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Analysis of serine proteases from marine sponges by 2-D zymography.
Electrophoresis. 2007 Feb; 28(3):429-36.E

Abstract

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.

Authors+Show Affiliations

Departamento de Química, Facultad de Ciencias y Tecnología, Universidad de Carabobo, Valencia, Venezuela. jwilkesm@uc.edu.veNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17195259

Citation

Wilkesman, Jeff G., and Heinz C. Schröder. "Analysis of Serine Proteases From Marine Sponges By 2-D Zymography." Electrophoresis, vol. 28, no. 3, 2007, pp. 429-36.
Wilkesman JG, Schröder HC. Analysis of serine proteases from marine sponges by 2-D zymography. Electrophoresis. 2007;28(3):429-36.
Wilkesman, J. G., & Schröder, H. C. (2007). Analysis of serine proteases from marine sponges by 2-D zymography. Electrophoresis, 28(3), 429-36.
Wilkesman JG, Schröder HC. Analysis of Serine Proteases From Marine Sponges By 2-D Zymography. Electrophoresis. 2007;28(3):429-36. PubMed PMID: 17195259.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Analysis of serine proteases from marine sponges by 2-D zymography. AU - Wilkesman,Jeff G, AU - Schröder,Heinz C, PY - 2006/12/30/pubmed PY - 2007/5/11/medline PY - 2006/12/30/entrez SP - 429 EP - 36 JF - Electrophoresis JO - Electrophoresis VL - 28 IS - 3 N2 - 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. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/17195259/Analysis_of_serine_proteases_from_marine_sponges_by_2_D_zymography_ L2 - https://doi.org/10.1002/elps.200600332 DB - PRIME DP - Unbound Medicine ER -