Zymography Principles.

Zymography, the detection, identification, and even quantification of enzyme activity fractionated by gel electrophoresis, has received increasing attention in the last years, as revealed by the number of articles published. A number of enzymes are routinely detected by zymography, especially with clinical interest. This introductory chapter reviews the major principles behind zymography. New advances of this method are basically focused towards two-dimensional zymography and transfer zymography as will be explained in the rest of the chapters. Some general considerations when performing the experiments are outlined as well as the major troubleshooting and safety issues necessary for correct development of the electrophoresis.

Two-Dimensional Zymography of Proteases from Steatotic Duck Liver.

Protease activity present in liver cells with steatosis can be electrophoretically characterized. Zymographic techniques allow semi-quantitative results, successfully detecting cathepsin and metalloprotease activity using polyacrylamide gels copolymerized with gelatin and quantified by densitometry. By using specific inhibitors, the identity of the proteases can be confirmed. 2D zymography allows the determination of both M r. and pI of the metalloprotease and cathepsin activity present in the homogenates. The analysis of liver proteases activities in force fed ducks may elucidate the mechanisms behind steatosis development.

Detection of Guaiacol Peroxidase on Electrophoretic Gels.

It is possible to analyze peroxidase (POD) from different vegetable sources by electrophoresis. Zymography, i.e., a SDS-PAGE method to detect enzyme activity, is used to specifically detect POD activity and to visualize the total protein profile. For this purpose, we describe how a radish homogenate is prepared and submitted first to electrophoresis, and then, the POD activity present in the gel is reactivated and selectively stained using guaiacol as substrate. After scanning the gel, the same gel is further stained with Coomassie blue to determine the whole protein profile of the sample.

Sequential Detection of Thermophilic Lipase and Protease by Zymography.

Lipase and protease present in cell-free fractions of thermophilic Bacillus sp. cultures were analyzed by polyacrylamide gel (PAG) electrophoresis. After run, the gel is electrotransferred to another PAG copolymerized with glycerol tributyrate, olive oil, and gelatin. This multi-substrate gel was incubated first for lipase detection, until bands appeared, and then stained with Coomassie for protease detection. Advantages of this sequential procedure are the detection of two different enzyme activities on a single PAG, beside time and resource saving.

Zymography Detection of a Bacterial Extracellular Thermoalkaline Esterase/Lipase Activity.

Lipases are esterases that occur widely in nature, yet those with commercial relevance are exclusively from microbial origin. Glycerol and long-chain fatty acids are the products after hydrolysis of esters bonds in saponifiable lipids catalyzed by lipases. In this work, we describe lipase/esterase activity contained in cell-free fractions from thermophilic bacteria, cultured in medium containing olive oil. Analysis of the cell-free fractions by electrotransference zymography, using tributyrin as substrate, revealed bands corresponding to lipase activity. The method is simple, fast, and inexpensive.

Guaiacol peroxidase zymography for the undergraduate laboratory.

This laboratory exercise presents a novel way to introduce undergraduate students to the specific detection of enzymatic activity by electrophoresis. First, students prepare a crude peroxidase extract and then analyze the homogenate via electrophoresis. Zymography, that is, a SDS-PAGE method to detect enzyme activity, is used to specifically detect peroxidase activity and furthermore, to analyze the total protein profile. After the assay, students may estimate the apparent molecular mass of the enzyme and discuss its structure. After the 4-h experiment, students gain knowledge concerning biological sample preparation, gel preparation, electrophoresis, and the importance of specific staining procedures for the detection of enzymatic activity.

Enhancement of sequential zymography technique for the detection of thermophilic lipases and proteases.

Analysis of lipases and proteases present in cell-free fractions of thermophilic Bacillus sp. cultures were performed in an enhanced sequential zymography method. After the PAGE run, the gel was electrotransferred to another polyacrylamide gel containing a mixture of glycerol tributyrate, olive oil and gelatin. After transference, this substrate-mix gel was incubated for lipase detection, until bands appeared, and later stained with CBB for protease detection. Assets are, besides detecting two enzymes on a single gel, time and material saving.

Advances in zymography techniques and patents regarding protease analysis.

Detection of enzymatic activity on gel electrophoresis, namely zymography, is a technique that has received increasing attention in the last 10 years, according to the number of articles published. A growing amount of enzymes, mainly proteases, are now routinely detected by zymography. Detailed analytical studies are beginning to be published, as well as new patents have been developed. This new article updates the information covered in our last review, condensing the recent publications dealing with the identification of proteolytic enzymes in electrophoretic gel supports and its variations. The new advances of this method are basically focused towards two dimensional zymography and transfer zymography. Though comparatively fewer patents have been published, they basically coincide in the study of matrix metalloproteases. The tendency is foreseen to be very productive in the area of zymoproteomics, combining electrophoresis and mass spectrometry for the analysis of proteases.

