Two‐dimensional zymography for analysis of proteolytic enzymes in human pure pancreatic juice

Kaino, Seiji; Furui, Toshifumi; Hatano, Satoko; Kaino, Miyuki; Okita, Kiwamu; Nakamura, Kazuyuki

doi:10.1002/elps.1150190530

Cited by 13 publications

(12 citation statements)

References 27 publications

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“…However, similar studies only concerning the zymographic technique have been published [29][30][31], but no efforts have been made to observe the effects of denaturing conditions on proteolytic patterns, either in 1-D or 2-D zymography.…”

Section: Discussionmentioning

confidence: 95%

Analysis of serine proteases from marine sponges by 2‐D zymography

Wilkesman

Schröder

2007

Electrophoresis

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Proteolytic activities isolated from the marine demosponges Geodia cydonium and Suberites domuncula were analyzed by 2-D zymography, a technique that combines IEF and zymography. After purification, a 200 kDa proteolytically active protein band was obtained from G. cydonium when analyzed in gelatin copolymerized 1-D zymograms. The enzymatic activity was quantified using alpha-N-benzoyl-D-arginine p-nitroanilide (BAPNA) as a substrate and corresponded to a serine protease. The protease activity was resistant to urea and SDS. DTT and 2-mercaptoethanol (2-ME) did not significantly change the protease activity, but induced a shift in molecular mass of the proteolytic band to lower M(r) values as detected by zymography. Under mild denaturing conditions, lower M(r) bands (<200 kDa) were identified in 1-D zymograms, suggesting that the protease is composed of subunits which retain the catalytic activity. After 2-D zymography, the protease from G. cydonium revealed a pI of 8.0 and an M(r) shift from 200 to 66 kDa. To contrast these results, a cytosolic sample from S. domuncula was analyzed. The proteolytic activity of this sponge after 2-D zymography corresponded to an M(r) of 40 kDa and a pI of 4.0. The biological function of both sponge proteases is not yet known. This study demonstrates that mild denaturing conditions required for IEF may alter the interpretation of the 2-D zymography, and care must be taken during sample preparation.

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Section: Discussionmentioning

confidence: 95%

Analysis of serine proteases from marine sponges by 2‐D zymography

Wilkesman

Schröder

2007

Electrophoresis

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“…They are used in many clinical investigations and research activities of fluids and tissues [46,47]. We applied the lowest and highest concentrations of the respective inhibitor suggested by the supplier and investigated whether there was any effect onto the protein patterns by determination of qualitative and semiquantitative differences.…”

Section: Resultsmentioning

confidence: 99%

“…To date, differences in the protein patterns of exocrine pancreatic juice in 2-D PAGE of patients with pancreatic cancer or chronic pancreatitis and healthy subjects have been reported [28][29][30]. Mostly isoelectric focusing based on the Ampholine technique [30,31] and 2-D electrophoresis were performed [32,33]. These studies preferred Pefabloc SC [33] and aprotinin [34] for the inhibition of proteases.…”

Section: Introductionmentioning

confidence: 99%

“…Mostly isoelectric focusing based on the Ampholine technique [30,31] and 2-D electrophoresis were performed [32,33]. These studies preferred Pefabloc SC [33] and aprotinin [34] for the inhibition of proteases. However, due to the complexity of pancreatic protein patterns, and the presence of many proteolytic enzymes, an improved and faster preparation protocol was mandatory, in particularly for the preparation for mass spectrometry.…”

Section: Introductionmentioning

confidence: 99%

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Autoimmune pancreatic disease: Preparation of pancreatic juice for proteome analysis

et al. 2001

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The identification of pancreatic proteins is generally hampered by the high content and activity of proteases produced by this organ. The aim of this work was the development of a protocol for the analysis of pancreatic juice by two-dimensional (2-D) gel electrophoresis allowing consistent and reproducible protein analysis encompassed by high-resolution protein 2-D maps and subtle protein spot recognition without substantial losses due to proteases. Immobilized pH gradient (IPG) strips were used for the first dimension, the second dimension was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the key step was the sample preparation technique. Improvements were achieved by using several protease inhibitors (phenylmethylsulfonyl fluoride, aprotinin, L-1-chloro-3-[4-tosyl-amido]-7-amino-2-heptanine (TLCK)-HCI, Complete) to prevent degradation of the proteins. The application of different pH-ranges was a valuable step for getting an overview of the expressed protein pattern. These investigations resulted in well-resolved 2-D maps with a high reproducibility.

