RESUMO
During shrimp larval development, changes occur in molecular components. Enzyme activity and mRNA expression of proteinases were assayed in Penaeus vannamei during larval development, which consists of 5 nauplius stages, 3 protozoeal stages, 3 mysis stages, and 12 postlarval stages. Trypsin activity reached a maximum at the beginning of postlarval stages 1 and 2, and significantly decreased in subsequent postlarval stages. Chymotrypsin activity increased at the third protozoeal stage, then significantly decreased in subsequent stages. Identification of proteinase by mass spectrometry and inhibitors allowed us to track their appearance in zymograms and to distinguish between isoenzymes. Chymotrypsin BI and BII had a distinguishing pattern of appearance during larval development, which could compensate for the reduction in trypsin activity. The mRNA content of isotrypsin 21, chymotrypsin 1, and zinc proteinase was differentially expressed in larvae. Zinc proteinase and chymotrypsin 1 mRNA were expressed at a basal content at the beginning of the protozoeal stages, increased by the end of the mysis stages and onward, while isotrypsin 21 mRNA had a peak at mysis stage 3. Transcript changes reflect transcriptional regulation of the proteinases tested. Proteinase mRNA in tissues, other than the digestive gland, suggests potentially different roles besides digestion during ontogeny.
Assuntos
Penaeidae/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Regulação Enzimológica da Expressão Gênica , Larva , Penaeidae/genética , Peptídeo Hidrolases/genética , RNA Mensageiro/genéticaRESUMO
Chymotrypsin from shrimp, Penaeus californiensis, was compared to Bos taurus chymotrypsin, and its structure-function relationship was studied. Catalytic efficiency toward synthetic substrate is lower, but it has a broad specificity and higher activity toward protein substrates, including collagen. It is active at pH 4-10 and fully active up to 50 °C for 2 h and at least nine days at room temperature. The activation peptide is twice as long as bovine chymotrypsinogen, has less disulfide bridges, and is a single polypeptide. Only one activation step is necessary from chymotrypsinogen to the mature enzyme. Postmortem implications in muscle softening and melanisation, resistance to temperature and pH and efficiency with proteinaceous substrates make chymotrypsin useful as a biotechnological tool in food processing. This makes shrimp processing wastes useful as a material for production of fine reagents.
Assuntos
Proteínas de Artrópodes/química , Quimotripsina/química , Penaeidae/enzimologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Biocatálise , Bovinos , Quimotripsina/genética , Sistema Digestório/química , Sistema Digestório/enzimologia , Estabilidade Enzimática , Dados de Sequência Molecular , Penaeidae/química , Alinhamento de SequênciaRESUMO
Three isotrypsins from digestive gland of Penaeus vannamei were purified and characterized by molecular, biochemical and kinetic parameters. Purified isotrypsins A, B, and C are glycoproteins with molecular masses between 30.2 and 32.9 kDa, and, therefore similar to other trypsins. The isoelectric points are anionic and different among the three isotrypsins: pH 3.5 for isotrypsin A, pH 3.0 for isotrypsin B, and pH 4.5 for isotrypsin C. Differences in the NH(2)-terminal amino acid sequences allowed us to define three different protein entities that match isotrypsins previously deduced by cDNA. Isoform C has higher physiological efficiency and specific activity, lower K(m), and requires higher concentrations of Ca(+2) to reach the same activity as the other two isotrypsins.
