Kinetic characterization of inosine monophosphate dehydrogenase of Leishmania donovani

Adenine Aminohydrolase from Leishmania donovani

“…The differential centrifugation protocol that was employed has been shown previously to yield intact glycosomes (80,81). The AAH co-sedimented with APRT, a cytosolic marker (13), whereas IMPDH, a known glycosomal enzyme (57), was associated exclusively with the organellar (45,000 ϫ g) pellet (Fig. 4A).…”

“…Monospecific polyclonal antibodies raised against purified recombinant L. donovani APRT, HGPRT, and XPRT proteins in rabbits have been described (9 -11). Inosine monophosphate dehydrogenase (IMPDH) antiserum was produced in guinea pigs as reported previously (57), and mouse antiserum against amastigote-specific A2 protein (58) was generously provided by Dr. Greg Matlashewski from McGill University Faculty of Medicine. Mouse monoclonal anti-␣-tubulin antibody (DM1A) was obtained from EMD Chemicals (Gibbstown, NJ).…”

Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

“…Many other vital components of the purine salvage pathway, however, including HGPRT, XPRT, and IMPDH (11,12,27), are sequestered within the glycosome. The targeting of HGPRT, XPRT, and IMPDH to the glycosome is mediated by an archetypical COOH-terminal triad, peroxisomal targeting signal 1 (11,12,27), which is lacking in ADSS and ASL. ADSS and ASL also lack the well characterized NH 2 -terminal peroxisomal targeting signal 2 (67).…”

“…The TOPO TA Cloning kit, pCR2.1-TOPO vector, Champion TM pET200/D-TOPO expression vector, and BL21 Star TM (DE3) One Shot TM competent cells, as well as the goat anti-rabbit Oregon Green-, goat anti-mouse Oregon Green-, goat anti-mouse Alexa Fluor 568-, and goat anti-guinea pig rhodamine red-conjugated secondary fluorescent antibodies, were bought from Invitrogen. IMPDH antiserum was produced in guinea pigs as reported (27), and mouse monoclonal anti-␣-tubulin (DM1A) antibody was obtained from EMD Millipore (Billerica, MA). Mouse monoclonal antihuman influenza hemagglutinin (HA) and mouse monoclonal (JL-8) anti-GFP primary antibodies were bought from SigmaAldrich and BD Biosciences, respectively.…”

“…The immunofluorescence assay was performed on L. donovani promastigotes as described (11,12,27,48) using a 1:1000 dilution of recombinant anti-ASL antibody and a 1:10,000 dilution of secondary goat anti-rabbit Oregon Green-conjugated antibody (Invitrogen).…”