Detection of enzymes dehydrogenases and proteases inBrugia malayi filarial parasites |
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| Authors: | Y P Bhandary K N Krithika Sandeep Kulkarni M V R Reddy B C Harinath |
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| Institution: | (1) Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, 442 102 Wardha, Maharashtra, India;(2) Jamnalal Bajaj Tropical Disease Research Centre, Mahatma Gandhi Institute of Medical Sciences, Sevagram, 442 102 Wardha, Maharashtra, India |
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| Abstract: | Lymphatic filariasis caused mainly by infection fromW. bancrofti andB. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis ofB. malayi mf, infective larval and adult worm lysates for the activity of enzymes led to the demonstration of activities of three key
enzymes of carbohydrate metabolism viz., Malate dehydrogenase (MDH), Malic enzyme (ME) and Glucose-6-phosphate dehydrogenase
(G6PDH) in all the three stages of the parasite. The specific activity of all the three dehydrogenases was significantly high
in mf lysate compared to their activity in lysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel
to their activity inlysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel electrophoresis (PAGE)
using 7.5% non-gradient gel showed the presence of two isoforms of each of the three enzymes (MDH, ME & G6PDH) in mf lysate,
while only one form of each enzyme was present in L3 larval and adult worm lysates. Further proteolytic enzyme activity was demonstrated both in microfilarial and infective larval
lysates ofB. malayi. While both mf and L3 larval lysates showed optimal protease activity at alkaline pH of 9.0, the mf lysate showed increased activity also at pH
3.0. The infective larval lysate was markedly inhibited by Tosylamide-L-Phenylalanine chloromethyl ketone (TPCK), a thiol
protease inhibitor, while the protease activity in mf lysate was significantly inhibited by both TPCK and a serine protease
inhibitor Phenyl Methyl Sulphonyl Flouride (PMSF). In sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),
using gelatin copolymerized gel, the microfilarial lysate showed 3 protease molecules of 40 kDa, 180 kDa and 200 kDa and the
L3 larval lysate had 6 protease molecules of 18, 25, 37, 49, 70 and 200 kDa size. |
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