RESUMO
The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 24, was investigated. Then, the variables studied were polyethylene glycol (PEG) molar mass (MPEG), concentrations of PEG (CPEG) and citrate (CCIT), and pH. The responses analyzed were the partition coefficient (K), activity yield (Y) and purification factor (PF). The thermodynamic parameters of the ATPS partition were estimated as a function of temperature. ATPS was able to pre-purify the protease (PF = 1.6) and obtained 84% activity yield. The thermodynamic parameters ΔG°m (-10.89 kJ mol-1), ΔHm (-5.0 kJ mol-1) and partition ΔSm (19.74 J mol-1 K-1) showed that the preferential migration of almost all protein contaminants of the crude extract to the salt-rich phase, while the preferred protease was the PEG rich phase. The extracted enzyme presents optimum temperature and pH at range of 40-50 °C and 9.0-11.0, respectively. Moreover, the enzyme was identified as serine protease based on inhibition profile. ATPS showed the satisfactory performance as the first step for Aspergillus tamarii Kita UCP1279 protease pre-purification.
Assuntos
Aspergillus/enzimologia , Polietilenoglicóis/química , Serina Proteases/biossíntese , Serina Proteases/isolamento & purificação , Citrato de Sódio/química , Termodinâmica , Água/química , Concentração de Íons de Hidrogênio , Íons/farmacologia , Metais/farmacologia , Peso Molecular , Inibidores de Proteases/farmacologia , Transdução de Sinais/efeitos dos fármacos , TemperaturaRESUMO
Prophenoloxidase activating factors (PPAFs) are a group of clip domain serine proteinases that can convert prophenoloxidase (pro-PO) to the active form of phenoloxidase (PO), causing melanization of pathogens. Here, two full-length PPAF cDNAs from Scylla paramamosain (SpPPAF1 and SpPPAF2) were cloned and characterized. The full-length SpPPAF1 cDNA was 1677 bp in length, including a 5'-untranslated region (UTR) of 52 bp, an open reading frame (ORF) of 1131 bp coding for a polypeptide of 376 amino acids, and a 3'-UTR of 494 bp. The full-length SpPPAF2 cDNA was 1808 bp in length, including a 5'-UTR of 88 bp, an ORF of 1125 bp coding for a polypeptide of 374 amino acids, and a 3'-UTR of 595 bp. The estimated molecular weight of SpPPAF1 and SpPPAF2 was 38.43 and 38.56 kDa with an isoelectric point of 7.54 and 7.14, respectively. Both SpPPAF1 and SpPPAF2 proteins consisted of a signal peptide, a characteristic structure of clip domain, and a carboxyl-terminal trypsin-like serine protease domain. Expression analysis by qRT-PCR showed that SpPPAF1 mRNA was mainly expressed in the gill, testis, and hemocytes, and SpPPAF2 mRNA was mainly expressed in hemocytes. In addition, SpPPAF1 and SpPPAF2 mRNA was expressed in a time-dependent manner after Vibrio parahaemolyticus challenge. The results showed that expression of both SpPPAF1 and SpPPAF2 was related to the bacterial challenge but the expression patterns differed. These findings suggest that SpPPAF is a serine proteinase and may be involved in the pro-PO activation pathway of the crab innate immune system.
Assuntos
Braquiúros/metabolismo , Catecol Oxidase/biossíntese , Precursores Enzimáticos/biossíntese , Serina Proteases/biossíntese , Sequência de Aminoácidos , Animais , Braquiúros/genética , Catecol Oxidase/genética , Clonagem Molecular/métodos , DNA Complementar/genética , Ativação Enzimática , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Estrutura Terciária de Proteína , Serina Proteases/química , Serina Proteases/genética , Serina Proteases/metabolismo , TranscriptomaRESUMO
Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.
Assuntos
Acanthamoeba/enzimologia , Acanthamoeba/virologia , Interações Hospedeiro-Parasita , Mimiviridae/crescimento & desenvolvimento , Serina Proteases/biossíntese , Esporos de Protozoários/crescimento & desenvolvimento , Acanthamoeba/crescimento & desenvolvimentoRESUMO
Somatic angiotensin-I converting enzyme (sACE) is a broadly distributed peptidase which plays a role in blood pressure and electrolyte homeostasis by the conversion of angiotensin I into angiotensin II. N-domain isoforms (nACE) with 65 and 90 kDa have been described in body fluids, tissues and mesangial cells (MC), and a 90 kDa nACE has been described only in spontaneously hypertensive rats. The aim of this study was to investigate the existence of proteolytic enzymes that may act in the hydrolysis of sACE generating nACEs in MC. After the confirmation of the presence of ACE sheddases in Immortalized MC (IMC), we purified and characterized these enzymes using fluorogenic substrates specifically designed for ACE sheddases. Purified enzyme identified as a serine protease by N-terminal sequence was able to generate nACE. In the present study, we described for the first time the presence of ACE sheddases in IMC, identified as serine proteases able to hydrolyze sACE in vitro. Further investigations are necessary to elucidate the mechanisms responsible for the expression and regulation of ACE sheddases in MC and their roles in the generation of nACEs, especially the 90 kDa form possibly related to hypertension.
