their potential as bioindicators regarding environmental contamination. Aquat. Toxicol. 2012;110–111:99–106. doi: 10.1016/j.aquatox.2011.12.014. [DOI] [PubMed] [Google Scholar]
  15. Hellman U., Wernstedt C., Gonez J., Heldin C. H. Improvement of an “In-Gel” digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal. Biochem. 1995;224:451–455. doi: 10.1006/abio.1995.1070. [DOI] [PubMed] [Google Scholar]
  16. Hirashita T., Iwashita Y., Ohta M., Komori Y., Eguchi H., Yada K., Kitano S. Expression of matrix metalloproteinase-7 is an unfavorable prognostic factor in intrahepatic cholangiocarcinoma. J. Gastrointest. Surg. 2012;16:842–848. doi: 10.1007/s11605-011-1813-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hornbuckle W. T. BC. Gastrointestinal function. In: Kaneko J., Harvey J., Bruss M., editors. Clinical Biochemistry of Domestic Animals. San Diego, CA: Academic Press; 1997. pp. 367–406. [Google Scholar]
  18. Iyer R. P., Patterson N. L., Fields G. B., Lindsey M. L. The history of matrix metalloproteinases: Milestones, myths, and misperceptions. Am. J. Physiol. Heart Circ. Physiol. 2012;303:H919–H930. doi: 10.1152/ajpheart.00577.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jiang, N., N. S. Tan, B. Ho, and J. L. Ding. 2007. Azocoll protease activity assay. Protocol Exchange, 10.
  20. Karasov W. H., Hume I. D. Vertebrate gastrointestinal system. In: Dantzler W. H., editor. Handbook of Physiology. New York, NY: Oxford University Press; 1997. pp. 407–480. [Google Scholar]
  21. Karasov W. H., Martínez del Rio C., Caviedes-Vidal E. Ecological physiology of diet and digestive systems. Annu. Rev. Physiol. 2011;73:69–93. doi: 10.1146/annurev-physiol-012110-142152. [DOI] [PubMed] [Google Scholar]
  22. Kirimlioğlu H., Türkmen I., Başsüllü N., Dirican A., Karadağ N., Kirimlioğlu V. The expression of matrix metalloproteinases in intrahepatic cholangiocarcinoma, hilar (Klatskin tumor), middle and distal extrahepatic cholangiocarcinoma, gallbladder cancer, and ampullary carcinoma: Role of matrix metalloproteinases in tumor progression and prognosis. Turk. J. Gastroenterol. 2009;20:41–47. [PubMed] [Google Scholar]
  23. Klasing K. C. Wallingford, UK: CAB International; 1998. Comparative Avian Nutrition. [Google Scholar]
  24. Matrixscience. 2013. Mascot database search: MS/MS results interpretation. Accessed Mar. 24, 2013. http://www.matrixscience.com/help/interpretation_help.html.