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“…A nonlinear pH 3-10 IPG strip was rehydrated with this sample for 16 h at 25C and subjected to isoelectric focusing 19 . Post-run, the strip was equilibrated with 0.05 M Tris-HCl, pH 8.8 containing 30% glycerol and placed on a 12.5% polyacrylamide gel using 1% agarose.…”

Section: Two-dimensional Zymographymentioning

confidence: 99%

Application of Transverse Urea Gradient Zymography for Structural and Functional Characterization of Proteolytic Enzymes

Sharma

Bhattacharyya

2016

Current Science

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Inactivation of an enzyme as it begins to unfold, along with the conformational perturbations which follow, can provide an insight into dynamics of the unfolding pathway. Urea gradient electrophoresis combined with zymography is a sensitive technique which provides a continuous visual profile of a proteolytic enzyme undergoing denaturation and inactivation simultaneously. Trypsin has been used as a reference protease to validate and standardize the method by correlating inactivation profile generated in zymography with a solution state assay. Stem bromelain, a cysteine endopeptidase was used as a case study to evaluate this methodology. The method highlighted the effect rendered by the substrate on the stability of the proteolytic domain of the enzyme, as it undergoes urea-induced unfolding. Transverse urea gradient zymography combined with molecular modelling of stem bromelain, where the disulphide bonds have been reduced, indicated that the evolutionary retention of Cys 23 -Cys 63 could be attributed to localized stabilization imparted by this bond to the catalytic site. This method encompasses various dimensions to extend the understanding of structure-function relationship in denaturant-induced unfolding pathways of proteases.Keywords: Protein unfolding, stem bromelain, urea gradient, zymography.TRANSVERSE urea gradient zymography (TUGZ) is an extended protocol based on the incorporation of transverse urea gradient in substrate polyacrylamide gels. Urea gradient electrophoresis provides a qualitative estimate of urea-induced conformational transitions whereas zymography identifies latent proteolytic activity under denaturing conditions [1][2][3][4][5][6] . Thus, the combination of two standard techniques can provide a comparative analysis of the unfolding behaviour of proteases having complex structures under variable conditions. There are fragmented reports where TUGZ was applied to verify stability of proteases against urea denaturation or to characterize proteolytic activity in a test sample 7,8 . In order to monitor the functional changes resulting from denaturant-induced unfolding, well-studied proteases such as trypsin, chymotrypsin and collagenase were selected which could validate and establish the general applicability of this method 9,10 . Poststandardization, the method was used to evaluate the behaviour of stem bromelain in the presence of urea.Crude extracts from the stem and fruit of pineapple plant (Ananas comosus) are called 'stem bromelain' and 'fruit bromelain' respectively. These bromelains contain a number of cysteine proteases having similar physiochemical properties along with other enzymes like peroxidase and enzyme inhibitors. These cysteine proteases are also called stem bromelain (SB) and fruit bromelain respectively, according to their sources. These cysteine proteases have highest abundance in the source where one component dominates. SB, a cysteine protease of 23.8 kDa, belongs to the ( + ) protein family 11 . It is the major enzyme present in pineapple stem extract. Some of th...

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Two‐dimensional zymography for analysis of proteolytic enzymes in human pure pancreatic juice

Cited by 13 publications

References 27 publications

Analysis of serine proteases from marine sponges by 2‐D zymography

Analysis of serine proteases from marine sponges by 2‐D zymography

Autoimmune pancreatic disease: Preparation of pancreatic juice for proteome analysis

Application of Transverse Urea Gradient Zymography for Structural and Functional Characterization of Proteolytic Enzymes

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