Assuntos
Hipertensão/enzimologia , Peptidil Dipeptidase A/metabolismo , Proteólise , Serina Proteases/metabolismo , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Animais , Pressão Sanguínea , Humanos , Hipertensão/metabolismo , Células Mesangiais/enzimologia , Células Mesangiais/metabolismo , Peptidil Dipeptidase A/biossíntese , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/metabolismo , Ratos , Serina Proteases/biossínteseRESUMO
The composition and toxicological profile of the venom of the rattlesnake Crotalus simus in Mexico was analyzed at the subspecies and individual levels. Venoms of the subspecies C. s. simus, C. s. culminatus and C. s. tzabcan greatly differ in the expression of the heterodimeric neurotoxin complex 'crotoxin', with highest concentrations in C. s. simus, followed by C. s. tzabcan, whereas the venom of C. s. culminatus is almost devoid of this neurotoxic PLA2. This explains the large variation in lethality (highest in C. s. simus, which also exerts higher myotoxicity). Coagulant activity on plasma and fibrinogen occurs with the venoms of C. s. simus and C. s. tzabcan, being absent in C. s. culminatus which, in turn, presents higher crotamine-like activity. Proteomic analysis closely correlates with toxicological profiles, since the venom of C. s. simus has high amounts of crotoxin and of serine proteinases, whereas the venom of C. s. culminatus presents higher amounts of metalloproteinases and crotamine. This complex pattern of intraspecies venom variation provides valuable information for the diagnosis and clinical management of envenoming by this species in Mexico, as well as for the preparation of venom pools for the production and quality control of antivenoms. BIOLOGICAL SIGNIFICANCE: This study describes the variation in venom composition and activities of the three subspecies of Crotalus simus from Mexico. Results demonstrate that there is a notorious difference in these venoms, particularly regarding the content of the potent neurotoxic phospholipase A2 complex 'crotoxin'. In addition, other differences were observed regarding myotoxic and coagulant activities, and expression of the myotoxin 'crotamine'. These findings have implications in, at least, three levels: (a) the adaptive role of variations in venom composition; (b) the possible differences in the clinical manifestations of envenomings by these subspecies in Mexico; and (c) the design of venom mixtures for the preparation of antivenoms effective in the neutralization of the venoms of the three subspecies.
Assuntos
Crotalus/metabolismo , Crotoxina , Regulação da Expressão Gênica/fisiologia , Proteoma , Animais , Crotoxina/análise , Crotoxina/biossíntese , México , Fosfolipases A2/análise , Fosfolipases A2/biossíntese , Proteoma/análise , Proteoma/metabolismo , Proteômica , Serina Proteases/análise , Serina Proteases/biossíntese , Especificidade da EspécieRESUMO
Paecilomyces lilacinus (LPS 876) efficiently degraded keratin in chicken feather during submerged cultivation producing extracellular proteases. Characterization of crude protease activity was done including its compatibility in commercial detergents. Optimum pH and temperature were 10.0 and 60 °C, respectively. Protease activity was enhanced by Ca²âº but was strongly inhibited by PMSF and by Hg²âº suggesting the presence of thiol-dependent serine proteases. The crude protease showed extreme stability toward non-ionic (Tween 20, Tween 85, and Triton X-100) and anionic (SDS) surfactants, and relative stability toward oxidizing agent (H2O2 and sodium perborate). In addition, it showed excellent stability and compatibility with various solid and liquid commercial detergents from 30 to 50 °C. The enzyme preparation retained more than 95% of its initial activity with solid detergents (Ariel™ and Drive™) and 97% of its original activity with a liquid detergent (Ace™) after pre-incubation at 40 °C. The protective effect of polyols (propylene glycol, PEG 4000, and glycerol) on the heat inactivation was also examined and the best results were obtained with glycerol from 50 to 60 °C. Considering its promising properties, P. lilacinus enzymatic preparation may be considered as a candidate for use in biotechnological processes (i.e., as detergent additive) and in the processing of keratinous wastes.
Assuntos
Detergentes/farmacologia , Resíduos Industriais , Queratinas/metabolismo , Paecilomyces/metabolismo , Serina Proteases/biossíntese , Serina Proteases/metabolismo , Animais , Galinhas , Misturas Complexas/química , Misturas Complexas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Espaço Extracelular/enzimologia , Plumas , Temperatura Alta , Concentração de Íons de Hidrogênio , Metais/farmacologia , Oxidantes/farmacologia , Paecilomyces/citologia , Paecilomyces/enzimologia , Paecilomyces/crescimento & desenvolvimento , Serina Proteases/química , Inibidores de Serina Proteinase/farmacologiaRESUMO
Keratinases are exciting proteolytic enzymes that display the capability to degrade the insoluble protein keratin. These enzymes are produced by diverse microorganisms belonging to the Eucarya, Bacteria, and Archea domains. Keratinases display a great diversity in their biochemical and biophysical properties. Most keratinases are optimally active at neutral to alkaline pH and 40-60 degrees Celsius, but examples of microbial keratinolysis at alkalophilic and thermophilic conditions have been well documented. Several keratinases have been associated to the subtilisin family of serine-type proteases by analysis of their protein sequences. Studies with specific substrates and inhibitors indicated that keratinases are often serine or metalloproteases with preference for hydrophobic and aromatic residues at the P1 position. Keratinolytic enzymes have several current and potential applications in agroindustrial, pharmaceutical, and biomedical fields. Their use in biomass conversion into biofuels may address the increasing concern on energy conservation and recycling.