Crithidia guilhermei: gelatin- and haemoglobin-degrading extracellular metalloproteinases
ABSTRACT The extracellular metalloproteinases of the insect trypanosomatid Crithidia guilhermei were characterized through the incorporation of different protein substrates (gelatin, casein, haemoglobin, and bovine serum albumin) into SDS-PAGE. Two gelatinases (60 and 80 kDa) showed ability to degrade casein as well and a 67-kDa enzyme presented the broadest specificity since it was also able to degrade casein and haemoglobin. Besides the 67-kDa extracellular proteinases detected on haemoglobin-SDS-PAGE, a 43-kDa haemoglobinase was only observed with this substrate. All C. guilhermei proteinases were incapable of using bovine serum albumin. C. guilhermei was also grown in four different culture media and the best proteinase production was reached using yeast extract-peptone medium containing glucose as the major carbon source. The results point to the importance of the use of distinct culture media and proteinaceous substrates on the characterization of extracellular proteolytic activities in trypanosomatids, since alterations in growth conditions and methods of detection could lead to distinct proteolytic profiles.
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ABSTRACT: Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.Brazilian Journal of Microbiology 10/2011; 42(4):1384-9. DOI:10.1590/S1517-838220110004000020 · 0.45 Impact Factor
Gel Electrophoresis - Advanced Techniques, 04/2012; , ISBN: 978-953-51-0457-5
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ABSTRACT: Cellular and extracellular peptidase profiles from non-infected and Trypanosoma cruzi-infected hepatocyte cell cultures were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing different copolymerized proteins as substrates. A 100 kDa metallopeptidase activity was detected in the cellular extracts and in the culture supernatant fluids of both systems, had the ability to exclusively degrade gelatin. However, non-infected hepatocytes produced an additional extracellular metallopeptidase of 85 kDa. In the non-infected and in the infected hepatocytes, a cysteine peptidase migrating in gelatin-SDS-PAGE at 60 kDa presented the broadest specificity, since it was also able to hydrolyze casein and hemoglobin. The 100 kDa component was only detected at alkaline pH and predominantly expressed in non-infected hepatocytes. Conversely, the 60 kDa cysteine peptidase was only observed in acidic condition and its production was robustly augmented in T. cruzi-infected cells, probably due to the cysteine peptidase synthesized by the parasites, as corroborated by immunoblotting assay using anti-cruzipain antibody. Collectively, these results suggest that peptidases may be involved in the interaction process between T. cruzi and hepatocytes in vitro.06/2008; 4(2):97-102.