Protease analysis by zymography: a review on techniques and patents.

Zymography, the detection of enzymatic activity on gel electrophoresis, has been a technique described in the literature for at least in the past 50 years. Although a diverse amount of enzymes, especially proteases, have been detected, advances and improvements have been slower in comparison with other molecular biology, biotechnology and chromatography techniques. Most of the reviews and patents published focus on the technique as an element for enzymatic testing, but detailed analytical studies are scarce. Patents referring to zymography per se are few and the technique itself is hardly an important issue in titles or keywords in many scientific publications. This review covers a small condensation of the works published so far dealing with the identification of proteolytic enzymes in electrophoretic gel supports and its variations like 2-D zymography, real-time zymography, and in-situ zymography. Moreover, a scope will be given to visualize the new tendencies of this method, regarding substrates used and activity visualization. What to expect from zymography in the near future is also approached.

Protein kinase CK1 from Trypanosoma cruzi.

A protein kinase activity, which uses casein as a substrate, has been purified to homogeneity from the epimastigote stage of Trypanosoma cruzi, by sequential chromatography on Q sepharose, heparin sepharose, phenyl sepharose, and alpha-casein agarose. An apparent molecular weight of 36,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography and sedimentation analyses demonstrated that the purified native enzyme is a monomer with a sedimentation coefficient of 2.9 S. The hydrodynamic parameters indicated that the shape of the protein is globular with a frictional ratio f/f(o) = 1.36 and a Stokes radius of 27.7 A. When two selective peptide substrates for protein kinases CK1 and CK2 were used (RRKDLHDDEEDEAM. SITA and RRRADDSDDDDD, respectively), the purified kinase was shown to predominantly phosphorylate the CK1-specific peptide. Additionally, the enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, a specific inactivator of CK1s from mammals. Based on these results, we concluded that the purified kinase corresponds to a parasite CK1.

Trypanosoma cruzi: in vitro phosphorylation of tubulin by a protein kinase CK2-like enzyme.

One predominant 55-kDa polypeptide was phosphorylated in vitro in Trypanosoma cruzi homogenates prepared from three differentiation stages: epimastigotes, trypomastigotes, and spheromastigotes. Anti-alpha and anti-beta tubulin monoclonal antibodies immunoprecipitated the phosphorylated 55-kDa polypeptide from epimastigote extracts. Phosphoserine was the only residue phosphorylated in vitro in the 55-kDa polypeptide and in immunoprecipitated alpha tubulin. The phosphorylation of both the 55-kDa polypeptide and exogenously added casein was inhibited with GTP, heparin, and 2,3-bisphosphoglycerate in a dose-dependent manner, indicating the involvement of a CK2-like protein kinase. Moreover, when tubulin was isolated from an epimastigote homogenate by ultracentrifugation, followed by DEAE-Sephacel chromatography, a protein kinase that phosphorylated tubulin and casein co-purified with this cytoskeletal component. This result suggests an association between tubulin and its corresponding protein kinase in T. cruzi.

Biochemical and enzymatic characterization of a partially purified casein kinase-1 like activity from Trypanosoma cruzi.

Two protein kinase activities that use casein as a substrate, Q-I and Q-II, were identified in the epimastigote stage of Trypanosoma cruzi upon chromatography on Q-Sepharose. Q-I was purified further through concanavalin A-sepharose (Q-I*) to remove any trace of the contaminating protease cruzipain. The optimal activity for Q-I* was obtained at pH 8.0, 25 degreesC, 5 mM MgCl(2) and 75 mM NaCl. The size and pI of Q-I* were determined to be 33-36 kDa and 9.6, respectively. When two selective peptide substrates for casein kinases (CKs) (P1: RRKDLHDDEEDEAMSITA for CK1 and P2: RRRADDSDDDDD for CK2) were used, Q-I* was shown to specifically phosphorylate P1. Kinetic studies showed that Q-I* has a K(m) of 5.3 +/- 0.34 mg/ml for casein, 157.6 +/- 5.3 microM for P1 and 35.9 +/- 3.9 microM for ATP. The enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7) or 1-(5-chloroisoquinoline-8-sulfonyl) (CKI-8), two inactivators of mammalian CKs. CKI-7 behaved as a competitive inhibitor with respect to ATP, with a K(I) of 75-100 microM. Treatment with high concentrations of polylysine or heparin also resulted in a significant inhibition of Q-I*. Two well-known activators of mammalian CKs, spermine and spermidine, were also tested. Spermine and spermidine activated Q-I* in a dose-dependent manner. Based on the following characteristics: (1) the ionic strength required for elution from anion-exchange resins; (2) its molecular size and monomeric structure; (3) pI; (4) high level of specificity for P1; (5) inactivation by CKI-7 and CKI-8; and (6) insensitivity to GTP and low concentrations of heparin, we conclude that Q-I* belongs to the CK1 family of protein kinases.