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 proteinase 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. Index Descriptors and Abbreviations: Crithidia guilhermei; trypanosomatid; extracellular proteinases; culture media; protein substrates; sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE); dl-Dithiothreitol (DTT); trans-epoxysuccinyl- l-leucylamido-(4-guanidino)-butane (E-64); phenylmethyl-sulfonyl fluoride (PMSF); yeast extract–peptone–sucrose (YEP-Suc); yeast extract–peptone–glucose (YEP-Glu); yeast extract–peptone–glycerol (YEP-Gly); brain heart infusion (BHI).