Purificação, caracterização química e atividade de proteases e inibidores de proteases durante a germinação e em eventos pré e pós-germinativos de sementes de Parkia multijuga
During seed germination, storage proteins are accumulated and degraded to support the major metabolic events involved in development new cells and tissues. The mobilization of proteins occurs through the activity of proteases, which can be regulated through the presence of proteases inhibitors th...
| Autor principal: | Chevreuil, Larissa Ramos |
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| Grau: | Tese |
| Idioma: | por |
| Publicado em: |
Instituto Nacional de Pesquisas da Amazônia - INPA
2020
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| Assuntos: | |
| Acesso em linha: |
https://repositorio.inpa.gov.br/handle/1/12823 http://lattes.cnpq.br/8899452024207330 |
| Resumo: |
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During seed germination, storage proteins are accumulated and degraded to support
the major metabolic events involved in development new cells and tissues. The
mobilization of proteins occurs through the activity of proteases, which can be
regulated through the presence of proteases inhibitors that prevent premature
hydrolysis of the storage proteins. The objective of this research was to investigate
the activity of proteases and protease inhibitors during germination and pre-and postgermination
of seeds of Parkia multijuga and purify trypsin inhibitors. Thereby, the
seeds after breaking dormancy, they were germinated in plastic pots containing
vermiculite and packed in germination chambers at 25ºC. The collection stages were
quiescent seeds (SQ), after 24 h of imbibition (EM), radicle protrusion (RA),
expansion of cotyledon node (NO) and issue shoot (PA). Proteins were extracted into
0.15 M NaCl. The activities of serine proteinases and trypsin and chymotrypsin
inhibitors were performed using BAPNA and azocasein as substrates. The activity of
cysteine proteases and papain and bromelain inhibitors using BANA and azocasein,
respectively. Trypsin inhibitors were purified by affinity chromatography usin trypsin-
Sepharose 4B and ion exchange on HiTrap DEAE FF. The purification and the
mobilization of proteins was performed by electrophoresis on SDS-PAGE and 2D
gels. During seed germination of P. multijuga observed a significant decrease in the
protein content after RA remaining until PA was confirmed by 2D gels, wich show
proteins degradation in the range of 35 to 75 kDa in PA, while 9 kDa proteins were
synthesized during EM. Regarding the inhibitory activity, serine protease inhibition
showed high stability during all stages studied, of which trypsin inhibition was 20
times higher than chymotrypsin. Inhibition of cysteine proteases is more variable,
observing for maximum inhibition of papain and bromelain in EM and NO,
respectively. The activities of serine and cysteine proteases presented themselves to
constant NO, with an increase in PA. The purification of trypsin inhibitors involved
two chromatographic steps, which were similar to the profiles obtained in all stages
studied, indicating the purification of the same molecule or isoinhibitors in different
stages studied. From the purification of trypsin inhibitors (PmTI1 and PmTI2) SQ,
these proteins are characterized by presenting against trypsin inhibitory specificity,
high stability against variation of temperature and pH and amino acid sequence
homology to the Bowman-type inhibitors Birk from other Fabaceae species. Thus,
serine and cysteine proteases inhibitors could participate in the regulation of
protease activity during germination and events pre and post-germination of seeds of
P. multijuga, with the trypsin inhibitors Bowman-Birk type present throughout the
process of formation of the seedling. |