  25. Morgunova E., Tuuttila A., Bergmann U., Tryggvason K. Structural insight into the complex formation of latent matrix metalloproteinase 2 with tissue inhibitor of metalloproteinase 2. Proc. Natl. Acad. Sci. USA. 2002;99:7414–7419. doi: 10.1073/pnas.102185399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Murphy G., Nagase H. Progress in matrix metalloproteinase research. Mol. Aspects Med. 2008;29:290–308. doi: 10.1016/j.mam.2008.05.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nagase H., Visse R. Drug Design of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease Applications. Hoboken, NJ: Wiley; 2009. Matrix metalloproteinases: An overview; pp. 487–517. [Google Scholar]
  28. Nagase H., Visse R., Murphy G. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc. Res. 2006;69:562–573. doi: 10.1016/j.cardiores.2005.12.002. [DOI] [PubMed] [Google Scholar]
  29. Nagy L., Johnson B. R., Hauschka P., Szabo S. Characterization of proteases and protease inhibitors in the rat stomach. Am. J. Physiol. 1997;272:G1151–G1158. doi: 10.1152/ajpgi.1997.272.5.G1151. [DOI] [PubMed] [Google Scholar]
  30. NRC. Washington, DC: Natl. Acad. Press; 1994. Nutrient Requirements of Poultry. [Google Scholar]
  31. Okada N., Ishida H., Murata N., Hashimoto D., Seyama Y., Kubota S. Matrix metalloproteinase-2 and -9 in bile as a marker of liver metastasis in colorectal cancer. Biochem. Biophys. Res. Commun. 2001;288:212–216. doi: 10.1006/bbrc.2001.5741. [DOI] [PubMed] [Google Scholar]
  32. Patterson M. L., Atkinson S. J., Knauper V., Murphy G. Specific collagenolysis by gelatinase A, MMP-2, is determined by the hemopexin domain and not the fibronectin-like domain. FEBS Lett. 2001;503:158–162. doi: 10.1016/s0014-5793(01)02723-5. [DOI] [PubMed] [Google Scholar]
  33. Piccard H., Van den Steen P. E., Opdenakker G. Hemopexin domains as multifunctional liganding modules in matrix metalloproteinases and other proteins. J. Leukoc. Biol. 2007;81:870–892. doi: 10.1189/jlb.1006629. [DOI] [PubMed] [Google Scholar]
  34. Rath N. C., Huff W. E., Huff G. R., Balog J. M., Xie H. Matrix metalloproteinase activities of turkey (Meleagris gallopavo) bile. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2001;130:97–105. doi: 10.1016/s1532-0456(01)00223-x. [DOI] [PubMed] [Google Scholar]
  35. Reiser J., Adair B., Reinheckel T. Specialized roles for cysteine cathepsins in health and disease. J. Clin. Invest. 2010;120:3421–3431. doi: 10.1172/JCI42918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rosenfeld J., Capdevielle J., Guillemot J. C., Ferrara P. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem. 1992;203:173–179. doi: 10.1016/0003-2697(92)90061-b. [DOI] [PubMed] [Google Scholar]
  37. Sekhon B. S. Matrix metalloproteinases—An overview. Res Rep Biol. 2010;2010:1–20. doi: 10.2147/RRB.S12043. [DOI] [Google Scholar]
  38. Sengupta N., MacDonald T. T. The role of matrix metalloproteinases in stromal/epithelial interactions in the gut. Physiology (Bethesda) 2007;22:401–409. doi: 10.1152/physiol.00027.2007. [DOI] [PubMed] [Google Scholar]
  39. Snoek-van Beurden P. A., Von den Hoff J. W. Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. Biotechniques. 2005;38:73–83. doi: 10.2144/05381RV01. [DOI] [PubMed] [Google Scholar]
  40. Syed I., Rathod J., Parmar M., Corcoran G. B., Ray S. D. Matrix metalloproteinase-9, -10, and -12, MDM2 and p53 expression in mouse liver during dimethylnitrosamine-induced oxidative stress and genomic injury. Mol. Cell. Biochem. 2012;365:351–361. doi: 10.1007/s11010-012-1277-z. [DOI] [PubMed] [Google Scholar]
  41. Trauner M., Boyer J. L. Bile salt transporters: molecular characterization, function, and regulation. Physiol. Rev. 2003;83:633–671. doi: 10.1152/physrev.00027.2002. [DOI] [PubMed] [Google Scholar]
  42. Tuchweber B., Yousef I., Ferland G., Perea A. Nutrition and bile formation. Nutr. Res. 1996;16:1041–1080. [Google Scholar]
  43. Wilder C. L., Park K. Y., Keegan P. M., Platt M. O. Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues. Arch. Biochem. Biophys. 2011;516:52–57. doi: 10.1016/j.abb.2011.09.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhang J. W., Gottschall P. E. Zymographic measurement of gelatinase activity in brain tissue after detergent extraction and affinity-support purification. J. Neurosci. Methods. 1997;76:15–20. doi: 10.1016/s0165-0270(97)00065-4. [DOI] [PubMed] [Google Scholar]

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