Identification of two possible serpins in porcine pulmonary extract.
Dias, S.C.1; Sakauchi, D.4; Netto, S.L.3; Raw, I.1; Iourtov, D.2 & Kubrusly, F.S.1
1Centro de Biotecnologia; 2 Laboratório de Produção de Vacina Recombinante contra Hepatite B; 3Laboratório de Bioquímica e Biofísica; 4 Serviço de Controle de Qualidade – 1,2,3,4 Instituto Butantan, São Paulo, Brasil
The regulation of proteolytic enzymes in tissues by endogenous inhibitors is a critical requirement in the maintenance of homeostasis. Proteolytic enzymes are essential for the survival of all kinds of organisms, and are encoded by about 2% of all genes. Despite their life-giving functions, enzymes that breakdown proteins are very damaging in living systems, so their activities need to be kept strictly under control. The survey of enzyme inhibitors in porcine pulmonary extracts was the main goal this work. To isolate these inhibitors we developed protocols using frozen pig lungs, saline extraction in triethanolamine buffer, extract clarification by CDR and centrifugation followed by TCA precipitation and centrifugation. One of the pulmonary extracts obtained had the pH adjusted to 7.8. The sample was centrifuged and concentrated by tangencial flow filtration with a 30 kDa membrane. SDS-PAGE of the pulmonary extract and 30 kDa concentrate revealed bands among 60 and 8 kDa, 22 and 8 kDa for 30kDa filtrate. All the samples inhibited trypsin and elastase, but the 30 kDa filtrate showed less inhibitory activity than other samples. The 30 kDa concentrate and the pulmonary extract showed one band of approximately 60 kDa, whereas 30 kDa filtrate none in the reverse zymography technique. Our current hypothesis is that there were two serine proteinase inhibitors in porcine pulmonary extract approximately 60 e 22 kDa bands. We are now carrying out new experiments to purify the 60 and 22 kDa band, to determine theirs molecular mass and theirs partial amino acid sequence. Meanwhile we performed the APTT (the activated partial thromboplastin time) assay to evaluate coagulation abnormalities as we postulate that the 60 kDa serpin could be an antithrombin III like. The preliminary results using normal human and guinea pig plasma with the 30 kDa concentrate showed an increase of 13 to 25% in the clotting time, respectively.
Supported by: CAPES, CNPq, SADIA and Fundação Butantan.
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