Analysis of serine proteases from marine sponges by 2-D zymography.
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.
Wilkesman
Jeff G
JG
Departamento de Química, Facultad de Ciencias y Tecnología, Universidad de Carabobo, Valencia, Venezuela. jwilkesm@uc.edu.ve
Schröder
Heinz C
HC
D000818:Animals; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004789:Enzyme Activation; D008623:Mercaptoethanol / Q000737:chemistry; D008970:Molecular Weight; D011161:Porifera / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000032:analysis / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013379:Substrate Specificity; D014508:Urea / Q000737:chemistry
D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D014508:Urea; D001586:Benzoylarginine Nitroanilide; D012697:Serine Endopeptidases; D004229:Dithiothreitol